MicroRNA-326 attenuates hepatic stellate cell activation and liver fibrosis by inhibiting TLR4 signaling

Hepatic fibrosis is a chronic inflammatory and reversible repair reaction of the liver under the continuous action of virus or various injuries. In this study, we aimed at identifying the role of miR-326 in the hepatic stellate cell (HSC) activation and liver fibrosis and its potential mechanism. In this study, the liver fibrosis mouse model was developed by injecting CCl₄. Liver tissue morphology was observed and the expression level of α-smooth muscle actin, collagen1α1 and miR-326 was measured. Target gene identification was performed by loss-of-function and gain-of-function. The effect of miR-326 on the expression level of the cytokines associated with the TLR4/MyD88/nuclear factor-κB (NF-κB) pathway was assessed in vitro and in vivo. We show that miR-326 was downregulated in CCl₄-induced fibrotic mice and activated HSCs. The target gene of miR-326 is TLR4. Moreover, miR-326 inhibited the activation of HSCs in vitro through TLR4/MyD88/NF-κB signaling. miR-326 attenuated hepatic fibrosis and inflammation of CCl₄-induced mice in vivo. Our results demonstrate for the first time that miR-326 inhibits HSC activation through TLR4/MyD88/NF-κB signaling. Furthermore, miR-326 plays critical roles in attenuating liver fibrosis and inflammation, suggesting the therapeutic potential of miRNAs.     

The role of AdipoR1 and AdipoR2 in liver fibrosis

Activation of the adiponectin (APN) signaling axis retards liver fibrosis. However, understanding of the role of AdipoR1 and AdipoR2 in mediating this response is still rudimentary. Here, we sought to elucidate the APN receptor responsible for limiting liver fibrosis by employing AdipoR1 and AdipoR2 knock-out mice in the carbon tetrachloride (CCl₄) model of liver fibrosis. In addition, we knocked down receptor function in primary hepatic stellate cells (HSCs) in vitro. Following the development of fibrosis, AdipoR1 and AdipoR2 KO mice had no quantitative difference in fibrosis by Sirius red staining. However, AdipoR2 KO mice had an enhanced fibrotic signature with increased Col1-α1, TGFß-1, TIMP-1, IL-10, MMP-2 and MMP-9. Knockdown of AdipoR1 or AdipoR2 in HSCs followed by APN treatment demonstrated that AdipoR1 and AdipoR2 did not affect proliferation or TIMP-1 gene expression, while AdipoR2 modulated Col1-α1 and α-SMA gene expression, HSC migration, and AMPK activity. These finding suggest that AdipoR2 is the major APN receptor on HSCs responsible for mediating its anti-fibrotic effects.     

Inhibition of hepatic damage and liver fibrosis by brain natriuretic peptide

Anti-fibrotic and organ protective effects of brain natriuretic peptide (BNP) have been reported. In this study, effects of BNP on liver fibrosis were examined in the carbon tetrachloride (CCl₄)-induced liver fibrosis model using BNP-transgenic (Tg) and wild-type (WT) mice. Twice-a-week intraperitoneal injections of CCl₄ for 8 weeks resulted in massive liver fibrosis, augmented transforming growth factor (TGF)-β₁ and type I procollagen α₁ chain (Col1a1) mRNA expression, and the hepatic stellate cell (HSC) activation in WT mice, all of which were significantly suppressed in Tg mice. These observations indicate that BNP inhibits liver fibrosis by attenuating the activation of HSCs.     

Hepatoprotective Effect of MMP-19 Deficiency in a Mouse Model of Chronic Liver Fibrosis

