Research Communication Tetramethylpyrazine Reduces Inflammation in Liver Fibrosis and Inhibits Inflammatory Cytokine Expression in Hepatic Stellate Cells by Modulating NLRP3 Inflammasome Pathway
Xiafei Wu1 Feng Zhang1,2,3 Xin Xiong1 Chunfeng Lu1 Naqi Lian1 Yin Lu1,2,3 Shizhong Zheng1,2,3*
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Department of Pharmacology, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China 2 National First-Class Key Discipline for Traditional Chinese Medicine of Nanjing University of Chinese Medicine, Nanjing, China 3 Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Material Medical, Nanjing University of Chinese Medicine, Nanjing, China
Abstract Hepatic fibrosis is concomitant with liver inflammation, which has been highlighted as significant treatment of chronic liver disease. We previously demonstrated that tetramethylpyrazine (TMP), the effective component of Ligusticum chuanxiong Hort, can inhibit the activation of HSCs and consequential antihepatic fibrosis. In this study, our work demonstrated that TMP improved liver histological architecture, decreased hepatic enzyme levels and attenuated collagen deposition in the rat fibrotic liver. In addition, TMP significantly protected the liver from CCl4-caused injury and fibrogenesis by suppressing inflammation with reducing levels of inflammatory
cytokines, including tumor necrosis factor-a (TNF-a), NLRP3, nuclear factor-kappa B (NF-jB) and interleukin-1b (IL-1b). Experiments in vitro showed that TMP inhibited inflammatory cytokine expression in HSCs associated with disrupting platelet-derived growth factor-b receptor (PDGF-bR)/NLRP3/ caspase1 pathway. These data collectively indicate that TMP can attenuate liver inflammation in liver fibrosis and possibly by targeting HSCs via PDGF-bR/NLRP3/caspase1 pathway. It provides novel mechanistic insights into TMP as a potential C 2015 IUBMB Life, therapeutic remedy for hepatic fibrosis. V 67(4):312–321, 2015
Keywords: tetramethylpyrazine; liver fibrosis; NLRP3 inflammasome; inflammation; hepatic stellate cells
Introduction Hepatic fibrosis represents a frequent event following various kinds of etiological factor in the liver, including autoimmune liver diseases, viral infection, and cholestasis. Firstly manifes-
C 2015 International Union of Biochemistry and Molecular Biology V
Volume 67, Number 4, April 2015, Pages 312–321 *Address correspondence to: Shizhong Zheng, Department of Pharmacology, College of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, China. Tel: 186-25-85811245. Fax: 186-25-86798188. E-mail: [email protected] Received 24 September 2014; Accepted 7 January 2015 DOI 10.1002/iub.1348 Published online 3 April 2015 in Wiley Online Library (wileyonlinelibrary.com)
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tations of liver inflammation, then quiescent Hepatic stellate cells (HSC) become activated and transdifferentiate into myofibroblast-like cells. It is characterized by excessive production and deposition of extracellular matrix (ECM) molecules (1). Recently, with the deepening research on hepatic fibrosis, it has been confirmed that in the development of fibrosis accompanies large amounts of inflammatory mediators and reactive oxygen species, including tumor necrosis factor-a (TNF-a), transforming growth factor-b (TGF-b), interleukin-1b (IL-1b) interleukin-6 (IL-6), interleukin-18 (IL-18) and so on. The persistence of this inflammation will cause progressive hepatic fibrosis and the development of cirrhosis (2). Chronic liver inflammation stimulates the progression of hepatic fibrosis, which can be weakened by immunosuppressive and anti-inflammatory therapies. In recent years, the role of NLRP3 inflammasome in hepatic injury is now attracting
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widespread attention. Inflammasomes are intracellular multiprotein complexes that act as platforms for the maturation and secretion of the proinflammatory cytokines. Over the past decades, many studies have focused on the role that the NLRP3 inflammasome has in the innate immune system as a sensor of pathogens and danger signals, and its activation mechanism has been widely investigated. It has been confirmed that the activation of NLRP3 is closely related with the congenital and acquired inflammatory disease, such as autoinflammatory disease, gout and type 2 diabetes mellitus. Research has demonstrated that unrestrained NLRP3 activation can results in severe liver inflammation, characterized by a predominantly neutrophilic infiltrate and HSC-activation with collagen deposition in the liver. However, the precise molecular mechanisms remain to be defined (3–5). Classic angiogenic mediators platelet-derived growth factor (PDGF) is the most potent mitogen of HSC and is likely to be an important mediator of the increased proliferation of the cells during the hepatic wound healing response (6,7), it can drive fibrogenic responses and may also foster a milieu that is permissive for the development of HCC (8). Previous research has shown that during hepatic fibrogenesis, PDGF is secreted by a variety of cell types as a response to injury, and many pro-inflammatory cytokines mediate their mitogenic effects via the autocrine release of PDGF (9). Moreover, previous research from our laboratory study provided in vitro evidence that PDGF could play a key role in HSC-based angiogenesis to promote hepatic fibrosis (10). Basic and clinical evidence has indicated the tight association between attenuation of liver inflammation and regression of liver fibrosis impulsive for development of potential antifibrotic therapies (11). Therefore, research identifying anti-inflammatory agents of liver that are innocuous is urgently needed. Recently, the natural product tetramethylpyrazine (TMP) has been reported to anti-inflammation reaction of patients with rheumatic heart disease (12). And animal studies also demonstrated that TMP played an anti-inflammation role in allergic asthma mice (13), suggesting that TMP as a potential therapeutics for anti-inflammation reaction. We previously reported that TMP can inhibit the activation of HSCs caused by Ang II in order to anti-hepatic fibrosis (14). Moreover, our latest investigations also showed that TMP can improve liver histology and attenuate fibrogenesis by down-regulating inflammatory cytokines (15). However, the underlying molecular mechanism is largely unknown. The current study established a CCl4-caused fibrosis model in rats to examine the relevance of inflammation attenuation to liver fibrosis reduction by TMP. And then the mechanisms by which TMP affected the inflammatory mediators expression in HSCs were elucidated. The obtained results provided novel insight into the mechanisms of TMP reduction in hepatic fibrosis.
Materials and Methods Reagents and Antibodies TMP was purchased from Sigma (St Louis, MO). Recombinant rat PDGF-BB was from Cell Sciences (Canton, MA). This cyto-
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kine was referred to as PDGF throughout the study. PDGF-bR blocker Imatinib was from Nanjing EnoGene Biotechnology (Nanjing, China). NLRP3 inhibitor Glyburide (Gly) was from CALBIOCHEM (Shanghai, China). Primary antibodies against aSMA, a1(I) procollagen, and fibronectin were from Epitomics (San Francisco, CA). Primary antibody against PDGF-bR was from Epitomics (San Francisco, CA). The primary antibodies against cleaved-IL-1b was purchased from Cell Signaling Technology (Danvers, MA). Primary antibody against NLRP3 was from Novus Biologicals (Littleton, CO). Primary antibody against caspase1 was from Millipore (Burlington, MA). The primary antibodies against pro-IL-1b and pro-IL-18 were from Bioworld Technology (Nanjing, China). MSU was purchased from Sigma (St Louis, MO).
Animal Procedures and Treatments All experimental procedures were approved by the institutional and local committee on the care and use of animals of Nanjing University of Chinese Medicine (Nanjing, China), and all animals received humane care according to the National Institutes of Health (USA) guidelines. Male Sprague-Dawley rats (180–220 g bodyweight) were obtained from Nanjing Medical University (Nanjing, China). A mixture of CCl4 (0.1 mL/100 g body weight) and olive oil [1:1 (w/v)] was used to induce liver fibrosis in rats. Forty-eight rats were randomly divided into six groups (eight rats per group). Group 1 was the vehicle control in which rats were not administrated CCl4 or TMP but intraperitoneally (i.p.) injected with olive oil. Group 2 was the CCl4 group in which rats were i.p. injected with CCl4 without TMP treatment. Groups 3, 4, and 5 were treatment groups in which rats were i.p. injected with CCl4 and orally given TMP at 50, 100, and 200 mg/kg, respectively. Group 6 was the positive control in which rats were injected with CCl4 and treated with colchicine (Yifeng Pharmacy, Nanjing, China). Rats in groups 2 to 6 were i.p. injected with CCl4 every other day for 8 weeks. TMP was suspended in sterile phosphate buffered saline (PBS) and given once daily by gavage during weeks 5 to 8. Colchicine was suspended in sterile PBS and given once daily by gavage (0.1 mg/kg) during weeks 5 to 8. At the end of the experiment, rats were sacrificed after being anesthetized by i.p. pentobarbital (50 mg/kg). Blood was collected, and livers were isolated for calculation of liver/bodyweight ratio. A small portion of the liver was removed for histopathological and immunohistochemical studies by fixation with 10% formalin and subsequent embedment with paraffin. The remaining liver was cut inpieces and rapidly frozen with liquid nitrogen for extraction of total RNA and hepatic proteins.
Liver Histopathology Harvested liver tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Liver slices of 5 lm thick were prepared and stained with hematoxylin and eosin and masson’ strichrome stain by using standard methods. For sirius red collagen staining, thin sections were deparaffinized and stained with picro-sirius red for 1 h at room temperature. After washes, sections on the slides were dehydrated in100%
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ethanol and in xylene, and then they were mounted in Permount. Photographs were taken in a blinded fashion at random fields. Representative views of liver sections are shown.
