Ginsenoside Rg1 inhibits apoptosis by increasing autophagy via the AMPK/mTOR signaling in serum deprivation macrophages

Ginsenoside Rg1 inhibits apoptosis by increasing autophagy via the AMPK/mTOR signaling in serum... Abstract Ginsenoside Rg1 (Rg1) has been widely used in a broad range of cardiovascular and cerebral–vascular diseases because of its unique therapeutic properties. However, the underlying mechanisms of Rg1 in the treatment of atherosclerosis have not been fully explored. This study sought to determine the precise molecular mechanisms on how Rg1 might be involved in regulating apoptosis in serum deprivation-induced Raw264.7 macrophages and primary bone marrow-derived macrophages. Results demonstrated that Rg1 treatment effectively suppressed apoptosis and the expression of phosphorylation level of mTOR induced by serum deprivation in Raw264.7 macrophages; the expressions of autophagic flux-related proteins including Atg5, Beclin1, microtubule-associated protein 1 light chain 3 (LC3), p62/SQSMT1, and the phosphorylation level of AMPK were concomitantly up-regulated. 3-Methyl-adennine (3-MA), the most widely used autophagy inhibitor, strongly up-regulated the expression of cleaved caspase-3, and blocked the anti-apoptosis function of Rg1 in macrophages. Importantly, autophagic flux was activated by Rg1, while Beclin1 knockdown partially abolished the anti-apoptosis of Rg1. Moreover, compound C, an AMPK inhibitor, partially decreased the expressions of phosphorylation of mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1, which were increased by Rg1. AICAR, an AMPK inducer, promoted the protein expressions of phosphorylation of mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1. In conclusion, Rg1 significantly suppressed apoptosis induced by serum deprivation in macrophages. Furthermore, Rg1 could effectively induce the autophagic flux by attenuating serum deprivation-induced apoptosis in Raw264.7 macrophages through activating the AMPK/mTOR signaling pathway. ginsenoside Rg1, autophagy, apoptosis, AMPK/mTOR, macrophages Introduction Atherosclerosis (AS) is the most common lesion in cardiovascular system. It is well recognized to be the common pathological basis in many cardiovascular and cerebrovascular diseases. Furthermore, it represents one of the most severe diseases threatening the health of human beings [1]. Macrophage apoptosis is one of the important characteristics in the occurrence and development of atheromatous plaque. It has been reported that macrophage apoptosis plays different roles in the progression during different stages of the formation of atheromatous plaque [2]. One such stress that induces cell death is serum deprivation. Serum contains growth factors, hormones, attachment and spreading factors, minerals, trace elements, lipids, and various other factors that are necessary for cell growth, differentiation, transport, attachment, spreading, pH maintenance, and protease inhibition [3]. Therefore, serum withdrawal causes the cells to stop growing. Serum deprivation also induces apoptosis, and reactive oxygen species (ROS) generation contributes to the cell death in serum-starved cells [4,5]. Indeed, excessive production of cellular ROS and apoptosis induced by growth factor withdrawal have also been found in AS [6]. In the early stage of AS, receptors on monocyte/macrophage are activated and coupled with the accumulation of macrophage and lymphocytes in the lesion sites. This would promote the development of AS on pathological changes. Therefore, the development of plaque may be effectively controlled in the early stage of pathological change to prevent apoptosis so as to reduce the accumulation of macrophages in the lesion sites. In the advanced stage, defective efferocytosis results in the decline of autophagy level; hence, a large number of apoptotic macrophages can not be effectively removed, which would lead to the increase of lipid core necrosis in the advanced plaque, resulting in the instability of plaque increase, and acute cardiovascular events [7]. In addition, studies have also shown that macrophage autophagy is involved in the occurrence and development of AS, thus underscoring the importance of the stability of plaque and the development of AS [8]. Autophagy is a catabolic pathway widely existing in eukaryotic cells and it can monitor and timely remove senescent and degenerative proteins and organelles in cells so as to maintain normal intracellular homeostasis, and render a certain protection on cells [9]. The formation of autophagosome with a double-membraned structure is a hallmark feature for the occurrence of autophagy. After formation, the autophagosome fuses with lysosome, producing autophagolysosome that degrades the contents wrapped by it using lysosomal enzymes to maintain the renewal of proteins and organelles, as well as cell homeostasis. Autophagy maintains organelle quality control by disposing of dysfunctional or damaged cellular organelles [10]. It is a critical cellular process that generally protects cells and organisms from stressors, such as nutrient deprivation, ROS increasing, DNA damage, and hypoxia. In addition to its role in normal physiology, autophagy also plays a role in pathological processes, such as cancer, neurodegeneration, and cardiovascular diseases [10–12]. Mizushima et al. [13] transferred GFP-light chain 3 (LC3)-II into the primary macrophages from mice, and reported distinctive changes of autophagy in cells stimulated with promoting AS factors. The same phenomenon has also been confirmed in vivo; compared with those in the wild-type mice, expression levels of Atg5 and GFP-LC3 in macrophages of atheromatous plaque are lower in GFP-LC3/(LDLr)−/− mice with a high fat diet [14]. These data further suggest that autophagy indeed is involved in the occurrence and development of AS. Additionally, Quimet et al. [15] reported that autophagy can hydrolyze lipids stored in macrophages, promote cholesterol efflux in cells, and provide certain protection for macrophages. Therefore, it would be possible to provide new therapeutic strategies and targets for the prevention and treatment of AS through a better understanding of the regulatory mechanisms of macrophage autophagy-related signals during the progression of AS. Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK), a kind of serine/threonine protein kinase, is activated by an increase of the AMP/ATP ratio [16], hypoxia, and ROS production [17,18]. AMPK also promotes autophagy indirectly through the suppression of mTORC1 activity or directly by the phosphorylation and activation of ULK1, which leads to autophagy induction [19–21]. There is ample evidence supporting that AMPK signal pathway plays an important role in the induction of autophagy by inhibiting mTOR signaling pathway, exhibiting a positive regulation as reported in a recent study [22]. Studies have also shown that starvation induced autophagy might be monitored by AMPK and Sirtuin 1 (Sirt1) [23,24], which has potential functions in regulating the proliferation, survival and death of cancer cells. Rg1 is a major active component in Panax notoginsengs saponins (Fig. 1). Rg1 has been shown to have effects on anti-oxidation, anti-senescence, anti-inflammation, and the relief of nerve function damage [25]. It plays broad roles in the pharmacology of the central nervous system, cardiovascular, endocrine system, and immune system. Rg1 also provides protection on cardiovascular system, such as anti-myocardial hypertrophy, anti-AS, and anti-acute myocardial ischemia. Studies have revealed that Rg1 can effectively inhibit macrophage-derived foam cell proliferation. Rg1 was shown to inhibit the excessive autophagy of myocardial cells in myocardial ischemia-reperfusion injury [26]. It has been reported that macrophage autophagy provides a certain protection on blood vessels in the early stage of AS; while in the advanced stage of AS, the loss of autophagy may cause macrophage apoptosis, efferocytosis defect, and the instability of plaque. In clinical practice, Rg1, as one of the major components in the compound preparation used for the treatment of cardiovascular diseases; however, there is only a modicum of information concerning its mechanisms in AS. Figure 1. View largeDownload slide Chemical structure of Rg1 Figure 1. View largeDownload slide Chemical structure of Rg1 Hence, this study was aimed to analyze the regulatory effects of Rg1 on the apoptosis of Raw264.7 macrophages induced by serum deprivation, and to find possible ways to reduce the unstable risk of plaque. Materials and Methods Chemicals and reagents Murine Raw264.7 macrophages were a generous gift from Professor Xiao-Chun Bai of the School of Basic Medical Sciences, Southern Medical University (Guangzhou, China). The Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco/BRL (Gaithersburg, USA). The fetal bovine serum (FBS) was obtained from Hyclone (Logan, USA). Immunoblot polyvinylidene difluoride membrane and the antibodies against β-actin, Bcl-2, Bax, cleaved caspase-3, p62/SQSMT1, AMPK and phosphor-AMPK, mTOR and phosphor-mTOR were purchased from Cell Signaling Technology (Danvers, USA). The antibodies against LC3, Beclin1, and Atg5 were obtained from Sigma (St Louis, USA). The TRITC-conjugated secondary antibody, horseradish peroxidase-conjugated secondary antibodies and ECL detection system were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). MDC stain was purchased from Leagene (Leagene, Beijing, China). Annexin V-FITC apoptosis detection kit was purchased from BD Bioscience (Franklin Lakes, USA). Granulocyte–macrophage colony stimulating factor (GM-CSF) was purchased from PeproTech (Rocky Hill, USA). 3-MA, compound C, AICAR, rapamycin, and chloroquine were purchased from MedChem Express (MCE, Monmouth Junction, USA). Lipofectamine 2000 was purchased from Invitrogen (Waltham, USA). StubRFP-SensGFP-LC3 lentivirus was purchased from Genechem (Genechem Co., Ltd, Shanghai, China). Unless otherwise mentioned, all other chemicals were purchased from Sigma. Ginsenoside Rg1 was purchased from Kunming Pharmaceutical Company (Kunming, China). Beclin1 siRNA was purchased from Cyagen (Guangzhou, China). Cells and treatments The murine Raw264.7 macrophages and Beclin1 siRNA Raw264.7 macrophages were cultured in DMEM containing 5% FBS and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin) at a density not exceeding 5 × 105 cells/ml and maintained at 37°C in a humidified incubator with 5% CO2. To harvest Raw264.7 macrophages, cells were trypsinized with 0.25% trypsin/EDTA in phosphate-buffered saline (PBS), then centrifuged (400 g for 10 min) and resuspended in serum-free DMEM. Cells were counted with a hemocytometer and trypan blue staining (0.4% trypan blue in PBS) showed more than 98% of the cells were viable. Primary bone marrow-derived macrophage (BMDM) were generated as described previously [27]. Lysis of red blood cells was performed using red blood cell lysis buffer (Beijing, Solarbio, China). Experiments in BMDM were carried out at a cell density of 5 × 105 cells/ml. BMDMs were cultured in DMEM supplemented with 3 μM GM-CSF, 10% FBS and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin). Rg1 (purity > 99%) at 50 μM was added at 1 h before serum deprivation stimulation. This time point was chosen to minimize the possibility of any direct interactions between Rg1 and serum deprivation. 