Treatment with 4-Methylpyrazole Modulated Stellate Cells and Natural Killer Cells and Ameliorated Liver Fibrosis in Mice

MethodsLiver fibrosis was induced by intraperitoneal injections of carbon tetrachloride (CCl₄) or bile duct ligation (BDL) for two weeks. To inhibit ADH3-mediated retinol metabolism, 10 μg 4-methylpyrazole (4-MP)/g of body weight was administered to mice treated with CCl₄ or subjected to BDL. The mice were sacrificed at week 2 to evaluate the regression of liver fibrosis. Liver sections were stained for collagen and α-smooth muscle actin (α-SMA). In addition, HSCs and NK cells were isolated from control and treated mice livers for molecular and immunological studies.ResultsTreatment with 4-MP attenuated CCl₄- and BDL-induced liver fibrosis in mice, without any adverse effects. HSCs from 4-MP treated mice depicted decreased levels of retinoic acids and increased retinol content than HSCs from control mice. In addition, the expression of α-SMA, transforming growth factor-β1 (TGF-β1), and type I collagen α1 was significantly reduced in the HSCs of 4-MP treated mice compared to the HSCs from control mice. Furthermore, inhibition of retinol metabolism by 4-MP increased interferon-γ production in NK cells, resulting in increased apoptosis of activated HSCs.ConclusionsBased on our data, we conclude that inhibition of retinol metabolism by 4-MP ameliorates liver fibrosis in mice through activation of NK cells and suppression of HSCs. Therefore, retinol and its metabolizing enzyme, ADH3, might be potential targets for therapeutic intervention of liver fibrosis.     

A Ribosomal S-6 Kinase–Mediated Signal to C/EBP-β Is Critical for the Development of Liver Fibrosis

Background

In response to liver injury, hepatic stellate cell (HSC) activation causes excessive liver fibrosis. Here we show that activation of RSK and phosphorylation of C/EBPβ on Thr217 in activated HSC is critical for the progression of liver fibrosis.

Methodology/Principal Findings

Chronic treatment with the hepatotoxin CCl₄ induced severe liver fibrosis in C/EBPβ^+/+ mice but not in mice expressing C/EBPβ-Ala217, a non-phosphorylatable RSK-inhibitory transgene. C/EBPβ-Ala217 was present within the death receptor complex II, with active caspase 8, and induced apoptosis of activated HSC. The C/EBPβ-Ala217 peptides directly stimulated caspase 8 activation in a cell-free system. C/EBPβ^+/+ mice with CCl₄-induced severe liver fibrosis, while continuing on CCl₄, were treated with a cell permeant RSK-inhibitory peptide for 4 or 8 weeks. The peptide inhibited RSK activation, stimulating apoptosis of HSC, preventing progression and inducing regression of liver fibrosis. We found a similar activation of RSK and phosphorylation of human C/EBPβ on Thr266 (human phosphoacceptor) in activated HSC in patients with severe liver fibrosis but not in normal livers, suggesting that this pathway may also be relevant in human liver fibrosis.

Conclusions/Significance

These data indicate that the RSK-C/EBPβ phosphorylation pathway is critical for the development of liver fibrosis and suggest a potential therapeutic target.     

Brivanib Attenuates Hepatic Fibrosis In Vivo and Stellate Cell Activation In Vitro by Inhibition of FGF,VEGF and PDGF Signaling

MethodsIn vivo, we induced liver fibrosis by bile duct ligation (BDL), chronic carbon tetrachloride (CCl₄), and chronic thioacetamide (TAA) administration. Liver fibrosis was examined by immunohistochemistry and Western immunoblotting. In vitro, we used LX-2 human hepatic stellate cells (HSCs) to assess the effect of brivanib on stellate cell proliferation and activation.ResultsAfter in vivo induction with BDL, CCl₄, and TAA, mice treated with brivanib showed reduced liver fibrosis and decreased expression of collagen Iα1 and α-smooth muscle actin in the liver. In vitro, brivanib decreased proliferation of HSCs induced by platelet-derived growth factor (PDGF), VEGF, and FGF. Brivanib also decreased stellate cell viability and inhibited PDGFBB-induced phosphorylation of its cognate receptor.ConclusionBrivanib reduces liver fibrosis in three different animal models and decreases human hepatic stellate cell activation. Brivanib may represent a novel therapeutic approach to treatment of liver fibrosis and prevention of liver cancer.     