Liver fibrosis is characterized by the deposition and increased turnover of extracellular matrix. This process is controlled by matrix metalloproteinases (MMPs), whose expression and activity dynamically change during injury progression. MMP-19, one of the most widely expressed MMPs, is highly expressed in liver; however, its contribution to liver pathology is unknown. The aim of this study was to elucidate the role of MMP-19 during the development and resolution of fibrosis by comparing the response of MMP-19-deficient (MMP19KO) and wild-type mice upon chronic liver CCl₄-intoxication. We show that loss of MMP-19 was beneficial during liver injury, as plasma ALT and AST levels, deposition of fibrillar collagen, and phosphorylation of SMAD3, a TGF-ß1 signaling molecule, were all significantly lower in MMP19KO mice. The ameliorated course of the disease in MMP19KO mice likely results from a slower rate of basement membrane destruction and ECM remodeling as the knockout mice maintained significantly higher levels of type IV collagen and lower expression and activation of MMP-2 after 4 weeks of CCl₄-intoxication. Hastened liver regeneration in MMP19KO mice was associated with slightly higher IGF-1 mRNA expression, slightly increased phosphorylation of Akt kinase, decreased TGF-ß1 mRNA levels and significantly reduced SMAD3 phosphorylation. In addition, primary hepatocytes isolated from MMP19KO mice showed impaired responsiveness towards TGF-ß1 stimulation, resulting in lower expression of Snail1 and vimentin mRNA. Thus, MMP-19-deficiency improves the development of hepatic fibrosis through the diminished replacement of physiological extracellular matrix with fibrotic deposits in the beginning of the injury, leading to subsequent changes in TGF-ß and IGF-1 signaling pathways.     

Aldehyde dehydrogenase 2 activation ameliorates CCl4‐induced chronic liver fibrosis in mice by up‐regulating Nrf2/HO‐1 antioxidant pathway

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is critical in the pathogenesis of alcoholic liver cirrhosis. However, the effect of ALHD2 on liver fibrosis remains to be further elucidated. This study aimed to demonstrate whether ALDH2 regulates carbon tetrachloride (CCl₄)‐induced liver fibrosis and to investigate the efficacy of Alda‐1, a specific activator of ALDH2, on attenuating liver fibrosis. ALDH2 expression was increased after chronic CCl₄ exposure. ALDH2 deficiency accentuated CCl₄‐induced liver fibrosis in mice, accompanied by increased expression of collagen 1α1, α‐SMA and TIMP‐1. Moreover, ALDH2 knockout triggered more ROS generation, hepatocyte apoptosis and impaired mitophagy after CCl₄ treatment. In cultured HSC‐T6 cells, ALDH2 knockdown by transfecting with lentivirus vector increased ROS generation and α‐SMA expression in an in vitro hepatocyte fibrosis model using TGF‐β1. ALDH2 overexpression by lentivirus or activation by Alda‐1 administration partly reversed the effect of TGF‐β1, whereas ALDH2 knockdown totally blocked the protective effect of Alda‐1. Furthermore, Alda‐1 administration protected against liver fibrosis in vivo, which might be mediated through up‐regulation of Nrf2/HO‐1 cascade and activation of Parkin‐related mitophagy. These findings indicate that ALDH2 deficiency aggravated CCl₄‐induced hepatic fibrosis through ROS overproduction, increased apoptosis and mitochondrial damage, whereas ALDH2 activation through Alda‐1 administration alleviated hepatic fibrosis partly through activation of the Nrf2/HO‐1 antioxidant pathway and Parkin‐related mitophagy, which indicate ALDH2 as a promising anti‐fibrotic target and Alda‐1 as a potential therapeutic agent in treating CCl₄‐induced liver fibrosis.     

Overexpression of c-myc in hepatocytes promotes activation of hepatic stellate cells and facilitates the onset of liver fibrosis

BackgroundLiver fibrosis is a consequence of chronic liver injury and can further progress to hepatocellular carcinoma (HCC). Fibrogenesis involves activation of hepatic stellate cells (HSC) and proliferation of hepatocytes upon liver injury. HCC is frequently associated with overexpression of the proto-oncogene c-myc. However, the impact of c-myc for initiating pathological precursor stages such as liver fibrosis is poorly characterized. In the present study we thus investigated the impact of c-myc for liver fibrogenesis.MethodsExpression of c-myc was measured in biopsies of patients with liver fibrosis of different etiologies by quantitative real-time PCR (qPCR). Primary HSC were isolated from mice with transgenic overexpression of c-myc in hepatocytes (alb-myc^tg) and wildtype (WT) controls and investigated for markers of cell cycle progression and fibrosis by qPCR and immunofluorescence microscopy. Liver fibrosis in WT and alb-myc^tg mice was induced by repetitive CCl₄ treatment.ResultsWe detected strong up-regulation of hepatic c-myc in patients with advanced liver fibrosis. In return, overexpression of c-myc in alb-myc^tg mice resulted in increased liver collagen deposition and induction of α-smooth-muscle-actin indicating HSC activation. Primary HSC derived from alb-myc^tg mice showed enhanced proliferation and accelerated transdifferentiation into myofibroblasts in vitro. Accordingly, fibrosis initiation in vivo after chronic CCl₄ treatment was accelerated in alb-myc^tg mice compared to controls.ConclusionOverexpression of c-myc is a novel marker of liver fibrosis in man and mice. We conclude that chronic induction of c-myc especially in hepatocytes has the potential to prime resident HSC for activation, proliferation and myofibroblast differentiation.     

