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Thymol alleviates lipopolysaccharide-stimulated inflammatory response via downregulation of RhoA-mediated NF-κB signalling pathway in human peritoneal mesothelial cells
Author’s Accepted Manuscript Thymol alleviates lipopolysaccharide-stimulated inflammatory response via downregulation of RhoA-mediated NF-κB signalling pathway in human peritoneal mesothelial cells Qinglian Wang, Fajuan Cheng, Ying Xu, Jing Zhang, Jianjun Qi, Xiang Liu, Rong Wang www.elsevier.com/locate/ejphar
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S0014-2999(18)30322-4 https://doi.org/10.1016/j.ejphar.2018.06.003 EJP71833
To appear in: European Journal of Pharmacology Received date: 21 February 2018 Revised date: 5 June 2018 Accepted date: 5 June 2018 Cite this article as: Qinglian Wang, Fajuan Cheng, Ying Xu, Jing Zhang, Jianjun Qi, Xiang Liu and Rong Wang, Thymol alleviates lipopolysaccharide-stimulated inflammatory response via downregulation of RhoA-mediated NF-κB signalling pathway in human peritoneal mesothelial cells, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Thymol alleviates lipopolysaccharide-stimulated inflammatory response via downregulation of RhoA-mediated NF-κB signalling pathway in human peritoneal mesothelial cells Qinglian Wang1, Fajuan Cheng1, Ying Xu1, Jing Zhang1, Jianjun Qi2, Xiang Liu1*, Rong Wang1* 1
Department of Nephrology, Shandong Provincial Hospital Affiliated to Shandong
University, Jinan, China 2
Department of Nephrology, Heze Municipal Hospital, Heze, China
[email protected] [email protected] *
Correspondence to: Xiang Liu & Rong Wang, Department of Nephrology,
Shandong Provincial Hospital Affiliated to Shandong University, 324 Jingwu Street, Jinan 250021, P.R. China. Tel.: +86-15168889155; +86-13791082272
Abstract Thymol is one of the most important dietary constituents in the thyme species and has been shown to possess anti-inflammatory properties both in vivo and in vitro. We investigated the protective effects of thymol on the lipopolysaccharide (LPS)-induced inflammatory responses in the human peritoneal mesothelial cell line (HMrSV5) to clarify the potential mechanism. HMrSV5 cells were stimulated with LPS in the presence or absence of thymol. Our results showed that thymol markedly suppressed the production of cytokines such as tumour necrosis factor α (TNF-α), interleukin 1
(IL)-6, monocyte chemoattractant protein 1 (MCP-1) and α-smooth muscle actin (α-SMA) in a dose-dependent manner. Western blot analysis indicated that RhoA and ROCK activation; Toll-like receptor 4 (TLR4) expression; and p65, IKK and IκBα phosphorylation were also inhibited by thymol. Moreover, siRNA knockdown of RhoA suppressed the expression of pro-inflammatory cytokines and phosphorylation of NF-κB p65 and IκBα proteins in LPS-stimulated HMrSV5 cells, but did not affect TLR4 expression. In conclusion, thymol inhibits LPS-induced inflammation in HMrSV5 cells by suppressing TLR4-mediated RhoA-dependent NF-κB signalling pathway. Our study suggests that thymol may be a promising therapeutic agent against peritonitis.
Key words
thymol; lipopolysaccharide (LPS); peritoneal mesothelial cells; RhoA/ROCK pathway; nuclear factor-kappa B (NF-κB)
1. INTRODUCTION Peritoneal dialysis is an important replacement therapeutic option for patients with end-stage renal disease (ESRD). Nevertheless, continuous exposure to bio-incompatible peritoneal dialysis fluid and episodes of peritonitis or hemoperitoneum induces acute and chronic inflammation that damages the peritoneal 2
membrane, which progressively undergoes fibrosis and eventually leads to ultrafiltration failure (Krediet, 1999; Krediet et al., 2000). Although the incidence rate of peritonitis has significantly decreased, it remains a major complication in peritoneal dialysis patients, leading to mesothelial cells damage and fibrosis (Campbell et al., 2015). In peritonitis, mesothelial cells are activated by pro-inflammatory cytokines and play critical roles in amplifying peritoneal inflammation by releasing many pro-inflammatory cytokines and mediators (Yung et al., 2006). Gram-negative bacteria are the most common pathogenic bacteria in peritoneal dialysis-related peritonitis. LPS derived from the Gram-negative bacteria is a major pathogenic factor that induces inflammation (Xiang et al., 2016). Thymol (2-isopropyl-5-methylphenol; Fig. 1), is one of the most important dietary constituents in the thyme species. It has been used in traditional medicine for many centuries because it possesses antioxidant, anti-inflammatory, antibacterial, antifungal, and antiseptic properties that are pharmacologically relevant (Nagoor Meeran et al., 2017). In vivo and in vitro studies related to LPS-induced inflammation show that thymol exerts anti-inflammatory action by suppressing pro-inflammatory signalling pathways and transcription factors such as NF-κB, p38 mitogen-activated protein kinases (MAPK), activator protein-1 (AP-1) and signal transducer and activator of transcription 3 (STAT-3) (Gholijani et al., 2016; Liang et al., 2014; Wan et al., 2018; Wu et al., 2017; Yao et al., 2017). However, the mechanisms underlying these protective effects of thymol are not fully understood. The Rho family of GTPases are implicated in a wide variety of cellular functions 3
including cell adhesion, cell motility and migration, growth control and cytokinesis, and the best known member of this family is RhoA (Etienne-Manneville and Hall, 2002; Wettschureck and Offermanns, 2002). The RhoA/ROCK signalling pathway is involved in LPS-induced inflammation and NF-κB activation (Kim et al., 2014; Meyer-Schwesinger et al., 2009; Montaner et al., 1998; Perona et al., 1997), which suggests a potential role in LPS-induced peritonitis. Inhibition of this pathway demonstrates strong anti-inflammatory effects. Rho kinase inhibition is effective against reperfusion injury of the liver (Kuroda et al., 2015; Shiotani et al., 2004), heart (Bao et al., 2004; Tratsiakovich et al., 2017), brain (Huang et al., 2017) and the lung (Ohata et al., 2017). It is also effective against acute injuries to the lung (Chen et al., 2015) and the kidneys (Meyer-Schwesinger et al., 2009; Wang et al., 2017). Partly, the anti-inflammatory effects are mediated by suppressing NF-κB activation (Chen et al., 2015; Huang et al., 2017; Meyer-Schwesinger et al., 2009; Segain et al., 2003). Although both thymol and the RhoA/ROCK pathway can inhibit NF-κB activation, it is unclear whether the RhoA/ROCK pathway contributes to the anti-inflammatory effect of thymol. In the present study, we aimed to clarify the potential anti-inflammatory effects of thymol in the LPS-induced inflammatory response in human peritoneal mesothelial cells and investigated the underlying molecular mechanisms.
2. MATERIALS AND METHODS 2.1 Chemicals and Reagents 4
A 20 mg/ml stock solution of Thymol (endotoxin-free, purity >99.9%; Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China), was prepared in ethanol and stored at -20˚C. The dilutions for working thymol solution did not exceed 0.1% ethanol in the medium. LPS (Escherichia coli 055:B5) purchased from Sigma Chemical Co. (St. Louis, MO, USA) was dissolved in PBS to obtain a 5 mg/ml stock solution and stored at -20˚C. Dulbecco's modified Eagle's medium (DMEM F12/1:1), fetal bovine serum (FBS), and trypsin/EDTA were purchased from HyClone (Logan, UT, USA). WST-8 cell proliferation and cytotoxicity assay kit was purchased from Dojindo (Kumamoto, Japan). RhoA Pull-down Activation Assay Biochem Kit (BK036) was purchased from Cytoskeleton, Inc. (Denver, USA). ELISA kits to assay human cell supernatant tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6) were purchased from R&D systems (CA, USA). Lipofectamine 2000 reagent was purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies against p-IκB kinase (IKK), p-IκBα and NF-κB-P65 were purchased from Cell Signaling Technology (CST, Beverly, USA). Antibodies against Rho-kinase, α-smooth muscle actin (α-SMA), monocyte chemoattractant protein-1 (MCP-1) and Toll-Like Receptor 4 (TLR-4) were purchased from Abcam (Abcam, UK). Other antibodies were obtained from Proteintech Biotechnology (Wuhan, China). β-actin and horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse antibodies were provided by Beyotime Biotechnology (Shanghai, China). All other chemicals were of reagent grade and endotoxin free.
