Parthenolide attenuated bleomycin-induced pulmonary fibrosis via the NF-κB/Snail signaling pathway

Parthenolide attenuated bleomycin-induced pulmonary fibrosis via the NF-κB/Snail signaling pathway Background: Parthenolide (PTL) is a natural molecule isolated from Tanacetum parthenium that exhibits excellent anti-inflammatory and antitumor activities. Pulmonary fibrosis (PF), especially idiopathic pulmonary fibrosis (IPF), is a chronic lung disease that lacks a proven effective therapy. The present study evaluated the therapeutic effect of PTL on PF. Methods: Serum-starved primary lung fibroblasts and HFL1 cells were treated with different doses of PTL, and cell viability and the migration rate were measured. Western blot analysis and a dual-luciferase assay were used to analyze the epithelial–mesenchymal transition (EMT)-related transcription factors influenced by PTL treatment in A549 cells and primary lung epithelial cells. Mice with bleomycin (BLM)-induced pulmonary fibrosis were treated with different doses of intragastric PTL, and pathological changes were evaluated using Hematoxylin-eosin (H&E) staining and immunohistochemical analysis. Results: Our results demonstrated that PTL reduced the cell viability and migration rate of lung fibroblasts and inhibited the expression of EMT-related transcription factors in lung epithelial cells. In vivo studies demonstrated that PTL attenuated BLM-induced pulmonary fibrosis and improved the body weight and pathological changes of BLM-treated mice. We further demonstrated that PTL attenuated BLM-induced PF primarily via inhibition of the NF-κB/Snail signaling pathway. Conclusion: These findings suggest that PTL inhibits EMT and attenuates BLM-induced PF via the NF-κB/Snail signaling pathway. PTL is a worthwhile candidate compound for pulmonary fibrosis therapy. Keywords: Parthenolide, Pulmonary fibrosis, NF-κB/Snail signaling pathway Background cyclophosphamide), are limited by low their efficacy and Pulmonary fibrosis (PF), especially idiopathic pulmonary severe side effects. The FDA recently approved two new fibrosis (IPF), is a chronic lung disease caused by several drugs, nintedanib and pirfenidone, to treat IPF. These factors. IPF exhibits a complex pathogenesis, but no drugs stabilize patients’ conditions well, but they do not effective treatment is available for IPF. The mortality reverse the progression of fibrosis. Both drugs produce rate of IPF is considerably increased in recent years, and side effects on the liver and skin, which limits their clin- it substantially threatens human health [1, 2]. Current ical application, especially in patients with liver problems treatments for IPF, such as immunosuppressants (e.g., [3, 4]. Recent research demonstrated that dexametha- sone (DEX) attenuated bleomycin (BLM)-induced lung fibrosis [5]. However, DEX treatment produces many * Correspondence: honggang.zhou@nankai.edu.cn side effects, such as growth retardation, hyperglycemia, Xiao-he Li and Ting Xiao contributed equally to this work. hypertension, myocardial hypertrophy, gastrointestinal State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, perforation, and neurological impairment [6–8]. There- Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People’s Republic of fore, new drugs with improved treatment efficacy and China fewer side effects are urgently needed. Tianjin Key Laboratory of Molecular Drug Research, Tianjin International Joint Academy of Biomedicine, Tianjin, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. Respiratory Research (2018) 19:111 Page 2 of 12 IPF is easily characterized by an excessive deposition supplied by Professor Wen Ning (Nankai University). of extracellular matrix (ECM), but the pathogenesis of The cells were cultured in a medium supplemented with IPF is not clear. Several hypotheses were proposed to 10% heat-inactivated (56 °C, 30 min) fetal calf serum explain the inner mechanisms, and epithelial–mesenchy- (HyClone, USA) and maintained at 37 °C with 5% CO2 mal transition (EMT) of alveolar epithelial cells (AECs) in a humidified atmosphere. received particular attention. Nuclear factor kappa-B (NF-κB) is an essential mediator of EMT. NF-κB pro- Isolation of primary fibroblasts and AECs motes the transcription of many inflammatory cytokines, Primary pulmonary fibroblasts isolated from NaCl/BLM- such as tumor necrosis factor α (TNF-α), interleukin treated mice were cultured in DMEM supplemented (IL) and transforming growth factor β (TGF-β), which with 10% FBS and antibiotics in 5% CO2 at 37 °C in a are highly associated with the progression of IPF, espe- humidified atmosphere as described previously [14]. cially TGF-β [9–11]. Therefore, it is essential to measure Cells at passages 3–4 were used for cell viability and these factors when evaluating the drug efficacy of IPF. wound healing assays. Primary AECs were isolated from Parthenolide (PTL) is a sesquiterpene lactone that is C57BL/6 J mice as previously described [15]. Newly isolated from the shoots of feverfew (Tanacetum parthe- isolated AECs were used for immunofluorescence and nium), and PTL is a traditional medicinal herb used for Western blotting assays. headaches and arthritis. Recent studies suggested that PTL is a useful antitumor and anti-inflammatory agent, Cell viability and wound-healing assays and it was tested in clinical studies for leukemia and Cell viability was determined using the MTT assay. Cells neurological tumors [12]. These biological activities of (5 × 10 cells/mL) were seeded in 96-well culture plates and PTL in tumor and inflammatory diseases primarily occur incubated overnight. Cells were treated with various con- via inhibition of NF-κB and the targeting of multiple centrations of PTL for 24 h. Cell viability was measured steps in the NF-κB signaling pathway. For example, PTL after the addition of MTT (20 μL) at 37 °C for 4 h. Di- binds an activator of NF-κB, IκB-kinase (IKK) [13]. methyl sulfoxide (150 μL) was added to dissolve the forma- However, PTL treatment of IPF and its pharmacological zan crystals. Optical density was measured at 570 nm using properties have not been reported. a microplate reader (Multiskan FC, Thermo Scientific, The present study found that PTL attenuated BLM- Waltham, MA, USA). induced EMT-related protein expression and inhibited Cells for the wound healing assay were grown on a 35- IPF-associated cytokines, which supports PTL as a mm dish to 100% confluency and scraped to form a 100- potential compound for IPF treatment. μm wound using sterile pipette tips. The cells were cultured in the presence or absence of PTL in serum- Methods free media for 24 h. Images of the cells were obtained at Reagents 24 h using a light microscope (Nikon, Japan). PTL (> 99%) was provided by Shangdeyaoyuan Co. (Tianjin, China). DEX sodium phosphate (> 98.5%) was purchased Immunofluorescence from Meilun Biological Technology Co. (Dalian, China), and Primary epithelial cells were fixed in 4% paraformalde- BLM sulfate (> 91%) was obtained from Meilun Biological hyde for 20 min, washed with PBS, permeabilized with Technology Co. (Dalian, China). The NF-κB, Snail, β-actin, 0.2% Triton X-100 in PBS, blocked with 5% BSA and in- GAPDH, E-cadherin, vimentin, MMP1, α-SMA and Col-1 cubated with E-cadherin and vimentin antibodies. Cells antibodies were purchased from Affinity Biosciences Co. were washed with PBS, and donkey anti-rabbit Fluor 555 (Beijing, China). The mouse TNF-α,mouse IL-4,mouse or donkey anti-mouse Fluor 488 secondary antibodies TGF-β1, and mouse interferon gamma ELISA Kits were (CWBIO, China) were used for immunofluorescence purchased from Meilian Biological Technology Co. visualization. The nucleus was labeled with DAPI (Solar- (Shanghai, China). Chlorine ammonia T (> 97.08%) and bio, China), and cells were photographed with a TCS p-dimethylaminobenzaldehyde (> 97.08%) were obtained SP5 confocal (Leica) microscope. from (> 99.71%). Reverse-4-hydroxy-L-proline (> 99.4%) was purchased from Bailingwei Technology Co. (Beijing, Dual luciferase assay China). Perchloric acid (> 70%) was obtained from AP1, STAT3, NF-κB, snail, slug and MYC promoters were