Hydroxyproline Examination The hydroxyproline levels in liver tissue and blood were determined by using a kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the protocol. Briefly, three small pieces of liver tissues randomly excised from the liver of every rat were hydrolyzed in 6 N HCl at 110 C for 24 h, and subsequently they were neutralized with NaOH. Isopropanol in citrate acetate-buffered chloramine T was added to aliquots of the hydrolysate, followed by the addition of Ehrlich reagent. The chemical reaction occurred in dark for 25 min at 60 C. After centrifugation, absorbance of the supernatant of each sample was read at 558 nm by using a 96-well plate spectrometer. Trans-hydroxyproline was used as the standard for quantification. Values were normalized to control.
Immunofluorescence Double Staining After deparaffinization, thin sections (5 lm) of the liver tissues (liver lobules and portal area) were blocked with 1% bovine serum albumin, and then they were incubated with primary antibodies overnight at 4 C. After three washes with PBS, sections on slides were incubated with secondary antibodies at room temperature for 1 h. Sections incubated with secondary antibodies alone were used as negative controls. Sections were viewed in a single plane under an MRC 1024 laser confocal microscope (Bio-Rad Laboratories). Representative views of liver sections with positive staining are shown.
Cell Isolation and Culture Conditions Primary HSCs were isolated from male Sprague-Dawley rats (200–250 g; Nanjing Medical University, Nanjing, China) according to a described protocol. All experimental procedures were approved by the institutional and local committee on the care and use of animals of Nanjing University of Chinese Medicine (Nanjing, China), and all animals received humane care according to the National Institutes of Health (USA) guidelines. All rats were maintained under a 12 h light/dark cycle at a controlled temperature (25 C) with free access to food and tap water. HSCs were cultured in DMEM (Invitrogen, Grand Island, NY) with 10% fetal bovine serum (Sijiqing Biological Engineering Materials, Hang Zhou, China), 1% antibiotics, and maintained at 37 C in a humidified atmosphere of 5% CO2 and 95% air. HSCs at passages 2 to 4 were used in experiments.
Real-Time PCR RNA isolation and real-time PCR were performed as we previously described (19). Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the invariant control. The following primers were used: pro-IL-1b(forward) 50 -GGCTTCCT TGTGCAAGTGTC-3, (reverse) 50 -TGTCGAGATGCTGCTG’TGAG3; pro-IL-18(forward) 50 -TTGACAAAAGAAACCCGCCT-3, (reverse) 50 -CCTGGCACACGTTTCTGAAA-30 ; GAPDH (forward) 50 -GACAT CAAGAAGGTGGTGAAGC-3, (reverse) 50 -TGTCATTGAGAGCAA TGCCAGC-3.
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Western Blot Analyses Whole-cell lysates were prepared using radioimmunoprecipitation analyses buffer supplemented with protease and/or phosphatase inhibitors. The protein levels were determined using a BCA assay kit (Pierce). Proteins (50 lg/well) were separated by SDS-polyacrylamide gel, transferred to a PVDF membrane (Millipore, Burlington, MA), blocked with 5% skim milk in Trisbuffered saline containing 0.1% Tween 20. Target proteins were detected by corresponding primary antibodies, and subsequently by horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using chemiluminescence reagent (Millipore, Burlington, MA). Equivalent loading was confirmed using an antibody against b-actin. The levels of target protein bands were densitometrically determined using Quantity Ones 4.4.1 (Bio-Rad Laboratories, Berkeley, CA). The variation in the density of bands was expressed as fold changes compared with the control in the blot after normalized to b-actin or the total protein in some experiments.
Enzyme-Linked Immunosorbent Assay (ELISA) The levels of each factor in liver tissues, blood and HSC culture supernatant were determined with an ELISA kit (Nanjing Jiancheng Bioengineering Institute) according to the protocol. For the experiments in vitro, HSCs (500,000 cells/well) were seeded in six-well plates and cultured in DMEM with 10% FBS for 24 h and then in low-serum medium (0.5%, v/v) for an additional 12 h. Hepatic stellate cells were treated with various reagents as indicated in specific experiments for 24 h. Briefly, samples of 100 lL were added to each well of the 96-well plates coated with antibody, followed by incubation for 2 h at room temperature. Working detector solution of 100 lL was loaded into each well, and the plates were incubated for an additional 1 h at room temperature before the addition of substrate solution of 100 lL. The reaction was stopped by adding stop solution of 50 lL. The absorbance was read at 450 nm wavelength. Values were normalized to control.
Statistical Analysis Data were presented as mean 6 SD, and results were analyzed using SPSS16.0 software. The significance of difference was determined by one-way ANOVA with the post hoc Dunnett’s test. Values of P < 0.05 were considered to be statistically significant.
Results TMP Protects the Liver Against CCl4-Induced Injury and Suppresses Hepatic Fibrogenesis in the Rat Model We initially evaluated the TMP effect on liver fibrotic injury in vivo. Gross examination showed that morphological changes pathologically occurred in the rat liver with CCl4 injection compared with the normal liver, but treatment with TMP improved the pathological changes in livers, and that colchicine as a positive control also effectively protected the rat liver from CCl4induced injury (Fig. 1A). TMP treatment and colchicine also significantly reduced the liver/body weight ratio, which was
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significantly elevated by CCl4 injection (Fig. 1A). CCl4 causes hepatic injury, including hepatocytic necrosis, steatosis, and inflammation. The effects of TMP on the protection of the liver from injury and fibrogenesis were initially evaluated by histological analyses. HE staining showed that TMP treatment resulted in remarkable improvement in liver histology evidenced by the ameliorated state of hepatic steatosis, necrosis, and fibrotic septa in the liver. To assess the impact of TMP on hepatic fibrogenesis caused by CCl4, liver sections were stained with Masson and picro-Sirius red for detecting the deposition of collagens, the results showing that collagens were severely deposited in the CCl4-injured liver accompanied by nodular formation, but were reduced by TMP dose-dependently in the liver of rats treated with TMP (Fig. 1B). Hydroxyproline is an amino acid found almost exclusively in collagens. Measurement of hepatic and blood hydroxyproline further indicated that collagen production was reduced by TMP in rats with liver fibrosis (Fig. 1C). Additional biochemical analyses of serum enzymes were performed to verify the role of TMP in the protection of the liver from injury. Our data demonstrated that TMP reduced serum levels of alkaline phosphatase, ALT and AST dose dependently in CCl4-treated rats (Fig. 1D). Furthermore, we examined the transcript and protein abundance of a-SMA, a (1) procollagen and fibronectin, three key markers of liver fibrosis. The results showed that TMP down-regulated the three markers at protein levels (Fig. 1E). As platelet-derived growth factor-b receptor (PDGF-bR) and transforming growth factor-b receptor 1 (TGFbRI) are the two key receptors transmitting pro-fibrogenic pathways (16,17), it was plausible to examine the effect of these two receptors to elucidate the underlying mechanisms of TMP in the suppression of hepatic fibrogenesis in the rat model. Immunofluorescent assay showed that TMP down-regulated of TGF-bRI and PDGF-bR (Fig. 1F). All these results confirmed that TMP suppressed hepatic fibrogenesis in the rat model.
TMP Suppresses Inflammation in the CCl4 Rat Model Inflammation is commonly associated with hepatic fibrogenesis during chronic liver diseases (18). To begin to explore the mechanisms underlying the protective effects of TMP, we proposed that TMP might protect the liver against CCl4-induced injury by suppressing inflammation in the liver. As shown in immunofluorescent assay, TMP decreased the expression of proinflammatory signal molecules TNF-a, NLRP3, NF-jB and IL-1b (Fig. 2A). Moreover, Elisa assays also demonstrated that liver and serum levels of TNF-a, IL-1b, IL-18, IL-6 and IL-8 were decreased by TMP dose-dependently (Fig. 2B). Further experiments demonstrated that the elevated expression of TNF-a, NF-jB, NLRP3, IL-1b in fibrotic liver was abolished by TMP at protein levels (Fig. 2C). These results indicated that TMP suppressed inflammation caused by CCl4, which might lead to the protection of the liver from injury.
TMP Inhibits the Expression of Proinflammatory Cytokine in HSCs Treated with PDGF To gain more insight into the effects and underlying mechanisms of TMP on the expression of proinflammatory cytokine
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of HSC, we extended our studies using PDGF, a potent mitogenic molecule responsible for HSC activation (9).In our PDGF experiments mimicking the in vivo status of HSCs, we observed that, PDGF up-regulated the expression of inflammatory cytokine (pro-IL-1b, pro-IL-18 and cleaved-IL-1b) dose-dependently (Figs. 3A–3C). Next, cell viability assay provided a rationale for TMP doses used in subsequent in vitro experiments. We found that TMP does-dependently inhibited PDGF-stimulated proliferation in HSCs and at 30 lM resulted in a significant inhibitory effect (Fig. 3D). Then, western blot assays demonstrated that TMP reduced the expression of inflammatory cytokine (pro-IL1b, pro-IL-18 and cleaved-IL-1b) dose-dependently (Fig. 3E). This result was confirmed by measuring inflammatory cytokine levels in HSC supernatant (Fig. 3F) and immunofluorescence (Fig. 3G).