5
2.2 Cell culture The human peritoneal mesothelial cell line (HMrSV5) was obtained from Professor Xueqing Yu of the First Affiliated Hospital of Sun Yat-sen University. It was established after infection of a fully characterized primary culture of human peritoneal mesothelial cells with an amphotropic recombinant retrovirus that encodes SV40 large-T Ag under the control of the Moloney virus long terminal repeat (LTR) and the gene conferring resistance to the G418 antibiotic. The HMrSV5 cells were cultured in DMEM F12/1:1 medium supplemented with 15% fetal bovine serum, streptomycin (100 μg/ml), and penicillin (100 units/ml) at 37°C in a 95% air/5% CO2 humidified atmosphere. When the cells reached confluence, they were rendered quiescent by incubating with 1% serum containing DMEM medium. The quiescent cells were then treated with LPS (3 μg/ml) or LPS plus thymol (10, 20, 40 μg/ml) for various times as indicated. Thymol was added 60 mins before addition of LPS. Detection of TNF-α, IL-6, MCP-1 and α-SMA was carried out after cells were incubated with LPS for 12 h, whereas activity of the RhoA/ROCK and TLR4/NF-kB signalling pathways was detected in cells incubated with LPS for 0.5 h.
2.3 RNA interference Small interference RNA (siRNA) targeting RhoA (si-RhoA: 5’-UCAAGCAUUUCUGUCCCAA-3’) and the negative control RNA probe (NC: 5’-AGUUCAACGACCAGUAGUC-3’) were designed and purchased from Takara (Dalian, China). HMrSV5 cells were seeded onto 6-well plates. When reached 6
approximately 60-70% confluency, they were transfected with Opti-MEM medium containing siRhoA/NC and Lipofectamine™ 2000 for 6 h. The medium was changed and the cells were grown further with fresh DMEM/F-12 medium for another 24 h. The transfected HMrSV5 cells were then treated with LPS (3 μg/ml) for 0.5 h or 12 h. The non-transfected cells grown in normal culture medium served as controls. The knockdown efficiency of si-RhoA in HMrSV5 cells was detected.
2.4 WST-8 assays The cell viability of all the control and experimental groups was evaluated (control, LPS, LPS plus Thymol (10, 20, 40 μg/ml), si-RhoA plus LPS, and NC plus LPS) using the WST-8 cell proliferation and cytotoxicity assay kit. Briefly, siRhoA-, NC- and non-transfected HMrSV5 cells were seeded in a 96-well plate and treated with LPS and thymol as described previously in 90 μl of complete medium for 24 h. Subsequently, 10 μL WST-8 solution was added to each well and incubated for an additional 2 h at 37˚C. The absorbance was read at 450 nm using a microplate reader. Cell viability of the experimental groups was normalized against untreated HMrSV5 cells.
2.5 RhoA pull-down assay RhoA pull-down assay was performed using a kit according to the manufacturer's instructions. Briefly, cell lysates were harvested rapidly on ice and centrifuged at 15,000 rpm for 5 min at 4°C. A total of 20 μl of the lysate was saved for protein 7
quantitation, and equivalent amounts of lysate protein (500 μg total protein) were incubated with a pre-determined amount of rhotekin-RBD beads at 4°C for 60 min with gentle rocking and then centrifuged at 15,000 rpm for 60 s at 4°C. The beads were washed and boiled for 2 min in 20 μl of 2× Laemmli sample buffer. The supernatant was resolved with 12% SDS-PAGE. Membranes were probed with anti-RhoA antibody (1 μg/ml; provided in the kit) and an HRP-conjugated secondary antibody. Protein bands were imaged using the ECL system
2.6 Western blot analysis Nuclear and cytoplasmic protein extracts from the same experiment were prepared using the kit according to manufacturer’s instructions (Beyotime Biotechnology, Shanghai, China) to detect nuclear and cytoplasmic NF-κB levels. Moreover, total protein extracted by RIPA was used to detect the other molecules. Protease and phosphatase inhibitor cocktail for general use (50×, Beyotime Biotechnology, Shanghai, China) were added in lysis buffer when protein extraction. Proteins (10 μg) were separated by 10-12% SDS-PAGE, and the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% skim milk for 1 h at room temperature and then incubated at 4°C overnight with the following primary antibodies: rabbit anti-Rho-kinase (1:1000; Abcam, UK), rabbit anti-p-IKK (1:1000; CST), rabbit anti-NF-κB-P65 (1:1000; CST), rabbit anti-p-IκBα (1:1000; CST), rabbit anti-α-SMA (1:300; Abcam, UK), rabbit anti-Histone 3 (1:1000; Proteintech), mouse anti-MCP-1 (1:1000; Abcam, 8
UK), rabbit anti-TLR4 (1:1000; Abcam, UK) and rabbit anti-GAPDH (1:1000; BOSTER, China). Then, the membranes were washed with TBST buffer and incubated with HRP-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibodies (1:5,000; Santa Cruz Biotechnology, Inc. Dallas, TX, USA) for 1 h at room temperature. Protein bands were detected using an ECL system and a Bio-Rad electrophoresis image analyser (Bio-Rad, Hercules, CA, USA). Histone3 (H3) and GAPDH were used as internal controls for nuclear and cytoplasmic proteins, respectively. Also GAPDH were used as internal controls for total protein.
2.7 Immunofluorescence HMrSV5 cells grown on glass coverslips were pretreated according to the experimental conditions. Then, they were fixed with 4% paraformaldehyde for 20 mins. After incubated with goat serum working solution to block nonspecific binding for 2 h in 37℃, cells were incubated with NF-κB P65 primary antibody at 4℃ overnight, Then, the cells were stained with DyLight549-conjugated secondary antibodies for 1 h at room temperature. The cells were incubated with 4, 6-diamidino-2-phenylindole (DAPI) for 5 min to stain the nuclei. Randomly selected fields were imaged using a fluorescent microscope.
2.8 Enzyme Linked Immunosorbent Assay (ELISA) The HMrSV5 cells were seeded in 6-well plates (4×105 cells/well) and pretreated with or without various concentrations of thymol (10, 20, 40 μg/mL) for 1 h. Then, 9
the cells were stimulated with LPS (3 μg/mL) for 12 h. The supernatant was collected and centrifuged at 1500 rpm for 3 min to measure the levels of TNF-α, IL-6 using ELISA kits according to the manufacturer’s instructions. The absorbance was read at 450 nm using a microplate reader. Cytokines TNF-α and IL-6 were calculated according to a standard curve set up by absorbance value.
2.9 Statistical analysis Data are presented as the mean ± S.D. unless stated otherwise. One-way ANOVA was used to determine the statistical difference significant differences between groups. The Dunnett’s test was used to perform multiple comparisons between the groups. A two-tailed P < 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS 20.0 software (SPSS Inc. Chicago, Illinois, USA).
3. RESULTS 3.1 LPS treatment activates RhoA in HMrSV5 cells RhoA has been identified as a mediator of pro-inflammatory responses and participates in LPS-induced inflammation. To assess RhoA activation in LPS-treated HMrSV5 cells, a RhoA pull-down activation assay was performed. First, we applied a time course analysis of HMrSV5 cells treated with 3 μg/ml LPS, and the result showed that RhoA activity was higher at 0.5 h compared with the control group (Fig. 2A). Subsequently, HMrSV5 cells treated for 0.5 h with different concentrations of 10
LPS showed that 3 μg/ml LPS stimulated maximal RhoA activity (Fig. 2B).