Jingchun Biological Technology Co. (Shanghai, China). cloned into the pGL6-TA luciferase reporter vector, and A549 cells were transfected with luciferase reporter plas- Cell culture mids using Lipofectamine (Invitrogen). Renilla-luciferase The human pulmonary epithelial A549 cell line was was used as an internal control. Cells were treated 1 d after obtained from KeyGen Biotech (Nanjing, China). The transfection with 5 μΜ (L) or 10 μM(H) PTLfor 24 h. human fetal lung fibroblast cell line HFL1 was kindly Cells were harvested, and the luciferase activity of cell Li et al. Respiratory Research (2018) 19:111 Page 3 of 12 lysates was determined using a luciferase assay system (Pro- a photomicroscope (Olympus, Tokyo, Japan) for micro- mega) as described by the manufacturer. Total light emis- scopic examination of morphological changes and fibro- sion was measured using a Luminoskan Ascent Reader sis evaluation (collagen fibers). System (Thermo, Massachusetts, USA). Immunohistochemistry BLM-induced PF in mice The tissue sections were pretreated in a microwave, Specific pathogen-free ICR mice (males) (body weights blocked and incubated using a series of antibodies, and 18–22 g) were purchased from the Laboratory Animal stained with DAB and hematoxylin. The results were Center, Academy of Military Medical Sciences of People’s captured using a microscope (Olympus, Japan). The in- Liberation Army (Beijing, China) and housed in groups of tensity and percentage of positive cells were measured. six under a regular 12-h light/dark cycle. Mice were accli- Multiplication (staining index) of intensity and percent- mated to laboratory conditions for one week prior to test- age scores was used to determine the results. ing at a constant temperature. Sixty mice were divided into six groups with 10 ani- Plasma collection mals per group according to body weight: control group, Mice were anesthetized, and a microhematocrit tube was BLM group, BLM + DEX group (0.45 mg/kg), BLM + introduced to the canthus of the orbit. The microhemato- PTL-H group (50 mg/kg), BLM + PTL-M group (25 mg/ crit tube was slightly advanced and rotated to allow blood kg), and BLM + PTL-L group (12.5 mg/kg). PF was flow into the lithium-heparin tube. Plasma was separated established in mice via a single intratracheal administra- from the cellular fraction via centrifugation at 3500 rpm tion of BLM at 5 mg/kg body weight. Different doses of for 10 min at 4 °C and stored at − 80 °C. PTL were intragastrically administered daily for four weeks beginning 7 days after BLM injury, and DEX was Bronchoalveolar lavage fluid (BALF) collection and cell used as the positive control. Control and model groups counts received an equal volume of vehicle (0.9% NaCl) using The tracheas of mice were cannulated and lavaged three the same schedule and route of administration. times with 1-ml sterile PBS at room temperature for Mouse body weights were recorded daily. Mice were BALF collection. Samples were centrifuged at 1000 rpm sacrificed on the 36th day using excess chloral hydrate for 5 min, and cell pellets were recovered in 1-ml sterile hydrochloride anesthesia. Blood was obtained for ELISA PBS. Cells were counted using a hemocytometer. Smears analyses, and whole lungs were removed and weighed. of BALF cells were stained with hematoxylin and eosin The right lungs were fixed in 10% formalin, dehydrated, and viewed under light microscopy to measure the and embedded in paraffin. The left lungs were used to de- inflammatory cell differential. termine hydroxyproline. The pulmonary coefficient was calculated using the following equation: lung weight/body TGF-β1, TNF-α and IL-4 assays weight × 100%. Plasma TGF-β1, TNF-α and IL-4 levels were assayed using ELISA Kits (Shanghai Enzyme-linked Biotechnol- Hydroxyproline assay ogy Co., Ltd., Shanghai, China). Assays were performed Collagen contents in left lungs of each group were mea- according to the manufacturer’s instructions. sured using a conventional hydroxyproline method [15]. The results were confirmed via measurement of samples Statistical analysis containing known amounts of purified collagen. Data are presented as the means ± standard deviation. Significant differences between treatment groups were Evaluation of pulmonary function detected using one-way ANOVA. All analyses were per- Mice were anesthetized with 10% chloral hydrate in formed using SPSS 17.0 statistical software. P < 0.05 was NaCl (i.p.) and transferred to a plethysmographic cham- considered statistically significant. ber for pulmonary function analyses using the Anires2005 system (Beijing Biolab, Beijing, China). This Results system automatically calculates and displays pulmonary PTL reduces cell viability and inhibits the migration of function parameters, including dynamic compliance and lung fibroblasts inspiratory and expiratory resistance. We determined the effect of PTL treatment (24 h) on the cell viability of primary pulmonary fibroblasts (PPF) Histopathological examination isolated from NaCl/BLM-treated mice and HFL1 cell Paraffin sections were prepared at a 4-μm thickness, lines using MTT assays (Fig. 1a, d and g). The half- stained with H&E and Masson’s trichrome using the maximal inhibitory concentrations (IC ) of PTL after manufacturers’ standard procedures, and observed under 24-h treatment in the PPF-NaCl, PPF-BLM and HFL1 Li et al. Respiratory Research (2018) 19:111 Page 4 of 12 Fig. 1 PTL reduces cell viability and migration of primary pulmonary fibroblasts (PPF) and HFL1 cells (a) The PPF isolated from NaCl-treated mice were treated with indicated dosages of PTL for 24 h (IC = 10.68 μM). PTL reduced the cell viability in a dose-dependent manner. b, c The PPF isolated from NaCl-treated mice were incubated in a medium containing PTL (0, 2.5, 5, 10, and 20 μM) for 24 h. Results showed that cell viability and migration were inhibited after incubation in different groups for 24 h. d The PPF isolated from BLM-treated mice were treated with indicated dosages of PTL for 24 h (IC = 26.01 μM). PTL reduced the cell viability in a dose-dependent manner. e, f The PPF isolated from BLM-treated mice were incubated in a medium containing PTL (0, 2.5, 5, 10, and 20 μM) for 24 h. g The HFL1 cells were treated with indicated dosages of PTL for 24 h (IC = 13.66 μM). PTL reduced the cell viability in a dose-dependent manner. h, i The HFL1 cells were incubated in a medium containing PTL (0, 2.5, 5, 10, and 20 μM) for 24 h cells were 10.68 μM, 26.01 μM and 13.66 μM, respect- microfilament structure of A549 cells. The PTL-H + TGF-β ively. These results indicate that PTL reduced the cell and PTL-L + TGF-β groups reversed the features of EMT viability of lung fibroblasts in a dose-dependent manner. to different degrees. The morphology changes and EMT The effect of PTL on the cellular migration of lung marker expression in TGF-β/PTL-treated primary AECs fibroblasts was determined using a wound-healing assay. exhibited the same results (Fig. 2b). We further evaluated Confluent cells were scraped with a sterile pipette tip, several EMT-related transcription factors, such as NF-κB, and the remaining cells were allowed to migrate to the Snail, AP-1, c-Myc, Slug, and Stat-3. We analyzed the activ- resulting gap in the absence or presence of PTL. Serum- ity of these transcription factors in A549 cells using a dual- starved PPF or HFL1 cells in the control group exhibited luciferase assay. PTL reduced the expression and activity of a narrow cell wound gap in the wound area 24 h after NF-κB and Snail, but did not influence the expression of wounding. Cells treated with PTL (2.5, 5, 10, or 20 μM) AP-1, c-Myc, Slug, and Stat-3 (Fig. 2c and d). exhibited relative delays in wound closure (Fig. 1b-c, e-f We verified the effect of PTL treatment on the expres- and h-i). sion of EMT-related transcription factors NF-κB and Snail in A549 cells and primary AECs. Western blot analysis re- PTL inhibits the expression of EMT-related transcription sults revealed that NF-κB and Snail levels increased sig- factors nificantly in the A549 cells and primary AECs after We researched the influence of PTL on the TGF-β1- exposure