TMP Reduces the Expression of Inflammatory cytokine Associated with Interference of the PDGF-bR-Mediated NLRP3/Caspase1 Pathway in HSCs Treated with PDGF Previous studies have shown that NLRP3 activation can results in severe liver inflammation (4). We postulated that PDGF-bR/ NLRP3/caspase1 pathway could be involved in TMP effect. Our current data demonstrated that the PDGF-enhanced phosphorylation of PDGF-bR and its downstream signal molecules NLRP3 and cleaved-caspase1, but inhibiting the expression of pro-caspase1 (Figs. 4A and 4B). Next, we examined the expression of theses signaling molecules when treated with TMP. Western blot (Figs. 4C and 4D) and immunofluorescence (Figs. 4E and 4F) showed they were diminished by TMP dosedependently, suggesting disruption of the pathway. Subsequently, PDGF-bR inhibitor Imatinib and NLRP3 inhibitor Gly were used to establish the link between TMP disruption of the pathway and reduction in inflammatory cytokine expression. MTS assays indicated appropriate doses of Gly at which cell viability was not affected but intracellular signaling events may be modulated (Fig. 4G), and the doses of Imatinib (10 lM) was determined from previous studies of our laboratory (19). Western blot assays showed that the TMP at the selected doses significantly abolished PDGF-increased p-PDGF-bR expression similar to Imatinib function (Fig. 4H). And it can also reduce NLRP3, cleaved-caspase1 and inflammatory cytokine protein expression mimicking the Gly and Imatinib effect (Fig. 4I). Finally, we found that NLRP3 agonist MSU can abolished the inhibitory effects of TMP on the key molecules in NLRP3 pathway (Fig. 4J). These data indicated that TMP reduced the expression of inflammatory cytokine associated with disruption of PDGF-bR/NLRP3/caspase1 pathway in activated HSC.
Discussion Hepatic fibrosis is a common outcome of a variety of chronic liver diseases, if without effective treatment, it can lead to severe hepatic dysfunctions and even life-threatening conditions such as liver cirrhosis and hepatocarcinoma (20).
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FIG 1
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TMP protects the rat liver from CCl4-induced injury. Rats were grouped as follows: group 1, vehicle control (no CCl4, no treatment); group 2, model group (with CCl4, no treatment); group 3, TMP (50 mg/kg) and CCl4-treated group; group 4, TMP (100 mg/kg) and CCl4-treated group; group 5, TMP (200mg/kg) and CCl4-treated group; group 6, colchicine (0.1 mg/kg) and CCl4-treated group. (A) Gross examination of rat livers with calculation of the liver/body weight ratio. Representative photographs are shown. (B) Liver sections were stained with hematoxylin and eosin, masson reagents and sirius red. Representative photographs are shown. (C) Measurement of hydroxyproline levels in liver and blood. (D) Determination of serum ALT, AST and ALP levels. (E) Western blot analyses of a-SMA, a(I)procollagen and fibronectin in liver tissues. (F) Liver sections were stained with immunofluorescence by using antibodies against TGF-bR1 and PDGF-bR. Representative blots were from three independent experiments. For the statistics of each panel in this figure, data are expressed as mean 6 SD (n 5 8/group); ## P < 0.01 versus group 1, ###P < 0.001 versus group 1, *P < 0.05 versus group 2, **P < 0.01 versus group 2, ***P < 0.001 versus group 2.
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FIG 2
TMP suppresses inflammation in rats with CCl4-caused fibrosis. Rats were grouped as follows: group 1, vehicle control (no CCl4, no treatment); group 2, model group (with CCl4, no treatment); group 3, TMP (50 mg/kg) and CCl4-treated group; group 4, TMP (100 mg/kg) and CCl4-treated group; group 5, TMP (200 mg/kg) and CCl4-treated group; group 6, colchicine (0.1 mg/kg) and CCl4-treated group. (A) Liver sections were stained with immunofluorescence by using antibodies against TNF-a, NLRP3, NF-KB and IL-1b. (B) ELISA measurement of TNF-a, IL-1b, IL-18, IL-6 and IL-8 levels in liver and serum. (C) Western blot analyses of TNF-a, NF-KB, NLRP3 and IL-1b. Representative blots were from three independent experiments. For the statistics of each panel in this figure, data are expressed as mean 6 SD (n 5 8/group); ##P < 0.01 versus group 1, ###P < 0.001 versus group 1, *P < 0.05 versus group 2, **P < 0.01 versus group 2.
Therefore, to explore the pathogenesis of liver fibrosis has attracted extensive attention from scholars at home and abroad. As HSCs are the key fibrogenic element in response to liver fibrogenesis, intervention of HSC biological behaviors can effectively prevent and treat liver fibrosis. Current researches suggest that hepatic inflammation is only one driver of hepatic fibrosis. In chronic liver injury, inflammatory cytokines can stimulate the activation and proliferation of HSC in order to promote the deposition of ECM. Conversely, HSCs exposured to inflammatory microenvironment can autocrine inflammatory factors to enhance the migration ability of HSCs and Inflammatory cells to the injury area, which exaggerate the hepatic fibrosis (21). In the current study, we evaluated the antifibrotic
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efficacy of TMP, the effective component of Ligusticum chuanxiong Hort, employing a classical animal model of liver fibrosis that causes necrosis of hepatocytes and induces inflammation (22). We herein demonstrated that TMP effectively improved liver histology, decreased serum levels of hepatic enzymes, and inhibited collagen production in the rat liver with injury and fibrogenesis caused by CCl4. In addition, we clearly showed that TMP inhibited inflammation in rat fibrotic liver. Examination of several key inflammatory cytokine downregulation suggesting that TMP inhibited the proinflammatory signaling pathways that aggravate liver inflammation. Moreover, previous investigations showed that PDGF is the most potent mitogen of HSC, it is released in high concentrations
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FIG 3
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TMP inhibits the expression of proinflammatory cytokine in HSCs treated with PDGF. HSCs were treated with DMSO (0.02%, w/ v) and PDGF for 24 h. (A–C) Real-time PCR analyses of pro-IL-1b and pro-IL-18 mRNA, *P < 0.05 versus control, **P < 0.01 versus control, ***P < 0.001 versus control (A). Western blot analyses of pro-IL-1b, pro-IL-18 and cleaved-IL-1b protein expression with densitometry, **P < 0.01 versus control, ***P < 0.001 versus control (B). ELISA measurement of cleaved-IL-1b and cleaved-IL-18 level in supernatant, *P < 0.05 versus control, **P < 0.01 versus control, ***P < 0.001 versus control (C).(D–G) HSCs were treated with DMSO (0.02%, w/v), PDGF (20 ng/mL) and TMP for 24 h. Cell viability was evaluated by MTS assay, # P < 0.05 versus DMSO, **P < 0.01 versus DMSO1PDGF (D). Western blot analyses of inflammatory cytokine expression, ## P < 0.01 versus DMSO, ###P < 0.001 versus DMSO, *P < 0.05 versus DMSO 1 PDGF, **P < 0.01 versus DMSO 1 PDGF, ***P < 0.001 versus DMSO 1 PDGF (E). ELISA measurement of inflammatory cytokine level in supernatant, ##P < 0.01 versus DMSO, ###P < 0.001 versus DMSO, *P < 0.05 versus DMSO1PDGF, **P < 0.01 versus DMSO 1 PDGF (F).Immunofluorescence by using antibody against cleaved-IL-1b (G). Representative blots were from three independent experiments.
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FIG 4
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TMP interrupts PDGF-bR/NLRP3/caspase1 pathway linking to reduced proinflammatory cytokine expression in HSCs. HSCs were treated with PDGF for 12 h. Western blot analyses were used to examine the p-PDGF-bR and PDGF-bR, ***P < 0.001 versus control (A). HSCs were treated with PDGF for 24 h. Western blot analyses were used to examine the NLRP3/caspase1 cascade *P < 0.05 versus control, **P < 0.01 versus control, ***P < 0.001 versus control (B). HSCs were treated with DMSO (0.02%, w/v) and TMP for 24 h prior to PDGF (20 ng/mL) stimulation for an additional 12 h. Western blot analyses of p-PDGF-bR and PDGF-bR, ###P < 0.001 versus DMSO, *P < 0.05 versus DMSO 1 PDGF, **P < 0.01 versus DMSO 1 PDGF (C). HSCs were treated with DMSO (0.02%, w/v), PDGF (20 ng/mL) and TMP for 24 h. Western blot analyses were used to examine the NLRP3/caspase1 cascade, ##P < 0.01 versus DMSO, ###P < 0.001 versus DMSO, *P< 0.05 versus DMSO 1 PDGF, **P < 0.01 versus DMSO 1 PDGF, ***P < 0.001 versus DMSO 1 PDGF (D). Immunofluorescence by using antibody against NLRP3 and cleaved- caspase1 (E and F). Subsequently, HSCs were treated with Gly for 24 h. Cell proliferation was assessed by an MTS assay, **P < 0.01 versus control, ***P < 0.001 versus control (G). HSCs were treated with DMSO (0.02%, w/v), TMP (20 lM), Gly (150 lM) and Imatinib (10 lM) for 24 h prior to PDGF(20 ng/mL) stimulation for an additional 12 h. Western blot analyses of p-PDGF-bR and PDGF-bR, ### P < 0.001 versus DMSO, ***P < 0.001 versus DMSO 1 PDGF (H). HSCs were treated with DMSO (0.02%, w/v), TMP (20 lM), Gly(150 lM) and Imatinib (10 lM) for 24 h. Western blot analyses were used to examine the NLRP3/caspase1 cascade, ### P < 0.001 versus DMSO, *P < 0.05 versus DMSO 1 PDGF, **P < 0.01 versus DMSO 1 PDGF, ***P < 0.001 versus DMSO1PDGF (I). HSCs were treated with DMSO (0.02%, w/v), TMP (20 lM) and MSU for 24 h. Western blot analyses were used to examine the NLRP3/caspase1 cascade, ###P < 0.001 versus DMSO, **P < 0.01 versus DMSO1PDGF, ***P < 0.001 versus DMSO 1 PDGF, & P < 0.05 versus PDGF 1 TMP, &&P < 0.01 versus PDGF1TMP, &&&P < 0.001 versus PDGF1TMP (J). Representative blots were from three independent experiments.