The direct profibrotic and indirect immune antifibrotic balance of blocking the cannabinoid 2 receptor

Cannabinoid 2 (CB2) receptors expressed on immune cells are considered to be antifibrogenic. Hepatic stellate cells (HSCs) directly interact with phagocytosis lymphocytes, but the nature of this interaction is obscure. We aimed to study the effects of CB2 receptors on hepatic fibrosis via their role in mediating immunity. Hepatic fibrosis was induced by carbon-tetrachloride (CCl(4)) administration in C57BL/6 wild-type (WT) and CB2 knockout (CB2(-/-)) mice. Irradiated animals were reconstituted with WT or CB2(-/-) lymphocytes. Lymphocytes from na?ve/fibrotic WT animals and healthy/cirrhotic hepatitis C virus were preincubated in vitro with or without CB2 antagonist, evaluated for proliferation and apoptosis, and then cocultured with primary mouse HSCs or a human HSC line (LX2), respectively. Lymphocyte phagocytosis was then evaluated. Following CCl(4)-administration, CB2(-/-) mice developed significant hepatic fibrosis but less necroinflammation. WT mice harbored decreased liver CD4(+) and NK(+) cells but increased CD8(+) subsets. Na?ve CB2(-/-) mice had significantly decreased T cell subsets. Adoptive transfer of CB2(-/-) lymphocytes led to decreased fibrosis in the irradiated WT recipient compared with animals receiving WT lymphocytes. Moreover, necroinflammation also tended to decrease. In vitro, a CB2-antagonist directly increased human HSC activation and increased apoptosis and decreased proliferation of mice/human T cells (healthy/fibrotic) and their phagocytosis. We concluded that CB2(-/-) lymphocytes exert an antifibrotic activity, whereas lack of CB2 receptor in HSCs promotes fibrosis. These findings broaden our understanding of cannabinoid signaling in hepatic fibrosis beyond their activity solely in HSCs.     

Induction of liver fibrosis by CCl4 mediates pathological alterations in the spleen and lymph nodes: The potential therapeutic role of propolis

In an animal models, carbon tetrachloride (CCl₄) is a carcinogenic agent that causes liver fibrosis. The current study aims to investigate whether induction in liver-fibrosis by CCl₄ in the mouse model could promote the initiation of fibrosis in lymph node and spleen due to sustained increase of inflammatory signals and also aimed to clarify the protective therapeutic effects of propolis. The male mice (BALB/c) were categorized into three experimental sets and each group involved 15 mice. Control group falls into first group; group-II and group-III were injected with CCl₄ to induce liver-fibrosis and oral supplementation with propolis was provided in group-III for 4-weeks. A major improvement with hepatic collagen and α-smooth muscle actin (α-SMA) production was aligned with the activation of liver fibrosis from CCl₄. Mice treated with CCl₄ exhibited collagen deposition towards liver sections, pathological alterations in spleen and lymph node architectures, and a significantly increase the circulation of both T&B cells in secondary lymphoid organs. Mechanically, the secondary lymphoid organs treated with CCl₄ in mice exposed a positive growth in α-SMA and collagen expression, increased in proinflammatory cytokine levels and a significant increase in TGF-β, NO and ROS levels. A manifest intensification in the expression of Nrf2, COX-2, and eNOS and upregulation of ASK1 and P38 phosphorylation. Interestingly, addition of propolis-treated CCl₄ mice, substantially suppressed deposition of liver collagen, repealed inflammatory signals and resorted CCl₄-mediated alterations in signaling cascades, thereby repairing the architectures of the secondary lymphoid organs. Our findings revealed benefits of propolis against fibrotic complications and enhancing secondary lymphoid organ architecture.     