Protective Effects of Polydatin from Polygonum cuspidatum against Carbon Tetrachloride-Induced Liver Injury in Mice

Polydatin is one of main compounds in Polygonum cuspidatum, a plant with both medicinal and nutritional value. The possible hepatoprotective effects of polydatin on acute liver injury mice induced by carbon tetrachloride (CCl₄) and the mechanisms involved were investigated. Intraperitoneal injection of CCl₄ (50 µl/kg) resulted in a significant increase in the levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and hepatic malondialdehyde (MDA), also a marked enhancement in the expression of hepatic tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and nuclearfactor-kappa B (NF-κB). On the other hand, decreased glutathione (GSH) content and activities of glutathione transferase (GST), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were observed following CCl₄ exposure. Nevertheless, all of these phenotypes were evidently reversed by preadministration of polydatin for 5 continuous days. The mRNA and protein expression levels of hepatic growth factor-beta1 (TGF-β₁) were enhanced further by polydatin. These results suggest that polydatin protects mice against CCl₄-induced liver injury through antioxidant stress and antiinflammatory effects. Polydatin may be an effective hepatoprotective agent and a promising candidate for the treatment of oxidative stress- and inflammation-related diseases.     

Dietary Supplementation of Blueberry Juice Enhances Hepatic Expression of Metallothionein and Attenuates Liver Fibrosis in Rats

Aim

To investigate the effect of blueberry juice intake on rat liver fibrosis and its influence on hepatic antioxidant defense.

Methods

Rabbiteye blueberry was used to prepare fresh juice to feed rats by daily gastric gavage. Dan-shao-hua-xian capsule (DSHX) was used as a positive control for liver fibrosis protection. Liver fibrosis was induced in male Sprague-Dawley rats by subcutaneous injection of CCl₄ and feeding a high-lipid/low-protein diet for 8 weeks. Hepatic fibrosis was evaluated by Masson staining. The expression of α-smooth muscle actin (α-SMA) and collagen III (Col III) were determined by immunohistochemical techniques. The activities of superoxide dismutase (SOD) and malondialdehyde (MDA) in liver homogenates were determined. Metallothionein (MT) expression was detected by real-time RT-PCR and immunohistochemical techniques.

Results

Blueberry juice consumption significantly attenuates CCl₄-induced rat hepatic fibrosis, which was associated with elevated expression of metallothionein (MT), increased SOD activity, reduced oxidative stress, and decreased levels of α-SMA and Col III in the liver.

Conclusion

Our study suggests that dietary supplementation of blueberry juice can augment antioxidative capability of the liver presumably via stimulating MT expression and SOD activity, which in turn promotes HSC inactivation and thus decreases extracellular matrix collagen accumulation in the liver, and thereby alleviating hepatic fibrosis.     

Mutant MMP-9 and HGF Gene Transfer Enhance Resolution of CCl4-Induced Liver Fibrosis in Rats: Role of ASH1 and EZH2 Methyltransferases Repression

Hepatocyte growth factor (HGF) gene transfer inhibits liver fibrosis by regulating aberrant cellular functions, while mutant matrix metalloproteinase-9 (mMMP-9) enhances matrix degradation by neutralizing the elevated tissue inhibitor of metalloproteinase-1 (TIMP-1). It was shown that ASH1 and EZH2 methyltransferases are involved in development of liver fibrosis; however, their role in the resolution phase of liver fibrosis has not been investigated. This study evaluated the role of ASH1 and EZH2 in two mechanistically different therapeutic modalities, HGF and mMMP-9 gene transfer in CCl₄ induced rat liver fibrosis. Liver fibrosis was induced in rats with twice a week intraperitoneal injection of CCl₄ for 8 weeks. Adenovirus vectors encoding mMMP-9 or HGF genes were injected through tail vein at weeks six and seven and were sacrificed one week after the second injection. A healthy animal group was likewise injected with saline to serve as a negative control. Rats treated with mMMP-9 showed significantly lower fibrosis score, less Sirius red stained collagen area, reduced hydroxyproline and ALT concentration, decreased transforming growth factor beta 1 (TGF-β1) mRNA and lower labeling indices of α smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) stained cells compared with HGF- or saline-treated rats. Furthermore, TIMP-1 protein expression in mMMP-9 group was markedly reduced compared with all fibrotic groups. ASH1 and EZH2 protein expression was significantly elevated in fibrotic liver and significantly decreased in mMMP-9- and HGF-treated compared to saline-treated fibrotic livers with further reduction in the mMMP-9 group. Conclusion: Gene transfer of mMMP-9 and HGF reduced liver fibrosis in rats. ASH1 and EZH2 methyltransferases are significantly reduced in mMMP-9 and HGF treated rats which underlines the central role of these enzymes during fibrogenesis. Future studies should evaluate the role of selective pharmacologic inhibitors of ASH1 and EZH2 in resolution of liver fibrosis.     