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during tissue repair and liver inflammatory processes, first from platelet alpha granules and subsequently by activated inflammatory cells, HSCs and Kupffer cells (6). In addition, in liver tissue obtained from patients with chronic liver diseases, expression of PDGF and its receptor subunits appears strictly correlated with the extent of necroinflammation and fibrosis (23). So, the activity of PDGF is closely related to the pathogenesis of liver fibrosis. Therefore, we established an PDGFinduced HSC activation model in vitro, which could mimic the HSC activation during liver inflammation. We then used this cellular model to evaluate the antifibrotic effects of TMP, a natural alkaloid that has been reported to anti-inflammation in the rheumatic heart disease and allergic asthma (12,13). In this report, traditional methods of assessing hepatic fibrosis, such as histopathological analysis, Western blot and hydroxyproline measurements, were performed to determine the effectiveness of TMP on the protection of the liver from CCl4-induced fibrogenesis. The number of eight rats in each group was chosen as the sample size in this report. Of course, if increasing the number of animals per group might make our data be more accurate. Our current data demonstrated that TMP significantly reduced the size stained with Sirius red in the liver, and results from examination of the level of hepatic hydroxyproline indicated that the CCl4-elevated levels of hepatic hydroxyproline were significantly reduced by TMP. Moreover, western blot assays showed that the expression of a-SMA, a1(I) procollagen, and fibronectin in the rat fibrotic liver were effectively down-regulated. In addition, our data demonstrated that TMP down-regulated of TGF-bRI and PDGF-bR, the two key receptors transmitting pro-fibrogenic pathways by immunofluorescent. We know that inflammation is commonly associated with hepatic fibrogenesis during chronic liver diseases (24). CCl4 is metabolized in the liver by cytochrome P450 into the free radical CCl3, it attacks hepatocytes and promotes inflammatory responses in the liver (25). In the present study, we confirmed the enhanced inflammatory responses in the CCl4-injured liver demonstrated by significantly increased inflammatory cytokine levels. However, the results from immunofluorescence double staining and western blot showed that TMP protected the liver against CCl4-induced injury by suppressing inflammation in the liver. The latest study expresses that inflammasome activation has been recently recognized to play a central role in the development of chronic hepatic disease (26). Inflammasomes are multi-protein cytoplasmic complexes that serve as pattern recognition receptors (27). NLRP3, the most well studies Nod like receptor, forms a complexes comprised of adaptor proteins such as the apoptosis associated speck like protein (ASC), and the serine protease caspase-1(Casp1). NLRP3 inflammasome activation governs the cleavage and activation of Casp1 resulting inmaturation of effector pro-inflammatory cytokines such as pro-IL-1b and pro-IL-18. Previous studies have demonstrated that inflammasome components were present in HSC, it could regulate a variety of HSC functions, and were required for the development of liver fibrosis (28). Therefore, we
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extrapolated that TMP might ameliorate HSC-driven liver inflammation through depressing NLRP3 inflammasome. We next aimed at elucidating the mechanisms by which TMP regulated the expression of inflammatory cytokine in HSCs. The present study suggested that PDGF-increased expression of inflammatory cytokine were associated with activation of the PDGF-bR/NLRP3/caspase1 pathway in cultured HSCs. Our data further demonstrated that TMP reduction of inflammatory cytokine and critical signaling molecules expression. Next, use of specific inhibitors of key signaling molecules provided evidence that TMP reduction in inflammatory cytokine expression was associated with blockade of PDGF-bR-mediated NLRP3/ caspase1 pathway in PDGF-treated HSCs. It could support the current speculation that interruption of NLRP3 might play a causal role in TMP inhibition of HSC-driven inflammation. Although more analyses are needed to firmly confirm the molecular pathways underlying TMP effects, the present studies highlighted the potential to target PDGF-bR/NLRP3/caspase1 pathway for intervention of inflammation responses during chronic liver injury. TMP is a well-characterized natural product, and its pharmacokinetic profiles have been demonstrated by many studies. It has a favorable bioavailability in vivo and could strengthen the physiological relevance of the TMP concentrations used in the current study. In the present study, we found that TMP could suppress inflammation through PDGF-bR/NLRP3/caspase1 pathway. However, our results do not exclude any other mechanisms involved in the anti-inflammatory of TMP. It as a natural small-molecule compound may have multiple targets and mechanisms within cells. Moreover, whether TMP would suppress inflammation through targeting in Kupffer cells and hepatocytes remain unknown. In summary, the present study demonstrated that TMP ameliorated liver inflammation in vivo. Mechanistic investigations revealed that block PDGF-bR-mediated NLRP3/caspase1 pathway linking to inhibition of inflammatory cytokine expression in HSCs. We anticipate that these novel discoveries could be impulsive for developing TMP as an antifibrotic agent.
Acknowledgements The financial support was from National Natural Science Foundation of China (81270514, 30873424), Doctoral Discipline Foundation of Ministry of Education of China (20103237110010), Project for Supporting Jiangsu Provincial Talents in Six Fields (2009-B-010), and the “Eleven-Five” National Science and Technology Supporting Program (2008BAI51B02).
References [1] Friedman, S. L. (2003) Liver fibrosis – from bench to bedside. J. Hepatol. 38(Suppl 1), S38–S53. [2] Czaja, A. J. and Carpenter, H. A. (2004) Progressive fibrosis during corticosteroid therapy of autoimmune hepatitis. Hepatology 39, 1631–1638.