Adenosine 2A Receptor Antagonist Prevented and Reversed Liver Fibrosis in a Mouse Model of Ethanol-Exacerbated Liver Fibrosis

The effect of moderate alcohol consumption on liver fibrosis is not well understood, but evidence suggests that adenosine may play a role in mediating the effects of moderate ethanol on tissue injury. Ethanol increases the concentration of adenosine in the liver. Adenosine 2A receptor (A2AR) activation is known to enhance hepatic stellate cell (HSC) activation and A2AR deficient mice are protected from fibrosis in mice. Making use of a novel mouse model of moderate ethanol consumption in which female C57BL/6J mice were allowed continued access to 2% (vol/vol) ethanol (11% calories) or pair-fed control diets for 2 days, 2 weeks or 5 weeks and superimposed with exposure to CCl₄, we tested the hypothesis that moderate ethanol consumption increases fibrosis in response to carbon tetrachloride (CCl₄) and that treatment of mice with an A2AR antagonist prevents and/or reverses this ethanol-induced increase in liver fibrosis. Neither the expression or activity of CYP2E1, required for bio-activation of CCl₄, nor AST and ALT activity in the plasma were affected by ethanol, indicating that moderate ethanol did not increase the direct hepatotoxicity of CCl₄. However, ethanol feeding enhanced HSC activation and exacerbated liver fibrosis upon exposure to CCl_4. This was associated with an increased sinusoidal angiogenic response in the liver. Treatment with A2AR antagonist both prevented and reversed the ability of ethanol to exacerbate liver fibrosis.

Conclusion

Moderate ethanol consumption exacerbates hepatic fibrosis upon exposure to CCl₄. A2AR antagonism may be a potential pharmaceutical intervention to decrease hepatic fibrosis in response to ethanol.     

TNFR1-mediated signaling is important to induce the improvement of liver fibrosis by bone marrow cell infusion

The importance of TNF-α signals mediated by tumor necrosis factor receptor type 1 (TNFR1) in inflammation and fibrosis induced by carbon tetrachloride (CCl₄), and in post-injury liver regeneration including a GFP/CCl₄ model developed as a liver repair model by bone marrow cell (BMC) infusion, was investigated. In mice in which TNFR1 was suppressed by antagonist administration or by knockout, liver fibrosis induced by CCl₄ was significantly decreased. In these mice, intrahepatic macrophage infiltration and TGF-β1 expression were reduced and stellate cell activity was decreased; however, expression of MMP-9 was also decreased. With GFP-positive BMC (TNFR1 wild-type, WT) infusion in these mice, fibrosis proliferation, including host endogenous intrahepatic macrophage infiltration, TGF-β1 expression and stellate cell activity, increased significantly. There was no significant increase of MMP-9 expression. In this study, TNFR1 in hosts had a promoting effect on CCl₄-induced hepatotoxicity and fibrosis, whereas BMC infusion in TNFR1 knockout mice enhanced host-derived intrahepatic inflammation and fibrosis proliferation. These findings differed from those in WT recipient mice, in which improvement in inflammation and fibrosis with BMC infusion had previously been reported. TNFR1-mediated signaling might be important to induce the improvement of liver fibrosis by bone marrow cell infusion.     

Hepatocyte Produced Matrix Metalloproteinases Are Regulated by CD147 in Liver Fibrogenesis

Background

The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC.

Methods

Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl₄ induced liver injury using ãCD147 antibody intervention.

Results

In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl₄ treated mice compared to controls.

Conclusion

We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by future anti-fibrogenic agents.     

Bleomycin-induced lung fibrosis in IL-4-overexpressing and knockout mice

The role of IL-4 in the development of lung fibrosis is as yet unclear. Bleomycin (Bleo) or saline (Sal) was injected intratracheally into three groups of C57BL/6J mice: transgenic animals that overexpressed IL-4 (IL-4 TG, n = 14), mice with a targeted knockout mutation of the IL-4 gene (IL-4 KO, n = 11), and wild-type (WT, n = 13) mice. At 14 days, lung fibrosis was evaluated by hydroxyproline measurement and by quantitative image analysis of fibrosis fraction and alveolar wall area fraction. Bronchoalveolar lavage cell counts in all Bleo-treated groups demonstrated an increased percentage of lymphocytes with a corresponding decrease in the percentage of macrophages. Comparing Bleo- to Sal-treated controls within each group of mice showed increases in all lung fibrosis parameters in IL-4 KO and WT, but not in any of the parameters in IL-4 TG mice. The severity of Bleo-induced fibrotic response was decreased in overexpressed IL-4 TG compared with IL-4 KO mice. These data negate a critical profibrotic role for IL-4 in Bleo-induced lung fibrosis.     

