Matrine Exerts Antidepressant-Like Effects on Mice: Role of the Hippocampal PI3K/Akt/mTOR Signaling

Matrine Exerts Antidepressant-Like Effects on Mice: Role of the Hippocampal PI3K/Akt/mTOR Signaling Background: Current antidepressants in clinical use always take weeks or even months to exert full therapeutic effects, and sometimes have serious side effects. Thus, it is very necessary to develop novel antidepressants with better efficacy and fewer adverse effects. The present study focused on investigating the antidepressant potential of matrine and its possible mechanisms of action. Methods: The forced swim test, tail suspension test, and chronic unpredictable mild stress model of depression were used to reveal the antidepressant-like effects of matrine on mice. Western blotting, immunohistochemistry, and lentivirus were further used together to explore the antidepressant mechanism of matrine. Results: It was found that matrine exhibited significant antidepressant actions in the forced swim test and tail suspension test without affecting the locomotor activity of mice. Chronic matrine administration fully reversed the chronic unpredictable mild stress-induced depressive-like symptoms in forced swim test, tail suspension test, and sucrose preference test. After that, western blotting analysis revealed that chronic matrine treatment restored the decreasing effects of chronic unpredictable mild stress on the PI3K/Akt/mammalian target of rapamycin signaling in hippocampus, but not prefrontal cortex. Furthermore, pharmacological and genetic blockade of the PI3K/Akt/mammalian target of rapamycin signaling in hippocampus abolished the antidepressant actions of matrine on mice. Conclusions: Taken together, matrine produces antidepressant-like effects on mice via promoting the hippocampal PI3K/Akt/ mammalian target of rapamycin signaling. Keywords: chronic unpredictable mild stress, depression, hippocampus, matrine, mTOR Introduction Depression is one of the leading public health problems in the et  al., 2008). Currently, most antidepressants used in clinical world, and antidepressants are among the most commonly practice elevate the level of monoaminergic neurotransmit- prescribed medications (Bowden, 2005; Shelton, 2007; Krishnan ters in brain, particularly serotonin and norepinephrine (Berton Received: December 18, 2017; Revised: February 27, 2018; Accepted: April 6, 2018 © The Author(s) 2018. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 1 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 2 | International Journal of Neuropsychopharmacology, 2018 Significance Statement This study shows that matrine possess antidepressant-like effects that are mediated by activating the PI3K/Akt/mTOR signaling in hippocampus. Its significance is as follows: (1) Extending the knowledge of matrine’s pharmacological effects; (2) providing a potential antidepressant with better efficacy or fewer side effects; (3) further proving that the central mTOR signaling is a good target for developing novel antidepressants. et al., 2006; Hirschfeld, 2012). However, it always takes weeks or relative humidity) for 1 week with free access to food and even months for these antidepressant drugs to exert their full water. Behavioral experiments were carried out during the therapeutic effects, and many patients are resistant to them light phase. All experimental procedures were conducted in (Berton et  al., 2006; Hirschfeld, 2012). Therefore, it is necessary accordance with the Guide for the Care and Use of Laboratory to develop novel antidepressants based on different pharmaco- Animals (8th edition, Institute of Laboratory Animal Resources logical targets. on Life Sciences, National Research Council, National Academy Mammalian target of rapamycin (mTOR) is a large serine/ of Sciences, Washington DC). threonine kinase that regulates initiation of protein translation (Abe et al., 2010). Activation of mTOR by phosphatidylinositol-3 Materials kinase (PI3K)/protein kinase B (AKT) results in its phosphoryl- ation at serine 2448, thereby promoting its downstream mol- Matrine (purity  >98%) and fluoxetine were bought from ecules, p70 ribosomal protein S6 kinase (p70S6K) and eukaryotic Sigma and dissolved in 1% DMSO in 0.9% saline. LY294002 initiation factor 4E-binding protein 1 (4E-BP-1). Then, p70S6K was purchased from Tocris, rapamycin was purchased from and 4E-BP-1 promote initiation of protein translation for syn- Target Mol, and they were dissolved in 1% DMSO in ACSF. aptic protein synthesis (Hay et  al., 2004; Dann et  al., 2007; The doses of matrine (15, 30, 60, and 120  mg/kg), fluox- Hashimoto, 2011). Recent studies support the hypothesis that etine (20  mg/kg), LY294002 (10  nmol/site), and rapamycin major depressive disorder may be a consequence of deficiency (0.2 nmol/site) were chosen based on previous reports (Jiang in the mTOR signaling (Hashimoto, 2011; Ludka et  al., 2016). It et  al., 2015; Gong et  al., 2016; Ludka et  al., 2016; Jiang et  al., has also been demonstrated that upregulation of the mTOR 2017). Matrine and fluoxetine were i.p. injected in a volume signaling in prefrontal cortex (PFC) and hippocampus leads to of 10 mL/kg. LY294002 and rapamycin were intracerebroven- rapid and consistent antidepressant-like effects (Li et al., 2010; tricularly (i.c.v.) infused. Hashimoto, 2011; Zhou et al., 2014). Matrine, a quinolizidine alkaloid compound extracted from FST the root of Sophorae flavescens, is known for its various pharma- cological activities. Matrine has been shown to have antiinflam- FST was carried out as previously described (Jiang et al., 2015; matory, antiallergic, antivirus, antitumor, and cardiovascular Jiang et al., 2017). Briefly, each mouse was individually placed protective effects (Li et al., 2010Fu et  ; al., 2014; Liu et al., 2014; in a glass cylinder (15  cm diameter, 25  cm height) filled with Kan et  al., 2015; Wu et  al., 2017). Recently, more and more 10  cm of 25°C ± 1°C water and forced to swim for 6 minutes. matrine-induced pharmacologic effects on the central nervous The immobility duration of the last 4 minutes was recorded system have been reported. For example, Gong et  al. reported by an investigator blind to the study. Water was exchanged that matrine had neuroprotective effects on a mouse model of between 2 trails. The time during which each mouse was float- vincristine-induced neuropathic pain (Gong et al., 2016). Zhang ing in water without struggling and only making minor move- et al. showed that matrine improved cognitive impairments in a ments to keep its nose above the water was regarded as the rat model of Alzheimer’s disease (Zhang et al., 2015). Meng et al. immobility time. showed that matrine also had neuroprotective effects against MPTP-induced Parkinson’s disease (Meng et al., 2017). Here, we TST noticed that in 2017, matrine had been demonstrated to have promoting effects on the PI3K/Akt/mTOR signaling in corpus TST was carried out as previously described (Jiang et  al., 2015, callosum (Liu et  al., 2017). Thus, we assume that matrine may 2017). Briefly, each mouse was suspended on the shelf, which possess antidepressant-like effects via activating mTOR. In this was 70 cm above the ground, for 6 minutes. A tape was placed study, we investigated this assumption using various methods, at about 1.5 cm away from the tip of tail. The immobility time including the forced swimming test (FST), tail suspension test during which each mouse was completely motionless was (TST), and chronic unpredictable mild stress (CUMS) model of recorded for the last 4 minutes. The observer was unaware of depression. mice grouping. Materials and Methods Locomotor Activity Test Animals As reported (Jiang et al., 20152017 , ), briefly, each mouse was indi- Adult male C57BL/6J mice (8 weeks old) were obtained from vidually placed in the middle of a wooden box (50× 50 × 40  cm) the Experimental Animal Center of Medical College, Nantong that consisted of 25 equal squares on the floor (10× 10  cm). The University. Before use, the mice were housed under stand- number of squares each mouse crossed was recorded for 5 min- ard conditions (12-hour-light/-dark cycle; lights on from utes by an observer unaware of mice grouping. The apparatus 7:00 am to 7:00 pm; 2°C ±1°C ambient temper ature; 55% ± 10% was cleaned after each test. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wu et al. | 3 PVDF membranes. The transferred membranes were blocked in CUMS 5% bovine serum albumin and then incubated with different In this procedure, mice were subjected to a random sequence primary antibodies overnight at 4°C. Primary antibodies against of unpredictable and mild stressors for 8 weeks. The stressors mTOR (1:500; Abcam), p-mTOR (Ser2448, 1:500; Abcam), 4E-BP-1 are listed as follows: (1) food or water deprivation for 24 hours; (1:500; Cell Signaling), p-4E-BP-1 (Thr37/46, 1:500; Cell Signaling), (2) damp sawdust for 12 hours; (3) restraint for 2 hours; (4) cage p70S6K (1:1000; Cell Signaling), p-p70S6K (Thr389, 1:1000; Cell shaking for 15 minutes; (5) inversion of light/dark cycle; (6) 45°C Signaling), AKT(1:500; Cell Signaling), p-AKT (Ser473, 1:500; cage tilting in empty cage for 12 hours; (7) tail pinch for 5 min- Cell Signaling), and β-actin (1:5000; Immunoway) were used. utes. Control mice were left undisturbed in home cages in a After incubated with goat anti-rabbit or goat anti-mouse HRP- separate room. Repeated administration of vehicle/fluoxetine/ conjugated secondary antibody for 2 hours at room tempera- matrine/inhibitors was given daily during the last 2 weeks. After ture, the blots were detected by enhanced chemiluminescence. CUMS and drug treatments, FST, TST, and the sucrose preference test were performed. Immunohistochemistry To analyze hippocampal neurogenesis, mice were anesthe- Sucrose Preference Test tized using 0.5% pentobarbital sodium (50 mg/kg, i.p.) and then For this test, mice were first adapted to 1% sucrose solution transcardially perfused with normal saline followed by 4% for 48 hours, then deprived of water and food for 24 hours, and paraformaldehyde in 0.01 M phosphate buffer saline (PBS). The finally given 2 preweighed bottles containing 1% sucrose solu- brains were postfixed in 4% paraformaldehyde overnight and tion and tap water, respectively. All the mice were housed indi- dehydrated in 30% sucrose solution for 2 days. Serial sections of vidually during this test, and the index of sucrose preference 25 μm were cut throughout hippocampi using a freezing micro- was calculated as a percentage of the volume of sucrose con- tome (Leica) and preserved in 0.01 M PBS. After that, the sections sumed/total volume of liquid consumed. were permeabilized with 0.3% Triton X-100 in PBS (30 minutes, first) and 3% bovine serum albumin in PBS (30 minutes, second) Intracerebroventricular (i.c.v.) Injections at room temperature. Then the sections were incubated with rat polyclonal anti-doublecortin (DCX, 1:100; Cell Signaling) primary As reported (Jiang et  al., 2016, 2017), each mouse was deeply antibody under 4°C overnight. The sections were further incu- anesthetized with 0.5% pentobarbital sodium (50  mg/kg, i.p.) bated in fluorescein isothiocyanate-labeled horse anti-rabbit and placed in a stereotaxic device (Stoelting). Scalp skin was IgG (1:50; Thermo Fisher) for 2 hours at room temperature. After shaved with clippers and disinfected using iodine. After opening washing, the sections were coverslipped and observed using a the scalp skin and exposing the skull, infusion cannulas were confocal fluorescence microscope (Leica). Examination of the implanted into the left lateral brain ventricle (AP = −0.2  mm, DCX-positive (DCX+) cells was confined to dentate gyrus (DG), ML = +1.0  mm, DV = +2.3  mm) and cemented. The mice were especially in the granule cell layer (GCL), including the subgran- allowed to recover for 3 d.  Osmotic minipumps were used to ular zone of hippocampus that was defined as a 2-cell body-wide deliver 0.3 μL/min of LY294002 or rapamycin or 1% DMSO in zone along the border between GCL and hilus. Quantifications ACSF (final volume, 3 μL/mouse). of the DCX+ cells were respectively conducted from 1-in-6 ser - ies of hippocampal sections spaced at 150 μm and spanning the Lentivirus Microinfusion rostrocaudal extent of DG bilaterally. Every DCX+ cell within the GCL and subgranular zone was counted. This was done according to previous reports (Jiang et al., 2014). Briefly, each mouse was deeply anesthetized with 0.5% pento- Statistical Analysis barbital sodium (50  mg/kg, i.p.) and placed in a stereotaxic device. After exposing the skull, 5 µL microsyringes (Hamilton) All data were analyzed using SPSS 13.0 software (SPSS Inc.). were inserted into hippocampus bilaterally to deliver lenti- Multiple group comparisons were performed using 1-way or virus. The location is as follows: AP = -2.3  mm, ML = ±1.6  mm, 2-way ANOVA, as appropriate. P < .05 was considered statistic- DV = +1.9  mm. Infusion of lentivirus was performed bilaterally ally significant. at a rate of 0.3 µL/min (1.5  µL/side). The mice were allowed to recover for 3 d. RESULTS LV-mTOR-shRNA-EGFP and LV-Scrambled-shRNA- EGFP used in this study were produced and provided by Effects of Matrine on the Immobility of Mice in FST Genechem Co., Ltd. The titers were adjusted to 6× 10 TU/ and TST mL. The sequences for mTOR-shRNA and Scrambled-shRNA were 5’-GGCCTATGGTCGAGATTTA-3’ and 5’-TTCTCCGAACGTGT As a first step of this study, FST and TST were used. Figure 1A CACGT-3’, respectively. and B showed the effects of matrine on the immobility dur - ation of mice in FST and TST, respectively. It was found that both matrine and fluoxetine significantly reduced the immo- Western Blotting bility of mice in FST and TST compared with the control mice The hippocampus and PFC tissues were dissected and imme- (n = 10). For the FST data, ANOVA indicated a significant main diately homogenized in NP-40 lysis buffer containing protease effect of drug treatment [F(5, 54) = 28.592, P < .01]. For the TST inhibitor cocktail for 30 minutes. Then the homogenates were data, ANOVA also revealed a significant main effect of drug centrifuged at 12 000 × g for 30 minutes at 4°C. The supernatants treatment [F(5, 54) = 33.017, P < .01]. Detailed analysis showed were collected on ice, and protein concentrations were assayed that compared with the control mice, 15  mg/kg of matrine using the BCA method. Then 30 µg of protein samples was sepa- treatment moderately decreased the immobility, 30  mg/kg rated by SDS-PAGE gels. After that, proteins were transferred to and 60  mg/kg of matrine treatment significantly decreased Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 4 | International Journal of Neuropsychopharmacology, 2018 Figure 1. Antidepressant-like effects of matrine in forced swim test (FST) and tail suspension test (TST). Naive mice were i.p. injected with a single dose of vehicle, fluoxetine (20 mg/kg), or matrine (15, 30, 60, or 120 mg/kg). The behavioral tests were conducted 30 minutes after the injection. Different groups of mice were used for these tests. (A) The matrine-treated mice had significantly less immobility than the vehicle-treated mice in FST. (B) The matrine-treated mice also showed significantly less immobility than the vehicle-treated mice in TST. (C) All groups of mice displayed similar locomotor activity in the locomotor activity test. Data are expressed as means ± SEM (n = 10); *P < .05, **P < .01; n.s., no significance. Comparisons were made by 1-way ANOVA followed by posthoc LSD test. the immobility, while 120  mg/kg matrine produced a similar reversed the promoting effects of CUMS on the immobility effect to 60 mg/kg matrine. Therefore, 30 mg/kg and 60 mg/kg of mice in TST (n = 10). ANOVA revealed a significant inter- were selected as the doses for matrine in the following stud- action [F(3, 72) = 42.045, P < .01] with significant effects of CUMS ies. Besides, Figure  1C showed that matrine treatment did [F(1, 72) = 54.134, P < .01] and drug treatment [F(3, 72)= 38.578, not affect the locomotor activity of mice (n = 10), and ANOVA P < .01]. Figure  2D illustrates the sucrose preference data. showed no significant effects of drug treatment [F(3, 36)= 0.715, Also, we found that chronic injection of matrine protected P = .386]. Together, matrine may have antidepressant-like against the CUMS-induced anhedonia behavior (n = 10). ANOVA effects on mice. revealed a significant interaction [F(3, 72)= 39.606, P < .01] with significant effects of CUMS [F(1, 72) = 51.552, P < .01] and drug treatment [F(3, 72) = 29.506, P < .01]. Thus, matrine indeed pos- Effects of Matrine on the Depressive-Like Behaviors sessed antidepressant-like effects on mice. of Mice in the CUMS Model of Depression Depression is also accompanied with decreased hippocam- Next, the CUMS model of depression was used to assay the pal neurogenesis (Santarelli et  al., 2003). Here, we examined antidepressant effects of matrine, with FST, TST, and the whether matrine prevented the CUMS-induced effects on sucrose preference test used to evaluate the CUMS-induced neurogenesis using DCX immuohistochemistry. It is known that depressive-like behaviors (Reid et  al., 1997; Shields, 2006). DCX is a microtubule-associated protein that serves as a marker Figure 2B illustrates the FST data. It was found that CUMS sig- of neurogenesis because of its transient expression in new- nificantly enhanced the immobility of mice, while matrine born neurons (Brown et al., 2003). As shown in Figure 3, chronic administration fully restored this change (n = 10). ANOVA matrine administration fully restored the decreasing effects of revealed a significant interaction [F(3, 72)= 33.097, P < .01] CUMS on the number of DCX+ cells in DG (n= 5). ANOVA revealed with significant effects of CUMS [F(1, 72) = 43.767, P < .01] and a significant interaction [F(3, 17)= 55.397, P < .01] with significant drug treatment [F(3, 72)= 26.143, P < .01]. Figure  2C illustrates effects of CUMS [F(1, 17)= 74.356, P < .01] and drug treatment the TST data, and similarly, matrine treatment significantly [F(3, 17) = 41.479, P < .01]. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wu et al. | 5 Figure 2. Antidepressant-like effects of matrine on the chronic unpredictable mild stress (CUMS) model of depression. Mice were exposed to 8 weeks of CUMS and received daily injection of vehicle, fluoxetine (20 mg/kg), or matrine (30 or 60 mg/kg) during the last 2 weeks. The behavioral tests were then conducted. (A) Schematic timeline of experimental procedures. (B) CUMS + matrine mice spent significantly less immobility than that of CUMS + vehicle mice in forced swim test (FST). (C) CUMS + matrine mice spent significantly less immobility than that of CUMS + vehicle mice in tail suspension test (TST). (D) CUMS + matrine mice displayed signifi- cantly higher sucrose preference than that of CUMS + vehicle mice. Data are expressed as means ± SEM (n = 10); *P < .05, **P < .01; n.s., no significance. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. 24) = 23.628, P < .01]. For the p-4E-BP-1 data, ANOVA indicated a Effects of Matrine on the Central PI3K/AKT/mTOR significant interaction [F(2, 24)= 40.042, P < .01] with significant Signaling in CUMS-Stressed Mice effects of CUMS [F(1, 24)= 56.278, P < .01] and drug treatment After the behavioral tests, western blotting experiments were [F(2, 24) = 33.712, P < .01]. For the p-p70S6K data, ANOVA indicated performed to detect the effects of matrine on levels of p-AKT a significant interaction [F(2, 24)= 48.135, P < .01] with significant (Ser473), AKT, p-mTOR (Ser2448), mTOR, p-4E-BP-1 (Thr37/46), effects of CUMS [F(1, 24)= 62.592, P < .01] and drug treatment 4E-BP-1, p-p70S6K (Thr389), p70S6K, and β-actin in both hippo- [F(2, 24) = 37.388, P < .01]. Matrine administration did not affect campus and PFC. Figure  4B illustrates the PFC data. Although the level of the central PI3K/AKT/mTOR signaling in naive mice CUMS significantly decreased the expression of p-AKT, p-mTOR, (n = 5). In contrast, the levels of total AKT, mTOR, 4E-BP-1, and p-4E-BP-1, and p-p70S6K in PFC, chronic matrine administra- p70S6K were unchanged among all groups (n= 5). Therefore, the tion did not restore these changes (n = 5). Figure  4A illustrates antidepressant-like effects of matrine on mice may involve the the hippocampus data, and we can see that chronic matrine PI3K/AKT/mTOR signaling in hippocampus. administration fully reversed the decreased p-AKT, p-mTOR, p-4E-BP-1, and p-p70S6K expression in the hippocampus of Pharmacological Inhibition of the PI3K/AKT/mTOR CUMS-stressed mice (n = 5). For the p-AKT data, ANOVA indicated Signaling Prevented the Antidepressant-Like Effects a significant interaction [F(2, 24)= 35.329, P < .01] with significant of Matrine on Mice effects of CUMS [F(1, 24)= 47.856, P < .01] and drug treatment [F(2, 24) = 23.084, P < .01]. For the p-mTOR data, ANOVA indicated a To test whether the PI3K/AKT/mTOR signaling is really neces- significant interaction [F(2, 24)= 32.592, P < .01] with significant sary for the effects of matrine, selective inhibitors of AKT effects of CUMS [F(1, 24)= 44.648, P < .01] and drug treatment [F(2, (LY294002) and mTOR (rapamycin) were used. In this study, Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 6 | International Journal of Neuropsychopharmacology, 2018 Figure  3. Antidepressant-like effects of matrine on hippocampal neurogenesis in chronic unpredictable mild stress (CUMS)-stressed mice. (A) Representative con- focal microscopic images showed the localization of doublecortin (DCX) in the dentate gyrus (DG) region. Scale bar is 150  μm for representative images and 75 μm for enlarged images, respectively. (B) Statistical analysis revealed the enhancing effects of matrine on the amount of DCX-positive cells in DG of CUMS-stressed mice. Data are expressed as means ± SEM (n = 5), **P < .01. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. these inhibitors were i.c.v. infused. As a first step, naive mice and rapamycin all blocked the decreasing effects of matrine were first infused with LY294002 or rapamycin and then on the immobility of the stressed mice in TST (n= 10). Figure 5F treated with matrine (60  mg/kg) followed by FST or TST. For showed that matrine could not restore the decreased sucrose LY294002, although its infusion alone produced no effects in preference of the stressed mice under existence of LY294002 FST or TST (n = 10), its pretreatment significantly blocked the or rapamycin (n = 10). Moreover, western blotting and immu- antidepressant-like effects of matrine in FST [ANOVA: matrine , nohistochemistry experiments were done. In parallel with the F(1, 36) = 28.598, P < .01; LY294002, F(1, 36) = 17.269, P < .01; inter - behavioral data, matrine could not reverse the decreased p-AKT, action, F(1, 36) = 22.146, P < .01; Figure  5B] and TST [ANOVA: p-mTOR, p-4E-BP-1, p-p70S6K expressions, and neurogenesis in matrine, F(1, 36) = 34.487, P < .01; LY294002, F(1, 36) = 20.479, hippocampus of the stressed mice under existence of LY294002 P < .01; interaction, F(1, 36)= 25.524, P < .01; Figure  5C]. Similarly, or rapamycin (n = 5, Figures 6 and 7). for rapamycin, although its treatment alone induced no effects (n = 10), it fully prevented the effects of matrine in FST [ANOVA: Genetic Knockdown of mTOR in Hippocampus matrine, F(1, 36) = 38.207, P < .01; LY294002, F(1, 36) = 21.443, P < .01; Abolished the Antidepressant-Like Effects of Matrine interaction, F(1, 36) = 27.288, P < .01; Figure 5B] and TST [ANOVA: on Mice matrine, F(1, 36) = 36.087, P < .01; LY294002, F(1, 36) = 28.123, P < .01; interaction, F(1, 36) = 19.115, P < .01; Figure 5C]. Furthermore, we generated an EGFP-containing lentiviral As a second step, CUMS-stressed mice were cotreated with vector that selectively expressed short hairpin RNA against matrine and LY294002/rapamycin followed by the behavioral mTOR (LV-mTOR-shRNA-EGFP). LV-mTOR-shRNA-EGFP or tests.F igure 5D showed that both LY294002 and rapamycin pre- LV-Scrambled-shRNA-EGFP was infused into bilateral hippo- vented the reducing effects of matrine on the immobility of the campus of mice by stereotaxic injection, and after 14 d, numer - stressed mice in FST (n = 10). Figure  5E showed that LY294002 ous EGFP positive cells and decreased mTOR expression were Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wu et al. | 7 Figure 4. Chronic matrine treatment reversed chronic unpredictable mild stress (CUMS)-induced decrease in the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling in hippocampus, but not prefrontal cortex (PFC). (A) Matrine treatment fully restored CUMS-induced decrease of p-AKT, p-mTOR, p-4E-BP-1, and p-p70S6K expressions in hippocampus, with the total AKT, mTOR, 4E-BP-1, p70S6K, and β-actin levels unchanged. (B) Matrine pro- duced no effects on the levels of p-AKT, p-mTOR, p-4E-BP-1, and p-p70S6K in PFC of CUMS-stressed mice. Data are expressed as means ± SEM (n = 5), *P < .05, **P < .01; n.s., no significance. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 8 | International Journal of Neuropsychopharmacology, 2018 Figure 5. Pharmacological inhibition of the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling prevented the antidepressant effects of matrine on mouse behaviors. (A) Schematic timeline of experimental procedures. (B) LY294002/rapamycin and matrine (60 mg/kg) were given 60 and 30 minutes before the forced swim test (FST), respectively. Both LY294002 and rapamycin prevented the reducing effects of matrine on the immobility of naive mice in FST (n = 10). (C) LY294002/rapamycin and matrine (60 mg/kg) were given 60 and 30 minutes before the tail suspension test (TST), respectively. Both LY294002 and rapamycin prevented the decreasing effects of matrine on the immobility of naive mice in TST (n= 10). (D) Both LY294002 and rapamycin blocked the reducing effects of matrine (60 mg/kg) on the immobility of CUMS-stressed mice in FST (n= 10). (E) Both LY294002 and rapamycin blocked the decreasing effects of matrine (60 mg/kg) on the immobility of CUMS-stressed mice in TST (n = 10). (F) Both LY294002 and rapamycin abolished the reversing effects of matrine (60 mg/kg) on the sucrose preference of CUMS-stressed mice (n= 10). Data are expressed as means ± SEM (n = 10); *P < .05, **P < .01; n.s., no significance. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. observed in hippocampus (n = 4; Figure  8A). It was found that P < .01; interaction, F(1, 36)= 34.288, P < .01; n = 10, Figure  8D]. mTOR knockdown in hippocampus fully abolished the antide- Moreover, the mTOR-knockdown mice were subjected to CUMS pressant-like effects of matrine in FST [ANOVA: matrine, F(1, and matrine administration followed by the behavioral tests. 36) = 33.395, P < .01; mTOR-shRNA, F(1, 36) = 26.493, P < .01; inter - Figure  8E and F revealed that mTOR knockdown in hippocam- action, F(1, 36) = 31.109, P < .01; n = 10, Figure 8C] and TST [ANOVA: pus completely abolished the decreasing effects of matrine matrine, F(1, 36) = 42.029, P < .01; mTOR-shRNA, F(1, 36) = 37.432, on the immobility of the stressed mice in FST (n= 10) and TST Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wu et al. | 9 Figure 6. Pharmacological inhibition of the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling prevented the reversing effects of matrine on hippocampal neurogenesis in the stressed mice. (A) Representative confocal microscopic images showed the localization of dou- blecortin (DCX) in dentate gyrus (DG) region. Scale bar is 150  μm for representative images and 75 μm for enlarged images, respectively. (B) Statistical analysis showed that the promoting effects of matrine (60 mg/kg) on the amount of DCX-positive cells in chronic unpredictable mild stress (CUMS)-stressed mice were fully blocked by infusion of LY294002 and rapamycin. Data are expressed as means ± SEM (n = 5); *P < .05, **P < .01; n.s., no significance. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. (n = 10), respectively. Figure  8G revealed that matrine could not antidepressant-like actions of matrine, as these neurological restore the decreased sucrose preference of the stressed mice disorders are always accompanied with depression (Li et  al., under existence of mTOR-shRNA (n = 10). Collectively, the PI3K/ 2014; Goto et al., 2018; Laumet et al., 2017; Sampath et al., 2017). AKT/mTOR signaling in hippocampus is necessary for the anti- In this study, we first detected the effects of matrine using FST depressant actions of matrine. and TST, as the 2 tests have high predictive validity for detecting potential antidepressant activities and are widely used (Porsolt et al., 1977; Steru et al., 1985). We found that a single injection of Discussion matrine produced a significant reduction of immobility in both The major findings of this study are as follows. First, matrine has FST and TST. Moreover, matrine treatment had no effects on the locomotor activity of mice, indicating that the matrine-induced antidepressant-like effects in multiple animal models screen- ing for antidepressant activities, including the CUMS paradigm, reduction of immobility was not due to locomotor abnormal- ity. We further used the CUMS model to validate the effects of FST, and TST. Second, the antidepressant-like effects of matrine require the PI3K/Akt/mTOR signaling in hippocampus. Together, matrine, as the CUMS model has excellent validity modulating the pathogenesis of depression (Papp et  al., 1996; Reid et  al., these data indicate that matrine could be a potential and novel antidepressant. 1997). As a result, chronic administration of matrine fully ame- liorated the behavioral deficits of CUMS-stressed mice. Matrine was considered in our study because of knowledge that it had promoting effects on the PI3K/Akt/mTOR signaling mTOR is a serine/threonine protein kinase that modulates a lot of physiological processes including cell proliferation, neur - in brain (Liu et al., 2017), while mTOR is closely involved in the pathophysiology of depression (Abelaira et  al., 2014). Besides, onal survival, and synaptic plasticity (Hay et al., 2004 Tr; amutola et al., 2017). It has also been demonstrated that mTOR is involved previous studies have demonstrated that matrine has protect- ing and improving effects on neuropathic pain, Alzheimer’s in many diseases, such as diabetes, tremor, and Alzheimer’s dis- ease (Dann et al., 2007). Since Li et al. reported in 2010 that keta- disease, and Parkinson’s disease (Zhang et  al., 2015; Gong et al., 2016; Meng et al., 2017). These reports may also imply the mine produced rapid antidepressant effects via activating the Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 10 | International Journal of Neuropsychopharmacology, 2018 Figure 7. Usage of LY294002 and rapamycin prevented the reversing effects of matrine on the hippocampal phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling. (A) Representative images of the western blotting results. (B) Quantitative analysis revealed that matrine (60 mg/kg) could not restore the decreased p-AKT, p-mTOR, p-4E-BP-1, and p-p70S6K expressions in the hippocampus of chronic unpredictable mild stress (CUMS)-stressed mice under existence of LY294002 or rapamycin (n= 5). Data are expressed as means ± SEM; *P < .05, **P < .01; n.s., no significance. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. mTOR pathway in PFC (Li et al., 2010), mTOR has been a popular signaling in neurons, implying that the pharmacological effects target in depression research. Until now, numerous studies have of matrine in body were complex. explored the role of mTOR in depression and also found that In addition, this study is also the first evidence showing many antidepressants used in clinical practice (fluoxetine, escit- the effects of matrine on hippocampal neurogenesis, which alopram, paroxetine, etc.) have promoting effects on mTOR activ- is interesting and may imply a proneurogenic compound. ity in hippocampus (Park et al., 2014; Liu et al., 2015). Here, our Matrine shall modulate hippocampal neurogenesis through results showed that matrine could reverse the CUMS-induced the PI3K/Akt/mTOR pathway, as the correlation between mTOR effects on the PI3K/Akt/mTOR signaling in hippocampus, but and neurogenesis has been welldemonstrated (Lee, 2015 ; not PFC. It is interesting that the effects of matrine on the mTOR Tee et al., 2016), and more importantly, inhibition of the PI3K/ system are region selective, while currently there are no persua- Akt/mTOR system fully antagonized its effects on hippocam- sive explanations for this finding, and more profound studies pal neurogenesis. are required in the future. More importantly, both pharmaco- Due to a prevalent belief that “natural shall be better,”- cur logical and genetic inhibition of the mTOR system abolished the rently a lot of public interests have focused on developing and antidepressant actions of matrine, further proving our assump- identifying novel antidepressant medications derived from tion. However, as the neurobiology of depression is very com- plant materials or natural products. As an important compo- plex, involving a lot of factors like brain-derived neurotrophic nent in Sophorae flavescens, matrine has wide-ranging biological factor, cAMP-response element binding protein, serotonin, and effects and many reveal positive therapeutic indices. Our study mTOR (Shelton, 2007; Krishnan et  al., 2008; Albert et  al., 2012), is the first evidence showing that matrine has antidepressant- so it can not conclude that some other targets are also involved like efficacy via promoting the hippocampal PI3K/Akt/mTOR in the antidepressant actions of matrine. For this, we will per - signaling. Meanwhile, it should be recognized that matrine is form further research using more selective inhibitors and found to be neurotoxic (Wang et al., 2010), which can be a major shRNAs. It should be also noticed that matrine had inhibiting limiting feature for its CNS use. Anyhow, this study extends the effects on the PI3K/Akt/mTOR signaling in lung cancer cells and knowledge of matrine’s pharmacological effects and sheds light acute myeloid leukaemia cells (Niu et al., 2014W ; u et al., 2017) , on the development of novel antidepressants with better effi- while here we found a positive effect of matrine on this cacy and fewer side effects. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wu et al. | 11 Figure 8. Genetic knockdown of mammalian target of rapamycin (mTOR) in hippocampus by mTOR-shRNA abolishes the antidepressant effects of matrine on mice. (A) Fluorescence of a fixed brain section which expressed LV-mTOR-shRNA-EGFP in the hippocampus 2 weeks after its stereotactic injection. Scale bar is 400  μm. Western blotting analysis showed the efficacy of mTOR-shRNA (n= 4). (B) Schematic timeline of experimental procedures. (C) mTOR-shRNA fully abolished the reducing effects of matrine (60 mg/kg) on the immobility of naive mice in the forced swim test (FST) (n= 10). (D) mTOR-shRNA also blocked the decreasing effects of matrine (60 mg/ kg) on the immobility of naive mice in the tail suspension test (TST) (n = 10). (E) mTOR-shRNA completely abolished the reducing effects of matine (60 mg/kg) on the immobility of chronic unpredictable mild stress (CUMS)-stressed mice in FST (n = 10). (F) mTOR-shRNA also blocked the decreasing effects of matrine (60 mg/kg) on the immobility of CUMS-stressed mice in TST (n = 10). (G) mTOR-shRNA completely antagonized the restoring effects of matrine (60  mg/kg) on the sucrose prefer - ence of CUMS-stressed mice (n = 10). Data are expressed as means ± SEM; **P < .01; n.s., no significance. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 12 | International Journal of Neuropsychopharmacology, 2018 Jiang B, Wang YJ, Wang H, Song L, Huang C, Zhu Q, Wu F, Zhang W Acknowledgments (2017) Antidepressant-like effects of fenofibrate in mice via This study was supported by Innovation and Demonstration the hippocampal brain-derived neurotrophic factor signal- Projects of Nantong Social Science and Technology (HS2011024). ling pathway. Br J Pharmacol 174:177–194. Kan QC, Lv P, Zhang XJ, Xu YM, Zhang GX, Zhu L (2015) Matrine protects neuro-axon from CNS inflammation-induced injury. Statement of Interest Exp Mol Pathol 98:124–130. None. Krishnan V, Nestler EJ (2008) The molecular neurobiology of depression. Nature 455:894–902. 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Matrine Exerts Antidepressant-Like Effects on Mice: Role of the Hippocampal PI3K/Akt/mTOR Signaling