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[3] Henao-Mejia, J., Elinav, E., Jin, C., Hao, L., Mehal, W. Z., et al. (2012) Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature 482, 179–185. [4] Ganz, M., Csak, T., Nath, B., and Szabo, G. (2011) Lipopolysaccharide induces and activates the Nalp3 inflammasome in the liver. World J. Gastroenterol.17, 4772–4778. [5] Imaeda, A. B., Watanabe, A., Sohail, M. A., Mahmood, S., Mohamadnejad, M., et al. (2009) Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome. J. Clin. Invest. 119, 305–314. [6] Galli, A., Crabb, D., Price, D., Ceni, E., Salzano, R., et al. (2000) Peroxisome proliferator-activated receptor gamma transcriptional regulation is involved in platelet-derived growth factor-induced proliferation of human hepatic stellate cells. Hepatology 31, 101–108. [7] Pinzani, M. (2002) PDGF and signal transduction in hepatic stellate cells. Front. Biosci. 7, d1720–d1726. [8] Friedman, S. L. (2010) Evolving challenges in hepatic fibrosis. Nat. Rev. Gastroenterol. Hepatol. 7, 425–436. [9] Bonner, J. C. (2004) Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor. Rev. 15, 255–273. [10] Zhang, F., Kong, D., Chen, L., Zhang, X., Lian, N., et al. (2014) Peroxisome proliferator-activated receptor-gamma interrupts angiogenic signal transduction by transrepression of platelet-derived growth factor-beta receptor in hepatic stellate cells. J. Cell. Sci. 127, 305–314. [11] Czaja, A. J. (2014) Hepatic inflammation and progressive liver fibrosis in chronic liver disease. World J. Gastroenterol. 20, 2515–2532. [12] Chen, Y. J., Huang, C. S., Wang, F., Gong, J. Y., and Pan, Z. H. (2014) Effect of ligustrazine hydrochloride on coagulation reaction and inflammation reaction in single valve replacement patients with rheumatic heart disease undergoing cardiopulmonary bypass. Chin. J. Integr. Tradit.West. Med. 34, 531–535. [13] Luo, Y. C., Xiang, Q. W., and Wang, Q. (2008) Effect of ligustrazine on expression of RhoA mRNA, ROCK-II protein in the lung and airway inflammation of allergic asthma model mice. Chin. J. Pediatrics 46, 868–869. [14] Zhang, X., Zhang, F., Kong, D., Wu, X., Lian, N., et al. (2014) Tetramethylpyrazine inhibits angiotensin II-induced activation of hepatic stellate cells associated with interference of platelet-derived growth factor beta receptor pathways. FEBS J. 281, 2754–2768. [15] Chen, W., Chen, W. X., Jin, Y., and Lu, Y. M. (2010) Effect of ligustrazine on the expression of TNF-a, IL-4 and IFN-c in rats with liver fibrosis. World Clin. Drug. 31, 153–156. [16] Bachem, M. G., Melchior, R., and Gressner, A. M. (1989) The role of thrombocytes in liver fibrogenesis: effects of platelet lysate and thrombocyte-
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derived growth factors on the mitogenic activity and glycosaminoglycan synthesis of cultured rat liver fat storing cells. J. Clin. Chem. Clin. Biochem. 27, 555–565. Gressner, A. M., Weiskirchen, R., Breitkopf, K., and Dooley, S. (2002) Roles of TGF-beta in hepatic fibrosis. Front. Biosci. 7, d793–807. Marra, F. (2002) Chemokines in liver inflammation and fibrosis. Front. Biosci. 7, d1899–1914. Zhang, F., Zhang, Z., Chen, L., Kong, D., Zhang, X., et al. (2014) Curcumin attenuates angiogenesis in liver fibrosis and inhibits angiogenic properties of hepatic stellate cells. J. Cell. Mol. Med. 18, 1392–1406. Ishikawa, T., Banas, A., Hagiwara, K., Iwaguro, H., and Ochiya, T. (2010) Stem cells for hepatic regeneration: the role of adipose tissue derived mesenchymal stem cells. Curr. Stem. Cell. Res. Ther. 5, 182–189. Solis-Herruzo, J. A., de la Torre, P., and Munoz-Yague, M. T. (2003) Hepatic stellate cells (HSC): architects of hepatic fibrosis. Rev. Esp. Enferm. Dig. 95, 438–439, 436–437. Basu, S. (2003) Carbon tetrachloride-induced lipid peroxidation: eicosanoid formation and their regulation by antioxidant nutrients. Toxicology 189, 113–127. Pinzani, M., Milani, S., Herbst, H., DeFranco, R., Grappone, C., et al. (1996) Expression of platelet-derived growth factor and its receptors in normal human liver and during active hepatic fibrogenesis. Am. J. Pathol. 148, 785– 800. Handa, P. and Kowdley, K. V. (2013) Chemokines: potent mediators of hepatic inflammation and fibrosis in chronic liver diseases. Ann. Hepatol. 13, 152–154. Burk, R. F., Lane, J. M., and Patel, K. (1984) Relationship of oxygen and glutathione in protection against carbon tetrachloride-induced hepatic microsomal lipid peroxidation and covalent binding in the rat. Rationale for the use of hyperbaric oxygen to treat carbon tetrachloride ingestion. J. Clin. Invest. 74, 1996–2001. Negash, A. A., Ramos, H. J., Crochet, N., Lau, D. T., Doehle, B., et al. (2013) IL-1beta production through the NLRP3 inflammasome by hepatic macrophages links hepatitis C virus infection with liver inflammation and disease. PLoS Pathog. 9, e1003330. Martinon, F., Burns, K., and Tschopp, J. (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol. Cell. 10, 417–426. Watanabe, A., Sohail, M. A., Gomes, D. A., Hashmi, A., Nagata, J., et al. (2009) Inflammasome-mediated regulation of hepatic stellate cells. Am. J. Physiol. Gastrointest. Liver. Physiol. 296, G1248–G1257.
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Xiafei Wu1 Feng Zhang1,2,3 Xin Xiong1 Chunfeng Lu1 Naqi Lian1 Yin Lu1,2,3 Shizhong Zheng1,2,3*
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Department of Pharmacology, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China 2 National First-Class Key Discipline for Traditional Chinese Medicine of Nanjing University of Chinese Medicine, Nanjing, China 3 Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Material Medical, Nanjing University of Chinese Medicine, Nanjing, China
Abstract Hepatic fibrosis is concomitant with liver inflammation, which has been highlighted as significant treatment of chronic liver disease. We previously demonstrated that tetramethylpyrazine (TMP), the effective component of Ligusticum chuanxiong Hort, can inhibit the activation of HSCs and consequential antihepatic fibrosis. In this study, our work demonstrated that TMP improved liver histological architecture, decreased hepatic enzyme levels and attenuated collagen deposition in the rat fibrotic liver. In addition, TMP significantly protected the liver from CCl4-caused injury and fibrogenesis by suppressing inflammation with reducing levels of inflammatory
cytokines, including tumor necrosis factor-a (TNF-a), NLRP3, nuclear factor-kappa B (NF-jB) and interleukin-1b (IL-1b). Experiments in vitro showed that TMP inhibited inflammatory cytokine expression in HSCs associated with disrupting platelet-derived growth factor-b receptor (PDGF-bR)/NLRP3/ caspase1 pathway. These data collectively indicate that TMP can attenuate liver inflammation in liver fibrosis and possibly by targeting HSCs via PDGF-bR/NLRP3/caspase1 pathway. It provides novel mechanistic insights into TMP as a potential C 2015 IUBMB Life, therapeutic remedy for hepatic fibrosis. V 67(4):312–321, 2015
Keywords: tetramethylpyrazine; liver fibrosis; NLRP3 inflammasome; inflammation; hepatic stellate cells
Introduction Hepatic fibrosis represents a frequent event following various kinds of etiological factor in the liver, including autoimmune liver diseases, viral infection, and cholestasis. Firstly manifes-
C 2015 International Union of Biochemistry and Molecular Biology V
Volume 67, Number 4, April 2015, Pages 312–321 *Address correspondence to: Shizhong Zheng, Department of Pharmacology, College of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, China. Tel: 186-25-85811245. Fax: 186-25-86798188. E-mail: [email protected] Received 24 September 2014; Accepted 7 January 2015 DOI 10.1002/iub.1348 Published online 3 April 2015 in Wiley Online Library (wileyonlinelibrary.com)
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tations of liver inflammation, then quiescent Hepatic stellate cells (HSC) become activated and transdifferentiate into myofibroblast-like cells. It is characterized by excessive production and deposition of extracellular matrix (ECM) molecules (1). Recently, with the deepening research on hepatic fibrosis, it has been confirmed that in the development of fibrosis accompanies large amounts of inflammatory mediators and reactive oxygen species, including tumor necrosis factor-a (TNF-a), transforming growth factor-b (TGF-b), interleukin-1b (IL-1b) interleukin-6 (IL-6), interleukin-18 (IL-18) and so on. The persistence of this inflammation will cause progressive hepatic fibrosis and the development of cirrhosis (2). Chronic liver inflammation stimulates the progression of hepatic fibrosis, which can be weakened by immunosuppressive and anti-inflammatory therapies. In recent years, the role of NLRP3 inflammasome in hepatic injury is now attracting
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widespread attention. Inflammasomes are intracellular multiprotein complexes that act as platforms for the maturation and secretion of the proinflammatory cytokines. Over the past decades, many studies have focused on the role that the NLRP3 inflammasome has in the innate immune system as a sensor of pathogens and danger signals, and its activation mechanism has been widely investigated. It has been confirmed that the activation of NLRP3 is closely related with the congenital and acquired inflammatory disease, such as autoinflammatory disease, gout and type 2 diabetes mellitus. Research has demonstrated that unrestrained NLRP3 activation can results in severe liver inflammation, characterized by a predominantly neutrophilic infiltrate and HSC-activation with collagen deposition in the liver. However, the precise molecular mechanisms remain to be defined (3–5). Classic angiogenic mediators platelet-derived growth factor (PDGF) is the most potent mitogen of HSC and is likely to be an important mediator of the increased proliferation of the cells during the hepatic wound healing response (6,7), it can drive fibrogenic responses and may also foster a milieu that is permissive for the development of HCC (8). Previous research has shown that during hepatic fibrogenesis, PDGF is secreted by a variety of cell types as a response to injury, and many pro-inflammatory cytokines mediate their mitogenic effects via the autocrine release of PDGF (9). Moreover, previous research from our laboratory study provided in vitro evidence that PDGF could play a key role in HSC-based angiogenesis to promote hepatic fibrosis (10). Basic and clinical evidence has indicated the tight association between attenuation of liver inflammation and regression of liver fibrosis impulsive for development of potential antifibrotic therapies (11). Therefore, research identifying anti-inflammatory agents of liver that are innocuous is urgently needed. Recently, the natural product tetramethylpyrazine (TMP) has been reported to anti-inflammation reaction of patients with rheumatic heart disease (12). And animal studies also demonstrated that TMP played an anti-inflammation role in allergic asthma mice (13), suggesting that TMP as a potential therapeutics for anti-inflammation reaction. We previously reported that TMP can inhibit the activation of HSCs caused by Ang II in order to anti-hepatic fibrosis (14). Moreover, our latest investigations also showed that TMP can improve liver histology and attenuate fibrogenesis by down-regulating inflammatory cytokines (15). However, the underlying molecular mechanism is largely unknown. The current study established a CCl4-caused fibrosis model in rats to examine the relevance of inflammation attenuation to liver fibrosis reduction by TMP. And then the mechanisms by which TMP affected the inflammatory mediators expression in HSCs were elucidated. The obtained results provided novel insight into the mechanisms of TMP reduction in hepatic fibrosis.