3.2 LPS treatment activates NF-κB in HMrSV5 cells Transcription factor NF-κB is a critical regulator of gene expression during LPS-induced inflammation. Hence we performed western blot analysis to analyse the activation of NF-κB in LPS-treated HMrSV5 cells. First, we set a short time course with a concentration of 3 μg/ml LPS. As expected, NF-κB p65 in the cytoplasm was significantly decreased after treatment for 0.5 h with LPS compared with the control group (Fig. 3A1). In addition, NF-κB p65 in the nucleus was significantly increased after treatment with LPS for 0.5 h (Fig. 3A2). That means there was a rapid translocation of activated NF-κB from the cytoplasm to the nucleus as quickly as 0.5 h in HMrSV5 cells stimulated by LPS. Second, concentration course experiments using this stimulation duration showed that 3 μg/ml LPS significantly activated NF-κB compared with the control group (Fig. 3B). Therefore, 3 μg/ml was the proper concentration of LPS to use in subsequent studies.
3.3 Effect of thymol and si-RNA on HMrSV5 cell viability To assess the potential cytotoxicity of thymol and si-RNA on HMrSV5 cells, we used the WST-8 assay. These results showed that cell viability was not affected by thymol or si-RhoA administration plus LPS. (Fig. 4)
3.4 Effects of thymol on the cytokines level in LPS-treated HMrSV5 cells 11
LPS exposure induces a strong increase in cytokine expression via the TLR-4-NF-κB signalling pathways. Therefore, we investigated the LPS-induced cytokine response to observe the role of thymol. The expression of the cytokines TNF-α, IL-6, MCP-1, and α-SMA was detected by ELISA and western blot. The results indicated that LPS treatment significantly increased the expression of TNF-α, IL-6, MCP-1, and α-SMA (Fig. 5A1 and 5A2). Moreover, pre-treatment with thymol (10, 20, and 40 μg/mL) for 1 h prior to LPS stimulation decreased the levels of cytokines in a dose-dependent manner (Fig. 5A). This demonstrates that thymol inhibits the induction of pro-inflammatory cytokines by LPS.
3.5 Effect of thymol on TLR4 mediated NF-κB pathway activation To clarify the anti-inflammatory molecular mechanism of thymol, we assessed the activation of TLR4/NF-κB pathway. Since TLR4 plays an important role in the LPS-induced inflammatory response, we analysed its expression by western blot. The result indicated that the expression of TLR4 was remarkably increased after LPS exposure; however, it was dose-dependently downregulated by thymol administration (Fig. 5B).
Moreover, effect of thymol on the NF-κB signalling pathway was also assessed. Compared with the LPS group, the phosphorylation of IKK and IκBα proteins in the cytoplasm (Fig. 5B) and nuclear NF-κB p65 levels (Fig. 5C) was significantly inhibited by thymol in a dose-dependent manner. Conversely, NF-κB p65 levels in the cytoplasm increased in an opposite trend. To confirm these results, 12
immunofluorescence assays were performed to determine if translocation of NF-κB p65 from the cytoplasm to the nucleus was suppressed. As shown in Fig. 7, the expression of NF-κB-p65 was significantly decreased in the nucleus by thymol administration.
3.6 Effect of thymol on RhoA/ROCK pathway activation The RhoA/ROCK signalling pathway is reported involving in the LPS-induced NF-κB activation during inflammation. Furthermore, we analysed the effect of thymol on the activation of this pathway in LPS-treated HMrSV5 cells and observed that thymol inhibited the expression of RhoA and ROCK in a dose-dependent manner (Fig. 5D).
3.7 Effect of si-RhoA on cytokines levels Previous results demonstrate that thymol inhibits LPS-induced inflammatory response in HMrSV5 cells partly by suppressing TLR4/NF-κB and RhoA/ROCK signalling pathways. To further evaluate if the anti-inflammatory effect of thymol was mediated by inhibiting the RhoA pathway, we knocked down RhoA in HMrSV5 cells using siRNA against RhoA (si-RhoA). Subsequently, we observed that si-RhoA significantly decreased RhoA expression (Fig. 6A). This correlates with downregulation of cytokines (Fig. 6B) upon LPS treatment, suggesting that inhibition of the RhoA/ROCK signalling pathway is central to the anti-inflammatory mechanism of thymol in LPS-treated HMrSV5 cells. 13
3.8 Effect of si-RhoA on TLR4 mediated NF-κB pathway activation We further evaluated the effects of si-RhoA on the TLR4/NF-κB pathway in LPS-treated HMrSV5 cells. The results show that the expression of TLR4 was not affected by si-RhoA compared to the LPS group (Fig. 6C). However, si-RhoA markedly decreased the phosphorylation of IκBα in the cytoplasm (Fig. 6C). It also decreased the levels of NF-κB-p65 in the nucleus (Fig. 6D), but increased the levels of NF-κB-p65 in the cytoplasm (Fig. 6C). As shown in Fig. 7, immunofluorescence assay confirmed that nuclear translocation of NF-κB p65 was significantly decreased in siRhoA-transfected HMrSV5 cells.
4. DISCUSSION Peritoneal mesothelial cells are key regulators of peritoneal homeostasis, and participate in peritoneal immune and inflammatory responses by protecting against the invading microbes (Aroeira et al., 2007; Yung and Chan, 2009). Thymol is a colourless crystalline monoterpene phenol that has been used in traditional medicine and possesses anti-inflammatory properties (Nagoor Meeran et al., 2017). In this study, we demonstrate that thymol exerts beneficial effects in LPS-induced inflammatory responses of HMrSV5 cells by modulating the NF-κB signalling pathway via RhoA. Peritoneal injury activates not only macrophages and neutrophils but also mesothelial cells, which are the main sources of pro-inflammatory cytokines and 14
fibrotic mediators in response to external stimuli (Bertoli et al., 1999; Devuyst et al., 2010; Yung et al., 2006). During peritonitis, LPS activates mesothelial cells through the Toll-like receptors (TLRs), resulting in the activation of the NF-kB pathway and subsequent secretion of numerous inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8 and MCP-1(Kato et al., 2004; Topley et al., 1993; Yung et al., 2006). TNF-α and IL-1β are two key components of the innate immune response (Lahouassa et al., 2007; Topley et al., 1993). IL-6 is a key player in modulating inflammation and a major effector of the acute phase reaction; its synthesis is induced by TNF-α and IL-1β in peritoneal mesothelial cells (Bellingan et al., 1996). In combination with the soluble IL-6 receptor, IL-6 promotes the synthesis and secretion of MCP-1 (Hurst et al., 2001). These pro-inflammatory factors play different functional roles in different phases of the inflammation response (Devuyst et al., 2010). In this study, we found that the expression of TNF-α, IL-6 and MCP-1 cytokines was markedly increased after LPS stimulation. However, pre-treatment with thymol inhibited the production of these cytokines in a dose-dependent manner. These results are in accordance with the findings in RAW 264.7 cells (Wu et al., 2017) and mouse mammary epithelial cells (Liang et al., 2014). We also found α-SMA increased markedly after LPS stimulation. It is well known that peritoneal inflammation could induce epithelial-mesenchymal transition (EMT) of mesothelial cells, then mesothelial cells undergo a transition from an epithelial phenotype to a mesenchymal phenotype (Aroeira et al., 2007). α-SMA is a marker of mesenchymal cells such as myofibroblasts. EMT is a complicated process and we 15
only detected the expression of α-SMA in this study. Our results suggest that thymol inhibited the inflammatory response induced by LPS, also might prevent EMT of mesothelial cells. Therefore, further studies are necessary to verify if thymol inhibits inflammation-induced peritoneal fibrosis. NF-κB is a transcription factor that regulates immune cell functions and inflammatory responses. When activated, NF-κB translocates to the nucleus and mediates the expression of target genes (Hoffmann and Baltimore, 2006; Srivastava and Ramana, 2009). TLR4 is the member of TLR family that is recognized and activated by LPS, and plays an important role in innate immunity (Kawai and Akira, 2010; O'Neill et al., 2013). TLR4/ NF-κB signalling pathway plays a major role in the induction of inflammatory cytokines by LPS (Kuzmich et al., 2017). In recent studies, TLR4 and NF-κB contribute to the anti-inflammatory mechanisms of thymol (Gholijani et al., 2016; Khosravi and Erle, 2016; Liang et al., 2014; Wan et al., 2018; Wu et al., 2017; Yao et al., 2017). In the present study, we demonstrated that thymol inhibits the upregulation of TLR4 and the phosphorylation of IKK, IκBα and p65 in a dose-dependent manner in LPS-stimulated HMrSV5 cells. This suggests that the anti-inflammatory role of thymol is mediated by inhibition of the TLR4/ NF-κB signalling pathway. Furthermore, previous studies have shown that the activation of NF-κB is regulated by the RhoA/ROCK signalling pathway during LPS-induced inflammation (Chen et al., 2015; Meyer-Schwesinger et al., 2009; Qin et al., 2014). ROCK inhibition markedly reduces the production of pro-inflammatory cytokines and 16
chemokines. Several studies have demonstrated that expression of TNF-α, IL-1β, IL-6 and IL-8 cytokines is reduced by ROCK inhibitor. (Chen et al., 2015; Meyer-Schwesinger et al., 2009; Segain et al., 2003). Therefore, we sought to investigate if thymol inhibits the activation of TLR4/NF-κB signalling via RhoA signalling pathway. Our study demonstrates that silencing RhoA decreases production of inflammatory cytokines and the phosphorylation of p65 and IκBα proteins in LPS-induced HMrSV5 cells. However, the upregulation of TLR4 is not altered. These results suggest that the transmembrane receptor TLR4 is upstream of RhoA and NF-κB locates downstream of RhoA. Therefore, we postulate that LPS activate the TLR4 receptor, which leads to the activation of NF-κB via RhoA in HMrSV5 cells. In conclusion, our study shows that thymol inhibits the inflammatory response induced by LPS in HMrSV5 cells. Therefore, TLR4-mediated RhoA-dependent NF-κB signalling pathway is relevant to the anti-inflammatory effects of thymol. Our findings suggest that thymol is a potential therapeutic agent against peritoneal dialysis-related peritonitis.