3-MA (5 mM), a specific autophagy inhibitor, was used to determine if autophagy is involved in Rg1’s action on Raw264.7 macrophages. Compound C (10 μM), an AMPK inhibitor and AICAR (250 μM), an AMPK inducer were used to determine if the AMPK signal pathway is involved in Rg1’s action on Raw264.7 macrophages. StubRFP-SensGFP-LC3 assay The Raw264.7 macrophages (4 × 104 cells/ml) were plated on the coverslip of the 96-well plate and incubated with the StubRFP-SensGFP-LC3 lentivirus (MOI = 10, 1 × 107 TU/ml). New medium was changed after 10 h of incubation. After 72 h of infection, all images were captured with a fluorescence microscope (Olympus, Tokyo, Japan). The puncta of each cell were counted for each sample. The GFP signal is sensitive to the acidic conditions of the lysosome lumen, whereas mRFP is more stable. Therefore, colocalization of both GFP and mRFP fluorescence indicates a compartment that has not fused with a lysosome. In contrast, an mRFP signal without GFP indicates a compartment fused with a lysosome. Thus, autophagic flux was then measured by confocal counting of GFP+/mRFP+ (yellow) and GFP−/mRFP+ (red) puncta. siRNA transfection assay Beclin1 siRNA was used for transient transfection of Raw264.7 macrophages with Lipofectamine 2000 to suppress the expression of targeted gene. After 72 h of the initial transfection and treatment, cell samples were collected and analyzed using western blot analysis to confirm the expression of proteins. Flow cytometry Annexin V-FITC apoptosis detection kit was used to measure apoptosis of each group of Raw264.7 macrophages according to the manufacturer’s protocol. Both the supernatant and adherent Raw264.7 macrophages were collected and incubated with 100 μl of 1× binding buffer containing 5 μl of Annexin V-FITC and 5 μl of PI. Within 1 h after incubation in the dark at room temperature for 15 min, Raw264.7 macrophages were analyzed by flow cytometry. Three independent experiments were performed. Double immunofluorescence labeling Raw264.7 macrophages or BMDMs derived from various treatments were fixed with 4% paraformaldehyde in 0.1 M PBS for 20 min. After rinse with PBS, the coverslips with adherent cells were used for double immunofluorescence labeling. Raw264.7 macrophages were incubated with DAPI plus goat anti-rabbit LC3 (dilution 1:500) or cleaved caspase-3 (dilution 1:200). Subsequently, the cells were incubated with TRITC-conjugated secondary antibody for 1 h at 37°C. For negative controls, a set of culture slides were incubated under similar conditions without the primary antibodies. All images were captured with a fluorescence microscope (Olympus). Results are representative of three independent experiments. Quantification of cell death using Hoechst PI nuclear staining and fluorescence microscopy Hoechst PI nuclear staining was carried out as previously described [28] with slight modifications. Briefly, Raw264.7 macrophages (5 × 105 cells/ml) were incubated for 15 min at 37°C with Hoechst 33342 dye (10 mg/ml in PBS), centrifuged, washed once with PBS, and then resuspended at an approximate density of 1 × 106 cells/ml. PI (50 mg/ml in PBS) was added just before microscopy. Raw264.7 macrophages were visualized using an Olympus microscope (Olympus) equipped with a fluorescent light source and a UV-2A filter cube with excitation wavelength of 330–380 nm and barrier filter of 420 nm. Cell morphology was scored as follows: (i) ,viable cells had blue-stained nuclei with smooth appearance; 2, viable apoptotic cells had blue-stained nuclei with multiple bright specks of condensed chromatin; 3, non-viable apoptotic cells had red-stained nuclei with either multiple bright specks of fragmented chromatin or one or more spheres of condensed chromatin (significantly more compact than normal nuclei); and 4, non-viable necrotic cells had red-stained, smooth and homogeneous nuclei that were about the same size as normal (control) nuclei. Samples were randomized and examined after blinding. At least 200 cells were counted for each treatment. Experiments were repeated at least three times. Monodansylcadaverine staining The autofluorescent agent monodansylcadaverine (MDC) was introduced as a specific autophagolysosome marker to analyze the autophagic process. After treatment, Raw264.7 macrophages were stained with MDC (50 μM) at 37°C for 40 min. After incubation, Raw264.7 macrophages were washed three times with PBS, fixed with 5% paraformaldehyde, and immediately observed under a fluorescence microscope. Western blot analysis Raw264.7 macrophages were plated in 6-well plates at a density of 5 × 105 cells per well and cultured for 24 h. The cells were further incubated in FBS-free medium for at least 1 h before treatments. Stimulated Raw264.7 macrophages were harvested with ice-cold PBS and centrifuged at 12,000 g for 5 min at 4°C. Raw264.7 macrophages were lysed in ice-cold lysis buffer containing 2.5 mM Tris–HCl, pH 6.8, 25% glycerol, 2% sodium dodecyl sulfate (SDS), 0.01% bromphenol blue, and 5% β-mercaptoethanol. Cell lysates were centrifuged at 12,000 g for 5 min at 4°C, and then the supernatants were collected. Protein content was determined by using the BCA protein assay (Pierce, Rockford, USA). Equal amounts of protein (50 μg) were subject to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto immunoblot polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 2 h at room temperature, and then incubated separately with goat anti-rabbit antibodies against AMPK and phospho-AMPK, mTOR and phosphor-mTOR, β-actin, Bcl-2, Bax, cleaved caspase-3, Atg5, Beclin1, LC3, and p62/SQSMT1, antibodies (1:1000 dilution) that recognize different molecules under study at 4°C overnight. The membranes were washed three times with TBS-T, and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Membranes were washed again for three times with TBS-T and developed using the ECL detection system. Signals were exposed to Fuji Medical X-Ray Film (Fuji Photo Film Co., Ltd, Karagawa, Japan). Statistical analysis Statistical analysis of the data was carried out by one-way analysis of variance (ANOVA) followed by Scheffe’s post hoc test, using SPSS (SPSS Inc., Chicago, USA). Data were shown as the mean ± SEM (standard error of mean) obtained from three independent experiments. A value of P < 0.05 was considered significant. Results Rg1 inhibits apoptosis induced by serum deprivation in Raw264.7 macrophages A recent in vivo study reported that in the advanced stage of AS, macrophage apoptosis may promote atheromatous plaque necrosis, resulting in the increasing plaque instability [29]. In order to determine the regulatory effects of Rg1 on AS, the inhibition of Rg1 on Raw264.7 macrophage apoptosis was first investigated. Serum deprivation was used to treat Raw264.7 macrophages for different time (0, 12, 24, 36, 48, and 72 h). Results with Hochest33342/PI double fluorescence staining revealed that the fluorescence of cell nucleus was noticeably enhanced. There was increased incidence of karyopyknosis and karyoclasis in prolonged serum deprivation (0–72 h) (Fig. 2A). Under the condition of serum deprivation, different concentrations (20, 50, 100, and 200 μM) of Rg1 were used to pre-treat the cells for 48 h. Hochest33342/PI double fluorescence staining showed that compared with those in the serum deprivation treatment group, the fluorescence of cell nucleus was obviously reduced. Concurrently, karyopyknosis along with karyoclasis was decreased 48 h after treatment with different concentrations (20, 50, 100, and 200 μM) of Rg1 (P < 0.05) (Fig. 2B). Meanwhile, western blot analysis showed that the expression of Bax was significantly down-regulated; however, the expression of Bcl-2 was up-regulated significantly (P < 0.05) (Fig. 2C,D). Flow cytometry results also showed that the rate of apoptosis was significantly decreased with different concentrations (20, 50, 100, and 200 μM) of Rg1, and 50 μM of Rg1 was found to be the most effective (P < 0.05). The labels Annexin V-FITC and PI were used to identify different cell populations as follows: viable cells (low Annexin V-FITC and low PI), early apoptotic cells (high Annexin V-FITC and low PI), and late apoptotic cells (high PI and high Annexin V-FITC). The total apoptotic cells include both early and late apoptotic cells. The rate of apoptosis was higher in the serum deprivation group (P < 0.05) (Fig. 2E,F) and Rg1 was found to be the most potent at a concentration of 50 μM. Thus, in the subsequent experimental analysis in serum deprivation, 50 μM of Rg1 was chosen to treat cells for different time (36, 48, and 72 h). Western blot analysis indicated that in comparison to those treated with serum deprivation alone, the expression of cleaved caspase-3 diminished significantly at different time points (P < 0.05) (Fig. 2G). These results confirmed that serum deprivation could induce apoptosis in Raw264.7 macrophages. Rg1 at different concentrations (20, 50, 100, and 200 μM) could inhibit apoptosis, among which 50 μM Rg1 was found to exert the most drastic inhibitory effect. Figure 2. View largeDownload slide Rg1 inhibits apoptosis induced by serum deprivation in Raw264.7 macrophages (A,B) The morphology of cell nucleus as examined by Hochest33342/PI double immunofluorescence labeling. The hyperchromatic nuclei as well as the nuclear condensation are increased consistently in serum deprivation medium at different time intervals (0, 12, 24, 36, 48, and 72 h) that is attenuated by 48 h of treatment with Rg1 (20, 50, 100, and 200 μM). (C) Raw264.7 macrophages with serum deprivation were treated with or without Rg1 (20, 50, 100, and 200 μM) for 48 h. Western blot analysis shows protein expression levels of Bcl-2 and Bax. (D) The ratios of Bcl-2 and Bax were quantified by densitometry based on immunoblot images from (C). (E) The apoptosis was examined by flow cytometry. The down-regulated apoptosis by serum deprivation is suppressed by Rg1 (20, 50, 100, and 200 μM). Compared with the cells pretreated with Rg1 (20, 50, 100, and 200 μM), the apoptosis rate of control cells increased markedly in serum deprivation medium at 48 h. (F) The apoptosis ratio of Raw264.7 macrophages determined by flow cytometry from (E). (G) Raw264.7 macrophages incubated with serum deprivation treated with or without 50 μM Rg1 at different time intervals (36, 48, and 72 h). The relative protein levels were quantified by scanning densitometry and normalized to β-actin. The data are shown as the mean ± SEM of data from three independent experiments. #P < 0.05 compared with control alone; *P < 0.05 compared with serum deprivation alone. Figure 2. View largeDownload slide Rg1 inhibits apoptosis induced by serum deprivation in Raw264.7 macrophages (A,B) The morphology of cell nucleus as examined by Hochest33342/PI double immunofluorescence labeling. The hyperchromatic nuclei as well as the nuclear condensation are increased consistently in serum deprivation medium at different time intervals (0, 12, 24, 36, 48, and 72 h) that is attenuated by 48 h of treatment with Rg1 (20, 50, 100, and 200 μM). (C) Raw264.7 macrophages with serum deprivation were treated with or without Rg1 (20, 50, 100, and 200 μM) for 48 h. Western blot analysis shows protein expression levels of Bcl-2 and Bax. (D) The ratios of Bcl-2 and Bax were quantified by densitometry based on immunoblot images from (C). (E) The apoptosis was examined by flow cytometry. The down-regulated apoptosis by serum deprivation is suppressed by Rg1 (20, 50, 100, and 200 μM). Compared with the cells pretreated with Rg1 (20, 50, 100, and 200 μM), the apoptosis rate of control cells increased markedly in serum deprivation medium at 48 h. (F) The apoptosis ratio of Raw264.7 macrophages determined by flow cytometry from (E). (G) Raw264.7 macrophages incubated with serum deprivation treated with or without 50 μM Rg1 at different time intervals (36, 48, and 72 h). The relative protein levels were quantified by scanning densitometry and normalized to β-actin. The data are shown as the mean ± SEM of data from three independent experiments. #P < 0.05 compared with control alone; *P < 0.05 compared with serum deprivation alone. Rg1 up-regulates autophagy inhibited by serum deprivation in Raw264.7 macrophages It has been suggested that Rg1 exerts a regulatory effect on autophagy of myocardial cells [26]. Therefore, we explored whether Rg1 could regulate autophagy in Raw264.7 macrophages. Under serum deprivation, Raw264.7 macrophages were treated with different concentrations (20, 50, 100, and 200 μM) of Rg1. Western blot analysis showed that compared with those in the pure serum deprivation group, 48 h after treatment with 50 and 100 μM Rg1, the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1 were significantly increased (P < 0.05), and 50 μM of Rg1 was found to be the most effective dose (Fig. 3A,B). To confirm the basic level of autophagy in the blank control group, the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1 were measured with or without 1 h treatment with rapamycin (1 μΜ) or chloroquine (20 μΜ). There was no difference in the autophagic protein expression levels among these groups (P > 0.05; Fig. 3C,D). The results of MDC fluorescence staining showed that in comparison to those in the pure serum deprivation group, the incidence of autophagosomes in Rg1-treated cells was markedly increased and with enhanced fluorescence (P < 0.05; Fig. 3E,F). In serum deprivation, 50 μM of Rg1 was added at different time intervals (36, 48, and 72 h). Western blot analysis indicated that compared with those in the pure serum deprivation group, the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1 were all significantly increased (P < 0.05; Fig. 3G,H). These data suggested that the autophagy level was inhibited after prolonged serum deprivation in Raw264.7 macrophages. On the other hand, when Raw264.7 macrophages were treated with 50 or 100 μM Rg1 for 48 h, the decreased autophagy was reversed, and 50 μM of Rg1 was found to be more effective. Figure 3. View largeDownload slide Rg1 enhances autophagy down-regulated by serum deprivation in Raw264.7 macrophages (A) Raw264.7 macrophages with serum deprivation were treated without or with 20, 50, 100, and 200 μM Rg1 for 48 h. Western blot shows the protein expression levels of Atg5, Beclin1, LC3, and P62/SQSMT1. (B) The ratios of Atg5, Beclin1, LC3, and P62/SQSMT1 were quantified by densitometry based on immunoblot images from (A). (C) Raw264.7 macrophages treated without or with rapamycin (1 μΜ) or chloroquine (20 μΜ) for 48 h. Western blot shows the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1. (D) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (C). (E) MDC staining of the autophagosome. The down-regulated autophagosome by serum deprivation is suppressed by Rg1. (F) Autophagic rate of Raw264.7 macrophages determined by MDC from (E). (G) Raw264.7 macrophages with serum deprivation were treated with or without 50 μM Rg1 for different time (36, 48, and 72 h). Western blot shows the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1. (H) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (G). The relative