to TGF-β.NF-κB and Snail levels decreased induced EMT process in lung epithelial cells to further in- slightly in the TGF-β + PTL-L group and decreased vestigate its effects on the pathological mechanisms of PF. severely in the TGF-β + PTL-H group (Fig. 2e-h). The Figure 2a shows that low doses of TGF-β,aninducer of expression levels of Snail and NF-κB decreased in a dose- EMT, resulted in extended pseudopodia and changes in the dependent manner. Li et al. Respiratory Research (2018) 19:111 Page 5 of 12 Fig. 2 PTL inhibits TGF-β1-induced EMT through inhibiting NF-κB/Snail expression in lung epithelial cells. a Typical images of A549 cells in the Control group, TGF-β group, PTL-L + TGF-β group, and PTL-H + TGF-β group under an optical microscope. b Typical images of primary lung epithelial cells in the Control group, TGF-β group, PTL-L + TGF-β group, and PTL-H + TGF-β group under an optical microscope and immunofuorescence staining of E-Cadherin (green) and Vimentin (red) was performed. The nucleus was staining with DAPI. c, d Expression levels of NF-κB, Snail, AP-1, c-Myc, Slug and Stat-3 were assessed using dual-luciferase assay. e-h After TGF-β/PTL treatment, NF-κB and Snail were evaluated using Western blot analysis. β-actin was used as a loading control. Data are presented as means of three experiments; error bars represent standard deviation, *P <0.05, **P <0.01 PTL attenuates the BLM-induced PF in mice injured mice with high/middle PTL doses significantly re- Mice were intragastrically administered with or without duced collagen content compared to mice treated with PTL after BLM injection to investigate the effects of DEX (Fig. 3b). The protein levels of collagen (Col1) and α- PTL on BLM-induced lung fibrosis. A protective effect SMA exhibited similar results (Fig. 3c). The lung coeffi- of PTL was observed on weight loss. Body weight loss was cients were higher in the lung tissues of the model group significantly attenuated in mice treated with PTL compared than the other groups, and the lung coefficient of the mice to the model group (Fig. 3a). Treatment of bleomycin- treated with PTL was considerably reduced compared to Li et al. Respiratory Research (2018) 19:111 Page 6 of 12 Fig. 3 PTL attenuates the BLM-induced pulmonary fibrosis in mice. a Body weights (g) of animals after PTL treatment. b Hydroxyproline content in lung tissues. c Western blot analysis of type I collagen (Col1) and α-SMA in lung tissues was performed. GAPDH was used as a loading control. d Lung coefficient levels in the mouse model of BLM-induced PF. e-g Pulmonary function parameters including inspiratory resistance, expiratory resistance and pulmonary dynamic compliance among different groups were compared. Data are expressed as mean ± SD, *P <0.05, **P < 0.01,***P <0.001 the model group (Fig. 3d). The attenuated fibrosis in PTL- mice, and this effect was dose-dependent. Our assay results treated mice was further supported by improved pulmon- demonstrated that PTL significantly reduced the inflamma- ary function, which was observed as a decreased inspiratory tory response in bleomycin-injured mice in a dose- resistance, expiratory resistance and increased dynamic dependent manner. compliance compared to BLM-treated mice (Fig. 3e-g). Collectively, these in vivo data indicate that PTL attenuated Effects of PTL on histopathological changes in vivo bleomycin-induced pulmonary fibrosis in mice. Lung tissues were pink and soft under the normal condi- tions, and the lungs of mice in the bleomycin-treated PTL inhibits the inflammatory responses of PF model group were hard, shrunken in size, and dark in Persistent inflammation exists in an early stage, and it color. Treatment with PTL remarkably improved the drives fibrotic progression in bleomycin-induced fibrosis. lung condition (Fig. 5a). Histopathological changes in However, the effect of inflammation in fibrogenesis is argu- mouse lung tissues were evaluated using H&E staining. able. Inflammatory cell numbers in BALF and inflammatory The lung tissue specimens of the BLM-induced mice cytokines in plasma were evaluated to determine whether treated with PTL exhibited marked improvements in PTL altered inflammatory responses. Notably, cell differen- inflammation and fibrosis (Fig. 5b and c). The BLM- tiation analysis of BALF revealed that the increase in mac- injured lungs exhibited fibroplasia, inflammatory cell in- rophages, lymphocytes and neutrophils was attenuated in filtration, thickened alveolar walls, destroyed and disor- the high/middle PTL dose groups compared to the model dered alveoli, and stenosed or partially collapsed alveolar group (Fig. 4a-c). TGF-β,TNF-α and IL-4 levels in plasma spaces (Fig. 5b and d). The lungs of the animals treated were detected using ELISA. Levels of the inflammatory with PTL revealed significantly reduced infiltration of cytokines TGF-β,TNF-α and IL-4 were increased in the inflammatory cells, edema, thrombosis, and structure plasma of the model group compared to the other groups. destruction compared to the model group. Masson’s The levels of these inflammatory cytokines decreased sig- trichrome staining was used to examine the collagen de- nificantly in mice treated with PTL compared to untreated position and distribution and assess fibrosis in lung Li et al. Respiratory Research (2018) 19:111 Page 7 of 12 Fig. 4 PTL attenuates BLM-induced lung inflammatory responses. a-c The different inflammatory cell counts in BALF (a:Macrophages, b: Lymphocytes, c: Neutrophils) were determined by standard morphologic criteria; (d-f) The expression levels of TGF-β, TNF-α and IL-4 in plasma of each group were evaluated with ELISA assay. All data are presented as means of three experiments; error bars represent standard deviation (*P < 0.05, **P <0.01) Fig. 5 Effects of PTL on histopathological change in vivo. a Gross appearance of the lungs under the stereoscopic microscope. b, d Photomicrographs of lung sections stained with hematoxylin–eosin (HE). c, e Masson trichrome staining of collagen on lung sections. (*P <0.05, **P <0.01) Li et al. Respiratory Research (2018) 19:111 Page 8 of 12 tissues. Significant collagen deposition, particularly revealed that PTL increased MMP1 levels and decreased around the bronchus, was observed in BLM-injured lung Col-1 levels in lung tissues in a dose-dependent manner tissues compared to their respective controls (Fig. 5c (Fig. 6). and e). The PTL-treated groups exhibited a significant The expression levels of NF-κB and Snail were also decrease in collagen deposition compared to the model assessed using immunohistochemical staining. The re- group, and PTL-H was better than PTL-L. sults demonstrated that NF-κB and Snail levels de- creased significantly compared to the model group in a dose-dependent manner (Fig. 7). PTL inhibited EMT via PTL attenuates the BLM-induced EMT-related protein the NF-κB/Snail pathway in lung tissues. These results expression and inhibits cytokine production of PF are consistent with the cell experimental results. The present study analyzed several EMT-related proteins to determine whether PTL affected the conversion of AECs to Mechanism research on PTL and large data analysis using fibroblasts (FBs). PTL increased epithelial cell markers, such online database as E-cad, and reduced the vimentin and α-SMA (marker of The STRING database was used to examine several types mesenchymal cells) expression (Fig. 6). These results suggest of interactions between the control and PTL treatment that PTL increased epithelial cell markers and reduced mes- groups. Gene Ontology (GO) analysis was performed to enchymal cell markers. The effect of PTL was better than analyze the function of differentially expressed mRNAs DEX. PTL also inhibits Col-1 and MMP1, which are PF cy- using the GO categories. GO categories are derived from tokines. Immunohistochemical staining of Col-1 and MMP1 Gene Ontology (http://www.geneontology.org), and the Fig. 6 PTL attenuates the BLM-induced expression of EMT-related protein. a Representative immunohistochemical staining of lung sections showing E-cadherin, vimentin, MMP1, α-SMA and Col-1 staining. b