CONFLICTS OF INTERESTS Authors declare that there are no conflicts of interest.
ACKNOWLEDGMENTS This work was supported by the grant to Xiang Liu from Projects of Medical and Health Technology Development Program in Shandong, China (Grant No. 2013WSB01025) and grants to Fajuan Cheng from Shandong Provincial Natural Science Foundation (Grant No. ZR2016HB09) and National Natural Science 17
Foundation (Grant No. 81601421). We are grateful to Professor Xueqing Yu of the First Affiliated Hospital of Sun Yat-sen University for providing the human peritoneal membrane cell line (HMrSV5).
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by attenuation of NF-kappaB p65 signaling. Am J Physiol Renal Physiol 296, F1088-1099. Montaner, S., Perona, R., Saniger, L., Lacal, J.C., 1998. Multiple signalling pathways lead to the activation of the nuclear factor kappaB by the Rho family of GTPases. J Biol Chem 273, 12779-12785. Nagoor Meeran, M.F., Javed, H., Al Taee, H., Azimullah, S., Ojha, S.K., 2017. Pharmacological Properties and Molecular Mechanisms of Thymol: Prospects for Its Therapeutic Potential and Pharmaceutical Development. Front Pharmacol 8, 380. O'Neill, L.A., Golenbock, D., Bowie, A.G., 2013. The history of Toll-like receptors - redefining innate immunity. Nat Rev Immunol 13, 453-460. Ohata, K., Chen-Yoshikawa, T.F., Menju, T., Miyamoto, E., Tanaka, S., Takahashi, M., Motoyama, H., Hijiya, K., Aoyama, A., Date, H., 2017. Protective Effect of Inhaled Rho-Kinase Inhibitor on Lung Ischemia-Reperfusion Injury. Ann Thorac Surg 103, 476-483. Perona, R., Montaner, S., Saniger, L., Sanchez-Perez, I., Bravo, R., Lacal, J.C., 1997. Activation of the nuclear factor-kappaB by Rho, CDC42, and Rac-1 proteins. Genes Dev 11, 463-475. Qin, L., Qin, S., Zhang, Y., Zhang, C., Ma, H., Li, N., Liu, L., Wang, X., Wu, R., 2014. p120 modulates LPS-induced NF-kappaB activation partially through RhoA in bronchial epithelial cells. Biomed Res Int 2014, 932340. Segain, J.P., Raingeard de la Bletiere, D., Sauzeau, V., Bourreille, A., Hilaret, G., Cario-Toumaniantz, C., Pacaud, P., Galmiche, J.P., Loirand, G., 2003. Rho kinase blockade prevents inflammation via nuclear factor kappa B inhibition: evidence in Crohn's disease and experimental colitis. Gastroenterology 124, 1180-1187. Shiotani, S., Shimada, M., Suehiro, T., Soejima, Y., Yosizumi, T., Shimokawa, H., Maehara, Y., 2004. Involvement of Rho-kinase in cold ischemia-reperfusion injury after liver transplantation in rats. 21
Transplantation 78, 375-382. Srivastava, S.K., Ramana, K.V., 2009. Focus on molecules: nuclear factor-kappaB. Exp Eye Res 88, 2-3. Topley, N., Jorres, A., Luttmann, W., Petersen, M.M., Lang, M.J., Thierauch, K.H., Muller, C., Coles, G.A., Davies, M., Williams, J.D., 1993. Human peritoneal mesothelial cells synthesize interleukin-6: induction by IL-1 beta and TNF alpha. Kidney Int 43, 226-233. Tratsiakovich, Y., Kiss, A., Gonon, A.T., Yang, J., Sjoquist, P.O., Pernow, J., 2017. Inhibition of Rho kinase protects from ischaemia-reperfusion injury via regulation of arginase activity and nitric oxide synthase in type 1 diabetes. Diab Vasc Dis Res 14, 236-245. Wan, L., Meng, D., Wang, H., Wan, S., Jiang, S., Huang, S., Wei, L., Yu, P., 2018. Preventive and Therapeutic Effects of Thymol in a Lipopolysaccharide-Induced Acute Lung Injury Mice Model. Inflammation 41, 183-192. Wang, Y., Zhang, H., Yang, Z., Miao, D., Zhang, D., 2017. Rho Kinase Inhibitor, Fasudil, Attenuates Contrast-induced Acute Kidney Injury. Basic Clin Pharmacol Toxicol. Wettschureck, N., Offermanns, S., 2002. Rho/Rho-kinase mediated signaling in physiology and pathophysiology. J Mol Med (Berl) 80, 629-638. Wu, H., Jiang, K., Yin, N., Ma, X., Zhao, G., Qiu, C., Deng, G., 2017. Thymol mitigates lipopolysaccharide-induced endometritis by regulating the TLR4- and ROS-mediated NF-kappaB signaling pathways. Oncotarget 8, 20042-20055. Xiang, N.L., Liu, J., Liao, Y.J., Huang, Y.W., Wu, Z., Bai, Z.Q., Lin, X., Zhang, J.H., 2016. Abrogating ClC-3 Inhibits LPS-induced Inflammation via Blocking the TLR4/NF-kappaB Pathway. Sci Rep 6, 27583. Yao, L., Hou, G., Wang, L., Zuo, X.S., Liu, Z., 2017. Protective effects of thymol on LPS-induced acute lung injury in mice. Microb Pathog 116, 8-12. 22
Yung, S., Chan, T.M., 2009. Intrinsic cells: mesothelial cells -- central players in regulating inflammation and resolution. Perit Dial Int 29 Suppl 2, S21-27. Yung, S., Li, F.K., Chan, T.M., 2006. Peritoneal mesothelial cell culture and biology. Perit Dial Int 26, 162-173.