protein levels were quantified by scanning densitometry and normalized to β-actin. Data are shown as the mean ± SEM from three independent experiments. #P < 0.05 compared with control alone; *P < 0.05 compared with serum deprivation alone. Figure 3. View largeDownload slide Rg1 enhances autophagy down-regulated by serum deprivation in Raw264.7 macrophages (A) Raw264.7 macrophages with serum deprivation were treated without or with 20, 50, 100, and 200 μM Rg1 for 48 h. Western blot shows the protein expression levels of Atg5, Beclin1, LC3, and P62/SQSMT1. (B) The ratios of Atg5, Beclin1, LC3, and P62/SQSMT1 were quantified by densitometry based on immunoblot images from (A). (C) Raw264.7 macrophages treated without or with rapamycin (1 μΜ) or chloroquine (20 μΜ) for 48 h. Western blot shows the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1. (D) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (C). (E) MDC staining of the autophagosome. The down-regulated autophagosome by serum deprivation is suppressed by Rg1. (F) Autophagic rate of Raw264.7 macrophages determined by MDC from (E). (G) Raw264.7 macrophages with serum deprivation were treated with or without 50 μM Rg1 for different time (36, 48, and 72 h). Western blot shows the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1. (H) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (G). The relative protein levels were quantified by scanning densitometry and normalized to β-actin. Data are shown as the mean ± SEM from three independent experiments. #P < 0.05 compared with control alone; *P < 0.05 compared with serum deprivation alone. Rg1 inhibits apoptosis through activating autophagy in macrophages Rg1 was found to inhibit apoptosis in Raw264.7 macrophages, but it remains to be determined if there exist interactions between apoptosis and autophagy in Raw264.7 macrophages and BMDMs. Western blot analysis indicated that compared with those in the serum deprivation+Rg1 group, after addition of autophagy inhibitor 3-MA, the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1 were significantly reduced (P < 0.05; Fig. 4A,B). StubRFP-SensGFP-LC3 assay showed that compared with that in the serum deprivation (48 h) +Rg1 (50 μM) group without 3-MA, LC3 puncta and autophagy marker expression were found to be significantly reduced in the serum deprivation (48 h)+Rg1 (50 μM) group with 3-MA (P < 0.05; Fig. 4C,D). The results of Hochest33342/PI double fluorescence staining indicated that the cell nucleus fluorescence was enhanced significantly; karyopyknosis and karyoclasis were concurrently increased significantly. Moreover, apoptosis and necrosis of the red-stained cell nucleus were detected during the advanced stage in the serum deprivation+Rg1+3-MA group, as compared with those in the serum deprivation+Rg1 group (Fig. 4E). Apoptosis was measured with or without the addition of 3-MA by flow cytometry. The labels Annexin V-FITC and PI were used to identify different cell populations as follows: viable cells (low Annexin V-FITC and low PI), early apoptotic cells (high Annexin V-FITC and low PI), and late apoptotic cells (high PI and high Annexin V-FITC). The total apoptotic cells include both early and late apoptotic cells. The rate of apoptosis was lower in the serum deprivation+Rg1 group than in the serum deprivation group (P < 0.05). Administration of 3-MA increased the rate of apoptosis in both groups (P < 0.05; Fig. 4F,G). Densitometry of the immunoblots also confirmed that the expression of cleaved caspase-3, a critical executer of apoptosis, was significantly increased after treatment with Rg1 (50 μM) for 48 h. Rg1 inhibited the expression level of cleaved caspase-3 activation, but after addition of the autophagy inhibitor 3-MA, in comparison with that in the serum deprivation+Rg1 group, the inhibitory effects of Rg1 on cleaved caspase-3 activation level were significantly reversed (P < 0.05; Fig. 4H). The same results of the expression of cleaved capase-3 were also shown in BMDM with double immunofluorescence labeling (Fig. 4I,J). It is well accepted that autophagy plays a crucial role in the anti-apoptotic activity of Rg1 and the up-regulation of autophagic flux can prevent apoptotic activation. To further determine the role of autophagy in the effect of apoptosis, Beclin1 siRNA was used. Compared with negative control, Beclin1 siRNA both increased the levels of Bax and cleaved caspase-3 and decreased the level of Bcl-2 in different Beclin1 siRNA-treated groups. The control siRNA appeared to decrease the apoptotic level, which, however, was not statistically significant (Fig. 4K,L). Taken together, all these results suggested that promoting autophagic flux might participate in the anti-apoptotic activity of Rg1 on macrophages against serum deprivation. Figure 4. View largeDownload slide Autophagy is involved in Rg1-inhibited apoptosis in macrophages Macrophages with serum deprivation were treated with 50 μM Rg1 for 48 h in the absence or presence of 3-MA (5 mM). (A) Protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1 determined by western blot analysis. (B) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (A). (C) Representative of the StubRFP-SensGFP-LC3 assay images of autophagy flux. Raw264.7 macrophages were infected with StubRFP-SensGFP-LC3. (D) The number of autolysosome (red) and autophagosome (yellow) puncta were quantified based on StubRFP-SensGFP-LC3 assay images from (C). (E) The morphology of cell nucleus as examined by Hochest33342/PI double immunofluorescence labeling. (F) The apoptosis as examined by flow cytometry. (G) The apoptosis ratio of Raw264.7 macrophages determined by flow cytometry from (F). (H) The protein expression level of cleaved caspase-3 determined by western blot analysis in Raw264.7 macrophages. (I) The protein expression level of cleaved caspase-3 determined by double immunofluorescence labeling in BMDMs. The up-regulated protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 by Rg1 were suppressed by 3-MA, and the down-regulated protein expression of cleaved caspase-3 by Rg1 was suppressed by 3-MA. (J) The average mean value of fluorescence staining with cleaved caspase-3 were quantified by densitometry based on immunofluorescence images from (I). (K) Raw264.7 macrophages and Beclin1 siRNA Raw264.7 macrophages with serum deprivation were treated with or without 50 μM Rg1 for 48 h. Proteins from cell lysates were analyzed by western blotting for Bax, Bcl-2, cleaved caspase-3, and β-actin. (L) The ratios of Bax, Bcl-2, cleaved caspase-3, and β-actin were quantified by densitometry based on immunoblot images from panel K. Data are shown as the mean ± SEM from three independent experiments. #P < 0.05 compared with control alone. *P < 0.05 compared with serum deprivation alone. ΔP < 0.05 compared with Rg1+serum deprivation. **P < 0.05 compared between control alone and Beclin1 siRNA control alone; ##P < 0.05 compared between serum deprivation alone and Beclin1 siRNA serum deprivation alone; ΔΔP < 0.05 compared between Rg1+serum deprivation and Beclin1 siRNA Rg1+serum deprivation. Figure 4. View largeDownload slide Autophagy is involved in Rg1-inhibited apoptosis in macrophages Macrophages with serum deprivation were treated with 50 μM Rg1 for 48 h in the absence or presence of 3-MA (5 mM). (A) Protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1 determined by western blot analysis. (B) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (A). (C) Representative of the StubRFP-SensGFP-LC3 assay images of autophagy flux. Raw264.7 macrophages were infected with StubRFP-SensGFP-LC3. (D) The number of autolysosome (red) and autophagosome (yellow) puncta were quantified based on StubRFP-SensGFP-LC3 assay images from (C). (E) The morphology of cell nucleus as examined by Hochest33342/PI double immunofluorescence labeling. (F) The apoptosis as examined by flow cytometry. (G) The apoptosis ratio of Raw264.7 macrophages determined by flow cytometry from (F). (H) The protein expression level of cleaved caspase-3 determined by western blot analysis in Raw264.7 macrophages. (I) The protein expression level of cleaved caspase-3 determined by double immunofluorescence labeling in BMDMs. The up-regulated protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 by Rg1 were suppressed by 3-MA, and the down-regulated protein expression of cleaved caspase-3 by Rg1 was suppressed by 3-MA. (J) The average mean value of fluorescence staining with cleaved caspase-3 were quantified by densitometry based on immunofluorescence images from (I). (K) Raw264.7 macrophages and Beclin1 siRNA Raw264.7 macrophages with serum deprivation were treated with or without 50 μM Rg1 for 48 h. Proteins from cell lysates were analyzed by western blotting for Bax, Bcl-2, cleaved caspase-3, and β-actin. (L) The ratios of Bax, Bcl-2, cleaved caspase-3, and β-actin were quantified by densitometry based on immunoblot images from panel K. Data are shown as the mean ± SEM from three independent experiments. #P < 0.05 compared with control alone. *P < 0.05 compared with serum deprivation alone. ΔP < 0.05 compared with Rg1+serum deprivation. **P < 0.05 compared between control alone and Beclin1 siRNA control alone; ##P < 0.05 compared between serum deprivation alone and Beclin1 siRNA serum deprivation alone; ΔΔP < 0.05 compared between Rg1+serum deprivation and Beclin1 siRNA Rg1+serum deprivation. Rg1 promotes autophagy and inhibits apoptosis through AMPK/mTOR pathway in Raw264.7 macrophages Mammalian target of rapamycin 1 (mTORC1) is a well-documented inhibitor of autophagy. Inhibition of AMPK induces autophagy by inhibition of mTORC1. AMPK phosphorylation at threonine 172 (T172) in the α-subunit is a key mechanism in mediation of AMPK activation [20,21]. To evaluate the roles of AMPK and mTOR in groups exposed to serum deprivation, different concentrations (20, 50, 100, and 200 μM) of Rg1 were used to treat macrophages for 48 h under the condition of serum deprivation. Western blot analysis showed that decreased phosphorylation of AMPK and mTOR in serum deprivation group was reversed, and 50 μM of Rg1 was found to be the most effective dose (P < 0.05; Fig. 5A,B). In order to explore the correlation among Rg1, AMPK/mTOR signal pathway and the level of autophagy in Raw264.7 macrophages, compound C, or AICAR was used to pre-treat Raw264.7 macrophages. Treatments with compound C or AICAR in blank control groups could not obviously reverse the levels of p-mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1 protein expression (P > 0.05; Fig. 5C,D). Compared with those in the serum deprivation (48 h)+Rg1 (50 μM) treatment group, in cells pretreated with compound C, the levels of p-mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1 protein expression up-regulated by Rg1 were significantly reversed by compound C (P < 0.05). When cells were pretreated with AICAR, the expressions of p-mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1 were further up-regulated (P < 0.05; Fig. 5E,F). Immunofluorescence double labeling showed the same result of LC3 in cells as that in western blot analysis, which were statistically significant (P < 0.05; Fig. 5G,H). The above data showed that Rg1 promotes autophagy and inhibits apoptosis induced by serum deprivation through the AMPK/mTOR pathway. Figure 5. View largeDownload slide AMPK/mTOR is required for Rg1-inhibited apoptosis induced by serum deprivation in Raw264.7 macrophages (A) Raw264.7 macrophages lysates of various treatment were subject to western blot analysis by using antibodies specific for total AMPK, phosphor-AMPK, total mTOR, and phosphor-mTOR. The relative protein levels were quantified by scanning densitometry and normalized to total AMPK or total mTOR, respectively. (B) The ratios of total AMPK, phosphor-AMPK, total mTOR, and phosphor-mTOR were quantified by densitometry based on immunoblot images from (A). (C) Raw264.7 macrophages incubated without or with compound C (10 mM) or AICAR (250 μM) for 48 h. (D) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (C). (E) Raw264.7 macrophages with serum deprivation were treated with 50 μM Rg1 for 48 h in absence or presence of compound C (10 mM) or AICAR (250 μM). Western blots of the protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1. (F) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (E). (G) The corresponding immunofluorescence images for protein expression of LC3. (H) The numbers of LC3 puncta ratio based on immunofluorescence images from (G). The AMPK inhibitor compound C reverses the Rg1-promoted protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 via activation of mTOR, and the AMPK inducer AICAR fosters the Rg1-promoted protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 through suppression of AMPK/mTOR. Data are shown as the mean ± SEM from three independent experiments. #P < 0.05 compared with control alone; *P < 0.05 compared with serum deprivation alone; ΔP < 0.05 compared with Rg1+serum deprivation. Figure 5. View largeDownload slide AMPK/mTOR is required for Rg1-inhibited apoptosis induced by serum deprivation in Raw264.7 macrophages (A) Raw264.7 macrophages lysates of various treatment were subject to western blot analysis by using antibodies specific for total AMPK, phosphor-AMPK, total