Expression levels of makers were evaluated by index of immunohistochemical staining. The number of positively stained cells in each group was calculated from twenty different 400× magnified fields under a microscope. The data represent the mean ± standard deviation (SD), n = 10 per group. *, P <0.05, **P < 0.01 vs. model group Li et al. Respiratory Research (2018) 19:111 Page 9 of 12 Fig. 7 PTL attenuates the BLM-induced expression of NF-κBand Snail. a Immunohistochemical staining for NF-κB and Snail in lung sections. b Expression levels of NF-κB and Snail were evaluated by index of immunohistochemical staining. The number of positively stained cells was calculated from twenty different 400× magnified fields under a microscope. The data represent the mean ± standard deviation (SD), n = 10 per group. *, P <0.05, **P <0.01 vs. model group categories include three integrated networks of defined Europe [18]. PTL from Magnolia grandiflora exhibited terms that describe gene properties (molecular function, antitumor properties for the first time in 1973 [19]. The cellular component and biological process). PTL influ- biological properties of PTL were primarily attributed to enced many functions, including inflammatory responses the strong inhibition of NF-κB and the targeting of vari- and proliferation (Fig. 8a). We also analyzed the molecular ous steps within the NF-κB signaling pathway [20, 21]. function, cell structure, and biological processes of the ex- The inflammatory response during the initial phase of pression profiling chip on the GO website after PTL treat- PF damages the ECM and produces numerous FBs via ment and compared the results with the control groups activation of the repair mechanism. The relationship be- (Fig. 8b). These biological processes are closely related to tween PF and cytokines, particularly TNF-α and TGF-β, pulmonary fibrosis. attracted considerable attention in recent years. These cytokines promote inflammation progression [22–24]. Discussion NF-κB is commonly activated to protect against organ- PTL is a natural molecule that was originally isolated isms, but disorder in its activation is related to chronic from the shoots of feverfew (Tanacetum parthenium). inflammation [25]. PTL inhibited the activities of TNF- PTL exhibits excellent anti-inflammatory and antitumor α, TGF-β, and NF-κB in the present study. activities [16, 17]. The first written records of the anti- Our studies evaluated the role of PTL in PF. The inflammatory effect of PTL were provided in 1597 in results demonstrated that PTL repressed BLM-induced Li et al. Respiratory Research (2018) 19:111 Page 10 of 12 Fig. 8 Mechanism research on the PTL effects. a-b GO analysis of PTL in affecting biological processes. c A model showing the role of PTL in BLM-induced EMT pulmonary fibrosis in mice. PTL inhibited EMT of Our results demonstrated that NF-κB and Snail expres- AECs, which upregulate epithelial marker expression sion levels and activities decreased following PTL treat- and downregulate the expression of mesenchymal ment. Therefore, PTL may inhibit the NF-κB signaling markers. We evaluated the influence of PTL on AP-1, pathway and exhibit proinflammatory effects during PF. NF-κB, STAT-3, Snail, Slug and c-Myc expression. PTL FBs are important in the structural formation process only downregulated NF-κB and Snail. The NF-κB path- and maintaining the function of pulmonary tissues [26]. way regulates Snail expression via transcriptional and The cross talk of FBs and AECs promotes fibrosis. FBs post-translational mechanisms. NF-κB binds to the hu- proliferate continuously as a result of multiple factors, man Snail promoter and increases Snail transcription. such as the stimulating action of cytokines [27–29]. Li et al. Respiratory Research (2018) 19:111 Page 11 of 12 Therefore, an effective approach to inhibit FB proliferation Availability of data and materials Data available on request from the authors. in PF should be urgently identified [30]. The present re- sults demonstrated that PTL inhibited the proliferation Authors’ contributions and migration of primary pulmonary fibroblasts and (I) Conception and design: HL, HZ; (II) Administrative support: HL, JG; (III) Collection and assembly of data: XL, XT, JY and YQ; (V) Data analysis and HFL1 cells in a dose-dependent manner. interpretation: XL, XT; (VI) Manuscript writing: All authors; (VII) Final approval EMT is an indispensable step in numerous diseases, of manuscript: All authors. All authors read and approved the final manuscript. and it induces cell changes involved in pathological pro- Ethics approval and consent to participate cesses, such as fibrosis [31, 32]. EMT plays a pivotal role The study was approved by the Institutional Ethical Committee of Tianjin in the development of PF. Lung epithelial cells are a fre- International Joint Academy of Biomedicine (No. SYXK 2017–0003). quent target of injury, a driver of normal repair, and a Competing interests key element in the pathobiology of fibrotic lung diseases. The authors declare that they have no competing interests. One important aspect of epithelial cells is their capacity to respond to microenvironmental cues by undergoing Publisher’sNote EMT. EMT regulates a series of critical signaling elements Springer Nature remains neutral with regard to jurisdictional claims in that produce proinflammatory signals and cause cell in- published maps and institutional affiliations. jury. EMT is not the widespread conversion of epithelial Received: 16 January 2018 Accepted: 6 May 2018 cells to FBs, but it is a graded response whereby epithelial cells reversibly acquire mesenchymal features and enhance References the capacity for mesenchymal cross talk [33]. Repeated in- 1. Foster MW, et al. 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Blocking follistatin-like 1 attenuates bleomycin-induced PF:Pulmonary fibrosis;PPF:Primary pulmonary fibroblasts; PTL: Parthenolide; TGF- pulmonary fibrosis in mice. J Exp Med. 2015;212(2):235–52. β: Transforming growth factor β;TNF-α: Tumor necrosis factor α; α-SMA: α-smooth 16. Zhang S, et al. Anti-cancer potential of sesquiterpene lactones: bioactivity and muscle actin molecular mechanisms. Curr Med Chem Anticancer Agents. 2005;5:239–49. 17. Kwok BH, et al. The anti-inflammatory natural product parthenolide from Funding the medicinal herb feverfew directly binds to and inhibits IkappaB kinase. This study was supported by Tianjin science and technology innovation system and Chem Biol. 2001;8:759–66. the condition of platform construction plan [Grant 14TXSYJC00572], Innovation fund 18. Knight DW. Feverfew: chemistry and biological activity. Nat Prod Rep. for technology based firms [Grant 12ZXCXSY06500] and [Grant 12ZXCXSY07200], 1995;12:271–6. Open fund project of State Key Laboratory of Medicinal Chemical Biology [Grant 19. Wiedhopf RM, et al. Tumor inhibitory agent from Magnolia grandiflora 2018128]. (Magnoliaceae). I. Parthenolide. J Pharm Sci. 1973;62:345. Li et al. Respiratory Research (2018) 19:111 Page 12 of 12 20. Jia QQ, et al. Sesquiterpene lactones and their derivatives inhibit high glucose-induced NF-κB activation and MCP-1 and TGF-β1 expression in rat mesangial cells. Molecules. 2013;18(10):13061–77. 21. Kim HY, et al. Balsalazide potentiates Parthenolide-mediated inhibition of nuclear factor-κB signaling in HCT116 human colorectal Cancer cells. Intest Res. 2015;13(3):233–41. 22. Driscoll KE, Maurer JK. Cytokine and growth factor release by alveolar macrophages: potential biomarkers of pulmonary toxicity. Toxicol Pathol. 1991;19(1):398–405. 23. Savici D, et al. Silica increases tumor necrosis factor (TNF) production, in part, by upregulating the TNF promoter. Exp Lung Res. 1994;102(6):613–25. 24. Bartram U, Speer CP. The role of transforming growth factor beta in lung development and disease. Chest. 2004;125(2):754. 25. Doyle SL, et al. Nuclear factor κB2 p52 protein has a role in antiviral immunity through IκB kinase -dependent induction of Sp1 protein and interleukin 15. J Biol Chem. 2013;288(35):25066. 26. Sivakumar P, et al. Into the matrix: targeting fibroblasts in pulmonary fibrosis. Curr Opin Pulm Med. 2012;18(5):462–9. 