Fig. 1 Chemical structure of thymol. Fig. 2 LPS treatment activates RhoA in HMrSV5 cells. A) RhoA pull-down activity assay in HMrSV5 cells treated with 3 μg/mL LPS over time. B) Increasing concentration course of RhoA activation in HMrSV5 cells treated with LPS for 0.5 h. (Data are mean ± S.D.; *P < 0.05 vs control, **P < 0.01 vs control, n = 3). Fig. 3 LPS treatment activates NF-κB pathway in HMrSV5 cells. A) Detection of cytoplasmic (A1) and nucleus (A2) NF-κB P65 after treatment with 3 μg/ml LPS over time. B) Detection of cytoplasmic (B1) and nuclear (B2) NF-κB P65 after exposure to increasing concentrations of LPS for 0.5 h. (Data are mean ± S.D.; *P < 0.05 vs control, n = 3). Fig. 4 Cell viability assays. The HMrSV5 cells were cultured with different concentrations of thymol or si-RhoA or si-NC in the presence of 3 μg/mL LPS for 24 h. WST-8 assay shows that the viability of all the experimental groups was similar. The values are presented as the mean ± S.D. (n = 4). Fig. 5 Thymol suppresses cytokines expression TLR4/NF-κB signalling and RhoA pathway activation. A) IL-6 and TNF-α levels in the supernatants of 23
LPS-stimulated HMrSV5 cells were detected by ELISA. α-SMA and MCP-1 were detected by western blot. B) TLR-4 protein expression and the phosphorylation of the NF-κB p65, IκBα and IKK in cytoplasm were detected by western blot, while GAPDH served as an internal control. C) Nuclear NF-κB P65 protein expression was detected by western blot, while H3 served as internal control. D) RhoA-GTP was detected by pull down assay, and ROCK was detected by western blot. con indicates the normal control group; LPS indicates LPS-stimulated group; 10, 20, and 40 refer to LPS plus 10 μg/ml, 20 μg/ml or 40 μg/ml thymol. (Data are mean ± S.D.; *P < 0.05 vs control, # P < 0.05 vs LPS. n = 3). Fig. 6 Effects of RhoA silencing on cytokine expression and NF-κB pathway activation. A) Knockdown efficiency of si-RhoA was detected by western blot. (Data are mean ± S.D.; *P < 0.05 vs si-NC, n=3) B) IL-6, TNF-α and MCP-1, α-SMA levels were respectively detected by ELISA or western blot. C) TLR-4 protein expression and the phosphorylation of the NF-κB p65 and IκBα in cytoplasm were detected by western blot. D) Nuclear NF-κB P65 protein expression was detected by western blot, while H3 is used as the internal control. con indicated the normal control group. LPS indicates the LPS-stimulated group. si-RhoA indicates LPS plus si-RhoA group and NC indicates si-NC plus LPS group. (Data are mean ± S.D.; *P < 0.05 vs control, # P < 0.05 vs LPS. n = 3). Fig. 7 Effects of thymol and RhoA silencing on NF-κB p65 translocation into the nucleus using immunofluorescence assays. Translocation of NF-κB p65 into the nucleus was obviously suppressed by thymol and si-RhoA. 24
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S0014-2999(18)30322-4 https://doi.org/10.1016/j.ejphar.2018.06.003 EJP71833
To appear in: European Journal of Pharmacology Received date: 21 February 2018 Revised date: 5 June 2018 Accepted date: 5 June 2018 Cite this article as: Qinglian Wang, Fajuan Cheng, Ying Xu, Jing Zhang, Jianjun Qi, Xiang Liu and Rong Wang, Thymol alleviates lipopolysaccharide-stimulated inflammatory response via downregulation of RhoA-mediated NF-κB signalling pathway in human peritoneal mesothelial cells, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Thymol alleviates lipopolysaccharide-stimulated inflammatory response via downregulation of RhoA-mediated NF-κB signalling pathway in human peritoneal mesothelial cells Qinglian Wang1, Fajuan Cheng1, Ying Xu1, Jing Zhang1, Jianjun Qi2, Xiang Liu1*, Rong Wang1* 1
Department of Nephrology, Shandong Provincial Hospital Affiliated to Shandong
University, Jinan, China 2
Department of Nephrology, Heze Municipal Hospital, Heze, China
[email protected] [email protected] *
Correspondence to: Xiang Liu & Rong Wang, Department of Nephrology,
Shandong Provincial Hospital Affiliated to Shandong University, 324 Jingwu Street, Jinan 250021, P.R. China. Tel.: +86-15168889155; +86-13791082272
Abstract Thymol is one of the most important dietary constituents in the thyme species and has been shown to possess anti-inflammatory properties both in vivo and in vitro. We investigated the protective effects of thymol on the lipopolysaccharide (LPS)-induced inflammatory responses in the human peritoneal mesothelial cell line (HMrSV5) to clarify the potential mechanism. HMrSV5 cells were stimulated with LPS in the presence or absence of thymol. Our results showed that thymol markedly suppressed the production of cytokines such as tumour necrosis factor α (TNF-α), interleukin 1
(IL)-6, monocyte chemoattractant protein 1 (MCP-1) and α-smooth muscle actin (α-SMA) in a dose-dependent manner. Western blot analysis indicated that RhoA and ROCK activation; Toll-like receptor 4 (TLR4) expression; and p65, IKK and IκBα phosphorylation were also inhibited by thymol. Moreover, siRNA knockdown of RhoA suppressed the expression of pro-inflammatory cytokines and phosphorylation of NF-κB p65 and IκBα proteins in LPS-stimulated HMrSV5 cells, but did not affect TLR4 expression. In conclusion, thymol inhibits LPS-induced inflammation in HMrSV5 cells by suppressing TLR4-mediated RhoA-dependent NF-κB signalling pathway. Our study suggests that thymol may be a promising therapeutic agent against peritonitis.
Key words
thymol; lipopolysaccharide (LPS); peritoneal mesothelial cells; RhoA/ROCK pathway; nuclear factor-kappa B (NF-κB)
1. INTRODUCTION Peritoneal dialysis is an important replacement therapeutic option for patients with end-stage renal disease (ESRD). Nevertheless, continuous exposure to bio-incompatible peritoneal dialysis fluid and episodes of peritonitis or hemoperitoneum induces acute and chronic inflammation that damages the peritoneal 2
membrane, which progressively undergoes fibrosis and eventually leads to ultrafiltration failure (Krediet, 1999; Krediet et al., 2000). Although the incidence rate of peritonitis has significantly decreased, it remains a major complication in peritoneal dialysis patients, leading to mesothelial cells damage and fibrosis (Campbell et al., 2015). In peritonitis, mesothelial cells are activated by pro-inflammatory cytokines and play critical roles in amplifying peritoneal inflammation by releasing many pro-inflammatory cytokines and mediators (Yung et al., 2006). Gram-negative bacteria are the most common pathogenic bacteria in peritoneal dialysis-related peritonitis. LPS derived from the Gram-negative bacteria is a major pathogenic factor that induces inflammation (Xiang et al., 2016). Thymol (2-isopropyl-5-methylphenol; Fig. 1), is one of the most important dietary constituents in the thyme species. It has been used in traditional medicine for many centuries because it possesses antioxidant, anti-inflammatory, antibacterial, antifungal, and antiseptic properties that are pharmacologically relevant (Nagoor Meeran et al., 2017). In vivo and in vitro studies related to LPS-induced inflammation show that thymol exerts anti-inflammatory action by suppressing pro-inflammatory signalling pathways and transcription factors such as NF-κB, p38 mitogen-activated protein kinases (MAPK), activator protein-1 (AP-1) and signal transducer and activator of transcription 3 (STAT-3) (Gholijani et al., 2016; Liang et al., 2014; Wan et al., 2018; Wu et al., 2017; Yao et al., 2017). However, the mechanisms underlying these protective effects of thymol are not fully understood. The Rho family of GTPases are implicated in a wide variety of cellular functions 3
including cell adhesion, cell motility and migration, growth control and cytokinesis, and the best known member of this family is RhoA (Etienne-Manneville and Hall, 2002; Wettschureck and Offermanns, 2002). The RhoA/ROCK signalling pathway is involved in LPS-induced inflammation and NF-κB activation (Kim et al., 2014; Meyer-Schwesinger et al., 2009; Montaner et al., 1998; Perona et al., 1997), which suggests a potential role in LPS-induced peritonitis. Inhibition of this pathway demonstrates strong anti-inflammatory effects. Rho kinase inhibition is effective against reperfusion injury of the liver (Kuroda et al., 2015; Shiotani et al., 2004), heart (Bao et al., 2004; Tratsiakovich et al., 2017), brain (Huang et al., 2017) and the lung (Ohata et al., 2017). It is also effective against acute injuries to the lung (Chen et al., 2015) and the kidneys (Meyer-Schwesinger et al., 2009; Wang et al., 2017). Partly, the anti-inflammatory effects are mediated by suppressing NF-κB activation (Chen et al., 2015; Huang et al., 2017; Meyer-Schwesinger et al., 2009; Segain et al., 2003). Although both thymol and the RhoA/ROCK pathway can inhibit NF-κB activation, it is unclear whether the RhoA/ROCK pathway contributes to the anti-inflammatory effect of thymol. In the present study, we aimed to clarify the potential anti-inflammatory effects of thymol in the LPS-induced inflammatory response in human peritoneal mesothelial cells and investigated the underlying molecular mechanisms.