mTOR, and phosphor-mTOR. The relative protein levels were quantified by scanning densitometry and normalized to total AMPK or total mTOR, respectively. (B) The ratios of total AMPK, phosphor-AMPK, total mTOR, and phosphor-mTOR were quantified by densitometry based on immunoblot images from (A). (C) Raw264.7 macrophages incubated without or with compound C (10 mM) or AICAR (250 μM) for 48 h. (D) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (C). (E) Raw264.7 macrophages with serum deprivation were treated with 50 μM Rg1 for 48 h in absence or presence of compound C (10 mM) or AICAR (250 μM). Western blots of the protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1. (F) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (E). (G) The corresponding immunofluorescence images for protein expression of LC3. (H) The numbers of LC3 puncta ratio based on immunofluorescence images from (G). The AMPK inhibitor compound C reverses the Rg1-promoted protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 via activation of mTOR, and the AMPK inducer AICAR fosters the Rg1-promoted protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 through suppression of AMPK/mTOR. Data are shown as the mean ± SEM from three independent experiments. #P < 0.05 compared with control alone; *P < 0.05 compared with serum deprivation alone; ΔP < 0.05 compared with Rg1+serum deprivation. Discussion Macrophage apoptosis in atherosclerotic lesions is almost certainly multifactorial, and it is likely that at least some of the death inducers are different in early versus late lesions. Despite this complexity and the paucity of in vivo data, several proapoptotic factors have been suggested based on cell culture models that attempt to mimic conditions in atherosclerotic lesions. Examples include high concentrations of oxidized LDL, oxysterols, TNF-α, Fas ligand, nitric oxide, growth factor withdrawal, hypoxia/ATP depletion, and intracellular accumulation of unesterified, or ‘free’ cholesterol [29]. However, the results of this study are based on serum deprivation to mimic macrophage apoptosis in advanced atherosclerotic lesions in vitro, which may be first suggested in macrophages. Nutrition is indispensable for cell survival and proliferation [30]. Thus, loss of nutrients caused by serum starvation in cells could induce formation of ROS, resulting in cell death [31]. In fact, serum deprivation is a vigorous stimulus for the induction of energy limiting stresses. These stresses are caused by elevation of ROS production and apoptosis [32]. Serum used in cell culture is a mixture of essential proteins and various factors for cell growth and proliferation [33]. Therefore, serum deprivation causes cellular apoptosis, and serum deprivation is widely used to investigate induction of apoptosis as well as related signal transduction pathways [34–36]. In this study, macrophages incubated in media without serum for the indicated time periods were induced to undergo apoptosis. These conditions closely mimic the pathophysiological conditions in advanced atherosclerotic lesions, wherein oxidative stress plays key roles in the induction of cell death caused by serum deprivation. Our results confirmed that Rg1 significantly reduces apoptosis in macrophages by promoting autophagic flux, but not by impairing autophagic flux. Several protein complexes have been found to regulate autophagy induction, autophagosomes formation and maturation into autolysosomes. The PtdIns3K-III/BENC1 complex is necessary for autophagosome initiation. Lipidation and redistribution of the cytoplasmic protein LC3 towards the phagophore contributes to its elongation around the cargo to be engulfed; and an autophagic receptor like p62/SQSTM1 allows cargo recognition. Upon autolysosome maturation, lysosomal catabolic enzymes degrade its content, including the autophagic receptor p62/SQSTM1, providing the cells with building blocks in order to maintain the energy status. When the autophagic flux is impaired, the cargo is not degraded leading to an accumulation of p62/SQSTM1 [37]. We have shown that 3-MA, an autophagic inhibitor, could reverse the inhibitory effect of Rg1 on apoptosis of Raw264.7 macrophages and BMDM induced by serum deprivation. The same phenomenon was also confirmed in Beclin1 siRNA Raw264.7 macrophages. The expression levels of apoptosis-related proteins in Raw264.7 macrophages are significantly lower in Beclin1 siRNA Raw264.7 macrophages with Rg1 treatment. In addition, inhibiting AMPK with compound C suppressed the phosphorylation of mTOR and the expressions of autophagy-related proteins, including Atg5, Beclin1, LC3, and p62/SQSMT1. Moreover, AICAR was found to upregulate the phosphorylation of mTOR and the expressions of autophagy-related proteins, such as Atg5, Beclin1, LC3, and p62/SQSMT1, ultimately. All these results suggest that Rg1 induces autophagy, which effectively down-regulates apoptosis of macrophage induced by serum deprivation, through activating AMPK/mTOR signal pathway. We reported previously that Rg1 induced autophagy, which contributed to inhibit inflammatory responses generated by OXLDL-induced Raw264.7 macrophages This study has extended our previous study and suggested that Rg1 could upregulate the expressions of autophagy-related proteins (Atg5, Beclin1, LC3, and p62/SQSMT1) in Raw264.7 macrophages under serum deprivation. In the present study, 50 μM of Rg1 was found to be the most effective dose, which increased the expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 under serum deprivation for 48 h. The expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 were higher in the 50 μM Rg1 group than in the 20, 100, and 200 μM Rg1 groups, demonstrating that 50 μM of Rg1 is the optimal dose to promote autophagy in Raw264.7 macrophages; hence, any dosage deviating from this may dilute the effect of Rg1 in promoting autophagy. It has been reported that, in the advanced stage of AS, decreased autophagy and defected efferocytosis result in impaired clearance of apoptotic macrophages, leading to further inflammation and promotion of lipid core necrosis accompanied by increases in unstable factors of plaque [38]. Studies have indicated that after gene silencing or knockout Atg5 in macrophages, apoptosis in macrophages and oxidative stress response mediated by NADPH oxidase were enhanced [8,39]. Our results further showed that the autophagy inhibitor 3-MA reduces the expressions of autophagy-related proteins induced by Rg1 in Raw264.7 macrophages and counteracts the anti-apoptotic role of Rg1, thus, supporting that Rg1 protects against apoptosis by promoting autophagy in macrophages. However, many studies by others have reported that serum deprivation does not inhibit autophagy; rather, it induces autophagy as a survival mechanism [40]. A possible explanation for the discrepancy may be attributed to the possibility that regulation of autophagy might be a time-dependent event during serum deprivation. Thus, in a shorter duration (within 24 h), the process occurs in parallel with the well-known early steps in autophagy induction (AMPK activation, mTOR dephosphorylation, increased conjugation of LC3 to phosphatidylethanolamine to form LC3II, recruitment of LC3II to autophagosomal membranes), and a subsequent decrease in p62/SQSMT1. Protein p62/SQSMT1 is involved in the assembly of autophagosome cargoes [41]. However, the process may play a protective role when the serum deprivation time is prolonged (e.g. over 36 h). The protective function may be reversed to promote apoptosis through downregulating autophagy level via inhibiting AMPK signaling as shown in the present study. Another explanation would be that during starvation, cells can enhance amino acid uptake and synthesis through the general amino acid control (GAAC) pathway, whereas nonessential cellular contents are recycled by autophagy. Chen et al. [42] reported that serum/glutamine starvation activates the GAAC pathway, which up-regulates amino acid transporters, leading to increased amino acid uptake. This elevates the intracellular amino acid level, which in turn reactivates mTOR and suppresses autophagy. Knockdown of the activating transcription factor 4, the major transcription factor in the GAAC pathway, or of SLC7A5, a leucine transporter, impaired mTOR reactivation and induced much higher levels of autophagy. Besides, Razani et al. [43] confirmed that the lesion sizes and necrotic areas were larger in arteries of Atg5fl/flLysmcre+/−/LDLr−/− mice than those in the wild-type control group. A large number of apoptotic cells, activated caspase-3, DHE, and p47 were detected in the plaque of Atg5fl/flLysmcre+/−/LDLr−/− mice with the use of TUNEL experiment. Therefore, in LDLr−/− mice, macrophage autophagy defect increases apoptosis and oxidative stress reaction of macrophages in plaque, and promotes atheromatous plaque necrosis. Liao et al. [44] showed that macrophage apoptosis and defective macrophage apoptosis can promote necrosis on atherosclerotic old plaque, and autophagy can inhibit apoptosis in macrophages and efferocytosis defect, which plays an important role in the process of AS pathological changes. The AMPK/mTOR pathway plays an important role in autophagy regulation in response to stress and glucose starvation. Both are reported to maintain renal tubular homeostasis and are involved in autophagy induced by I/R in renal tubular cell injury [21,45]. There is mounting evidence [46,47] indicating that AMPK/mTOR signal pathway plays an important role in the induction of autophagy. Hence, inhibiting AMPK/mTOR signal pathway can further inhibit the expressions of macrophage-related proteins inhibited by serum deprivation; enhancing AMPK/mTOR signal pathway can reverse the expressions of macrophage-related proteins inhibited by serum deprivation. As observed, Rg1 is able to activate AMPK/mTOR signal pathway inhibited by serum deprivation in macrophages. This is not consistent with results by others [26], which showed that Rg1 can inhibit AMPK phosphorylation and reduce autophagy in H9c2 myocardial cells. This conflicting result may be attributed to different cell sources and different experimental paradigms used. We confirmed that pretreatment of Raw264.7 macrophages with compound C followed by Rg1 inhibited the expressions of autophagy-related proteins (Atg5, Beclin1, LC3, and p62/SQSMT1). Additionally, AICAR pretreatment further enhanced the expressions of autophagy-related proteins (Atg5, Beclin1, LC3, and p62/SQSMT1). Autophagy is up-regulated by the activation of AMPK and inhibition of mTOR [48]. LC3 and autophagy-related genes 5–12 (Atg5–12) are required for the formation of the double-membrane autophagosomes. The autophagosome formation can be detected by measuring the conversion of LC3-I (unconjugated cytosolic form) to LC3II (autophagosomal membrane-associated phosphatidylethanolamine-conjugated form) [10,49,50]. Additionally, the p62/SQSTM1 protein, also known as sequestosome-1, recognizes cellular components marked for degradation and targets them for autophagy by the virtue of its ubiquitin association domain (UBA) and a LC3-interacting region (LIR) [51]. Impairment of autophagic clearance results in the build-up of p62/SQSTM1 and cellular dysfunction [52]. Studies have suggested that there may exist some complex and cross regulatory mechanisms between autophagy and apoptosis, including independent regulation, coordinated regulation, and antagonistic regulation [53]. Therefore, the regulatory function of Rg1 in autophagy and apoptosis may rely on the interaction of different signal pathways. It was found that Rg1 could reverse the diminished expression levels of autophagy-related proteins (Atg5, Beclin1, LC3, and p62/SQSMT1), suggesting that AMPK/mTOR activated by Rg1 might regulate autophagy and apoptosis. Along with this, we also showed that preconditioning macrophages with 3-MA followed by Rg1 results in decreased expressions of autophagy-related proteins (Atg5, Beclin1, LC3, and p62/SQSMT1), but increased expression of cleaved caspase-3. In summary, our results indicate that Rg1 positively regulates the autophagy process through an association with the AMPK/mTOR signaling pathway. Autophagy inhibits apoptosis and plays a protective role under conditions of serum deprivation in macrophage as demonstrated in the present experimental paradigm in Raw264.7 macrophages (Fig. 6). Importantly, we have also shown that AMPK pathway is one of the signaling pathways by which Rg1 exerts its anti-apoptotic effects. It can be concluded that Rg1 exerts its protective effects to inhibit apoptosis by upregulating autophagy due to activation of AMPK/mTOR channel on the macrophages and this study may be helpful for the design of novel therapeutic strategies for the treatment of AS. Figure 6. View largeDownload slide Rg1-mediated inhibition of apoptosis by activating autophagy through AMPK/mTOR in response to serum deprivation in macrophages Figure 6. 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Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Biochimica et Biophysica Sinica Oxford University Press

Ginsenoside Rg1 inhibits apoptosis by increasing autophagy via the AMPK/mTOR signaling in serum deprivation macrophages