Toll like receptor 2 knock-out attenuates carbon tetrachloride (CCl4)-induced liver fibrosis by downregulating MAPK and NF-κB signaling pathways

Innate immune signaling associated with Toll-like receptors (TLRs) is a key pathway involved in the progression of liver fibrosis. In this study, we reported that TLR2 is required for hepatic fibrogenesis induced by carbon tetrachloride (CCl₄). After CCl₄ treatment, TLR2^−/− mice had reduced liver enzyme levels, diminished collagen deposition, decreased inflammatory infiltration and impaired activation of hepatic stellate cells (HSCs) than wild type (WT) mice. Furthermore, after CCl₄ treatment, TLR2^−/− mice demonstrated downregulated expression of profibrotic and proinflammatory genes and impaired mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NF-κB) activation than WT mice. Collectively, our data indicate that TLR2 deficiency protects against CCl₄-induced liver fibrosis.     

PGE2 induces apoptosis of hepatic stellate cells and attenuates liver fibrosis in mice by downregulating miR-23a-5p and miR-28a-5p

MicroRNAs (miRNAs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver diseases, whose end stage is hepatic fibrosis, a major global health burden. Pharmacological strategies for prevention or treatment of hepatic fibrosis are still limited, what makes it necessary to establish a better understanding of the molecular mechanisms underlying its pathogenesis. In this context, we have recently shown that cyclooxygenase-2 (COX-2) expression in hepatocytes restricts activation of hepatic stellate cells (HSCs), a pivotal event in the initiation and progression of hepatic fibrosis. Here, we evaluated the role of COX-2 in the regulation of a specific set of miRNAs on a mouse model of CCl₄ and bile duct ligation (BDL)-induced liver fibrosis. Our results provide evidence that COX-2 represses miR-23a-5p and miR-28-5p expression in HSC. The decrease of miR-23a-5p and miR-28-5p expression promotes protection against fibrosis by decreasing the levels of pro-fibrogenic markers α-SMA and COL1A1 and increasing apoptosis of HSC. Moreover, we demonstrate that serum levels of miR-28-5p are decreased in patients with chronic liver disease. These results suggest a protective effect exerted by COX-2-derived prostanoids in the process of hepatofibrogenesis.     

CDH11 promotes liver fibrosis via activation of hepatic stellate cells

Liver fibrosis, an important health condition associated with chronic liver injury that provides a permissive environment for cancer development, is characterized by the persistent deposition of extracellular matrix components that are mainly derived from activated hepatic stellate cells (HSCs). CDH11 belongs to a group of transmembrane proteins that are principally located in adherens junctions. CDH11 mediates homophilic cell-to-cell adhesion, which may promote the development of cirrhosis. The goal of this study was to determine whether CDH11 regulates liver fibrosis and to examine its mechanism by focusing on HSC activation. Here we demonstrate that CDH11 expression is elevated in human and mouse fibrotic liver tissues and that CDH11 mediates the profibrotic response in activated HSCs. Our data indicate that CDH11 regulates the TGFβ-induced activation of HSCs. Moreover, cells from CDH11 deficient mice displayed decreased HSC activation in vitro, and CDH11 deficient mice developed liver fibrogenesis in response to chronic damage induced by CCl₄ administration. In addition, CDH11 expression was positively correlated with liver fibrosis in patients with cirrhosis, and could therefore be a prognostic factor in patients with liver fibrosis. Collectively, our findings demonstrate that CDH11 promotes liver fibrosis by activating HSCs and may represent a potential target for anti-fibrotic therapies.     