2. MATERIALS AND METHODS 2.1 Chemicals and Reagents 4
A 20 mg/ml stock solution of Thymol (endotoxin-free, purity >99.9%; Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China), was prepared in ethanol and stored at -20˚C. The dilutions for working thymol solution did not exceed 0.1% ethanol in the medium. LPS (Escherichia coli 055:B5) purchased from Sigma Chemical Co. (St. Louis, MO, USA) was dissolved in PBS to obtain a 5 mg/ml stock solution and stored at -20˚C. Dulbecco's modified Eagle's medium (DMEM F12/1:1), fetal bovine serum (FBS), and trypsin/EDTA were purchased from HyClone (Logan, UT, USA). WST-8 cell proliferation and cytotoxicity assay kit was purchased from Dojindo (Kumamoto, Japan). RhoA Pull-down Activation Assay Biochem Kit (BK036) was purchased from Cytoskeleton, Inc. (Denver, USA). ELISA kits to assay human cell supernatant tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6) were purchased from R&D systems (CA, USA). Lipofectamine 2000 reagent was purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies against p-IκB kinase (IKK), p-IκBα and NF-κB-P65 were purchased from Cell Signaling Technology (CST, Beverly, USA). Antibodies against Rho-kinase, α-smooth muscle actin (α-SMA), monocyte chemoattractant protein-1 (MCP-1) and Toll-Like Receptor 4 (TLR-4) were purchased from Abcam (Abcam, UK). Other antibodies were obtained from Proteintech Biotechnology (Wuhan, China). β-actin and horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse antibodies were provided by Beyotime Biotechnology (Shanghai, China). All other chemicals were of reagent grade and endotoxin free.
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2.2 Cell culture The human peritoneal mesothelial cell line (HMrSV5) was obtained from Professor Xueqing Yu of the First Affiliated Hospital of Sun Yat-sen University. It was established after infection of a fully characterized primary culture of human peritoneal mesothelial cells with an amphotropic recombinant retrovirus that encodes SV40 large-T Ag under the control of the Moloney virus long terminal repeat (LTR) and the gene conferring resistance to the G418 antibiotic. The HMrSV5 cells were cultured in DMEM F12/1:1 medium supplemented with 15% fetal bovine serum, streptomycin (100 μg/ml), and penicillin (100 units/ml) at 37°C in a 95% air/5% CO2 humidified atmosphere. When the cells reached confluence, they were rendered quiescent by incubating with 1% serum containing DMEM medium. The quiescent cells were then treated with LPS (3 μg/ml) or LPS plus thymol (10, 20, 40 μg/ml) for various times as indicated. Thymol was added 60 mins before addition of LPS. Detection of TNF-α, IL-6, MCP-1 and α-SMA was carried out after cells were incubated with LPS for 12 h, whereas activity of the RhoA/ROCK and TLR4/NF-kB signalling pathways was detected in cells incubated with LPS for 0.5 h.
2.3 RNA interference Small interference RNA (siRNA) targeting RhoA (si-RhoA: 5’-UCAAGCAUUUCUGUCCCAA-3’) and the negative control RNA probe (NC: 5’-AGUUCAACGACCAGUAGUC-3’) were designed and purchased from Takara (Dalian, China). HMrSV5 cells were seeded onto 6-well plates. When reached 6
approximately 60-70% confluency, they were transfected with Opti-MEM medium containing siRhoA/NC and Lipofectamine™ 2000 for 6 h. The medium was changed and the cells were grown further with fresh DMEM/F-12 medium for another 24 h. The transfected HMrSV5 cells were then treated with LPS (3 μg/ml) for 0.5 h or 12 h. The non-transfected cells grown in normal culture medium served as controls. The knockdown efficiency of si-RhoA in HMrSV5 cells was detected.
2.4 WST-8 assays The cell viability of all the control and experimental groups was evaluated (control, LPS, LPS plus Thymol (10, 20, 40 μg/ml), si-RhoA plus LPS, and NC plus LPS) using the WST-8 cell proliferation and cytotoxicity assay kit. Briefly, siRhoA-, NC- and non-transfected HMrSV5 cells were seeded in a 96-well plate and treated with LPS and thymol as described previously in 90 μl of complete medium for 24 h. Subsequently, 10 μL WST-8 solution was added to each well and incubated for an additional 2 h at 37˚C. The absorbance was read at 450 nm using a microplate reader. Cell viability of the experimental groups was normalized against untreated HMrSV5 cells.
2.5 RhoA pull-down assay RhoA pull-down assay was performed using a kit according to the manufacturer's instructions. Briefly, cell lysates were harvested rapidly on ice and centrifuged at 15,000 rpm for 5 min at 4°C. A total of 20 μl of the lysate was saved for protein 7
quantitation, and equivalent amounts of lysate protein (500 μg total protein) were incubated with a pre-determined amount of rhotekin-RBD beads at 4°C for 60 min with gentle rocking and then centrifuged at 15,000 rpm for 60 s at 4°C. The beads were washed and boiled for 2 min in 20 μl of 2× Laemmli sample buffer. The supernatant was resolved with 12% SDS-PAGE. Membranes were probed with anti-RhoA antibody (1 μg/ml; provided in the kit) and an HRP-conjugated secondary antibody. Protein bands were imaged using the ECL system
2.6 Western blot analysis Nuclear and cytoplasmic protein extracts from the same experiment were prepared using the kit according to manufacturer’s instructions (Beyotime Biotechnology, Shanghai, China) to detect nuclear and cytoplasmic NF-κB levels. Moreover, total protein extracted by RIPA was used to detect the other molecules. Protease and phosphatase inhibitor cocktail for general use (50×, Beyotime Biotechnology, Shanghai, China) were added in lysis buffer when protein extraction. Proteins (10 μg) were separated by 10-12% SDS-PAGE, and the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% skim milk for 1 h at room temperature and then incubated at 4°C overnight with the following primary antibodies: rabbit anti-Rho-kinase (1:1000; Abcam, UK), rabbit anti-p-IKK (1:1000; CST), rabbit anti-NF-κB-P65 (1:1000; CST), rabbit anti-p-IκBα (1:1000; CST), rabbit anti-α-SMA (1:300; Abcam, UK), rabbit anti-Histone 3 (1:1000; Proteintech), mouse anti-MCP-1 (1:1000; Abcam, 8
UK), rabbit anti-TLR4 (1:1000; Abcam, UK) and rabbit anti-GAPDH (1:1000; BOSTER, China). Then, the membranes were washed with TBST buffer and incubated with HRP-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibodies (1:5,000; Santa Cruz Biotechnology, Inc. Dallas, TX, USA) for 1 h at room temperature. Protein bands were detected using an ECL system and a Bio-Rad electrophoresis image analyser (Bio-Rad, Hercules, CA, USA). Histone3 (H3) and GAPDH were used as internal controls for nuclear and cytoplasmic proteins, respectively. Also GAPDH were used as internal controls for total protein.