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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1672-9145
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1745-7270
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10.1093/abbs/gmx136
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Abstract

Abstract Ginsenoside Rg1 (Rg1) has been widely used in a broad range of cardiovascular and cerebral–vascular diseases because of its unique therapeutic properties. However, the underlying mechanisms of Rg1 in the treatment of atherosclerosis have not been fully explored. This study sought to determine the precise molecular mechanisms on how Rg1 might be involved in regulating apoptosis in serum deprivation-induced Raw264.7 macrophages and primary bone marrow-derived macrophages. Results demonstrated that Rg1 treatment effectively suppressed apoptosis and the expression of phosphorylation level of mTOR induced by serum deprivation in Raw264.7 macrophages; the expressions of autophagic flux-related proteins including Atg5, Beclin1, microtubule-associated protein 1 light chain 3 (LC3), p62/SQSMT1, and the phosphorylation level of AMPK were concomitantly up-regulated. 3-Methyl-adennine (3-MA), the most widely used autophagy inhibitor, strongly up-regulated the expression of cleaved caspase-3, and blocked the anti-apoptosis function of Rg1 in macrophages. Importantly, autophagic flux was activated by Rg1, while Beclin1 knockdown partially abolished the anti-apoptosis of Rg1. Moreover, compound C, an AMPK inhibitor, partially decreased the expressions of phosphorylation of mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1, which were increased by Rg1. AICAR, an AMPK inducer, promoted the protein expressions of phosphorylation of mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1. In conclusion, Rg1 significantly suppressed apoptosis induced by serum deprivation in macrophages. Furthermore, Rg1 could effectively induce the autophagic flux by attenuating serum deprivation-induced apoptosis in Raw264.7 macrophages through activating the AMPK/mTOR signaling pathway. ginsenoside Rg1, autophagy, apoptosis, AMPK/mTOR, macrophages Introduction Atherosclerosis (AS) is the most common lesion in cardiovascular system. It is well recognized to be the common pathological basis in many cardiovascular and cerebrovascular diseases. Furthermore, it represents one of the most severe diseases threatening the health of human beings [1]. Macrophage apoptosis is one of the important characteristics in the occurrence and development of atheromatous plaque. It has been reported that macrophage apoptosis plays different roles in the progression during different stages of the formation of atheromatous plaque [2]. One such stress that induces cell death is serum deprivation. Serum contains growth factors, hormones, attachment and spreading factors, minerals, trace elements, lipids, and various other factors that are necessary for cell growth, differentiation, transport, attachment, spreading, pH maintenance, and protease inhibition [3]. Therefore, serum withdrawal causes the cells to stop growing. Serum deprivation also induces apoptosis, and reactive oxygen species (ROS) generation contributes to the cell death in serum-starved cells [4,5]. Indeed, excessive production of cellular ROS and apoptosis induced by growth factor withdrawal have also been found in AS [6]. In the early stage of AS, receptors on monocyte/macrophage are activated and coupled with the accumulation of macrophage and lymphocytes in the lesion sites. This would promote the development of AS on pathological changes. Therefore, the development of plaque may be effectively controlled in the early stage of pathological change to prevent apoptosis so as to reduce the accumulation of macrophages in the lesion sites. In the advanced stage, defective efferocytosis results in the decline of autophagy level; hence, a large number of apoptotic macrophages can not be effectively removed, which would lead to the increase of lipid core necrosis in the advanced plaque, resulting in the instability of plaque increase, and acute cardiovascular events [7]. In addition, studies have also shown that macrophage autophagy is involved in the occurrence and development of AS, thus underscoring the importance of the stability of plaque and the development of AS [8]. Autophagy is a catabolic pathway widely existing in eukaryotic cells and it can monitor and timely remove senescent and degenerative proteins and organelles in cells so as to maintain normal intracellular homeostasis, and render a certain protection on cells [9]. The formation of autophagosome with a double-membraned structure is a hallmark feature for the occurrence of autophagy. After formation, the autophagosome fuses with lysosome, producing autophagolysosome that degrades the contents wrapped by it using lysosomal enzymes to maintain the renewal of proteins and organelles, as well as cell homeostasis. Autophagy maintains organelle quality control by disposing of dysfunctional or damaged cellular organelles [10]. It is a critical cellular process that generally protects cells and organisms from stressors, such as nutrient deprivation, ROS increasing, DNA damage, and hypoxia. In addition to its role in normal physiology, autophagy also plays a role in pathological processes, such as cancer, neurodegeneration, and cardiovascular diseases [10–12]. Mizushima et al. [13] transferred GFP-light chain 3 (LC3)-II into the primary macrophages from mice, and reported distinctive changes of autophagy in cells stimulated with promoting AS factors. The same phenomenon has also been confirmed in vivo; compared with those in the wild-type mice, expression levels of Atg5 and GFP-LC3 in macrophages of atheromatous plaque are lower in GFP-LC3/(LDLr)−/− mice with a high fat diet [14]. These data further suggest that autophagy indeed is involved in the occurrence and development of AS. Additionally, Quimet et al. [15] reported that autophagy can hydrolyze lipids stored in macrophages, promote cholesterol efflux in cells, and provide certain protection for macrophages. Therefore, it would be possible to provide new therapeutic strategies and targets for the prevention and treatment of AS through a better understanding of the regulatory mechanisms of macrophage autophagy-related signals during the progression of AS. Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK), a kind of serine/threonine protein kinase, is activated by an increase of the AMP/ATP ratio [16], hypoxia, and ROS production [17,18]. AMPK also promotes autophagy indirectly through the suppression of mTORC1 activity or directly by the phosphorylation and activation of ULK1, which leads to autophagy induction [19–21]. There is ample evidence supporting that AMPK signal pathway plays an important role in the induction of autophagy by inhibiting mTOR signaling pathway, exhibiting a positive regulation as reported in a recent study [22]. Studies have also shown that starvation induced autophagy might be monitored by AMPK and Sirtuin 1 (Sirt1) [23,24], which has potential functions in regulating the proliferation, survival and death of cancer cells. Rg1 is a major active component in Panax notoginsengs saponins (Fig. 1). Rg1 has been shown to have effects on anti-oxidation, anti-senescence, anti-inflammation, and the relief of nerve function damage [25]. It plays broad roles in the pharmacology of the central nervous system, cardiovascular, endocrine system, and immune system. Rg1 also provides protection on cardiovascular system, such as anti-myocardial hypertrophy, anti-AS, and anti-acute myocardial ischemia. Studies have revealed that Rg1 can effectively inhibit macrophage-derived foam cell proliferation. Rg1 was shown to inhibit the excessive autophagy of myocardial cells in myocardial ischemia-reperfusion injury [26]. It has been reported that macrophage autophagy provides a certain protection on blood vessels in the early stage of AS; while in the advanced stage of AS, the loss of autophagy may cause macrophage apoptosis, efferocytosis defect, and the instability of plaque. In clinical practice, Rg1, as one of the major components in the compound preparation used for the treatment of cardiovascular diseases; however, there is only a modicum of information concerning its mechanisms in AS. Figure 1. View largeDownload slide Chemical structure of Rg1 Figure 1. View largeDownload slide Chemical structure of Rg1 Hence, this study was aimed to analyze the regulatory effects of Rg1 on the apoptosis of Raw264.7 macrophages induced by serum deprivation, and to find possible ways to reduce the unstable risk of plaque. Materials and Methods Chemicals and reagents Murine Raw264.7 macrophages were a generous gift from Professor Xiao-Chun Bai of the School of Basic Medical Sciences, Southern Medical University (Guangzhou, China). The Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco/BRL (Gaithersburg, USA). The fetal bovine serum (FBS) was obtained from Hyclone (Logan, USA). Immunoblot polyvinylidene difluoride membrane and the antibodies against β-actin, Bcl-2, Bax, cleaved caspase-3, p62/SQSMT1, AMPK and phosphor-AMPK, mTOR and phosphor-mTOR were purchased from Cell Signaling Technology (Danvers, USA). The antibodies against LC3, Beclin1, and Atg5 were obtained from Sigma (St Louis, USA). The TRITC-conjugated secondary antibody, horseradish peroxidase-conjugated secondary antibodies and ECL detection system were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). MDC stain was purchased from Leagene (Leagene, Beijing, China). Annexin V-FITC apoptosis detection kit was purchased from BD Bioscience (Franklin Lakes, USA). Granulocyte–macrophage colony stimulating factor (GM-CSF) was purchased from PeproTech (Rocky Hill, USA). 3-MA, compound C, AICAR, rapamycin, and chloroquine were purchased from MedChem Express (MCE, Monmouth Junction, USA). Lipofectamine 2000 was purchased from Invitrogen (Waltham, USA). StubRFP-SensGFP-LC3 lentivirus was purchased from Genechem (Genechem Co., Ltd, Shanghai, China). Unless otherwise mentioned, all other chemicals were purchased from Sigma. Ginsenoside Rg1 was purchased from Kunming Pharmaceutical Company (Kunming, China). Beclin1 siRNA was purchased from Cyagen (Guangzhou, China). Cells and treatments The murine Raw264.7 macrophages and Beclin1 siRNA Raw264.7 macrophages were cultured in DMEM containing 5% FBS and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin) at a density not exceeding 5 × 105 cells/ml and maintained at 37°C in a humidified incubator with 5% CO2. To harvest Raw264.7 macrophages, cells were trypsinized with 0.25% trypsin/EDTA in phosphate-buffered saline (PBS), then centrifuged (400 g for 10 min) and resuspended in serum-free DMEM. Cells were counted with a hemocytometer and trypan blue staining (0.4% trypan blue in PBS) showed more than 98% of the cells were viable. Primary bone marrow-derived macrophage (BMDM) were generated as described previously [27]. Lysis of red blood cells was performed using red blood cell lysis buffer (Beijing, Solarbio, China). Experiments in BMDM were carried out at a cell density of 5 × 105 cells/ml. BMDMs were cultured in DMEM supplemented with 3 μM GM-CSF, 10% FBS and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin). Rg1 (purity > 99%) at 50 μM was added at 1 h before serum deprivation stimulation. This time point was chosen to minimize the possibility of any direct interactions between Rg1 and serum deprivation. 