27. Piek E, Heldin C, Dijke PT. Specificity, diversity, and regulation in TGF-β superfamily signaling. FASEB J. 1999;13(15):2105. 28. Nakamuta M, et al. Effects of fibril- or fixed-collagen on matrix metalloproteinase-1and tissue inhibitor of matrix metalloproteinase-1 production in the human hepatocyte cell line HLE. World J Gastroenterol. 2005;11(15):2264–8. 29. Pardo A, et al. Role of matrix metalloproteinases in the pathogenesis of idiopathic pulmonary fibrosis. Respir Res. 2016;17(1):1–10. 30. Darby IA, et al. The myofibroblast, a key cell in normal and pathological tissue repair. Cell Mol Life Sci. 2016;73(6):1145–57. 31. Corvol H, et al. Lung alveolar epithelium and interstitial lung disease. Int J Biochem Cell Biol. 2009;41(8–9):1643. 32. Lee JM, et al. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol. 2006;172(7):973. 33. Horowitz JC, Thannickal VJ. Epithelial-mesenchymal interactions in pulmonary fibrosis. Semin Respir Crit Care Med. 2006;27(6):600. 34. Ohbayashi M, et al. Involvement of epithelial-mesenchymal transition in methotrexate-induced pulmonary fibrosis. J Toxicol Sci. 2014;39(2):319–30. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Respiratory Research Springer Journals

Parthenolide attenuated bleomycin-induced pulmonary fibrosis via the NF-κB/Snail signaling pathway

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Abstract

Background: Parthenolide (PTL) is a natural molecule isolated from Tanacetum parthenium that exhibits excellent anti-inflammatory and antitumor activities. Pulmonary fibrosis (PF), especially idiopathic pulmonary fibrosis (IPF), is a chronic lung disease that lacks a proven effective therapy. The present study evaluated the therapeutic effect of PTL on PF. Methods: Serum-starved primary lung fibroblasts and HFL1 cells were treated with different doses of PTL, and cell viability and the migration rate were measured. Western blot analysis and a dual-luciferase assay were used to analyze the epithelial–mesenchymal transition (EMT)-related transcription factors influenced by PTL treatment in A549 cells and primary lung epithelial cells. Mice with bleomycin (BLM)-induced pulmonary fibrosis were treated with different doses of intragastric PTL, and pathological changes were evaluated using Hematoxylin-eosin (H&E) staining and immunohistochemical analysis. Results: Our results demonstrated that PTL reduced the cell viability and migration rate of lung fibroblasts and inhibited the expression of EMT-related transcription factors in lung epithelial cells. In vivo studies demonstrated that PTL attenuated BLM-induced pulmonary fibrosis and improved the body weight and pathological changes of BLM-treated mice. We further demonstrated that PTL attenuated BLM-induced PF primarily via inhibition of the NF-κB/Snail signaling pathway. Conclusion: These findings suggest that PTL inhibits EMT and attenuates BLM-induced PF via the NF-κB/Snail signaling pathway. PTL is a worthwhile candidate compound for pulmonary fibrosis therapy. Keywords: Parthenolide, Pulmonary fibrosis, NF-κB/Snail signaling pathway Background cyclophosphamide), are limited by low their efficacy and Pulmonary fibrosis (PF), especially idiopathic pulmonary severe side effects. The FDA recently approved two new fibrosis (IPF), is a chronic lung disease caused by several drugs, nintedanib and pirfenidone, to treat IPF. These factors. IPF exhibits a complex pathogenesis, but no drugs stabilize patients’ conditions well, but they do not effective treatment is available for IPF. The mortality reverse the progression of fibrosis. Both drugs produce rate of IPF is considerably increased in recent years, and side effects on the liver and skin, which limits their clin- it substantially threatens human health [1, 2]. Current ical application, especially in patients with liver problems treatments for IPF, such as immunosuppressants (e.g., [3, 4]. Recent research demonstrated that dexametha- sone (DEX) attenuated bleomycin (BLM)-induced lung fibrosis [5]. However, DEX treatment produces many * Correspondence: honggang.zhou@nankai.edu.cn side effects, such as growth retardation, hyperglycemia, Xiao-he Li and Ting Xiao contributed equally to this work. hypertension, myocardial hypertrophy, gastrointestinal State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, perforation, and neurological impairment [6–8]. There- Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People’s Republic of fore, new drugs with improved treatment efficacy and China fewer side effects are urgently needed. Tianjin Key Laboratory of Molecular Drug Research, Tianjin International Joint Academy of Biomedicine, Tianjin, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. Respiratory Research (2018) 19:111 Page 2 of 12 IPF is easily characterized by an excessive deposition supplied by Professor Wen Ning (Nankai University). of extracellular matrix (ECM), but the pathogenesis of The cells were cultured in a medium supplemented with IPF is not clear. Several hypotheses were proposed to 10% heat-inactivated (56 °C, 30 min) fetal calf serum explain the inner mechanisms, and epithelial–mesenchy- (HyClone, USA) and maintained at 37 °C with 5% CO2 mal transition (EMT) of alveolar epithelial cells (AECs) in a humidified atmosphere. received particular attention. Nuclear factor kappa-B (NF-κB) is an essential mediator of EMT. NF-κB pro- Isolation of primary fibroblasts and AECs motes the transcription of many inflammatory cytokines, Primary pulmonary fibroblasts isolated from NaCl/BLM- such as tumor necrosis factor α (TNF-α), interleukin treated mice were cultured in DMEM supplemented (IL) and transforming growth factor β (TGF-β), which with 10% FBS and antibiotics in 5% CO2 at 37 °C in a are highly associated with the progression of IPF, espe- humidified atmosphere as described previously [14]. cially TGF-β [9–11]. Therefore, it is essential to measure Cells at passages 3–4 were used for cell viability and these factors when evaluating the drug efficacy of IPF. wound healing assays. Primary AECs were isolated from Parthenolide (PTL) is a sesquiterpene lactone that is C57BL/6 J mice as previously described [15]. Newly isolated from the shoots of feverfew (Tanacetum parthe- isolated AECs were used for immunofluorescence and nium), and PTL is a traditional medicinal herb used for Western blotting assays. headaches and arthritis. Recent studies suggested that PTL is a useful antitumor and anti-inflammatory agent, Cell viability and wound-healing assays and it was tested in clinical studies for leukemia and Cell viability was determined using the MTT assay. Cells neurological tumors [12]. These biological activities of (5 × 10 cells/mL) were seeded in 96-well culture plates and PTL in tumor and inflammatory diseases primarily occur incubated overnight. Cells were treated with various con- via inhibition of NF-κB and the targeting of multiple centrations of PTL for 24 h. Cell viability was measured steps in the NF-κB signaling pathway. For example, PTL after the addition of MTT (20 μL) at 37 °C for 4 h. Di- binds an activator of NF-κB, IκB-kinase (IKK) [13]. methyl sulfoxide (150 μL) was added to dissolve the forma- However, PTL treatment of IPF and its pharmacological zan crystals. Optical density was measured at 570 nm using properties have not been reported. a microplate reader (Multiskan FC, Thermo Scientific, The present study found that PTL attenuated BLM- Waltham, MA, USA). induced EMT-related protein expression and inhibited Cells for the wound healing assay were grown on a 35- IPF-associated cytokines, which supports PTL as a mm dish to 100% confluency and scraped to form a 100- potential compound for IPF treatment. μm wound using sterile pipette tips. The cells were cultured in the presence or absence of PTL in serum- Methods free media for 24 h. Images of the cells were obtained at Reagents 24 h using a light microscope (Nikon, Japan). PTL (> 99%) was provided by Shangdeyaoyuan Co. (Tianjin, China). DEX sodium phosphate (> 98.5%) was purchased Immunofluorescence from Meilun Biological Technology Co. (Dalian, China), and Primary