Materials and Methods Reagents and Antibodies TMP was purchased from Sigma (St Louis, MO). Recombinant rat PDGF-BB was from Cell Sciences (Canton, MA). This cyto-
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kine was referred to as PDGF throughout the study. PDGF-bR blocker Imatinib was from Nanjing EnoGene Biotechnology (Nanjing, China). NLRP3 inhibitor Glyburide (Gly) was from CALBIOCHEM (Shanghai, China). Primary antibodies against aSMA, a1(I) procollagen, and fibronectin were from Epitomics (San Francisco, CA). Primary antibody against PDGF-bR was from Epitomics (San Francisco, CA). The primary antibodies against cleaved-IL-1b was purchased from Cell Signaling Technology (Danvers, MA). Primary antibody against NLRP3 was from Novus Biologicals (Littleton, CO). Primary antibody against caspase1 was from Millipore (Burlington, MA). The primary antibodies against pro-IL-1b and pro-IL-18 were from Bioworld Technology (Nanjing, China). MSU was purchased from Sigma (St Louis, MO).
Animal Procedures and Treatments All experimental procedures were approved by the institutional and local committee on the care and use of animals of Nanjing University of Chinese Medicine (Nanjing, China), and all animals received humane care according to the National Institutes of Health (USA) guidelines. Male Sprague-Dawley rats (180–220 g bodyweight) were obtained from Nanjing Medical University (Nanjing, China). A mixture of CCl4 (0.1 mL/100 g body weight) and olive oil [1:1 (w/v)] was used to induce liver fibrosis in rats. Forty-eight rats were randomly divided into six groups (eight rats per group). Group 1 was the vehicle control in which rats were not administrated CCl4 or TMP but intraperitoneally (i.p.) injected with olive oil. Group 2 was the CCl4 group in which rats were i.p. injected with CCl4 without TMP treatment. Groups 3, 4, and 5 were treatment groups in which rats were i.p. injected with CCl4 and orally given TMP at 50, 100, and 200 mg/kg, respectively. Group 6 was the positive control in which rats were injected with CCl4 and treated with colchicine (Yifeng Pharmacy, Nanjing, China). Rats in groups 2 to 6 were i.p. injected with CCl4 every other day for 8 weeks. TMP was suspended in sterile phosphate buffered saline (PBS) and given once daily by gavage during weeks 5 to 8. Colchicine was suspended in sterile PBS and given once daily by gavage (0.1 mg/kg) during weeks 5 to 8. At the end of the experiment, rats were sacrificed after being anesthetized by i.p. pentobarbital (50 mg/kg). Blood was collected, and livers were isolated for calculation of liver/bodyweight ratio. A small portion of the liver was removed for histopathological and immunohistochemical studies by fixation with 10% formalin and subsequent embedment with paraffin. The remaining liver was cut inpieces and rapidly frozen with liquid nitrogen for extraction of total RNA and hepatic proteins.
Liver Histopathology Harvested liver tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Liver slices of 5 lm thick were prepared and stained with hematoxylin and eosin and masson’ strichrome stain by using standard methods. For sirius red collagen staining, thin sections were deparaffinized and stained with picro-sirius red for 1 h at room temperature. After washes, sections on the slides were dehydrated in100%
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ethanol and in xylene, and then they were mounted in Permount. Photographs were taken in a blinded fashion at random fields. Representative views of liver sections are shown.
Hydroxyproline Examination The hydroxyproline levels in liver tissue and blood were determined by using a kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the protocol. Briefly, three small pieces of liver tissues randomly excised from the liver of every rat were hydrolyzed in 6 N HCl at 110 C for 24 h, and subsequently they were neutralized with NaOH. Isopropanol in citrate acetate-buffered chloramine T was added to aliquots of the hydrolysate, followed by the addition of Ehrlich reagent. The chemical reaction occurred in dark for 25 min at 60 C. After centrifugation, absorbance of the supernatant of each sample was read at 558 nm by using a 96-well plate spectrometer. Trans-hydroxyproline was used as the standard for quantification. Values were normalized to control.
Immunofluorescence Double Staining After deparaffinization, thin sections (5 lm) of the liver tissues (liver lobules and portal area) were blocked with 1% bovine serum albumin, and then they were incubated with primary antibodies overnight at 4 C. After three washes with PBS, sections on slides were incubated with secondary antibodies at room temperature for 1 h. Sections incubated with secondary antibodies alone were used as negative controls. Sections were viewed in a single plane under an MRC 1024 laser confocal microscope (Bio-Rad Laboratories). Representative views of liver sections with positive staining are shown.
Cell Isolation and Culture Conditions Primary HSCs were isolated from male Sprague-Dawley rats (200–250 g; Nanjing Medical University, Nanjing, China) according to a described protocol. All experimental procedures were approved by the institutional and local committee on the care and use of animals of Nanjing University of Chinese Medicine (Nanjing, China), and all animals received humane care according to the National Institutes of Health (USA) guidelines. All rats were maintained under a 12 h light/dark cycle at a controlled temperature (25 C) with free access to food and tap water. HSCs were cultured in DMEM (Invitrogen, Grand Island, NY) with 10% fetal bovine serum (Sijiqing Biological Engineering Materials, Hang Zhou, China), 1% antibiotics, and maintained at 37 C in a humidified atmosphere of 5% CO2 and 95% air. HSCs at passages 2 to 4 were used in experiments.
Real-Time PCR RNA isolation and real-time PCR were performed as we previously described (19). Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the invariant control. The following primers were used: pro-IL-1b(forward) 50 -GGCTTCCT TGTGCAAGTGTC-3, (reverse) 50 -TGTCGAGATGCTGCTG’TGAG3; pro-IL-18(forward) 50 -TTGACAAAAGAAACCCGCCT-3, (reverse) 50 -CCTGGCACACGTTTCTGAAA-30 ; GAPDH (forward) 50 -GACAT CAAGAAGGTGGTGAAGC-3, (reverse) 50 -TGTCATTGAGAGCAA TGCCAGC-3.
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Western Blot Analyses Whole-cell lysates were prepared using radioimmunoprecipitation analyses buffer supplemented with protease and/or phosphatase inhibitors. The protein levels were determined using a BCA assay kit (Pierce). Proteins (50 lg/well) were separated by SDS-polyacrylamide gel, transferred to a PVDF membrane (Millipore, Burlington, MA), blocked with 5% skim milk in Trisbuffered saline containing 0.1% Tween 20. Target proteins were detected by corresponding primary antibodies, and subsequently by horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using chemiluminescence reagent (Millipore, Burlington, MA). Equivalent loading was confirmed using an antibody against b-actin. The levels of target protein bands were densitometrically determined using Quantity Ones 4.4.1 (Bio-Rad Laboratories, Berkeley, CA). The variation in the density of bands was expressed as fold changes compared with the control in the blot after normalized to b-actin or the total protein in some experiments.
Enzyme-Linked Immunosorbent Assay (ELISA) The levels of each factor in liver tissues, blood and HSC culture supernatant were determined with an ELISA kit (Nanjing Jiancheng Bioengineering Institute) according to the protocol. For the experiments in vitro, HSCs (500,000 cells/well) were seeded in six-well plates and cultured in DMEM with 10% FBS for 24 h and then in low-serum medium (0.5%, v/v) for an additional 12 h. Hepatic stellate cells were treated with various reagents as indicated in specific experiments for 24 h. Briefly, samples of 100 lL were added to each well of the 96-well plates coated with antibody, followed by incubation for 2 h at room temperature. Working detector solution of 100 lL was loaded into each well, and the plates were incubated for an additional 1 h at room temperature before the addition of substrate solution of 100 lL. The reaction was stopped by adding stop solution of 50 lL. The absorbance was read at 450 nm wavelength. Values were normalized to control.
Statistical Analysis Data were presented as mean 6 SD, and results were analyzed using SPSS16.0 software. The significance of difference was determined by one-way ANOVA with the post hoc Dunnett’s test. Values of P < 0.05 were considered to be statistically significant.
Results TMP Protects the Liver Against CCl4-Induced Injury and Suppresses Hepatic Fibrogenesis in the Rat Model We initially evaluated the TMP effect on liver fibrotic injury in vivo. Gross examination showed that morphological changes pathologically occurred in the rat liver with CCl4 injection compared with the normal liver, but treatment with TMP improved the pathological changes in livers, and that colchicine as a positive control also effectively protected the rat liver from CCl4induced injury (Fig. 1A). TMP treatment and colchicine also significantly reduced the liver/body weight ratio, which was
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significantly elevated by CCl4 injection (Fig. 1A). CCl4 causes hepatic injury, including hepatocytic necrosis, steatosis, and inflammation. The effects of TMP on the protection of the liver from injury and fibrogenesis were initially evaluated by histological analyses. HE staining showed that TMP treatment resulted in remarkable improvement in liver histology evidenced by the ameliorated state of hepatic steatosis, necrosis, and fibrotic septa in the liver. To assess the impact of TMP on hepatic fibrogenesis caused by CCl4, liver sections were stained with Masson and picro-Sirius red for detecting the deposition of collagens, the results showing that collagens were severely deposited in the CCl4-injured liver accompanied by nodular formation, but were reduced by TMP dose-dependently in the liver of rats treated with TMP (Fig. 1B). Hydroxyproline is an amino acid found almost exclusively in collagens. Measurement of hepatic and blood hydroxyproline further indicated that collagen production was reduced by TMP in rats with liver fibrosis (Fig. 1C). Additional biochemical analyses of serum enzymes were performed to verify the role of TMP in the protection of the liver from injury. Our data demonstrated that TMP reduced serum levels of alkaline phosphatase, ALT and AST dose dependently in CCl4-treated rats (Fig. 1D). Furthermore, we examined the transcript and protein abundance of a-SMA, a (1) procollagen and fibronectin, three key markers of liver fibrosis. The results showed that TMP down-regulated the three markers at protein levels (Fig. 1E). As platelet-derived growth factor-b receptor (PDGF-bR) and transforming growth factor-b receptor 1 (TGFbRI) are the two key receptors transmitting pro-fibrogenic pathways (16,17), it was plausible to examine the effect of these two receptors to elucidate the underlying mechanisms of TMP in the suppression of hepatic fibrogenesis in the rat model. Immunofluorescent assay showed that TMP down-regulated of TGF-bRI and PDGF-bR (Fig. 1F). All these results confirmed that TMP suppressed hepatic fibrogenesis in the rat model.