3-MA (5 mM), a specific autophagy inhibitor, was used to determine if autophagy is involved in Rg1’s action on Raw264.7 macrophages. Compound C (10 μM), an AMPK inhibitor and AICAR (250 μM), an AMPK inducer were used to determine if the AMPK signal pathway is involved in Rg1’s action on Raw264.7 macrophages. StubRFP-SensGFP-LC3 assay The Raw264.7 macrophages (4 × 104 cells/ml) were plated on the coverslip of the 96-well plate and incubated with the StubRFP-SensGFP-LC3 lentivirus (MOI = 10, 1 × 107 TU/ml). New medium was changed after 10 h of incubation. After 72 h of infection, all images were captured with a fluorescence microscope (Olympus, Tokyo, Japan). The puncta of each cell were counted for each sample. The GFP signal is sensitive to the acidic conditions of the lysosome lumen, whereas mRFP is more stable. Therefore, colocalization of both GFP and mRFP fluorescence indicates a compartment that has not fused with a lysosome. In contrast, an mRFP signal without GFP indicates a compartment fused with a lysosome. Thus, autophagic flux was then measured by confocal counting of GFP+/mRFP+ (yellow) and GFP−/mRFP+ (red) puncta. siRNA transfection assay Beclin1 siRNA was used for transient transfection of Raw264.7 macrophages with Lipofectamine 2000 to suppress the expression of targeted gene. After 72 h of the initial transfection and treatment, cell samples were collected and analyzed using western blot analysis to confirm the expression of proteins. Flow cytometry Annexin V-FITC apoptosis detection kit was used to measure apoptosis of each group of Raw264.7 macrophages according to the manufacturer’s protocol. Both the supernatant and adherent Raw264.7 macrophages were collected and incubated with 100 μl of 1× binding buffer containing 5 μl of Annexin V-FITC and 5 μl of PI. Within 1 h after incubation in the dark at room temperature for 15 min, Raw264.7 macrophages were analyzed by flow cytometry. Three independent experiments were performed. Double immunofluorescence labeling Raw264.7 macrophages or BMDMs derived from various treatments were fixed with 4% paraformaldehyde in 0.1 M PBS for 20 min. After rinse with PBS, the coverslips with adherent cells were used for double immunofluorescence labeling. Raw264.7 macrophages were incubated with DAPI plus goat anti-rabbit LC3 (dilution 1:500) or cleaved caspase-3 (dilution 1:200). Subsequently, the cells were incubated with TRITC-conjugated secondary antibody for 1 h at 37°C. For negative controls, a set of culture slides were incubated under similar conditions without the primary antibodies. All images were captured with a fluorescence microscope (Olympus). Results are representative of three independent experiments. Quantification of cell death using Hoechst PI nuclear staining and fluorescence microscopy Hoechst PI nuclear staining was carried out as previously described [28] with slight modifications. Briefly, Raw264.7 macrophages (5 × 105 cells/ml) were incubated for 15 min at 37°C with Hoechst 33342 dye (10 mg/ml in PBS), centrifuged, washed once with PBS, and then resuspended at an approximate density of 1 × 106 cells/ml. PI (50 mg/ml in PBS) was added just before microscopy. Raw264.7 macrophages were visualized using an Olympus microscope (Olympus) equipped with a fluorescent light source and a UV-2A filter cube with excitation wavelength of 330–380 nm and barrier filter of 420 nm. Cell morphology was scored as follows: (i) ,viable cells had blue-stained nuclei with smooth appearance; 2, viable apoptotic cells had blue-stained nuclei with multiple bright specks of condensed chromatin; 3, non-viable apoptotic cells had red-stained nuclei with either multiple bright specks of fragmented chromatin or one or more spheres of condensed chromatin (significantly more compact than normal nuclei); and 4, non-viable necrotic cells had red-stained, smooth and homogeneous nuclei that were about the same size as normal (control) nuclei. Samples were randomized and examined after blinding. At least 200 cells were counted for each treatment. Experiments were repeated at least three times. Monodansylcadaverine staining The autofluorescent agent monodansylcadaverine (MDC) was introduced as a specific autophagolysosome marker to analyze the autophagic process. After treatment, Raw264.7 macrophages were stained with MDC (50 μM) at 37°C for 40 min. After incubation, Raw264.7 macrophages were washed three times with PBS, fixed with 5% paraformaldehyde, and immediately observed under a fluorescence microscope. Western blot analysis Raw264.7 macrophages were plated in 6-well plates at a density of 5 × 105 cells per well and cultured for 24 h. The cells were further incubated in FBS-free medium for at least 1 h before treatments. Stimulated Raw264.7 macrophages were harvested with ice-cold PBS and centrifuged at 12,000 g for 5 min at 4°C. Raw264.7 macrophages were lysed in ice-cold lysis buffer containing 2.5 mM Tris–HCl, pH 6.8, 25% glycerol, 2% sodium dodecyl sulfate (SDS), 0.01% bromphenol blue, and 5% β-mercaptoethanol. Cell lysates were centrifuged at 12,000 g for 5 min at 4°C, and then the supernatants were collected. Protein content was determined by using the BCA protein assay (Pierce, Rockford, USA). Equal amounts of protein (50 μg) were subject to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto immunoblot polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 2 h at room temperature, and then incubated separately with goat anti-rabbit antibodies against AMPK and phospho-AMPK, mTOR and phosphor-mTOR, β-actin, Bcl-2, Bax, cleaved caspase-3, Atg5, Beclin1, LC3, and p62/SQSMT1, antibodies (1:1000 dilution) that recognize different molecules under study at 4°C overnight. The membranes were washed three times with TBS-T, and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Membranes were washed again for three times with TBS-T and developed using the ECL detection system. Signals were exposed to Fuji Medical X-Ray Film (Fuji Photo Film Co., Ltd, Karagawa, Japan). Statistical analysis Statistical analysis of the data was carried out by one-way analysis of variance (ANOVA) followed by Scheffe’s post hoc test, using SPSS (SPSS Inc., Chicago, USA). Data were shown as the mean ± SEM (standard error of mean) obtained from three independent experiments. A value of P < 0.05 was considered significant. Results Rg1 inhibits apoptosis induced by serum deprivation in Raw264.7 macrophages A recent in vivo study reported that in the advanced stage of AS, macrophage apoptosis may promote atheromatous plaque necrosis, resulting in the increasing plaque instability [29]. In order to determine the regulatory effects of Rg1 on AS, the inhibition of Rg1 on Raw264.7 macrophage apoptosis was first investigated. Serum deprivation was used to treat Raw264.7 macrophages for different time (0, 12, 24, 36, 48, and 72 h). Results with Hochest33342/PI double fluorescence staining revealed that the fluorescence of cell nucleus was noticeably enhanced. There was increased incidence of karyopyknosis and karyoclasis in prolonged serum deprivation (0–72 h) (Fig. 2A). Under the condition of serum deprivation, different concentrations (20, 50, 100, and 200 μM) of Rg1 were used to pre-treat the cells for 48 h. Hochest33342/PI double fluorescence staining showed that compared with those in the serum deprivation treatment group, the fluorescence of cell nucleus was obviously reduced. Concurrently, karyopyknosis along with karyoclasis was decreased 48 h after treatment with different concentrations (20, 50, 100, and 200 μM) of Rg1 (P < 0.05) (Fig. 2B). Meanwhile, western blot analysis showed that the expression of Bax was significantly down-regulated; however, the expression of Bcl-2 was up-regulated significantly (P < 0.05) (Fig. 2C,D). Flow cytometry results also showed that the rate of apoptosis was significantly decreased with different concentrations (20, 50, 100, and 200 μM) of Rg1, and 50 μM of Rg1 was found to be the most effective (P < 0.05). The labels Annexin V-FITC and PI were used to identify different cell populations as follows: viable cells (low Annexin V-FITC and low PI), early apoptotic cells (high Annexin V-FITC and low PI), and late apoptotic cells (high PI and high Annexin V-FITC). The total apoptotic cells include both early and late apoptotic cells. The rate of apoptosis was higher in the serum deprivation group (P < 0.05) (Fig. 2E,F) and Rg1 was found to be the most potent at a concentration of 50 μM. Thus, in the subsequent experimental analysis in serum deprivation, 50 μM of Rg1 was chosen to treat cells for different time (36, 48, and 72 h). Western blot analysis indicated that in comparison to those treated with serum deprivation alone, the expression of cleaved caspase-3 diminished significantly at different time points (P < 0.05) (Fig. 2G). These results confirmed that serum deprivation could induce apoptosis in Raw264.7 macrophages. Rg1 at different concentrations (20, 50, 100, and 200 μM) could inhibit apoptosis, among which 50 μM Rg1 was found to exert the most drastic inhibitory effect. Figure 2. View largeDownload slide Rg1 inhibits apoptosis induced by serum deprivation in Raw264.7 macrophages (A,B) The morphology of cell nucleus as examined by Hochest33342/PI double immunofluorescence labeling. The hyperchromatic nuclei as well as the nuclear condensation are increased consistently in serum deprivation medium at different time intervals (0, 12, 24, 36, 48, and 72 h) that is attenuated by 48 h of treatment with Rg1 (20, 50, 100, and 200 μM). (C) Raw264.7 macrophages with serum deprivation were treated with or without Rg1 (20, 50, 100, and 200 μM) for 48 h. Western blot analysis shows protein expression levels of Bcl-2 and Bax. (D) The ratios of Bcl-2 and Bax were quantified by densitometry based on immunoblot images from (C). (E) The apoptosis was examined by flow cytometry. The down-regulated apoptosis by serum deprivation is suppressed by Rg1 (20, 50, 100, and 200 μM). Compared with the cells pretreated with Rg1 (20, 50, 100, and 200 μM), the apoptosis rate of control cells increased markedly in serum deprivation medium at 48 h. (F) The apoptosis ratio of Raw264.7 macrophages determined by flow cytometry from (E). (G) Raw264.7 macrophages incubated with serum deprivation treated with or without 50 μM Rg1 at different time intervals (36, 48, and 72 h). The relative protein levels were quantified by scanning densitometry and normalized to β-actin. The data are shown as the mean ± SEM of data from three independent experiments. #P < 0.05 compared with control alone; *P < 0.05 compared with serum deprivation alone. Figure 2. View largeDownload slide Rg1 inhibits apoptosis induced by serum deprivation in Raw264.7 macrophages (A,B) The morphology of cell nucleus as examined by Hochest33342/PI double immunofluorescence labeling. The hyperchromatic nuclei as well as the nuclear condensation are increased consistently in serum deprivation medium at different time intervals (0, 12, 24, 36, 48, and 72 h) that is attenuated by 48 h of treatment with Rg1 (20, 50, 100, and 200 μM). (C) Raw264.7 macrophages with serum deprivation were treated with or without Rg1 (20, 50, 100, and 200 μM) for 48 h. Western blot analysis shows protein expression levels of Bcl-2 and Bax. (D) The ratios of Bcl-2 and Bax were quantified by densitometry based on immunoblot images from (C). (E) The apoptosis was examined by flow cytometry. The down-regulated apoptosis by serum deprivation is suppressed by Rg1 (20, 50, 100, and 200 μM). Compared with the cells pretreated with Rg1 (20, 50, 100, and 200 μM), the apoptosis rate of control cells increased markedly in serum deprivation medium at 48 h. (F) The apoptosis ratio of Raw264.7 macrophages determined by flow cytometry from (E). (G) Raw264.7 macrophages incubated with serum deprivation treated with or without 50 μM Rg1 at different time intervals (36, 48, and 72 h). The relative protein levels were quantified by scanning densitometry and normalized to β-actin. The data are shown as the mean ± SEM of data from three independent experiments. #P < 0.05 compared with control alone; *P < 0.05 compared with serum deprivation alone. Rg1 up-regulates autophagy inhibited by serum deprivation in Raw264.7 macrophages It has been suggested that Rg1 exerts a regulatory effect on autophagy of myocardial cells [26]. Therefore, we explored whether Rg1 could regulate autophagy in Raw264.7 macrophages. Under serum deprivation, Raw264.7 macrophages were treated with different concentrations (20, 50, 100, and 200 μM) of Rg1. Western blot analysis showed that compared with those in the pure serum deprivation group, 48 h after treatment with 50 and 100 μM Rg1, the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1 were significantly increased (P < 0.05), and 50 μM of Rg1 was found to be the most effective dose (Fig. 3A,B). To confirm the basic level of autophagy in the blank control group, the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1 were measured with or without 1 h treatment with rapamycin (1 μΜ) or chloroquine (20 μΜ). There was no difference in the autophagic protein expression levels among these groups (P > 0.05; Fig. 3C,D). The results of MDC fluorescence staining showed that in comparison to those in the pure serum deprivation group, the incidence of autophagosomes in Rg1-treated cells was markedly increased and with enhanced fluorescence (P < 0.05; Fig. 3E,F). In serum deprivation, 50 μM of Rg1 was added at different time intervals (36, 48, and 72 h). Western blot analysis indicated that compared with those in the pure serum deprivation group, the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1 were all significantly increased (P < 0.05; Fig. 3G,H). These data suggested that the autophagy level was inhibited after prolonged serum deprivation in Raw264.7 macrophages. On the other hand, when Raw264.7 macrophages were treated with 50 or 100 μM Rg1 for 48 h, the decreased autophagy was reversed, and 50 μM of Rg1 was found to be more effective. Figure 3. View largeDownload slide Rg1 enhances autophagy down-regulated by serum deprivation in Raw264.7 macrophages (A) Raw264.7 macrophages with serum deprivation were treated without or with 20, 50, 100, and 200 μM Rg1 for 48 h. Western blot shows the protein expression levels of Atg5, Beclin1, LC3, and P62/SQSMT1. (B) The ratios of Atg5, Beclin1, LC3, and P62/SQSMT1 were quantified by densitometry based on immunoblot images from (A). (C) Raw264.7 macrophages treated without or with rapamycin (1 μΜ) or chloroquine (20 μΜ) for 48 h. Western blot shows the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1. (D) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (C). (E) MDC staining of the autophagosome. The down-regulated autophagosome by serum deprivation is suppressed by Rg1. (F) Autophagic rate of Raw264.7 macrophages determined by MDC from (E). (G) Raw264.7 macrophages with serum deprivation were treated with or without 50 μM Rg1 for different time (36, 48, and 72 h). Western blot shows the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1. (H) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (G). The