epithelial cells were fixed in 4% paraformalde- BLM sulfate (> 91%) was obtained from Meilun Biological hyde for 20 min, washed with PBS, permeabilized with Technology Co. (Dalian, China). The NF-κB, Snail, β-actin, 0.2% Triton X-100 in PBS, blocked with 5% BSA and in- GAPDH, E-cadherin, vimentin, MMP1, α-SMA and Col-1 cubated with E-cadherin and vimentin antibodies. Cells antibodies were purchased from Affinity Biosciences Co. were washed with PBS, and donkey anti-rabbit Fluor 555 (Beijing, China). The mouse TNF-α,mouse IL-4,mouse or donkey anti-mouse Fluor 488 secondary antibodies TGF-β1, and mouse interferon gamma ELISA Kits were (CWBIO, China) were used for immunofluorescence purchased from Meilian Biological Technology Co. visualization. The nucleus was labeled with DAPI (Solar- (Shanghai, China). Chlorine ammonia T (> 97.08%) and bio, China), and cells were photographed with a TCS p-dimethylaminobenzaldehyde (> 97.08%) were obtained SP5 confocal (Leica) microscope. from (> 99.71%). Reverse-4-hydroxy-L-proline (> 99.4%) was purchased from Bailingwei Technology Co. (Beijing, Dual luciferase assay China). Perchloric acid (> 70%) was obtained from AP1, STAT3, NF-κB, snail, slug and MYC promoters were Jingchun Biological Technology Co. (Shanghai, China). cloned into the pGL6-TA luciferase reporter vector, and A549 cells were transfected with luciferase reporter plas- Cell culture mids using Lipofectamine (Invitrogen). Renilla-luciferase The human pulmonary epithelial A549 cell line was was used as an internal control. Cells were treated 1 d after obtained from KeyGen Biotech (Nanjing, China). The transfection with 5 μΜ (L) or 10 μM(H) PTLfor 24 h. human fetal lung fibroblast cell line HFL1 was kindly Cells were harvested, and the luciferase activity of cell Li et al. Respiratory Research (2018) 19:111 Page 3 of 12 lysates was determined using a luciferase assay system (Pro- a photomicroscope (Olympus, Tokyo, Japan) for micro- mega) as described by the manufacturer. Total light emis- scopic examination of morphological changes and fibro- sion was measured using a Luminoskan Ascent Reader sis evaluation (collagen fibers). System (Thermo, Massachusetts, USA). Immunohistochemistry BLM-induced PF in mice The tissue sections were pretreated in a microwave, Specific pathogen-free ICR mice (males) (body weights blocked and incubated using a series of antibodies, and 18–22 g) were purchased from the Laboratory Animal stained with DAB and hematoxylin. The results were Center, Academy of Military Medical Sciences of People’s captured using a microscope (Olympus, Japan). The in- Liberation Army (Beijing, China) and housed in groups of tensity and percentage of positive cells were measured. six under a regular 12-h light/dark cycle. Mice were accli- Multiplication (staining index) of intensity and percent- mated to laboratory conditions for one week prior to test- age scores was used to determine the results. ing at a constant temperature. Sixty mice were divided into six groups with 10 ani- Plasma collection mals per group according to body weight: control group, Mice were anesthetized, and a microhematocrit tube was BLM group, BLM + DEX group (0.45 mg/kg), BLM + introduced to the canthus of the orbit. The microhemato- PTL-H group (50 mg/kg), BLM + PTL-M group (25 mg/ crit tube was slightly advanced and rotated to allow blood kg), and BLM + PTL-L group (12.5 mg/kg). PF was flow into the lithium-heparin tube. Plasma was separated established in mice via a single intratracheal administra- from the cellular fraction via centrifugation at 3500 rpm tion of BLM at 5 mg/kg body weight. Different doses of for 10 min at 4 °C and stored at − 80 °C. PTL were intragastrically administered daily for four weeks beginning 7 days after BLM injury, and DEX was Bronchoalveolar lavage fluid (BALF) collection and cell used as the positive control. Control and model groups counts received an equal volume of vehicle (0.9% NaCl) using The tracheas of mice were cannulated and lavaged three the same schedule and route of administration. times with 1-ml sterile PBS at room temperature for Mouse body weights were recorded daily. Mice were BALF collection. Samples were centrifuged at 1000 rpm sacrificed on the 36th day using excess chloral hydrate for 5 min, and cell pellets were recovered in 1-ml sterile hydrochloride anesthesia. Blood was obtained for ELISA PBS. Cells were counted using a hemocytometer. Smears analyses, and whole lungs were removed and weighed. of BALF cells were stained with hematoxylin and eosin The right lungs were fixed in 10% formalin, dehydrated, and viewed under light microscopy to measure the and embedded in paraffin. The left lungs were used to de- inflammatory cell differential. termine hydroxyproline. The pulmonary coefficient was calculated using the following equation: lung weight/body TGF-β1, TNF-α and IL-4 assays weight × 100%. Plasma TGF-β1, TNF-α and IL-4 levels were assayed using ELISA Kits (Shanghai Enzyme-linked Biotechnol- Hydroxyproline assay ogy Co., Ltd., Shanghai, China). Assays were performed Collagen contents in left lungs of each group were mea- according to the manufacturer’s instructions. sured using a conventional hydroxyproline method [15]. The results were confirmed via measurement of samples Statistical analysis containing known amounts of purified collagen. Data are presented as the means ± standard deviation. Significant differences between treatment groups were Evaluation of pulmonary function detected using one-way ANOVA. All analyses were per- Mice were anesthetized with 10% chloral hydrate in formed using SPSS 17.0 statistical software. P < 0.05 was NaCl (i.p.) and transferred to a plethysmographic cham- considered statistically significant. ber for pulmonary function analyses using the Anires2005 system (Beijing Biolab, Beijing, China). This Results system automatically calculates and displays pulmonary PTL reduces cell viability and inhibits the migration of function parameters, including dynamic compliance and lung fibroblasts inspiratory and expiratory resistance. We determined the effect of PTL treatment (24 h) on the cell viability of primary pulmonary fibroblasts (PPF) Histopathological examination isolated from NaCl/BLM-treated mice and HFL1 cell Paraffin sections were prepared at a 4-μm thickness, lines using MTT assays (Fig. 1a, d and g). The half- stained with H&E and Masson’s trichrome using the maximal inhibitory concentrations (IC ) of PTL after manufacturers’ standard procedures, and observed under 24-h treatment in the PPF-NaCl, PPF-BLM and HFL1 Li et al. Respiratory Research (2018) 19:111 Page 4 of 12 Fig. 1 PTL reduces cell viability and migration of primary pulmonary fibroblasts (PPF) and HFL1 cells (a) The PPF isolated from NaCl-treated mice were treated with indicated dosages of PTL for 24 h (IC = 10.68 μM). PTL reduced the cell viability in a dose-dependent manner. b, c The PPF isolated from NaCl-treated mice were incubated in a medium containing PTL (0, 2.5, 5, 10, and 20 μM) for 24 h. Results showed that cell viability and migration were inhibited after incubation in different groups for 24 h. d The PPF isolated from BLM-treated mice were treated with indicated dosages of PTL for 24 h (IC = 26.01 μM). PTL reduced the cell viability in a dose-dependent manner. e, f The PPF isolated from BLM-treated mice were incubated in a medium containing PTL (0, 2.5, 5, 10, and 20 μM) for 24 h. g The HFL1 cells were treated with indicated dosages of PTL for 24 h (IC = 13.66 μM). PTL reduced the cell viability in a dose-dependent manner. h, i The HFL1 cells were incubated in a medium containing PTL (0, 2.5, 5, 10, and 20 μM) for 24 h cells were 10.68 μM, 26.01 μM and 13.66 μM, respect- microfilament structure of A549 cells. The PTL-H + TGF-β ively. These results indicate that PTL reduced the cell and PTL-L + TGF-β groups reversed the features of EMT viability of lung fibroblasts in a dose-dependent manner. to different degrees. The morphology changes and EMT The effect of PTL on the cellular migration of lung marker expression in TGF-β/PTL-treated primary AECs fibroblasts was determined using a wound-healing