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Abstract

Background: Current antidepressants in clinical use always take weeks or even months to exert full therapeutic effects, and sometimes have serious side effects. Thus, it is very necessary to develop novel antidepressants with better efficacy and fewer adverse effects. The present study focused on investigating the antidepressant potential of matrine and its possible mechanisms of action. Methods: The forced swim test, tail suspension test, and chronic unpredictable mild stress model of depression were used to reveal the antidepressant-like effects of matrine on mice. Western blotting, immunohistochemistry, and lentivirus were further used together to explore the antidepressant mechanism of matrine. Results: It was found that matrine exhibited significant antidepressant actions in the forced swim test and tail suspension test without affecting the locomotor activity of mice. Chronic matrine administration fully reversed the chronic unpredictable mild stress-induced depressive-like symptoms in forced swim test, tail suspension test, and sucrose preference test. After that, western blotting analysis revealed that chronic matrine treatment restored the decreasing effects of chronic unpredictable mild stress on the PI3K/Akt/mammalian target of rapamycin signaling in hippocampus, but not prefrontal cortex. Furthermore, pharmacological and genetic blockade of the PI3K/Akt/mammalian target of rapamycin signaling in hippocampus abolished the antidepressant actions of matrine on mice. Conclusions: Taken together, matrine produces antidepressant-like effects on mice via promoting the hippocampal PI3K/Akt/ mammalian target of rapamycin signaling. Keywords: chronic unpredictable mild stress, depression, hippocampus, matrine, mTOR Introduction Depression is one of the leading public health problems in the et  al., 2008). Currently, most antidepressants used in clinical world, and antidepressants are among the most commonly practice elevate the level of monoaminergic neurotransmit- prescribed medications (Bowden, 2005; Shelton, 2007; Krishnan ters in brain, particularly serotonin and norepinephrine (Berton Received: December 18, 2017; Revised: February 27, 2018; Accepted: April 6, 2018 © The Author(s) 2018. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 1 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 2 | International Journal of Neuropsychopharmacology, 2018 Significance Statement This study shows that matrine possess antidepressant-like effects that are mediated by activating the PI3K/Akt/mTOR signaling in hippocampus. Its significance is as follows: (1) Extending the knowledge of matrine’s pharmacological effects; (2) providing a potential antidepressant with better efficacy or fewer side effects; (3) further proving that the central mTOR signaling is a good target for developing novel antidepressants. et al., 2006; Hirschfeld, 2012). However, it always takes weeks or relative humidity) for 1 week with free access to food and even months for these antidepressant drugs to exert their full water. Behavioral experiments were carried out during the therapeutic effects, and many patients are resistant to them light phase. All experimental procedures were conducted in (Berton et  al., 2006; Hirschfeld, 2012). Therefore, it is necessary accordance with the Guide for the Care and Use of Laboratory to develop novel antidepressants based on different pharmaco- Animals (8th edition, Institute of Laboratory Animal Resources logical targets. on Life Sciences, National Research Council, National Academy Mammalian target of rapamycin (mTOR) is a large serine/ of Sciences, Washington DC). threonine kinase that regulates initiation of protein translation (Abe et al., 2010). Activation of mTOR by phosphatidylinositol-3 Materials kinase (PI3K)/protein kinase B (AKT) results in its phosphoryl- ation at serine 2448, thereby promoting its downstream mol- Matrine (purity  >98%) and fluoxetine were bought from ecules, p70 ribosomal protein S6 kinase (p70S6K) and eukaryotic Sigma and dissolved in 1% DMSO in 0.9% saline. LY294002 initiation factor 4E-binding protein 1 (4E-BP-1). Then, p70S6K was purchased from Tocris, rapamycin was purchased from and 4E-BP-1 promote initiation of protein translation for syn- Target Mol, and they were dissolved in 1% DMSO in ACSF. aptic protein synthesis (Hay et  al., 2004; Dann et  al., 2007; The doses of matrine (15, 30, 60, and 120  mg/kg), fluox- Hashimoto, 2011). Recent studies support the hypothesis that etine (20  mg/kg), LY294002 (10  nmol/site), and rapamycin major depressive disorder may be a consequence of deficiency (0.2 nmol/site) were chosen based on previous reports (Jiang in the mTOR signaling (Hashimoto, 2011; Ludka et  al., 2016). It et  al., 2015; Gong et  al., 2016; Ludka et  al., 2016; Jiang et  al., has also been demonstrated that upregulation of the mTOR 2017). Matrine and fluoxetine were i.p. injected in a volume signaling in prefrontal cortex (PFC) and hippocampus leads to of 10 mL/kg. LY294002 and rapamycin were intracerebroven- rapid and consistent antidepressant-like effects (Li et al., 2010; tricularly (i.c.v.) infused. Hashimoto, 2011; Zhou et al., 2014). Matrine, a quinolizidine alkaloid compound extracted from FST the root of Sophorae flavescens, is known for its various pharma- cological activities. Matrine has been shown to have antiinflam- FST was carried out as previously described (Jiang et al., 2015; matory, antiallergic, antivirus, antitumor, and cardiovascular Jiang et al., 2017). Briefly, each mouse was individually placed protective effects (Li et al., 2010Fu et  ; al., 2014; Liu et al., 2014; in a glass cylinder (15  cm diameter, 25  cm height) filled with Kan et  al., 2015; Wu et  al., 2017). Recently, more and more 10  cm of 25°C ± 1°C water and forced to swim for 6 minutes. matrine-induced pharmacologic effects on the central nervous The immobility duration of the last 4 minutes was recorded system have been reported. For example, Gong et  al. reported by an investigator blind to the study. Water was exchanged that matrine had neuroprotective effects on a mouse model of between 2 trails. The time during which each mouse was float- vincristine-induced neuropathic pain (Gong et al., 2016). Zhang ing in water without struggling and only making minor move- et al. showed that matrine improved cognitive impairments in a ments to keep its nose above the water was regarded as the rat model of Alzheimer’s disease (Zhang et al., 2015). Meng et al. immobility time. showed that matrine also had neuroprotective effects against MPTP-induced Parkinson’s disease (Meng et al., 2017). Here, we TST noticed that in 2017, matrine had been demonstrated to have promoting effects on the PI3K/Akt/mTOR signaling in corpus TST was carried out as previously described (Jiang et  al., 2015, callosum (Liu et  al., 2017). Thus, we assume that matrine may 2017). Briefly, each mouse was suspended on the shelf, which possess antidepressant-like effects via activating mTOR. In this was 70 cm above the ground, for 6 minutes. A tape was placed study, we investigated this assumption using various methods, at about 1.5 cm away from the tip of tail. The immobility time including the forced swimming test (FST), tail suspension test during which each mouse was completely motionless was (TST), and chronic unpredictable mild stress (CUMS) model of recorded for the last 4 minutes. The observer was unaware of depression. mice grouping. Materials and Methods Locomotor Activity Test Animals As reported (Jiang et al., 20152017 , ), briefly, each mouse was indi- Adult male C57BL/6J mice (8 weeks old) were obtained from vidually placed in the middle of a wooden box (50× 50 × 40  cm) the Experimental Animal Center of Medical College, Nantong that consisted of 25 equal squares on the floor (10× 10  cm). The University. Before use, the mice were housed under stand- number of squares each mouse crossed was recorded for 5 min- ard conditions (12-hour-light/-dark cycle; lights on from utes by an observer unaware of mice grouping. The apparatus 7:00 am to 7:00 pm; 2°C ±1°C ambient temper ature; 55% ± 10% was cleaned after each test. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wu et al. | 3 PVDF membranes. The transferred membranes were blocked in CUMS 5% bovine serum albumin and then incubated with different In this procedure, mice were subjected to a random sequence primary antibodies overnight at 4°C. Primary antibodies against of unpredictable and mild stressors for 8 weeks. The stressors mTOR (1:500; Abcam), p-mTOR (Ser2448, 1:500; Abcam), 4E-BP-1 are listed as follows: (1) food or water deprivation for 24 hours; (1:500; Cell Signaling), p-4E-BP-1 (Thr37/46, 1:500; Cell Signaling), (2) damp sawdust for 12 hours; (3) restraint for 2 hours; (4) cage p70S6K (1:1000; Cell Signaling), p-p70S6K (Thr389, 1:1000; Cell shaking for 15 minutes; (5) inversion of light/dark cycle; (6) 45°C Signaling), AKT(1:500; Cell Signaling), p-AKT (Ser473, 1:500; cage tilting in empty cage for 12 hours; (7) tail pinch for 5 min- Cell Signaling), and β-actin (1:5000; Immunoway) were used. utes. Control mice were left undisturbed in home cages in a After incubated with goat anti-rabbit or goat anti-mouse HRP- separate room. Repeated administration of vehicle/fluoxetine/ conjugated secondary antibody for 2 hours at room tempera- matrine/inhibitors was given daily during the last 2 weeks. After ture, the blots were detected by enhanced chemiluminescence. CUMS and drug treatments, FST, TST, and the sucrose preference test were performed. Immunohistochemistry To analyze hippocampal neurogenesis, mice were anesthe- Sucrose Preference Test tized using 0.5% pentobarbital sodium (50 mg/kg, i.p.) and then For this test, mice were first adapted to 1% sucrose solution transcardially perfused with normal saline followed by 4% for 48 hours, then deprived of water and food for 24 hours, and paraformaldehyde in 0.01 M phosphate buffer saline (PBS). The finally given 2 preweighed bottles containing 1% sucrose solu- brains were postfixed in 4% paraformaldehyde overnight and tion and tap water, respectively. All the mice were housed indi- dehydrated in 30% sucrose solution for 2 days. Serial sections of vidually during this test, and the index of sucrose preference 25 μm were cut throughout hippocampi using a freezing micro- was calculated as a percentage of the volume of sucrose con- tome (Leica) and preserved in 0.01 M PBS. After that, the sections sumed/total volume of liquid consumed. were permeabilized with 0.3% Triton X-100 in PBS (30 minutes, first) and 3% bovine serum albumin in PBS (30 minutes, second) Intracerebroventricular (i.c.v.) Injections at room temperature. Then the sections were incubated with rat polyclonal anti-doublecortin (DCX, 1:100; Cell Signaling) primary As reported (Jiang et  al., 2016, 2017), each mouse was deeply antibody under 4°C overnight. The sections were further incu- anesthetized with 0.5% pentobarbital sodium (50  mg/kg, i.p.) bated in fluorescein isothiocyanate-labeled horse anti-rabbit and placed in a stereotaxic device (Stoelting). Scalp skin was IgG (1:50; Thermo Fisher) for 2 hours at room temperature. After shaved with clippers and disinfected using iodine. After opening washing, the sections were coverslipped and observed using a the scalp skin and exposing the skull, infusion cannulas were confocal fluorescence microscope (Leica). Examination of the implanted into the left lateral brain ventricle (AP = −0.2  mm, DCX-positive (DCX+) cells was confined to dentate gyrus (DG), ML = +1.0  mm, DV = +2.3  mm) and cemented. The mice were especially in the granule cell layer (GCL), including the subgran- allowed to recover for 3 d.  Osmotic minipumps were used to ular zone of hippocampus that was defined as a 2-cell body-wide deliver 0.3 μL/min of LY294002 or rapamycin or 1% DMSO in zone along the border between GCL and hilus. Quantifications ACSF (final volume, 3 μL/mouse). of the DCX+ cells were respectively conducted from 1-in-6 ser - ies of hippocampal sections spaced at 150 μm and spanning the Lentivirus Microinfusion rostrocaudal extent of DG bilaterally. Every DCX+ cell within the GCL and subgranular zone was counted. This was done according to previous reports (Jiang et al., 2014). Briefly, each mouse was deeply anesthetized with 0.5% pento- Statistical Analysis barbital sodium (50  mg/kg, i.p.) and placed in a stereotaxic device. After exposing the skull, 5 µL microsyringes (Hamilton) All data were analyzed using SPSS 13.0 software (SPSS Inc.). were inserted into hippocampus bilaterally to deliver lenti- Multiple group comparisons were performed using 1-way or virus. The location is as follows: AP = -2.3  mm, ML = ±1.6  mm, 2-way ANOVA, as appropriate. P < .05 was considered statistic- DV = +1.9  mm. Infusion of lentivirus was performed bilaterally ally significant. at a rate of 0.3 µL/min (1.5  µL/side). The mice were allowed to recover for 3 d. RESULTS LV-mTOR-shRNA-EGFP and LV-Scrambled-shRNA- EGFP used in this study were produced and provided by Effects of Matrine on the Immobility of Mice in FST Genechem Co., Ltd. The titers were adjusted to 6× 10 TU/ and TST mL. The sequences for mTOR-shRNA and Scrambled-shRNA were 5’-GGCCTATGGTCGAGATTTA-3’ and 5’-TTCTCCGAACGTGT As a first step of this study, FST and TST were used. Figure 1A CACGT-3’, respectively. and B showed the effects of matrine on the immobility dur - ation of mice in FST and TST, respectively. It was found that both matrine and fluoxetine significantly reduced the immo- Western Blotting bility of mice in FST and TST compared with the control mice The hippocampus and PFC tissues were dissected and imme- (n = 10). For the FST data, ANOVA indicated a significant main diately homogenized in NP-40 lysis buffer containing protease effect of drug treatment [F(5, 54) = 28.592, P < .01]. For the TST inhibitor cocktail for 30 minutes. Then the homogenates were data, ANOVA also revealed a significant main effect of drug centrifuged at 12 000 × g for 30 minutes at 4°C. The supernatants treatment [F(5, 54) = 33.017, P < .01]. Detailed analysis showed were collected on ice, and protein concentrations were assayed that compared with the control mice, 15  mg/kg of matrine using the BCA method. Then 30 µg of protein samples was sepa- treatment moderately decreased the immobility, 30  mg/kg rated by SDS-PAGE gels. After that, proteins were transferred to and 60  mg/kg of matrine treatment significantly decreased Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 4 | International Journal of Neuropsychopharmacology, 2018 Figure 1. Antidepressant-like effects of matrine in forced swim test (FST) and tail suspension test (TST). Naive mice were i.p. injected with a single dose of vehicle, fluoxetine (20 mg/kg), or matrine (15, 30, 60, or 120 mg/kg). The behavioral tests were conducted 30 minutes after the injection. Different groups of mice were used for these tests. (A) The matrine-treated mice had significantly less immobility than the vehicle-treated mice in FST. (B) The matrine-treated mice also showed significantly less immobility than the vehicle-treated mice in TST. (C) All groups of mice displayed similar locomotor activity in the locomotor activity test. Data are expressed as means ± SEM (n = 10); *P < .05, **P < .01; n.s., no significance. Comparisons were made by 1-way ANOVA followed by posthoc LSD test. the immobility, while 120  mg/kg matrine produced a similar reversed the promoting effects of CUMS on the immobility effect to 60 mg/kg matrine. Therefore, 30 mg/kg and 60 mg/kg of mice in TST (n = 10). ANOVA revealed a significant inter- were selected as the doses for matrine in the following stud- action [F(3, 72) = 42.045, P < .01] with significant effects of CUMS ies. Besides, Figure  1C showed that matrine treatment did [F(1, 72) = 54.134, P < .01] and drug treatment [F(3, 72)= 38.578, not affect the locomotor activity of mice (n = 10), and ANOVA P < .01]. Figure  2D illustrates the sucrose preference data. showed no significant effects of drug treatment [F(3, 36)= 0.715, Also, we found that chronic injection of matrine protected P = .386]. Together, matrine may have antidepressant-like against the CUMS-induced anhedonia behavior (n = 10). ANOVA effects on mice. revealed a significant interaction [F(3, 72)= 39.606, P < .01] with significant effects of CUMS [F(1, 72) = 51.552, P < .01] and drug treatment [F(3, 72) = 29.506, P < .01]. Thus, matrine indeed pos- Effects of Matrine on the Depressive-Like Behaviors sessed antidepressant-like effects on mice. of Mice in the CUMS Model of Depression Depression is also accompanied with decreased hippocam- Next, the CUMS model of depression was used to assay the pal neurogenesis (Santarelli et  al., 2003). Here, we examined antidepressant effects of matrine, with FST, TST, and the whether matrine prevented the CUMS-induced effects on sucrose preference test used to evaluate the CUMS-induced neurogenesis using DCX immuohistochemistry. It is known that depressive-like behaviors (Reid et  al., 1997; Shields, 2006). DCX is a microtubule-associated protein that serves as a marker Figure 2B illustrates the FST data. It was found that CUMS sig- of neurogenesis because of its transient expression in new- nificantly enhanced the immobility of mice, while matrine born neurons (Brown et al., 2003). As shown in Figure 3, chronic administration fully restored this change (n = 10). ANOVA matrine administration fully restored the decreasing effects of revealed a significant interaction [F(3, 72)= 33.097, P < .01] CUMS on the number of DCX+ cells in DG (n= 5). ANOVA revealed with significant effects of CUMS [F(1, 72) = 43.767, P < .01] and a significant interaction [F(3, 17)= 55.397, P < .01] with significant drug treatment [F(3, 72)= 26.143, P < .01]. Figure  2C illustrates effects of CUMS [F(1, 17)= 74.356, P < .01] and drug treatment the TST data, and similarly, matrine treatment significantly [F(3, 17) = 41.479, P < .01]. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wu et al. | 5 Figure 2. Antidepressant-like effects of matrine on the chronic unpredictable mild stress (CUMS) model of depression. Mice were exposed to 8 weeks of CUMS and received daily injection of vehicle, fluoxetine (20 mg/kg), or matrine (30 or 60 mg/kg) during the last 2 weeks. The behavioral tests were then conducted. (A) Schematic timeline of experimental procedures. (B) CUMS + matrine mice spent significantly less immobility than that of CUMS + vehicle mice in forced swim test (FST). (C) CUMS + matrine mice spent significantly less immobility than that of CUMS + vehicle mice in tail suspension test (TST). (D) CUMS + matrine mice displayed signifi- cantly higher sucrose preference than that of CUMS + vehicle mice. Data are expressed as means ± SEM (n = 10); *P < .05, **P < .01; n.s., no significance. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. 24) = 23.628, P < .01]. For the p-4E-BP-1 data, ANOVA indicated a Effects of Matrine on the Central PI3K/AKT/mTOR significant interaction [F(2, 24)= 40.042, P < .01] with significant Signaling in CUMS-Stressed Mice effects of CUMS [F(1, 24)= 56.278, P < .01] and drug treatment After the behavioral tests, western blotting experiments were [F(2, 24) = 33.712, P < .01]. For the p-p70S6K data, ANOVA indicated performed to detect the effects of matrine on levels of p-AKT a significant interaction [F(2, 24)= 48.135, P < .01] with significant (Ser473), AKT, p-mTOR (Ser2448), mTOR, p-4E-BP-1 (Thr37/46), effects of CUMS [F(1, 24)= 62.592, P < .01] and drug treatment 4E-BP-1, p-p70S6K (Thr389), p70S6K, and β-actin in both hippo- [F(2, 24) = 37.388, P < .01]. Matrine administration did not affect campus and PFC. Figure  4B illustrates the PFC data. Although the level of the central PI3K/AKT/mTOR signaling in naive mice CUMS significantly decreased the expression of p-AKT, p-mTOR, (n = 5). In contrast, the levels of total AKT, mTOR, 4E-BP-1, and p-4E-BP-1, and p-p70S6K in PFC, chronic matrine administra- p70S6K were unchanged among all groups (n= 5). Therefore, the tion did not restore these changes (n = 5). Figure  4A illustrates antidepressant-like effects of matrine on mice may involve the the hippocampus data, and we can see that chronic matrine PI3K/AKT/mTOR signaling in hippocampus. administration fully reversed the decreased p-AKT, p-mTOR, p-4E-BP-1, and p-p70S6K expression in the hippocampus of Pharmacological Inhibition of the PI3K/AKT/mTOR CUMS-stressed mice (n = 5). For the p-AKT data, ANOVA indicated Signaling Prevented the Antidepressant-Like Effects a significant interaction [F(2, 24)= 35.329, P < .01] with significant of Matrine on Mice effects of CUMS [F(1, 24)= 47.856, P < .01] and drug treatment [F(2, 24) = 23.084, P < .01]. For the p-mTOR data, ANOVA indicated a To test whether the PI3K/AKT/mTOR signaling is really neces- significant interaction [F(2, 24)= 32.592, P < .01] with significant sary for the effects of matrine, selective inhibitors of AKT effects of CUMS [F(1, 24)= 44.648, P < .01] and drug treatment [F(2, (LY294002) and mTOR (rapamycin) were used. In this study, Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 6 | International Journal of Neuropsychopharmacology, 2018 Figure  3. Antidepressant-like effects of matrine on hippocampal neurogenesis in chronic unpredictable mild stress (CUMS)-stressed mice. (A) Representative con- focal microscopic images showed the localization of doublecortin (DCX) in the dentate gyrus (DG) region. Scale bar is 150  μm for representative images and 75 μm for enlarged images, respectively. (B) Statistical analysis revealed the enhancing effects of matrine on the amount of DCX-positive cells in DG of CUMS-stressed mice. Data are expressed as means ± SEM (n = 5), **P < .01. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. these inhibitors were i.c.v. infused. As a first step, naive mice and rapamycin all blocked the decreasing effects of matrine were first infused with LY294002 or rapamycin and then on the immobility of the stressed mice in TST (n= 10). Figure 5F treated with matrine (60  mg/kg) followed by FST or TST. For showed that matrine could not restore the decreased sucrose LY294002, although its infusion alone produced no effects in preference of the stressed mice under existence of LY294002 FST or TST (n = 10), its pretreatment significantly blocked the or rapamycin (n = 10). Moreover, western blotting and immu- antidepressant-like effects of matrine in FST [ANOVA: matrine , nohistochemistry experiments were done. In parallel with the F(1, 36) = 28.598, P < .01; LY294002, F(1, 36) = 17.269, P < .01; inter - behavioral data, matrine could not reverse the decreased p-AKT, action, F(1, 36) = 22.146, P < .01; Figure  5B] and TST [ANOVA: p-mTOR, p-4E-BP-1, p-p70S6K expressions, and neurogenesis in matrine, F(1, 36) = 34.487, P < .01; LY294002, F(1, 36) = 20.479, hippocampus of the stressed mice under existence of LY294002 P < .01; interaction, F(1, 36)= 25.524, P < .01; Figure  5C]. Similarly, or rapamycin (n = 5, Figures 6 and 7). for rapamycin, although its treatment alone induced no effects (n = 10), it fully prevented the effects of matrine in FST [ANOVA: Genetic Knockdown of mTOR in Hippocampus matrine, F(1, 36) = 38.207, P < .01; LY294002, F(1, 36) = 21.443, P < .01; Abolished the Antidepressant-Like Effects of Matrine interaction, F(1, 36) = 27.288, P < .01; Figure 5B] and TST [ANOVA: on Mice matrine, F(1, 36) = 36.087, P < .01; LY294002, F(1, 36) = 28.123, P < .01; interaction, F(1, 36) = 19.115, P < .01; Figure 5C]. Furthermore, we generated an EGFP-containing lentiviral As a second step, CUMS-stressed mice were cotreated with vector that selectively expressed short hairpin RNA against matrine and LY294002/rapamycin followed by the behavioral mTOR (LV-mTOR-shRNA-EGFP). LV-mTOR-shRNA-EGFP or tests.F igure 5D showed that both LY294002 and rapamycin pre- LV-Scrambled-shRNA-EGFP was infused into bilateral hippo- vented the reducing effects of matrine on the immobility of the campus of mice by stereotaxic injection, and after 14 d, numer - stressed mice in FST (n = 10). Figure  5E showed that LY294002 ous EGFP positive cells and decreased mTOR expression were Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wu et al. | 7 Figure 4. Chronic matrine treatment reversed chronic unpredictable mild stress (CUMS)-induced decrease in the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling in hippocampus, but not prefrontal cortex (PFC). (A) Matrine treatment fully restored CUMS-induced decrease of p-AKT, p-mTOR, p-4E-BP-1, and p-p70S6K expressions in hippocampus, with the total AKT, mTOR, 4E-BP-1, p70S6K, and β-actin levels unchanged. (B) Matrine pro- duced no effects on the levels of p-AKT, p-mTOR, p-4E-BP-1, and p-p70S6K in PFC of CUMS-stressed mice. Data are expressed as means ± SEM (n = 5), *P < .05, **P < .01; n.s., no significance. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 8 | International Journal of Neuropsychopharmacology, 2018 Figure 5. Pharmacological inhibition of the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling prevented the antidepressant effects of matrine on mouse behaviors. (A) Schematic timeline of experimental procedures. (B) LY294002/rapamycin and matrine (60 mg/kg) were given 60 and 30 minutes before the forced swim test (FST), respectively. Both LY294002 and rapamycin prevented the reducing effects of matrine on the immobility of naive mice in FST (n = 10). (C) LY294002/rapamycin and matrine (60 mg/kg) were given 60 and 30 minutes before the tail suspension test (TST), respectively. Both LY294002 and rapamycin prevented the decreasing effects of matrine on the immobility of naive mice in TST (n= 10). (D) Both LY294002 and rapamycin blocked the reducing effects of matrine (60 mg/kg) on the immobility of CUMS-stressed mice in FST (n= 10). (E) Both LY294002 and rapamycin blocked the decreasing effects of matrine (60 mg/kg) on the immobility of CUMS-stressed mice in TST (n = 10). (F) Both LY294002 and rapamycin abolished the reversing effects of matrine (60 mg/kg) on the sucrose preference of CUMS-stressed mice (n= 10). Data are expressed as means ± SEM (n = 10); *P < .05, **P < .01; n.s., no significance. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. observed in hippocampus (n = 4; Figure  8A). It was found that P < .01; interaction, F(1, 36)= 34.288, P < .01; n = 10, Figure  8D]. mTOR knockdown in hippocampus fully abolished the antide- Moreover, the mTOR-knockdown mice were subjected to CUMS pressant-like effects of matrine in FST [ANOVA: matrine, F(1, and matrine administration followed by the behavioral tests. 36) = 33.395, P < .01; mTOR-shRNA, F(1, 36) = 26.493, P < .01; inter - Figure  8E and F revealed that mTOR knockdown in hippocam- action, F(1, 36) = 31.109, P < .01; n = 10, Figure 8C] and TST [ANOVA: pus completely abolished the decreasing effects of matrine matrine, F(1, 36) = 42.029, P < .01; mTOR-shRNA, F(1, 36) = 37.432, on the immobility of the stressed mice in FST (n= 10) and TST Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wu et al. | 9 Figure 6. Pharmacological inhibition of the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling prevented the reversing effects of matrine on hippocampal neurogenesis in the stressed mice. (A) Representative confocal microscopic images showed the localization of dou- blecortin (DCX) in dentate gyrus (DG) region. Scale bar is 150  μm for representative images and 75 μm for enlarged images, respectively. (B) Statistical analysis showed that the promoting effects of matrine (60 mg/kg) on the amount of DCX-positive cells in chronic unpredictable mild stress (CUMS)-stressed mice were fully blocked by infusion of LY294002 and rapamycin. Data are expressed as means ± SEM (n = 5); *P < .05, **P < .01; n.s., no significance. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. (n = 10), respectively. Figure  8G revealed that matrine could not antidepressant-like actions of matrine, as these neurological restore the decreased sucrose preference of the stressed mice disorders are always accompanied with depression (Li et  al., under existence of mTOR-shRNA (n = 10). Collectively, the PI3K/ 2014; Goto et al., 2018; Laumet et al., 2017; Sampath et al., 2017). AKT/mTOR signaling in hippocampus is necessary for the anti- In this study, we first detected the effects of matrine using FST depressant actions of matrine. and TST, as the 2 tests have high predictive validity for detecting potential antidepressant activities and are widely used (Porsolt et al., 1977; Steru et al., 1985). We found that a single injection of Discussion matrine produced a significant reduction of immobility in both The major findings of this study are as follows. First, matrine has FST and TST. Moreover, matrine treatment had no effects on the locomotor activity of mice, indicating that the matrine-induced antidepressant-like effects in multiple animal models screen- ing for antidepressant activities, including the CUMS paradigm, reduction of immobility was not due to locomotor abnormal- ity. We further used the CUMS model to validate the effects of FST, and TST. Second, the antidepressant-like effects of matrine require the PI3K/Akt/mTOR signaling in hippocampus. Together, matrine, as the CUMS model has excellent validity modulating the pathogenesis of depression (Papp et  al., 1996; Reid et  al., these data indicate that matrine could be a potential and novel antidepressant. 1997). As a result, chronic administration of matrine fully ame- liorated the behavioral deficits of CUMS-stressed mice. Matrine was considered in our study because of knowledge that it had promoting effects on the PI3K/Akt/mTOR signaling mTOR is a serine/threonine protein kinase that modulates a lot of physiological processes including cell proliferation, neur - in brain (Liu et al., 2017), while mTOR is closely involved in the pathophysiology of depression (Abelaira et  al., 2014). Besides, onal survival, and synaptic plasticity (Hay et al., 2004 Tr; amutola et al., 2017). It has also been demonstrated that mTOR is involved previous studies have demonstrated that matrine has protect- ing and improving effects on neuropathic pain, Alzheimer’s in many diseases, such as diabetes, tremor, and Alzheimer’s dis- ease (Dann et al., 2007). Since Li et al. reported in 2010 that keta- disease, and Parkinson’s disease (Zhang et  al., 2015; Gong et al., 2016; Meng et al., 2017). These reports may also imply the mine produced rapid antidepressant effects via activating the Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 10 | International Journal of Neuropsychopharmacology, 2018 Figure 7. Usage of LY294002 and rapamycin prevented the reversing effects of matrine on the hippocampal phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling. (A) Representative images of the western blotting results. (B) Quantitative analysis revealed that matrine (60 mg/kg) could not restore the decreased p-AKT, p-mTOR, p-4E-BP-1, and p-p70S6K expressions in the hippocampus of chronic unpredictable mild stress (CUMS)-stressed mice under existence of LY294002 or rapamycin (n= 5). Data are expressed as means ± SEM; *P < .05, **P < .01; n.s., no significance. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. mTOR pathway in PFC (Li et al., 2010), mTOR has been a popular signaling in neurons, implying that the pharmacological effects target in depression research. Until now, numerous studies have of matrine in body were complex. explored the role of mTOR in depression and also found that In addition, this study is also the first evidence showing many antidepressants used in clinical practice (fluoxetine, escit- the effects of matrine on hippocampal neurogenesis, which alopram, paroxetine, etc.) have promoting effects on mTOR activ- is interesting and may imply a proneurogenic compound. ity in hippocampus (Park et al., 2014; Liu et al., 2015). Here, our Matrine shall modulate hippocampal neurogenesis through results showed that matrine could reverse the CUMS-induced the PI3K/Akt/mTOR pathway, as the correlation between mTOR effects on the PI3K/Akt/mTOR signaling in hippocampus, but and neurogenesis has been welldemonstrated (Lee, 2015 ; not PFC. It is interesting that the effects of matrine on the mTOR Tee et al., 2016), and more importantly, inhibition of the PI3K/ system are region selective, while currently there are no persua- Akt/mTOR system fully antagonized its effects on hippocam- sive explanations for this finding, and more profound studies pal neurogenesis. are required in the future. More importantly, both pharmaco- Due to a prevalent belief that “natural shall be better,”- cur logical and genetic inhibition of the mTOR system abolished the rently a lot of public interests have focused on developing and antidepressant actions of matrine, further proving our assump- identifying novel antidepressant medications derived from tion. However, as the neurobiology of depression is very com- plant materials or natural products. As an important compo- plex, involving a lot of factors like brain-derived neurotrophic nent in Sophorae flavescens, matrine has wide-ranging biological factor, cAMP-response element binding protein, serotonin, and effects and many reveal positive therapeutic indices. Our study mTOR (Shelton, 2007; Krishnan et  al., 2008; Albert et  al., 2012), is the first evidence showing that matrine has antidepressant- so it can not conclude that some other targets are also involved like efficacy via promoting the hippocampal PI3K/Akt/mTOR in the antidepressant actions of matrine. For this, we will per - signaling. Meanwhile, it should be recognized that matrine is form further research using more selective inhibitors and found to be neurotoxic (Wang et al., 2010), which can be a major shRNAs. It should be also noticed that matrine had inhibiting limiting feature for its CNS use. Anyhow, this study extends the effects on the PI3K/Akt/mTOR signaling in lung cancer cells and knowledge of matrine’s pharmacological effects and sheds light acute myeloid leukaemia cells (Niu et al., 2014W ; u et al., 2017) , on the development of novel antidepressants with better effi- while here we found a positive effect of matrine on this cacy and fewer side effects. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wu et al. | 11 Figure 8. Genetic knockdown of mammalian target of rapamycin (mTOR) in hippocampus by mTOR-shRNA abolishes the antidepressant effects of matrine on mice. (A) Fluorescence of a fixed brain section which expressed LV-mTOR-shRNA-EGFP in the hippocampus 2 weeks after its stereotactic injection. Scale bar is 400  μm. Western blotting analysis showed the efficacy of mTOR-shRNA (n= 4). (B) Schematic timeline of experimental procedures. (C) mTOR-shRNA fully abolished the reducing effects of matrine (60 mg/kg) on the immobility of naive mice in the forced swim test (FST) (n= 10). (D) mTOR-shRNA also blocked the decreasing effects of matrine (60 mg/ kg) on the immobility of naive mice in the tail suspension test (TST) (n = 10). (E) mTOR-shRNA completely abolished the reducing effects of matine (60 mg/kg) on the immobility of chronic unpredictable mild stress (CUMS)-stressed mice in FST (n = 10). (F) mTOR-shRNA also blocked the decreasing effects of matrine (60 mg/kg) on the immobility of CUMS-stressed mice in TST (n = 10). (G) mTOR-shRNA completely antagonized the restoring effects of matrine (60  mg/kg) on the sucrose prefer - ence of CUMS-stressed mice (n = 10). Data are expressed as means ± SEM; **P < .01; n.s., no significance. Comparisons were made by 2-way ANOVA followed by posthoc Bonferroni’s test. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy028/4971202 by Ed 'DeepDyve' Gillespie user on 08 June 2018 12 | International Journal of Neuropsychopharmacology, 2018 Jiang B, Wang YJ, Wang H, Song L, Huang C, Zhu Q, Wu F, Zhang W Acknowledgments (2017) Antidepressant-like effects of fenofibrate in mice via This study was supported by Innovation and Demonstration the hippocampal brain-derived neurotrophic factor signal- Projects of Nantong Social Science and Technology (HS2011024). ling pathway. Br J Pharmacol 174:177–194. Kan QC, Lv P, Zhang XJ, Xu YM, Zhang GX, Zhu L (2015) Matrine protects neuro-axon from CNS inflammation-induced injury. Statement of Interest Exp Mol Pathol 98:124–130. None. Krishnan V, Nestler EJ (2008) The molecular neurobiology of depression. Nature 455:894–902. 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International Journal of NeuropsychopharmacologyOxford University Press

Published: Apr 16, 2018

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