relative protein levels were quantified by scanning densitometry and normalized to β-actin. Data are shown as the mean ± SEM from three independent experiments. #P < 0.05 compared with control alone; *P < 0.05 compared with serum deprivation alone. Figure 3. View largeDownload slide Rg1 enhances autophagy down-regulated by serum deprivation in Raw264.7 macrophages (A) Raw264.7 macrophages with serum deprivation were treated without or with 20, 50, 100, and 200 μM Rg1 for 48 h. Western blot shows the protein expression levels of Atg5, Beclin1, LC3, and P62/SQSMT1. (B) The ratios of Atg5, Beclin1, LC3, and P62/SQSMT1 were quantified by densitometry based on immunoblot images from (A). (C) Raw264.7 macrophages treated without or with rapamycin (1 μΜ) or chloroquine (20 μΜ) for 48 h. Western blot shows the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1. (D) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (C). (E) MDC staining of the autophagosome. The down-regulated autophagosome by serum deprivation is suppressed by Rg1. (F) Autophagic rate of Raw264.7 macrophages determined by MDC from (E). (G) Raw264.7 macrophages with serum deprivation were treated with or without 50 μM Rg1 for different time (36, 48, and 72 h). Western blot shows the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1. (H) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (G). The relative protein levels were quantified by scanning densitometry and normalized to β-actin. Data are shown as the mean ± SEM from three independent experiments. #P < 0.05 compared with control alone; *P < 0.05 compared with serum deprivation alone. Rg1 inhibits apoptosis through activating autophagy in macrophages Rg1 was found to inhibit apoptosis in Raw264.7 macrophages, but it remains to be determined if there exist interactions between apoptosis and autophagy in Raw264.7 macrophages and BMDMs. Western blot analysis indicated that compared with those in the serum deprivation+Rg1 group, after addition of autophagy inhibitor 3-MA, the protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1 were significantly reduced (P < 0.05; Fig. 4A,B). StubRFP-SensGFP-LC3 assay showed that compared with that in the serum deprivation (48 h) +Rg1 (50 μM) group without 3-MA, LC3 puncta and autophagy marker expression were found to be significantly reduced in the serum deprivation (48 h)+Rg1 (50 μM) group with 3-MA (P < 0.05; Fig. 4C,D). The results of Hochest33342/PI double fluorescence staining indicated that the cell nucleus fluorescence was enhanced significantly; karyopyknosis and karyoclasis were concurrently increased significantly. Moreover, apoptosis and necrosis of the red-stained cell nucleus were detected during the advanced stage in the serum deprivation+Rg1+3-MA group, as compared with those in the serum deprivation+Rg1 group (Fig. 4E). Apoptosis was measured with or without the addition of 3-MA by flow cytometry. The labels Annexin V-FITC and PI were used to identify different cell populations as follows: viable cells (low Annexin V-FITC and low PI), early apoptotic cells (high Annexin V-FITC and low PI), and late apoptotic cells (high PI and high Annexin V-FITC). The total apoptotic cells include both early and late apoptotic cells. The rate of apoptosis was lower in the serum deprivation+Rg1 group than in the serum deprivation group (P < 0.05). Administration of 3-MA increased the rate of apoptosis in both groups (P < 0.05; Fig. 4F,G). Densitometry of the immunoblots also confirmed that the expression of cleaved caspase-3, a critical executer of apoptosis, was significantly increased after treatment with Rg1 (50 μM) for 48 h. Rg1 inhibited the expression level of cleaved caspase-3 activation, but after addition of the autophagy inhibitor 3-MA, in comparison with that in the serum deprivation+Rg1 group, the inhibitory effects of Rg1 on cleaved caspase-3 activation level were significantly reversed (P < 0.05; Fig. 4H). The same results of the expression of cleaved capase-3 were also shown in BMDM with double immunofluorescence labeling (Fig. 4I,J). It is well accepted that autophagy plays a crucial role in the anti-apoptotic activity of Rg1 and the up-regulation of autophagic flux can prevent apoptotic activation. To further determine the role of autophagy in the effect of apoptosis, Beclin1 siRNA was used. Compared with negative control, Beclin1 siRNA both increased the levels of Bax and cleaved caspase-3 and decreased the level of Bcl-2 in different Beclin1 siRNA-treated groups. The control siRNA appeared to decrease the apoptotic level, which, however, was not statistically significant (Fig. 4K,L). Taken together, all these results suggested that promoting autophagic flux might participate in the anti-apoptotic activity of Rg1 on macrophages against serum deprivation. Figure 4. View largeDownload slide Autophagy is involved in Rg1-inhibited apoptosis in macrophages Macrophages with serum deprivation were treated with 50 μM Rg1 for 48 h in the absence or presence of 3-MA (5 mM). (A) Protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1 determined by western blot analysis. (B) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (A). (C) Representative of the StubRFP-SensGFP-LC3 assay images of autophagy flux. Raw264.7 macrophages were infected with StubRFP-SensGFP-LC3. (D) The number of autolysosome (red) and autophagosome (yellow) puncta were quantified based on StubRFP-SensGFP-LC3 assay images from (C). (E) The morphology of cell nucleus as examined by Hochest33342/PI double immunofluorescence labeling. (F) The apoptosis as examined by flow cytometry. (G) The apoptosis ratio of Raw264.7 macrophages determined by flow cytometry from (F). (H) The protein expression level of cleaved caspase-3 determined by western blot analysis in Raw264.7 macrophages. (I) The protein expression level of cleaved caspase-3 determined by double immunofluorescence labeling in BMDMs. The up-regulated protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 by Rg1 were suppressed by 3-MA, and the down-regulated protein expression of cleaved caspase-3 by Rg1 was suppressed by 3-MA. (J) The average mean value of fluorescence staining with cleaved caspase-3 were quantified by densitometry based on immunofluorescence images from (I). (K) Raw264.7 macrophages and Beclin1 siRNA Raw264.7 macrophages with serum deprivation were treated with or without 50 μM Rg1 for 48 h. Proteins from cell lysates were analyzed by western blotting for Bax, Bcl-2, cleaved caspase-3, and β-actin. (L) The ratios of Bax, Bcl-2, cleaved caspase-3, and β-actin were quantified by densitometry based on immunoblot images from panel K. Data are shown as the mean ± SEM from three independent experiments. #P < 0.05 compared with control alone. *P < 0.05 compared with serum deprivation alone. ΔP < 0.05 compared with Rg1+serum deprivation. **P < 0.05 compared between control alone and Beclin1 siRNA control alone; ##P < 0.05 compared between serum deprivation alone and Beclin1 siRNA serum deprivation alone; ΔΔP < 0.05 compared between Rg1+serum deprivation and Beclin1 siRNA Rg1+serum deprivation. Figure 4. View largeDownload slide Autophagy is involved in Rg1-inhibited apoptosis in macrophages Macrophages with serum deprivation were treated with 50 μM Rg1 for 48 h in the absence or presence of 3-MA (5 mM). (A) Protein expression levels of Atg5, Beclin1, LC3, and p62/SQSMT1 determined by western blot analysis. (B) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (A). (C) Representative of the StubRFP-SensGFP-LC3 assay images of autophagy flux. Raw264.7 macrophages were infected with StubRFP-SensGFP-LC3. (D) The number of autolysosome (red) and autophagosome (yellow) puncta were quantified based on StubRFP-SensGFP-LC3 assay images from (C). (E) The morphology of cell nucleus as examined by Hochest33342/PI double immunofluorescence labeling. (F) The apoptosis as examined by flow cytometry. (G) The apoptosis ratio of Raw264.7 macrophages determined by flow cytometry from (F). (H) The protein expression level of cleaved caspase-3 determined by western blot analysis in Raw264.7 macrophages. (I) The protein expression level of cleaved caspase-3 determined by double immunofluorescence labeling in BMDMs. The up-regulated protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 by Rg1 were suppressed by 3-MA, and the down-regulated protein expression of cleaved caspase-3 by Rg1 was suppressed by 3-MA. (J) The average mean value of fluorescence staining with cleaved caspase-3 were quantified by densitometry based on immunofluorescence images from (I). (K) Raw264.7 macrophages and Beclin1 siRNA Raw264.7 macrophages with serum deprivation were treated with or without 50 μM Rg1 for 48 h. Proteins from cell lysates were analyzed by western blotting for Bax, Bcl-2, cleaved caspase-3, and β-actin. (L) The ratios of Bax, Bcl-2, cleaved caspase-3, and β-actin were quantified by densitometry based on immunoblot images from panel K. Data are shown as the mean ± SEM from three independent experiments. #P < 0.05 compared with control alone. *P < 0.05 compared with serum deprivation alone. ΔP < 0.05 compared with Rg1+serum deprivation. **P < 0.05 compared between control alone and Beclin1 siRNA control alone; ##P < 0.05 compared between serum deprivation alone and Beclin1 siRNA serum deprivation alone; ΔΔP < 0.05 compared between Rg1+serum deprivation and Beclin1 siRNA Rg1+serum deprivation. Rg1 promotes autophagy and inhibits apoptosis through AMPK/mTOR pathway in Raw264.7 macrophages Mammalian target of rapamycin 1 (mTORC1) is a well-documented inhibitor of autophagy. Inhibition of AMPK induces autophagy by inhibition of mTORC1. AMPK phosphorylation at threonine 172 (T172) in the α-subunit is a key mechanism in mediation of AMPK activation [20,21]. To evaluate the roles of AMPK and mTOR in groups exposed to serum deprivation, different concentrations (20, 50, 100, and 200 μM) of Rg1 were used to treat macrophages for 48 h under the condition of serum deprivation. Western blot analysis showed that decreased phosphorylation of AMPK and mTOR in serum deprivation group was reversed, and 50 μM of Rg1 was found to be the most effective dose (P < 0.05; Fig. 5A,B). In order to explore the correlation among Rg1, AMPK/mTOR signal pathway and the level of autophagy in Raw264.7 macrophages, compound C, or AICAR was used to pre-treat Raw264.7 macrophages. Treatments with compound C or AICAR in blank control groups could not obviously reverse the levels of p-mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1 protein expression (P > 0.05; Fig. 5C,D). Compared with those in the serum deprivation (48 h)+Rg1 (50 μM) treatment group, in cells pretreated with compound C, the levels of p-mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1 protein expression up-regulated by Rg1 were significantly reversed by compound C (P < 0.05). When cells were pretreated with AICAR, the expressions of p-mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1 were further up-regulated (P < 0.05; Fig. 5E,F). Immunofluorescence double labeling showed the same result of LC3 in cells as that in western blot analysis, which were statistically significant (P < 0.05; Fig. 5G,H). The above data showed that Rg1 promotes autophagy and inhibits apoptosis induced by serum deprivation through the AMPK/mTOR pathway. Figure 5. View largeDownload slide AMPK/mTOR is required for Rg1-inhibited apoptosis induced by serum deprivation in Raw264.7 macrophages (A) Raw264.7 macrophages lysates of various treatment were subject to western blot analysis by using antibodies specific for total AMPK, phosphor-AMPK, total mTOR, and phosphor-mTOR. The relative protein levels were quantified by scanning densitometry and normalized to total AMPK or total mTOR, respectively. (B) The ratios of total AMPK, phosphor-AMPK, total mTOR, and phosphor-mTOR were quantified by densitometry based on immunoblot images from (A). (C) Raw264.7 macrophages incubated without or with compound C (10 mM) or AICAR (250 μM) for 48 h. (D) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (C). (E) Raw264.7 macrophages with serum deprivation were treated with 50 μM Rg1 for 48 h in absence or presence of compound C (10 mM) or AICAR (250 μM). Western blots of the protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1. (F) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (E). (G) The corresponding immunofluorescence images for protein expression of LC3. (H) The numbers of LC3 puncta ratio based on immunofluorescence images from (G). The AMPK inhibitor compound C reverses the Rg1-promoted protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 via activation of mTOR, and the AMPK inducer AICAR fosters the Rg1-promoted protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 through suppression of AMPK/mTOR. Data are shown as the mean ± SEM from three independent experiments. #P < 0.05 compared with control alone; *P < 0.05 compared with serum deprivation alone; ΔP < 0.05 compared with Rg1+serum deprivation. Figure 5. View largeDownload slide AMPK/mTOR is required for Rg1-inhibited apoptosis induced by serum deprivation in Raw264.7 macrophages (A) Raw264.7 macrophages lysates of various treatment were subject to western blot analysis