assay. exhibited the same results (Fig. 2b). We further evaluated Confluent cells were scraped with a sterile pipette tip, several EMT-related transcription factors, such as NF-κB, and the remaining cells were allowed to migrate to the Snail, AP-1, c-Myc, Slug, and Stat-3. We analyzed the activ- resulting gap in the absence or presence of PTL. Serum- ity of these transcription factors in A549 cells using a dual- starved PPF or HFL1 cells in the control group exhibited luciferase assay. PTL reduced the expression and activity of a narrow cell wound gap in the wound area 24 h after NF-κB and Snail, but did not influence the expression of wounding. Cells treated with PTL (2.5, 5, 10, or 20 μM) AP-1, c-Myc, Slug, and Stat-3 (Fig. 2c and d). exhibited relative delays in wound closure (Fig. 1b-c, e-f We verified the effect of PTL treatment on the expres- and h-i). sion of EMT-related transcription factors NF-κB and Snail in A549 cells and primary AECs. Western blot analysis re- PTL inhibits the expression of EMT-related transcription sults revealed that NF-κB and Snail levels increased sig- factors nificantly in the A549 cells and primary AECs after We researched the influence of PTL on the TGF-β1- exposure to TGF-β.NF-κB and Snail levels decreased induced EMT process in lung epithelial cells to further in- slightly in the TGF-β + PTL-L group and decreased vestigate its effects on the pathological mechanisms of PF. severely in the TGF-β + PTL-H group (Fig. 2e-h). The Figure 2a shows that low doses of TGF-β,aninducer of expression levels of Snail and NF-κB decreased in a dose- EMT, resulted in extended pseudopodia and changes in the dependent manner. Li et al. Respiratory Research (2018) 19:111 Page 5 of 12 Fig. 2 PTL inhibits TGF-β1-induced EMT through inhibiting NF-κB/Snail expression in lung epithelial cells. a Typical images of A549 cells in the Control group, TGF-β group, PTL-L + TGF-β group, and PTL-H + TGF-β group under an optical microscope. b Typical images of primary lung epithelial cells in the Control group, TGF-β group, PTL-L + TGF-β group, and PTL-H + TGF-β group under an optical microscope and immunofuorescence staining of E-Cadherin (green) and Vimentin (red) was performed. The nucleus was staining with DAPI. c, d Expression levels of NF-κB, Snail, AP-1, c-Myc, Slug and Stat-3 were assessed using dual-luciferase assay. e-h After TGF-β/PTL treatment, NF-κB and Snail were evaluated using Western blot analysis. β-actin was used as a loading control. Data are presented as means of three experiments; error bars represent standard deviation, *P <0.05, **P <0.01 PTL attenuates the BLM-induced PF in mice injured mice with high/middle PTL doses significantly re- Mice were intragastrically administered with or without duced collagen content compared to mice treated with PTL after BLM injection to investigate the effects of DEX (Fig. 3b). The protein levels of collagen (Col1) and α- PTL on BLM-induced lung fibrosis. A protective effect SMA exhibited similar results (Fig. 3c). The lung coeffi- of PTL was observed on weight loss. Body weight loss was cients were higher in the lung tissues of the model group significantly attenuated in mice treated with PTL compared than the other groups, and the lung coefficient of the mice to the model group (Fig. 3a). Treatment of bleomycin- treated with PTL was considerably reduced compared to Li et al. Respiratory Research (2018) 19:111 Page 6 of 12 Fig. 3 PTL attenuates the BLM-induced pulmonary fibrosis in mice. a Body weights (g) of animals after PTL treatment. b Hydroxyproline content in lung tissues. c Western blot analysis of type I collagen (Col1) and α-SMA in lung tissues was performed. GAPDH was used as a loading control. d Lung coefficient levels in the mouse model of BLM-induced PF. e-g Pulmonary function parameters including inspiratory resistance, expiratory resistance and pulmonary dynamic compliance among different groups were compared. Data are expressed as mean ± SD, *P <0.05, **P < 0.01,***P <0.001 the model group (Fig. 3d). The attenuated fibrosis in PTL- mice, and this effect was dose-dependent. Our assay results treated mice was further supported by improved pulmon- demonstrated that PTL significantly reduced the inflamma- ary function, which was observed as a decreased inspiratory tory response in bleomycin-injured mice in a dose- resistance, expiratory resistance and increased dynamic dependent manner. compliance compared to BLM-treated mice (Fig. 3e-g). Collectively, these in vivo data indicate that PTL attenuated Effects of PTL on histopathological changes in vivo bleomycin-induced pulmonary fibrosis in mice. Lung tissues were pink and soft under the normal condi- tions, and the lungs of mice in the bleomycin-treated PTL inhibits the inflammatory responses of PF model group were hard, shrunken in size, and dark in Persistent inflammation exists in an early stage, and it color. Treatment with PTL remarkably improved the drives fibrotic progression in bleomycin-induced fibrosis. lung condition (Fig. 5a). Histopathological changes in However, the effect of inflammation in fibrogenesis is argu- mouse lung tissues were evaluated using H&E staining. able. Inflammatory cell numbers in BALF and inflammatory The lung tissue specimens of the BLM-induced mice cytokines in plasma were evaluated to determine whether treated with PTL exhibited marked improvements in PTL altered inflammatory responses. Notably, cell differen- inflammation and fibrosis (Fig. 5b and c). The BLM- tiation analysis of BALF revealed that the increase in mac- injured lungs exhibited fibroplasia, inflammatory cell in- rophages, lymphocytes and neutrophils was attenuated in filtration, thickened alveolar walls, destroyed and disor- the high/middle PTL dose groups compared to the model dered alveoli, and stenosed or partially collapsed alveolar group (Fig. 4a-c). TGF-β,TNF-α and IL-4 levels in plasma spaces (Fig. 5b and d). The lungs of the animals treated were detected using ELISA. Levels of the inflammatory with PTL revealed significantly reduced infiltration of cytokines TGF-β,TNF-α and IL-4 were increased in the inflammatory cells, edema, thrombosis, and structure plasma of the model group compared to the other groups. destruction compared to the model group. Masson’s The levels of these inflammatory cytokines decreased sig- trichrome staining was used to examine the collagen de- nificantly in mice treated with PTL compared to untreated position and distribution and assess fibrosis in lung Li et al. Respiratory Research (2018) 19:111 Page 7 of 12 Fig. 4 PTL attenuates BLM-induced lung inflammatory responses. a-c The different inflammatory cell counts in BALF (a:Macrophages, b: Lymphocytes, c: Neutrophils) were determined by standard morphologic criteria; (d-f) The expression levels of TGF-β, TNF-α and IL-4 in plasma of each group were evaluated with ELISA assay. All data are presented as means of three experiments; error bars represent standard deviation (*P < 0.05, **P <0.01) Fig. 5 Effects of PTL on histopathological change in vivo. a Gross appearance of the lungs under the stereoscopic microscope. b, d Photomicrographs of lung sections stained with hematoxylin–eosin (HE). c, e Masson trichrome staining of collagen on lung sections. (*P <0.05, **P <0.01) Li et al. Respiratory Research (2018) 19:111 Page 8 of 12 tissues. Significant collagen deposition, particularly revealed that PTL increased MMP1 levels and decreased around the bronchus, was observed in BLM-injured lung Col-1 levels in lung tissues in a dose-dependent manner tissues compared to their respective controls (Fig. 5c (Fig. 6). and e). The PTL-treated groups exhibited a significant The expression levels of NF-κB and Snail were also decrease in collagen deposition compared to the model assessed using immunohistochemical staining. The re- group, and PTL-H was better than PTL-L. sults demonstrated that NF-κB and Snail levels de- creased significantly compared to the model group in a dose-dependent manner (Fig. 7). PTL inhibited EMT via PTL attenuates the BLM-induced EMT-related protein the NF-κB/Snail pathway in lung tissues. These results expression