TMP Suppresses Inflammation in the CCl4 Rat Model Inflammation is commonly associated with hepatic fibrogenesis during chronic liver diseases (18). To begin to explore the mechanisms underlying the protective effects of TMP, we proposed that TMP might protect the liver against CCl4-induced injury by suppressing inflammation in the liver. As shown in immunofluorescent assay, TMP decreased the expression of proinflammatory signal molecules TNF-a, NLRP3, NF-jB and IL-1b (Fig. 2A). Moreover, Elisa assays also demonstrated that liver and serum levels of TNF-a, IL-1b, IL-18, IL-6 and IL-8 were decreased by TMP dose-dependently (Fig. 2B). Further experiments demonstrated that the elevated expression of TNF-a, NF-jB, NLRP3, IL-1b in fibrotic liver was abolished by TMP at protein levels (Fig. 2C). These results indicated that TMP suppressed inflammation caused by CCl4, which might lead to the protection of the liver from injury.
TMP Inhibits the Expression of Proinflammatory Cytokine in HSCs Treated with PDGF To gain more insight into the effects and underlying mechanisms of TMP on the expression of proinflammatory cytokine
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of HSC, we extended our studies using PDGF, a potent mitogenic molecule responsible for HSC activation (9).In our PDGF experiments mimicking the in vivo status of HSCs, we observed that, PDGF up-regulated the expression of inflammatory cytokine (pro-IL-1b, pro-IL-18 and cleaved-IL-1b) dose-dependently (Figs. 3A–3C). Next, cell viability assay provided a rationale for TMP doses used in subsequent in vitro experiments. We found that TMP does-dependently inhibited PDGF-stimulated proliferation in HSCs and at 30 lM resulted in a significant inhibitory effect (Fig. 3D). Then, western blot assays demonstrated that TMP reduced the expression of inflammatory cytokine (pro-IL1b, pro-IL-18 and cleaved-IL-1b) dose-dependently (Fig. 3E). This result was confirmed by measuring inflammatory cytokine levels in HSC supernatant (Fig. 3F) and immunofluorescence (Fig. 3G).
TMP Reduces the Expression of Inflammatory cytokine Associated with Interference of the PDGF-bR-Mediated NLRP3/Caspase1 Pathway in HSCs Treated with PDGF Previous studies have shown that NLRP3 activation can results in severe liver inflammation (4). We postulated that PDGF-bR/ NLRP3/caspase1 pathway could be involved in TMP effect. Our current data demonstrated that the PDGF-enhanced phosphorylation of PDGF-bR and its downstream signal molecules NLRP3 and cleaved-caspase1, but inhibiting the expression of pro-caspase1 (Figs. 4A and 4B). Next, we examined the expression of theses signaling molecules when treated with TMP. Western blot (Figs. 4C and 4D) and immunofluorescence (Figs. 4E and 4F) showed they were diminished by TMP dosedependently, suggesting disruption of the pathway. Subsequently, PDGF-bR inhibitor Imatinib and NLRP3 inhibitor Gly were used to establish the link between TMP disruption of the pathway and reduction in inflammatory cytokine expression. MTS assays indicated appropriate doses of Gly at which cell viability was not affected but intracellular signaling events may be modulated (Fig. 4G), and the doses of Imatinib (10 lM) was determined from previous studies of our laboratory (19). Western blot assays showed that the TMP at the selected doses significantly abolished PDGF-increased p-PDGF-bR expression similar to Imatinib function (Fig. 4H). And it can also reduce NLRP3, cleaved-caspase1 and inflammatory cytokine protein expression mimicking the Gly and Imatinib effect (Fig. 4I). Finally, we found that NLRP3 agonist MSU can abolished the inhibitory effects of TMP on the key molecules in NLRP3 pathway (Fig. 4J). These data indicated that TMP reduced the expression of inflammatory cytokine associated with disruption of PDGF-bR/NLRP3/caspase1 pathway in activated HSC.
Discussion Hepatic fibrosis is a common outcome of a variety of chronic liver diseases, if without effective treatment, it can lead to severe hepatic dysfunctions and even life-threatening conditions such as liver cirrhosis and hepatocarcinoma (20).
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FIG 1
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TMP protects the rat liver from CCl4-induced injury. Rats were grouped as follows: group 1, vehicle control (no CCl4, no treatment); group 2, model group (with CCl4, no treatment); group 3, TMP (50 mg/kg) and CCl4-treated group; group 4, TMP (100 mg/kg) and CCl4-treated group; group 5, TMP (200mg/kg) and CCl4-treated group; group 6, colchicine (0.1 mg/kg) and CCl4-treated group. (A) Gross examination of rat livers with calculation of the liver/body weight ratio. Representative photographs are shown. (B) Liver sections were stained with hematoxylin and eosin, masson reagents and sirius red. Representative photographs are shown. (C) Measurement of hydroxyproline levels in liver and blood. (D) Determination of serum ALT, AST and ALP levels. (E) Western blot analyses of a-SMA, a(I)procollagen and fibronectin in liver tissues. (F) Liver sections were stained with immunofluorescence by using antibodies against TGF-bR1 and PDGF-bR. Representative blots were from three independent experiments. For the statistics of each panel in this figure, data are expressed as mean 6 SD (n 5 8/group); ## P < 0.01 versus group 1, ###P < 0.001 versus group 1, *P < 0.05 versus group 2, **P < 0.01 versus group 2, ***P < 0.001 versus group 2.
Liver Fibrosis and Inflammatory Cytokine Expression
FIG 2
TMP suppresses inflammation in rats with CCl4-caused fibrosis. Rats were grouped as follows: group 1, vehicle control (no CCl4, no treatment); group 2, model group (with CCl4, no treatment); group 3, TMP (50 mg/kg) and CCl4-treated group; group 4, TMP (100 mg/kg) and CCl4-treated group; group 5, TMP (200 mg/kg) and CCl4-treated group; group 6, colchicine (0.1 mg/kg) and CCl4-treated group. (A) Liver sections were stained with immunofluorescence by using antibodies against TNF-a, NLRP3, NF-KB and IL-1b. (B) ELISA measurement of TNF-a, IL-1b, IL-18, IL-6 and IL-8 levels in liver and serum. (C) Western blot analyses of TNF-a, NF-KB, NLRP3 and IL-1b. Representative blots were from three independent experiments. For the statistics of each panel in this figure, data are expressed as mean 6 SD (n 5 8/group); ##P < 0.01 versus group 1, ###P < 0.001 versus group 1, *P < 0.05 versus group 2, **P < 0.01 versus group 2.
Therefore, to explore the pathogenesis of liver fibrosis has attracted extensive attention from scholars at home and abroad. As HSCs are the key fibrogenic element in response to liver fibrogenesis, intervention of HSC biological behaviors can effectively prevent and treat liver fibrosis. Current researches suggest that hepatic inflammation is only one driver of hepatic fibrosis. In chronic liver injury, inflammatory cytokines can stimulate the activation and proliferation of HSC in order to promote the deposition of ECM. Conversely, HSCs exposured to inflammatory microenvironment can autocrine inflammatory factors to enhance the migration ability of HSCs and Inflammatory cells to the injury area, which exaggerate the hepatic fibrosis (21). In the current study, we evaluated the antifibrotic
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efficacy of TMP, the effective component of Ligusticum chuanxiong Hort, employing a classical animal model of liver fibrosis that causes necrosis of hepatocytes and induces inflammation (22). We herein demonstrated that TMP effectively improved liver histology, decreased serum levels of hepatic enzymes, and inhibited collagen production in the rat liver with injury and fibrogenesis caused by CCl4. In addition, we clearly showed that TMP inhibited inflammation in rat fibrotic liver. Examination of several key inflammatory cytokine downregulation suggesting that TMP inhibited the proinflammatory signaling pathways that aggravate liver inflammation. Moreover, previous investigations showed that PDGF is the most potent mitogen of HSC, it is released in high concentrations
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FIG 3
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TMP inhibits the expression of proinflammatory cytokine in HSCs treated with PDGF. HSCs were treated with DMSO (0.02%, w/ v) and PDGF for 24 h. (A–C) Real-time PCR analyses of pro-IL-1b and pro-IL-18 mRNA, *P < 0.05 versus control, **P < 0.01 versus control, ***P < 0.001 versus control (A). Western blot analyses of pro-IL-1b, pro-IL-18 and cleaved-IL-1b protein expression with densitometry, **P < 0.01 versus control, ***P < 0.001 versus control (B). ELISA measurement of cleaved-IL-1b and cleaved-IL-18 level in supernatant, *P < 0.05 versus control, **P < 0.01 versus control, ***P < 0.001 versus control (C).(D–G) HSCs were treated with DMSO (0.02%, w/v), PDGF (20 ng/mL) and TMP for 24 h. Cell viability was evaluated by MTS assay, # P < 0.05 versus DMSO, **P < 0.01 versus DMSO1PDGF (D). Western blot analyses of inflammatory cytokine expression, ## P < 0.01 versus DMSO, ###P < 0.001 versus DMSO, *P < 0.05 versus DMSO 1 PDGF, **P < 0.01 versus DMSO 1 PDGF, ***P < 0.001 versus DMSO 1 PDGF (E). ELISA measurement of inflammatory cytokine level in supernatant, ##P < 0.01 versus DMSO, ###P < 0.001 versus DMSO, *P < 0.05 versus DMSO1PDGF, **P < 0.01 versus DMSO 1 PDGF (F).Immunofluorescence by using antibody against cleaved-IL-1b (G). Representative blots were from three independent experiments.