by using antibodies specific for total AMPK, phosphor-AMPK, total mTOR, and phosphor-mTOR. The relative protein levels were quantified by scanning densitometry and normalized to total AMPK or total mTOR, respectively. (B) The ratios of total AMPK, phosphor-AMPK, total mTOR, and phosphor-mTOR were quantified by densitometry based on immunoblot images from (A). (C) Raw264.7 macrophages incubated without or with compound C (10 mM) or AICAR (250 μM) for 48 h. (D) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (C). (E) Raw264.7 macrophages with serum deprivation were treated with 50 μM Rg1 for 48 h in absence or presence of compound C (10 mM) or AICAR (250 μM). Western blots of the protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1. (F) The ratios of Atg5, Beclin1, LC3, and p62/SQSMT1 were quantified by densitometry based on immunoblot images from (E). (G) The corresponding immunofluorescence images for protein expression of LC3. (H) The numbers of LC3 puncta ratio based on immunofluorescence images from (G). The AMPK inhibitor compound C reverses the Rg1-promoted protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 via activation of mTOR, and the AMPK inducer AICAR fosters the Rg1-promoted protein expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 through suppression of AMPK/mTOR. Data are shown as the mean ± SEM from three independent experiments. #P < 0.05 compared with control alone; *P < 0.05 compared with serum deprivation alone; ΔP < 0.05 compared with Rg1+serum deprivation. Discussion Macrophage apoptosis in atherosclerotic lesions is almost certainly multifactorial, and it is likely that at least some of the death inducers are different in early versus late lesions. Despite this complexity and the paucity of in vivo data, several proapoptotic factors have been suggested based on cell culture models that attempt to mimic conditions in atherosclerotic lesions. Examples include high concentrations of oxidized LDL, oxysterols, TNF-α, Fas ligand, nitric oxide, growth factor withdrawal, hypoxia/ATP depletion, and intracellular accumulation of unesterified, or ‘free’ cholesterol [29]. However, the results of this study are based on serum deprivation to mimic macrophage apoptosis in advanced atherosclerotic lesions in vitro, which may be first suggested in macrophages. Nutrition is indispensable for cell survival and proliferation [30]. Thus, loss of nutrients caused by serum starvation in cells could induce formation of ROS, resulting in cell death [31]. In fact, serum deprivation is a vigorous stimulus for the induction of energy limiting stresses. These stresses are caused by elevation of ROS production and apoptosis [32]. Serum used in cell culture is a mixture of essential proteins and various factors for cell growth and proliferation [33]. Therefore, serum deprivation causes cellular apoptosis, and serum deprivation is widely used to investigate induction of apoptosis as well as related signal transduction pathways [34–36]. In this study, macrophages incubated in media without serum for the indicated time periods were induced to undergo apoptosis. These conditions closely mimic the pathophysiological conditions in advanced atherosclerotic lesions, wherein oxidative stress plays key roles in the induction of cell death caused by serum deprivation. Our results confirmed that Rg1 significantly reduces apoptosis in macrophages by promoting autophagic flux, but not by impairing autophagic flux. Several protein complexes have been found to regulate autophagy induction, autophagosomes formation and maturation into autolysosomes. The PtdIns3K-III/BENC1 complex is necessary for autophagosome initiation. Lipidation and redistribution of the cytoplasmic protein LC3 towards the phagophore contributes to its elongation around the cargo to be engulfed; and an autophagic receptor like p62/SQSTM1 allows cargo recognition. Upon autolysosome maturation, lysosomal catabolic enzymes degrade its content, including the autophagic receptor p62/SQSTM1, providing the cells with building blocks in order to maintain the energy status. When the autophagic flux is impaired, the cargo is not degraded leading to an accumulation of p62/SQSTM1 [37]. We have shown that 3-MA, an autophagic inhibitor, could reverse the inhibitory effect of Rg1 on apoptosis of Raw264.7 macrophages and BMDM induced by serum deprivation. The same phenomenon was also confirmed in Beclin1 siRNA Raw264.7 macrophages. The expression levels of apoptosis-related proteins in Raw264.7 macrophages are significantly lower in Beclin1 siRNA Raw264.7 macrophages with Rg1 treatment. In addition, inhibiting AMPK with compound C suppressed the phosphorylation of mTOR and the expressions of autophagy-related proteins, including Atg5, Beclin1, LC3, and p62/SQSMT1. Moreover, AICAR was found to upregulate the phosphorylation of mTOR and the expressions of autophagy-related proteins, such as Atg5, Beclin1, LC3, and p62/SQSMT1, ultimately. All these results suggest that Rg1 induces autophagy, which effectively down-regulates apoptosis of macrophage induced by serum deprivation, through activating AMPK/mTOR signal pathway. We reported previously that Rg1 induced autophagy, which contributed to inhibit inflammatory responses generated by OXLDL-induced Raw264.7 macrophages This study has extended our previous study and suggested that Rg1 could upregulate the expressions of autophagy-related proteins (Atg5, Beclin1, LC3, and p62/SQSMT1) in Raw264.7 macrophages under serum deprivation. In the present study, 50 μM of Rg1 was found to be the most effective dose, which increased the expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 under serum deprivation for 48 h. The expressions of Atg5, Beclin1, LC3, and p62/SQSMT1 were higher in the 50 μM Rg1 group than in the 20, 100, and 200 μM Rg1 groups, demonstrating that 50 μM of Rg1 is the optimal dose to promote autophagy in Raw264.7 macrophages; hence, any dosage deviating from this may dilute the effect of Rg1 in promoting autophagy. It has been reported that, in the advanced stage of AS, decreased autophagy and defected efferocytosis result in impaired clearance of apoptotic macrophages, leading to further inflammation and promotion of lipid core necrosis accompanied by increases in unstable factors of plaque [38]. Studies have indicated that after gene silencing or knockout Atg5 in macrophages, apoptosis in macrophages and oxidative stress response mediated by NADPH oxidase were enhanced [8,39]. Our results further showed that the autophagy inhibitor 3-MA reduces the expressions of autophagy-related proteins induced by Rg1 in Raw264.7 macrophages and counteracts the anti-apoptotic role of Rg1, thus, supporting that Rg1 protects against apoptosis by promoting autophagy in macrophages. However, many studies by others have reported that serum deprivation does not inhibit autophagy; rather, it induces autophagy as a survival mechanism [40]. A possible explanation for the discrepancy may be attributed to the possibility that regulation of autophagy might be a time-dependent event during serum deprivation. Thus, in a shorter duration (within 24 h), the process occurs in parallel with the well-known early steps in autophagy induction (AMPK activation, mTOR dephosphorylation, increased conjugation of LC3 to phosphatidylethanolamine to form LC3II, recruitment of LC3II to autophagosomal membranes), and a subsequent decrease in p62/SQSMT1. Protein p62/SQSMT1 is involved in the assembly of autophagosome cargoes [41]. However, the process may play a protective role when the serum deprivation time is prolonged (e.g. over 36 h). The protective function may be reversed to promote apoptosis through downregulating autophagy level via inhibiting AMPK signaling as shown in the present study. Another explanation would be that during starvation, cells can enhance amino acid uptake and synthesis through the general amino acid control (GAAC) pathway, whereas nonessential cellular contents are recycled by autophagy. Chen et al. [42] reported that serum/glutamine starvation activates the GAAC pathway, which up-regulates amino acid transporters, leading to increased amino acid uptake. This elevates the intracellular amino acid level, which in turn reactivates mTOR and suppresses autophagy. Knockdown of the activating transcription factor 4, the major transcription factor in the GAAC pathway, or of SLC7A5, a leucine transporter, impaired mTOR reactivation and induced much higher levels of autophagy. Besides, Razani et al. [43] confirmed that the lesion sizes and necrotic areas were larger in arteries of Atg5fl/flLysmcre+/−/LDLr−/− mice than those in the wild-type control group. A large number of apoptotic cells, activated caspase-3, DHE, and p47 were detected in the plaque of Atg5fl/flLysmcre+/−/LDLr−/− mice with the use of TUNEL experiment. Therefore, in LDLr−/− mice, macrophage autophagy defect increases apoptosis and oxidative stress reaction of macrophages in plaque, and promotes atheromatous plaque necrosis. Liao et al. [44] showed that macrophage apoptosis and defective macrophage apoptosis can promote necrosis on atherosclerotic old plaque, and autophagy can inhibit apoptosis in macrophages and efferocytosis defect, which plays an important role in the process of AS pathological changes. The AMPK/mTOR pathway plays an important role in autophagy regulation in response to stress and glucose starvation. Both are reported to maintain renal tubular homeostasis and are involved in autophagy induced by I/R in renal tubular cell injury [21,45]. There is mounting evidence [46,47] indicating that AMPK/mTOR signal pathway plays an important role in the induction of autophagy. Hence, inhibiting AMPK/mTOR signal pathway can further inhibit the expressions of macrophage-related proteins inhibited by serum deprivation; enhancing AMPK/mTOR signal pathway can reverse the expressions of macrophage-related proteins inhibited by serum deprivation. As observed, Rg1 is able to activate AMPK/mTOR signal pathway inhibited by serum deprivation in macrophages. This is not consistent with results by others [26], which showed that Rg1 can inhibit AMPK phosphorylation and reduce autophagy in H9c2 myocardial cells. This conflicting result may be attributed to different cell sources and different experimental paradigms used. We confirmed that pretreatment of Raw264.7 macrophages with compound C followed by Rg1 inhibited the expressions of autophagy-related proteins (Atg5, Beclin1, LC3, and p62/SQSMT1). Additionally, AICAR pretreatment further enhanced the expressions of autophagy-related proteins (Atg5, Beclin1, LC3, and p62/SQSMT1). Autophagy is up-regulated by the activation of AMPK and inhibition of mTOR [48]. LC3 and autophagy-related genes 5–12 (Atg5–12) are required for the formation of the double-membrane autophagosomes. The autophagosome formation can be detected by measuring the conversion of LC3-I (unconjugated cytosolic form) to LC3II (autophagosomal membrane-associated phosphatidylethanolamine-conjugated form) [10,49,50]. Additionally, the p62/SQSTM1 protein, also known as sequestosome-1, recognizes cellular components marked for degradation and targets them for autophagy by the virtue of its ubiquitin association domain (UBA) and a LC3-interacting region (LIR) [51]. Impairment of autophagic clearance results in the build-up of p62/SQSTM1 and cellular dysfunction [52]. Studies have suggested that there may exist some complex and cross regulatory mechanisms between autophagy and apoptosis, including independent regulation, coordinated regulation, and antagonistic regulation [53]. Therefore, the regulatory function of Rg1 in autophagy and apoptosis may rely on the interaction of different signal pathways. It was found that Rg1 could reverse the diminished expression levels of autophagy-related proteins (Atg5, Beclin1, LC3, and p62/SQSMT1), suggesting that AMPK/mTOR activated by Rg1 might regulate autophagy and apoptosis. Along with this, we also showed that preconditioning macrophages with 3-MA followed by Rg1 results in decreased expressions of autophagy-related proteins (Atg5, Beclin1, LC3, and p62/SQSMT1), but increased expression of cleaved caspase-3. In summary, our results indicate that Rg1 positively regulates the autophagy process through an association with the AMPK/mTOR signaling pathway. Autophagy inhibits apoptosis and plays a protective role under conditions of serum deprivation in macrophage as demonstrated in the present experimental paradigm in Raw264.7 macrophages (Fig. 6). Importantly, we have also shown that AMPK pathway is one of the signaling pathways by which Rg1 exerts its anti-apoptotic effects. It can be concluded that Rg1 exerts its protective effects to inhibit apoptosis by upregulating autophagy due to activation of AMPK/mTOR channel on the macrophages and this study may be helpful for the design of novel therapeutic strategies for the treatment of AS. Figure 6. View largeDownload slide Rg1-mediated inhibition of apoptosis by activating autophagy through AMPK/mTOR in response to serum deprivation in macrophages Figure 6. 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Acta Biochimica et Biophysica SinicaOxford University Press

Published: Feb 1, 2018

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