and inhibits cytokine production of PF are consistent with the cell experimental results. The present study analyzed several EMT-related proteins to determine whether PTL affected the conversion of AECs to Mechanism research on PTL and large data analysis using fibroblasts (FBs). PTL increased epithelial cell markers, such online database as E-cad, and reduced the vimentin and α-SMA (marker of The STRING database was used to examine several types mesenchymal cells) expression (Fig. 6). These results suggest of interactions between the control and PTL treatment that PTL increased epithelial cell markers and reduced mes- groups. Gene Ontology (GO) analysis was performed to enchymal cell markers. The effect of PTL was better than analyze the function of differentially expressed mRNAs DEX. PTL also inhibits Col-1 and MMP1, which are PF cy- using the GO categories. GO categories are derived from tokines. Immunohistochemical staining of Col-1 and MMP1 Gene Ontology (http://www.geneontology.org), and the Fig. 6 PTL attenuates the BLM-induced expression of EMT-related protein. a Representative immunohistochemical staining of lung sections showing E-cadherin, vimentin, MMP1, α-SMA and Col-1 staining. b Expression levels of makers were evaluated by index of immunohistochemical staining. The number of positively stained cells in each group was calculated from twenty different 400× magnified fields under a microscope. The data represent the mean ± standard deviation (SD), n = 10 per group. *, P <0.05, **P < 0.01 vs. model group Li et al. Respiratory Research (2018) 19:111 Page 9 of 12 Fig. 7 PTL attenuates the BLM-induced expression of NF-κBand Snail. a Immunohistochemical staining for NF-κB and Snail in lung sections. b Expression levels of NF-κB and Snail were evaluated by index of immunohistochemical staining. The number of positively stained cells was calculated from twenty different 400× magnified fields under a microscope. The data represent the mean ± standard deviation (SD), n = 10 per group. *, P <0.05, **P <0.01 vs. model group categories include three integrated networks of defined Europe [18]. PTL from Magnolia grandiflora exhibited terms that describe gene properties (molecular function, antitumor properties for the first time in 1973 [19]. The cellular component and biological process). PTL influ- biological properties of PTL were primarily attributed to enced many functions, including inflammatory responses the strong inhibition of NF-κB and the targeting of vari- and proliferation (Fig. 8a). We also analyzed the molecular ous steps within the NF-κB signaling pathway [20, 21]. function, cell structure, and biological processes of the ex- The inflammatory response during the initial phase of pression profiling chip on the GO website after PTL treat- PF damages the ECM and produces numerous FBs via ment and compared the results with the control groups activation of the repair mechanism. The relationship be- (Fig. 8b). These biological processes are closely related to tween PF and cytokines, particularly TNF-α and TGF-β, pulmonary fibrosis. attracted considerable attention in recent years. These cytokines promote inflammation progression [22–24]. Discussion NF-κB is commonly activated to protect against organ- PTL is a natural molecule that was originally isolated isms, but disorder in its activation is related to chronic from the shoots of feverfew (Tanacetum parthenium). inflammation [25]. PTL inhibited the activities of TNF- PTL exhibits excellent anti-inflammatory and antitumor α, TGF-β, and NF-κB in the present study. activities [16, 17]. The first written records of the anti- Our studies evaluated the role of PTL in PF. The inflammatory effect of PTL were provided in 1597 in results demonstrated that PTL repressed BLM-induced Li et al. Respiratory Research (2018) 19:111 Page 10 of 12 Fig. 8 Mechanism research on the PTL effects. a-b GO analysis of PTL in affecting biological processes. c A model showing the role of PTL in BLM-induced EMT pulmonary fibrosis in mice. PTL inhibited EMT of Our results demonstrated that NF-κB and Snail expres- AECs, which upregulate epithelial marker expression sion levels and activities decreased following PTL treat- and downregulate the expression of mesenchymal ment. Therefore, PTL may inhibit the NF-κB signaling markers. We evaluated the influence of PTL on AP-1, pathway and exhibit proinflammatory effects during PF. NF-κB, STAT-3, Snail, Slug and c-Myc expression. PTL FBs are important in the structural formation process only downregulated NF-κB and Snail. The NF-κB path- and maintaining the function of pulmonary tissues [26]. way regulates Snail expression via transcriptional and The cross talk of FBs and AECs promotes fibrosis. FBs post-translational mechanisms. NF-κB binds to the hu- proliferate continuously as a result of multiple factors, man Snail promoter and increases Snail transcription. such as the stimulating action of cytokines [27–29]. Li et al. Respiratory Research (2018) 19:111 Page 11 of 12 Therefore, an effective approach to inhibit FB proliferation Availability of data and materials Data available on request from the authors. in PF should be urgently identified [30]. The present re- sults demonstrated that PTL inhibited the proliferation Authors’ contributions and migration of primary pulmonary fibroblasts and (I) Conception and design: HL, HZ; (II) Administrative support: HL, JG; (III) Collection and assembly of data: XL, XT, JY and YQ; (V) Data analysis and HFL1 cells in a dose-dependent manner. interpretation: XL, XT; (VI) Manuscript writing: All authors; (VII) Final approval EMT is an indispensable step in numerous diseases, of manuscript: All authors. All authors read and approved the final manuscript. and it induces cell changes involved in pathological pro- Ethics approval and consent to participate cesses, such as fibrosis [31, 32]. EMT plays a pivotal role The study was approved by the Institutional Ethical Committee of Tianjin in the development of PF. Lung epithelial cells are a fre- International Joint Academy of Biomedicine (No. SYXK 2017–0003). quent target of injury, a driver of normal repair, and a Competing interests key element in the pathobiology of fibrotic lung diseases. The authors declare that they have no competing interests. One important aspect of epithelial cells is their capacity to respond to microenvironmental cues by undergoing Publisher’sNote EMT. EMT regulates a series of critical signaling elements Springer Nature remains neutral with regard to jurisdictional claims in that produce proinflammatory signals and cause cell in- published maps and institutional affiliations. jury. EMT is not the widespread conversion of epithelial Received: 16 January 2018 Accepted: 6 May 2018 cells to FBs, but it is a graded response whereby epithelial cells reversibly acquire mesenchymal features and enhance References the capacity for mesenchymal cross talk [33]. Repeated in- 1. Foster MW, et al. 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Bartram U, Speer CP. The role of transforming growth factor beta in lung development and disease. Chest. 2004;125(2):754. 25. Doyle SL, et al. Nuclear factor κB2 p52 protein has a role in antiviral immunity through IκB kinase -dependent induction of Sp1 protein and interleukin 15. J Biol Chem. 2013;288(35):25066. 26. Sivakumar P, et al. Into the matrix: targeting fibroblasts in pulmonary fibrosis. Curr Opin Pulm Med. 2012;18(5):462–9. 27. Piek E, Heldin C, Dijke PT. Specificity, diversity, and regulation in TGF-β superfamily signaling. FASEB J. 1999;13(15):2105. 28. Nakamuta M, et al. Effects of fibril- or fixed-collagen on matrix metalloproteinase-1and tissue inhibitor of matrix metalloproteinase-1 production in the human hepatocyte cell line HLE. World J Gastroenterol. 2005;11(15):2264–8. 29. Pardo A, et al. Role of matrix metalloproteinases in the pathogenesis of idiopathic pulmonary fibrosis. Respir Res. 2016;17(1):1–10. 30. Darby IA, et al. 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Respiratory ResearchSpringer Journals

Published: Jun 5, 2018

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