Liver Fibrosis and Inflammatory Cytokine Expression
FIG 4
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TMP interrupts PDGF-bR/NLRP3/caspase1 pathway linking to reduced proinflammatory cytokine expression in HSCs. HSCs were treated with PDGF for 12 h. Western blot analyses were used to examine the p-PDGF-bR and PDGF-bR, ***P < 0.001 versus control (A). HSCs were treated with PDGF for 24 h. Western blot analyses were used to examine the NLRP3/caspase1 cascade *P < 0.05 versus control, **P < 0.01 versus control, ***P < 0.001 versus control (B). HSCs were treated with DMSO (0.02%, w/v) and TMP for 24 h prior to PDGF (20 ng/mL) stimulation for an additional 12 h. Western blot analyses of p-PDGF-bR and PDGF-bR, ###P < 0.001 versus DMSO, *P < 0.05 versus DMSO 1 PDGF, **P < 0.01 versus DMSO 1 PDGF (C). HSCs were treated with DMSO (0.02%, w/v), PDGF (20 ng/mL) and TMP for 24 h. Western blot analyses were used to examine the NLRP3/caspase1 cascade, ##P < 0.01 versus DMSO, ###P < 0.001 versus DMSO, *P< 0.05 versus DMSO 1 PDGF, **P < 0.01 versus DMSO 1 PDGF, ***P < 0.001 versus DMSO 1 PDGF (D). Immunofluorescence by using antibody against NLRP3 and cleaved- caspase1 (E and F). Subsequently, HSCs were treated with Gly for 24 h. Cell proliferation was assessed by an MTS assay, **P < 0.01 versus control, ***P < 0.001 versus control (G). HSCs were treated with DMSO (0.02%, w/v), TMP (20 lM), Gly (150 lM) and Imatinib (10 lM) for 24 h prior to PDGF(20 ng/mL) stimulation for an additional 12 h. Western blot analyses of p-PDGF-bR and PDGF-bR, ### P < 0.001 versus DMSO, ***P < 0.001 versus DMSO 1 PDGF (H). HSCs were treated with DMSO (0.02%, w/v), TMP (20 lM), Gly(150 lM) and Imatinib (10 lM) for 24 h. Western blot analyses were used to examine the NLRP3/caspase1 cascade, ### P < 0.001 versus DMSO, *P < 0.05 versus DMSO 1 PDGF, **P < 0.01 versus DMSO 1 PDGF, ***P < 0.001 versus DMSO1PDGF (I). HSCs were treated with DMSO (0.02%, w/v), TMP (20 lM) and MSU for 24 h. Western blot analyses were used to examine the NLRP3/caspase1 cascade, ###P < 0.001 versus DMSO, **P < 0.01 versus DMSO1PDGF, ***P < 0.001 versus DMSO 1 PDGF, & P < 0.05 versus PDGF 1 TMP, &&P < 0.01 versus PDGF1TMP, &&&P < 0.001 versus PDGF1TMP (J). Representative blots were from three independent experiments.
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during tissue repair and liver inflammatory processes, first from platelet alpha granules and subsequently by activated inflammatory cells, HSCs and Kupffer cells (6). In addition, in liver tissue obtained from patients with chronic liver diseases, expression of PDGF and its receptor subunits appears strictly correlated with the extent of necroinflammation and fibrosis (23). So, the activity of PDGF is closely related to the pathogenesis of liver fibrosis. Therefore, we established an PDGFinduced HSC activation model in vitro, which could mimic the HSC activation during liver inflammation. We then used this cellular model to evaluate the antifibrotic effects of TMP, a natural alkaloid that has been reported to anti-inflammation in the rheumatic heart disease and allergic asthma (12,13). In this report, traditional methods of assessing hepatic fibrosis, such as histopathological analysis, Western blot and hydroxyproline measurements, were performed to determine the effectiveness of TMP on the protection of the liver from CCl4-induced fibrogenesis. The number of eight rats in each group was chosen as the sample size in this report. Of course, if increasing the number of animals per group might make our data be more accurate. Our current data demonstrated that TMP significantly reduced the size stained with Sirius red in the liver, and results from examination of the level of hepatic hydroxyproline indicated that the CCl4-elevated levels of hepatic hydroxyproline were significantly reduced by TMP. Moreover, western blot assays showed that the expression of a-SMA, a1(I) procollagen, and fibronectin in the rat fibrotic liver were effectively down-regulated. In addition, our data demonstrated that TMP down-regulated of TGF-bRI and PDGF-bR, the two key receptors transmitting pro-fibrogenic pathways by immunofluorescent. We know that inflammation is commonly associated with hepatic fibrogenesis during chronic liver diseases (24). CCl4 is metabolized in the liver by cytochrome P450 into the free radical CCl3, it attacks hepatocytes and promotes inflammatory responses in the liver (25). In the present study, we confirmed the enhanced inflammatory responses in the CCl4-injured liver demonstrated by significantly increased inflammatory cytokine levels. However, the results from immunofluorescence double staining and western blot showed that TMP protected the liver against CCl4-induced injury by suppressing inflammation in the liver. The latest study expresses that inflammasome activation has been recently recognized to play a central role in the development of chronic hepatic disease (26). Inflammasomes are multi-protein cytoplasmic complexes that serve as pattern recognition receptors (27). NLRP3, the most well studies Nod like receptor, forms a complexes comprised of adaptor proteins such as the apoptosis associated speck like protein (ASC), and the serine protease caspase-1(Casp1). NLRP3 inflammasome activation governs the cleavage and activation of Casp1 resulting inmaturation of effector pro-inflammatory cytokines such as pro-IL-1b and pro-IL-18. Previous studies have demonstrated that inflammasome components were present in HSC, it could regulate a variety of HSC functions, and were required for the development of liver fibrosis (28). Therefore, we
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extrapolated that TMP might ameliorate HSC-driven liver inflammation through depressing NLRP3 inflammasome. We next aimed at elucidating the mechanisms by which TMP regulated the expression of inflammatory cytokine in HSCs. The present study suggested that PDGF-increased expression of inflammatory cytokine were associated with activation of the PDGF-bR/NLRP3/caspase1 pathway in cultured HSCs. Our data further demonstrated that TMP reduction of inflammatory cytokine and critical signaling molecules expression. Next, use of specific inhibitors of key signaling molecules provided evidence that TMP reduction in inflammatory cytokine expression was associated with blockade of PDGF-bR-mediated NLRP3/ caspase1 pathway in PDGF-treated HSCs. It could support the current speculation that interruption of NLRP3 might play a causal role in TMP inhibition of HSC-driven inflammation. Although more analyses are needed to firmly confirm the molecular pathways underlying TMP effects, the present studies highlighted the potential to target PDGF-bR/NLRP3/caspase1 pathway for intervention of inflammation responses during chronic liver injury. TMP is a well-characterized natural product, and its pharmacokinetic profiles have been demonstrated by many studies. It has a favorable bioavailability in vivo and could strengthen the physiological relevance of the TMP concentrations used in the current study. In the present study, we found that TMP could suppress inflammation through PDGF-bR/NLRP3/caspase1 pathway. However, our results do not exclude any other mechanisms involved in the anti-inflammatory of TMP. It as a natural small-molecule compound may have multiple targets and mechanisms within cells. Moreover, whether TMP would suppress inflammation through targeting in Kupffer cells and hepatocytes remain unknown. In summary, the present study demonstrated that TMP ameliorated liver inflammation in vivo. Mechanistic investigations revealed that block PDGF-bR-mediated NLRP3/caspase1 pathway linking to inhibition of inflammatory cytokine expression in HSCs. We anticipate that these novel discoveries could be impulsive for developing TMP as an antifibrotic agent.
Acknowledgements The financial support was from National Natural Science Foundation of China (81270514, 30873424), Doctoral Discipline Foundation of Ministry of Education of China (20103237110010), Project for Supporting Jiangsu Provincial Talents in Six Fields (2009-B-010), and the “Eleven-Five” National Science and Technology Supporting Program (2008BAI51B02).
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