2.7 Immunofluorescence HMrSV5 cells grown on glass coverslips were pretreated according to the experimental conditions. Then, they were fixed with 4% paraformaldehyde for 20 mins. After incubated with goat serum working solution to block nonspecific binding for 2 h in 37℃, cells were incubated with NF-κB P65 primary antibody at 4℃ overnight, Then, the cells were stained with DyLight549-conjugated secondary antibodies for 1 h at room temperature. The cells were incubated with 4, 6-diamidino-2-phenylindole (DAPI) for 5 min to stain the nuclei. Randomly selected fields were imaged using a fluorescent microscope.
2.8 Enzyme Linked Immunosorbent Assay (ELISA) The HMrSV5 cells were seeded in 6-well plates (4×105 cells/well) and pretreated with or without various concentrations of thymol (10, 20, 40 μg/mL) for 1 h. Then, 9
the cells were stimulated with LPS (3 μg/mL) for 12 h. The supernatant was collected and centrifuged at 1500 rpm for 3 min to measure the levels of TNF-α, IL-6 using ELISA kits according to the manufacturer’s instructions. The absorbance was read at 450 nm using a microplate reader. Cytokines TNF-α and IL-6 were calculated according to a standard curve set up by absorbance value.
2.9 Statistical analysis Data are presented as the mean ± S.D. unless stated otherwise. One-way ANOVA was used to determine the statistical difference significant differences between groups. The Dunnett’s test was used to perform multiple comparisons between the groups. A two-tailed P < 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS 20.0 software (SPSS Inc. Chicago, Illinois, USA).
3. RESULTS 3.1 LPS treatment activates RhoA in HMrSV5 cells RhoA has been identified as a mediator of pro-inflammatory responses and participates in LPS-induced inflammation. To assess RhoA activation in LPS-treated HMrSV5 cells, a RhoA pull-down activation assay was performed. First, we applied a time course analysis of HMrSV5 cells treated with 3 μg/ml LPS, and the result showed that RhoA activity was higher at 0.5 h compared with the control group (Fig. 2A). Subsequently, HMrSV5 cells treated for 0.5 h with different concentrations of 10
LPS showed that 3 μg/ml LPS stimulated maximal RhoA activity (Fig. 2B).
3.2 LPS treatment activates NF-κB in HMrSV5 cells Transcription factor NF-κB is a critical regulator of gene expression during LPS-induced inflammation. Hence we performed western blot analysis to analyse the activation of NF-κB in LPS-treated HMrSV5 cells. First, we set a short time course with a concentration of 3 μg/ml LPS. As expected, NF-κB p65 in the cytoplasm was significantly decreased after treatment for 0.5 h with LPS compared with the control group (Fig. 3A1). In addition, NF-κB p65 in the nucleus was significantly increased after treatment with LPS for 0.5 h (Fig. 3A2). That means there was a rapid translocation of activated NF-κB from the cytoplasm to the nucleus as quickly as 0.5 h in HMrSV5 cells stimulated by LPS. Second, concentration course experiments using this stimulation duration showed that 3 μg/ml LPS significantly activated NF-κB compared with the control group (Fig. 3B). Therefore, 3 μg/ml was the proper concentration of LPS to use in subsequent studies.
3.3 Effect of thymol and si-RNA on HMrSV5 cell viability To assess the potential cytotoxicity of thymol and si-RNA on HMrSV5 cells, we used the WST-8 assay. These results showed that cell viability was not affected by thymol or si-RhoA administration plus LPS. (Fig. 4)
3.4 Effects of thymol on the cytokines level in LPS-treated HMrSV5 cells 11
LPS exposure induces a strong increase in cytokine expression via the TLR-4-NF-κB signalling pathways. Therefore, we investigated the LPS-induced cytokine response to observe the role of thymol. The expression of the cytokines TNF-α, IL-6, MCP-1, and α-SMA was detected by ELISA and western blot. The results indicated that LPS treatment significantly increased the expression of TNF-α, IL-6, MCP-1, and α-SMA (Fig. 5A1 and 5A2). Moreover, pre-treatment with thymol (10, 20, and 40 μg/mL) for 1 h prior to LPS stimulation decreased the levels of cytokines in a dose-dependent manner (Fig. 5A). This demonstrates that thymol inhibits the induction of pro-inflammatory cytokines by LPS.
3.5 Effect of thymol on TLR4 mediated NF-κB pathway activation To clarify the anti-inflammatory molecular mechanism of thymol, we assessed the activation of TLR4/NF-κB pathway. Since TLR4 plays an important role in the LPS-induced inflammatory response, we analysed its expression by western blot. The result indicated that the expression of TLR4 was remarkably increased after LPS exposure; however, it was dose-dependently downregulated by thymol administration (Fig. 5B).
Moreover, effect of thymol on the NF-κB signalling pathway was also assessed. Compared with the LPS group, the phosphorylation of IKK and IκBα proteins in the cytoplasm (Fig. 5B) and nuclear NF-κB p65 levels (Fig. 5C) was significantly inhibited by thymol in a dose-dependent manner. Conversely, NF-κB p65 levels in the cytoplasm increased in an opposite trend. To confirm these results, 12
immunofluorescence assays were performed to determine if translocation of NF-κB p65 from the cytoplasm to the nucleus was suppressed. As shown in Fig. 7, the expression of NF-κB-p65 was significantly decreased in the nucleus by thymol administration.
3.6 Effect of thymol on RhoA/ROCK pathway activation The RhoA/ROCK signalling pathway is reported involving in the LPS-induced NF-κB activation during inflammation. Furthermore, we analysed the effect of thymol on the activation of this pathway in LPS-treated HMrSV5 cells and observed that thymol inhibited the expression of RhoA and ROCK in a dose-dependent manner (Fig. 5D).
3.7 Effect of si-RhoA on cytokines levels Previous results demonstrate that thymol inhibits LPS-induced inflammatory response in HMrSV5 cells partly by suppressing TLR4/NF-κB and RhoA/ROCK signalling pathways. To further evaluate if the anti-inflammatory effect of thymol was mediated by inhibiting the RhoA pathway, we knocked down RhoA in HMrSV5 cells using siRNA against RhoA (si-RhoA). Subsequently, we observed that si-RhoA significantly decreased RhoA expression (Fig. 6A). This correlates with downregulation of cytokines (Fig. 6B) upon LPS treatment, suggesting that inhibition of the RhoA/ROCK signalling pathway is central to the anti-inflammatory mechanism of thymol in LPS-treated HMrSV5 cells. 13
3.8 Effect of si-RhoA on TLR4 mediated NF-κB pathway activation We further evaluated the effects of si-RhoA on the TLR4/NF-κB pathway in LPS-treated HMrSV5 cells. The results show that the expression of TLR4 was not affected by si-RhoA compared to the LPS group (Fig. 6C). However, si-RhoA markedly decreased the phosphorylation of IκBα in the cytoplasm (Fig. 6C). It also decreased the levels of NF-κB-p65 in the nucleus (Fig. 6D), but increased the levels of NF-κB-p65 in the cytoplasm (Fig. 6C). As shown in Fig. 7, immunofluorescence assay confirmed that nuclear translocation of NF-κB p65 was significantly decreased in siRhoA-transfected HMrSV5 cells.