Sirtuin3 deficiency exacerbates carbon tetrachloride−induced hepatic injury in mice

Sirtuin3 (SIRT3) plays an important role in maintaining normal mitochondrial function and alleviating oxidative stress. After carbon tetrachloride (CCl₄) administration, the expression of SIRT3 decreased in the liver of mice, which indicated that the SIRT3 might play a crucial role during chemical‐induced acute hepatic injury. To verify the hypothesis, CCl ₄ was given to induce acute hepatic injury in SIRT3 knockout (KO) mice and wild‐type (WT) mice. CCl ₄‐induced liver injury was more severe in SIRT3 KO mice compared with the WT mice. In addition, the oxidative stress induced by CCl ₄ was enhanced in the SIRT3 KO mice. Furthermore, the increased expression of dynamin‐related protein 1 was also aggravated in SIRT3 KO mice after CCl ₄ administration. In conclusion, our study demonstrated that SIRT3 deficiency exacerbated CCl ₄‐induced impairment of the liver in mice, and the mechanism might be related to enhanced oxidative stress.     

Deficiency in myeloid differentiation factor-2 and toll-like receptor 4 expression attenuates nonalcoholic steatohepatitis and fibrosis in mice

Toll-like receptor 4 (TLR4) and its coreceptor, myeloid differentiation factor-2 (MD-2), are key in recognition of lipopolysaccharide (LPS) and activation of proinflammatory pathways. Here we tested the hypothesis that TLR4 and its coreceptor MD-2 play a central role in nonalcoholic steatohepatitis (NASH) and liver fibrosis in nonalcoholic fatty liver disease. Mice of control genotypes and those deficient in MD-2 or TLR4 [knockout (KO)] received methionine choline-deficient (MCD) or methionine choline-supplemented (MCS) diet. In mice of control genotypes, MCD diet resulted in NASH, liver triglycerides accumulation, and increased thiobarbituric acid reactive substances, a marker of lipid peroxidation, compared with MCS diet. These features of NASH were significantly attenuated in MD-2 KO and TLR4 KO mice. Serum alanine aminotransferase, an indicator of liver injury, was increased in MCD diet-fed genotype controls but was attenuated in MD-2 KO and TLR4 KO mice. Inflammatory activation, indicated by serum TNF-α and nictoinamide adenine dinucleotide phosphate oxidase complex mRNA expression and activation, was significantly lower in MCD diet-fed MD-2 KO and TLR4 KO compared with corresponding genotype control mice. Markers of liver fibrosis [collagen by Sirius red and α-smooth muscle actin (SMA) staining, procollagen-I, transforming growth factor-β1, α-SMA, matrix metalloproteinase-2, and tissue inhibitor of matrix metalloproteinase-1 mRNA] were attenuated in MD-2 and TLR4 KO compared with their control genotype counterparts. In conclusion, our results demonstrate a novel, critical role for LPS recognition complex, including MD-2 and TLR4, through NADPH activation in liver steatosis, and fibrosis in a NASH model in mice.     

Participation of miR-200a in TGF-β1-mediated hepatic stellate cell activation

Hepatic stellate cell (HSC) activation is a pivotal event in the initiation and progression of hepatic fibrosis since it mediates transforming growth factor beta 1 (TGF-β1)-driven extracellular matrix (ECM) deposition. MicroRNAs (miRNAs), small non-coding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key factors to regulate cell proliferation, differentiation, and apoptosis. Although the function of miR-200a has been discussed in many cancers and fibrotic diseases, its role in hepatic fibrosis is still poorly understood. The aim of this study is to investigate whether miR-200a could attenuate hepatic fibrosis partly through Wnt/β-catenin and TGF-β-dependant mechanisms. Our study found that the expression of endogenous miR-200a was decreased in vitro in TGF-β1-induced HSC activation as well as in vivo in CCl₄-induced rat liver fibrosis. Overexpression of miR-200a significantly inhibited α-SMA activity and further affected the proliferation of TGF-β1-dependent activation of HSC. In addition, we identified β-catenin and TGF-β2 as two functional downstream targets for miR-200a. Interestingly, miR-200a specifically suppressed β-catenin in the protein level, whereas miR-200a-mediated suppression of TGF-β2 was shown on both mRNA and protein levels. Our results revealed the critical regulatory role of miR-200a in HSC activation and implied miR-200a as a potential candidate for therapy by deregulation of Wnt/β-catenin and TGFβ signaling pathways, at least in part, via decreasing the expression of β-catenin and TGF-β2.