4. DISCUSSION Peritoneal mesothelial cells are key regulators of peritoneal homeostasis, and participate in peritoneal immune and inflammatory responses by protecting against the invading microbes (Aroeira et al., 2007; Yung and Chan, 2009). Thymol is a colourless crystalline monoterpene phenol that has been used in traditional medicine and possesses anti-inflammatory properties (Nagoor Meeran et al., 2017). In this study, we demonstrate that thymol exerts beneficial effects in LPS-induced inflammatory responses of HMrSV5 cells by modulating the NF-κB signalling pathway via RhoA. Peritoneal injury activates not only macrophages and neutrophils but also mesothelial cells, which are the main sources of pro-inflammatory cytokines and 14
fibrotic mediators in response to external stimuli (Bertoli et al., 1999; Devuyst et al., 2010; Yung et al., 2006). During peritonitis, LPS activates mesothelial cells through the Toll-like receptors (TLRs), resulting in the activation of the NF-kB pathway and subsequent secretion of numerous inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8 and MCP-1(Kato et al., 2004; Topley et al., 1993; Yung et al., 2006). TNF-α and IL-1β are two key components of the innate immune response (Lahouassa et al., 2007; Topley et al., 1993). IL-6 is a key player in modulating inflammation and a major effector of the acute phase reaction; its synthesis is induced by TNF-α and IL-1β in peritoneal mesothelial cells (Bellingan et al., 1996). In combination with the soluble IL-6 receptor, IL-6 promotes the synthesis and secretion of MCP-1 (Hurst et al., 2001). These pro-inflammatory factors play different functional roles in different phases of the inflammation response (Devuyst et al., 2010). In this study, we found that the expression of TNF-α, IL-6 and MCP-1 cytokines was markedly increased after LPS stimulation. However, pre-treatment with thymol inhibited the production of these cytokines in a dose-dependent manner. These results are in accordance with the findings in RAW 264.7 cells (Wu et al., 2017) and mouse mammary epithelial cells (Liang et al., 2014). We also found α-SMA increased markedly after LPS stimulation. It is well known that peritoneal inflammation could induce epithelial-mesenchymal transition (EMT) of mesothelial cells, then mesothelial cells undergo a transition from an epithelial phenotype to a mesenchymal phenotype (Aroeira et al., 2007). α-SMA is a marker of mesenchymal cells such as myofibroblasts. EMT is a complicated process and we 15
only detected the expression of α-SMA in this study. Our results suggest that thymol inhibited the inflammatory response induced by LPS, also might prevent EMT of mesothelial cells. Therefore, further studies are necessary to verify if thymol inhibits inflammation-induced peritoneal fibrosis. NF-κB is a transcription factor that regulates immune cell functions and inflammatory responses. When activated, NF-κB translocates to the nucleus and mediates the expression of target genes (Hoffmann and Baltimore, 2006; Srivastava and Ramana, 2009). TLR4 is the member of TLR family that is recognized and activated by LPS, and plays an important role in innate immunity (Kawai and Akira, 2010; O'Neill et al., 2013). TLR4/ NF-κB signalling pathway plays a major role in the induction of inflammatory cytokines by LPS (Kuzmich et al., 2017). In recent studies, TLR4 and NF-κB contribute to the anti-inflammatory mechanisms of thymol (Gholijani et al., 2016; Khosravi and Erle, 2016; Liang et al., 2014; Wan et al., 2018; Wu et al., 2017; Yao et al., 2017). In the present study, we demonstrated that thymol inhibits the upregulation of TLR4 and the phosphorylation of IKK, IκBα and p65 in a dose-dependent manner in LPS-stimulated HMrSV5 cells. This suggests that the anti-inflammatory role of thymol is mediated by inhibition of the TLR4/ NF-κB signalling pathway. Furthermore, previous studies have shown that the activation of NF-κB is regulated by the RhoA/ROCK signalling pathway during LPS-induced inflammation (Chen et al., 2015; Meyer-Schwesinger et al., 2009; Qin et al., 2014). ROCK inhibition markedly reduces the production of pro-inflammatory cytokines and 16
chemokines. Several studies have demonstrated that expression of TNF-α, IL-1β, IL-6 and IL-8 cytokines is reduced by ROCK inhibitor. (Chen et al., 2015; Meyer-Schwesinger et al., 2009; Segain et al., 2003). Therefore, we sought to investigate if thymol inhibits the activation of TLR4/NF-κB signalling via RhoA signalling pathway. Our study demonstrates that silencing RhoA decreases production of inflammatory cytokines and the phosphorylation of p65 and IκBα proteins in LPS-induced HMrSV5 cells. However, the upregulation of TLR4 is not altered. These results suggest that the transmembrane receptor TLR4 is upstream of RhoA and NF-κB locates downstream of RhoA. Therefore, we postulate that LPS activate the TLR4 receptor, which leads to the activation of NF-κB via RhoA in HMrSV5 cells. In conclusion, our study shows that thymol inhibits the inflammatory response induced by LPS in HMrSV5 cells. Therefore, TLR4-mediated RhoA-dependent NF-κB signalling pathway is relevant to the anti-inflammatory effects of thymol. Our findings suggest that thymol is a potential therapeutic agent against peritoneal dialysis-related peritonitis.
CONFLICTS OF INTERESTS Authors declare that there are no conflicts of interest.
ACKNOWLEDGMENTS This work was supported by the grant to Xiang Liu from Projects of Medical and Health Technology Development Program in Shandong, China (Grant No. 2013WSB01025) and grants to Fajuan Cheng from Shandong Provincial Natural Science Foundation (Grant No. ZR2016HB09) and National Natural Science 17
Foundation (Grant No. 81601421). We are grateful to Professor Xueqing Yu of the First Affiliated Hospital of Sun Yat-sen University for providing the human peritoneal membrane cell line (HMrSV5).
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Fig. 1 Chemical structure of thymol. Fig. 2 LPS treatment activates RhoA in HMrSV5 cells. A) RhoA pull-down activity assay in HMrSV5 cells treated with 3 μg/mL LPS over time. B) Increasing concentration course of RhoA activation in HMrSV5 cells treated with LPS for 0.5 h. (Data are mean ± S.D.; *P < 0.05 vs control, **P < 0.01 vs control, n = 3). Fig. 3 LPS treatment activates NF-κB pathway in HMrSV5 cells. A) Detection of cytoplasmic (A1) and nucleus (A2) NF-κB P65 after treatment with 3 μg/ml LPS over time. B) Detection of cytoplasmic (B1) and nuclear (B2) NF-κB P65 after exposure to increasing concentrations of LPS for 0.5 h. (Data are mean ± S.D.; *P < 0.05 vs control, n = 3). Fig. 4 Cell viability assays. The HMrSV5 cells were cultured with different concentrations of thymol or si-RhoA or si-NC in the presence of 3 μg/mL LPS for 24 h. WST-8 assay shows that the viability of all the experimental groups was similar. The values are presented as the mean ± S.D. (n = 4). Fig. 5 Thymol suppresses cytokines expression TLR4/NF-κB signalling and RhoA pathway activation. A) IL-6 and TNF-α levels in the supernatants of 23
LPS-stimulated HMrSV5 cells were detected by ELISA. α-SMA and MCP-1 were detected by western blot. B) TLR-4 protein expression and the phosphorylation of the NF-κB p65, IκBα and IKK in cytoplasm were detected by western blot, while GAPDH served as an internal control. C) Nuclear NF-κB P65 protein expression was detected by western blot, while H3 served as internal control. D) RhoA-GTP was detected by pull down assay, and ROCK was detected by western blot. con indicates the normal control group; LPS indicates LPS-stimulated group; 10, 20, and 40 refer to LPS plus 10 μg/ml, 20 μg/ml or 40 μg/ml thymol. (Data are mean ± S.D.; *P < 0.05 vs control, # P < 0.05 vs LPS. n = 3). Fig. 6 Effects of RhoA silencing on cytokine expression and NF-κB pathway activation. A) Knockdown efficiency of si-RhoA was detected by western blot. (Data are mean ± S.D.; *P < 0.05 vs si-NC, n=3) B) IL-6, TNF-α and MCP-1, α-SMA levels were respectively detected by ELISA or western blot. C) TLR-4 protein expression and the phosphorylation of the NF-κB p65 and IκBα in cytoplasm were detected by western blot. D) Nuclear NF-κB P65 protein expression was detected by western blot, while H3 is used as the internal control. con indicated the normal control group. LPS indicates the LPS-stimulated group. si-RhoA indicates LPS plus si-RhoA group and NC indicates si-NC plus LPS group. (Data are mean ± S.D.; *P < 0.05 vs control, # P < 0.05 vs LPS. n = 3). Fig. 7 Effects of thymol and RhoA silencing on NF-κB p65 translocation into the nucleus using immunofluorescence assays. Translocation of NF-κB p65 into the nucleus was obviously suppressed by thymol and si-RhoA. 24
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