Transplanted human amniotic membrane-derived mesenchymal stem cells ameliorate carbon tetrachloride-induced liver cirrhosis in mouse

Background

Human amniotic membrane-derived mesenchymal stem cells (hAMCs) have the potential to reduce heart and lung fibrosis, but whether could reduce liver fibrosis remains largely unknown.

Methodology/Principal Findings

Hepatic cirrhosis model was established by infusion of CCl₄ (1 ml/kg body weight twice a week for 8 weeks) in immunocompetent C57Bl/6J mice. hAMCs, isolated from term delivered placenta, were infused into the spleen at 4 weeks after mice were challenged with CCl₄. Control mice received only saline infusion. Animals were sacrificed at 4 weeks post-transplantation. Blood analysis was performed to evaluate alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Histological analysis of the livers for fibrosis, hepatic stellate cells activation, hepatocyte apoptosis, proliferation and senescence were performed. The donor cell engraftment was assessed using immunofluorescence and polymerase chain reaction. The areas of hepatic fibrosis were reduced (6.2%±2.1 vs. control 9.6%±1.7, p<0.05) and liver function parameters (ALT 539.6±545.1 U/dl, AST 589.7±342.8 U/dl,vs. control ALT 139.1±138.3 U/dl, p<0.05 and AST 212.3±110.7 U/dl, p<0.01) were markedly ameliorated in the hAMCs group compared to control group. The transplantation of hAMCs into liver-fibrotic mice suppressed activation of hepatic stellate cells, decreased hepatocyte apoptosis and promoted liver regeneration. More interesting, hepatocyte senescence was depressed significantly in hAMCs group compared to control group. Immunofluorescence and polymerase chain reaction revealed that hAMCs engraftment into host livers and expressed the hepatocyte-specific markers, human albumin and α-fetoproteinran.

Conclusions/Significance

The transplantation of hAMCs significantly decreased the fibrosis formation and progression of CCl₄-induced cirrhosis, providing a new approach for the treatment of fibrotic liver disease.     

Oroxylin A Accelerates Liver Regeneration in CCI4-Induced Acute Liver Injury Mice

Introduction

Based on the previous research that oroxylin A can suppress inflammation, we investigated the hepatoprotective role of oroxylin A against CCl₄-induced liver damage in mice and then studied the possible alteration of the activities of cytokine signaling participating in liver regeneration. Wild type (WT) mice were orally administrated with oroxylin A (60 mg/kg) for 4 days after CCl₄ injection, the anti-inflammatory effects of oroxylin A were assessed directly by hepatic histology and indirectly by measuring serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Albumin. Proliferating cell nuclear antigen (PCNA) staining was performed to evaluate the role of oroxylin A in promoting hepatocyte proliferation. Serum IL-1β, TNF-α, IL-6 and IL-1Ra levels were measured by enzyme-linked immunosorbent assay (ELISA) and liver HGF, EGF, TNF-α, IL-6, IL-1Ra and IL-1β gene expression was determined by quantitative real-time PCR. The data indicated that the IL-6 and TNF-α mRNA of oroxylin A administered group significantly increased higher than the control within 12 hours after CCl₄ treatment. Meanwhile, oroxylin A significantly enhanced the expression of IL-1Ra at the early phase, which indicated that oroxylin A could facilitate the initiating events in liver regeneration by increasing IL-1Ra which acts as an Acute-Phase Protein (APP). In addition, a lethal CCl₄-induced acute liver failure model offers a survival benefit in oroxylin A treated WT mice. However, oroxylin A could not significantly improve the percent survival of IL-1RI^−/− mice with a lethal CCl₄-induced acute liver failure.

Conclusions

Our study confirmed that oroxylin A could strongly promote liver structural remodeling and functional recovery through IL-1Ra/IL-1RI signaling pathway. All these results support the possibility of oroxylin A being a therapeutic candidate for acute liver injury.     

Attenuation of CCl4-Induced Hepatic Fibrosis in Mice by Vaccinating against TGF-β1

Transforming growth factor β1 (TGF-β1) is the pivotal pro-fibrogenic cytokine in hepatic fibrosis. Reducing the over-produced expression of TGF-β1 or blocking its signaling pathways is considered to be a promising therapeutic strategy for hepatic fibrosis. In this study, we evaluated the feasibility of attenuating hepatic fibrosis by vaccination against TGF-β1 with TGF-β1 kinoids. Two TGF-β1 kinoid vaccines were prepared by cross-linking TGF-β1-derived polypeptides (TGF-β1²⁵–[41-65] and TGF-β1³⁰–[83-112]) to keyhole limpet hemocyanin (KLH). Immunization with the two TGF-β1 kinoids efficiently elicited the production of high-levels of TGF-β1-specific antibodies against in BALB/c mice as tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The antisera neutralized TGF-β1-induced growth-inhibition on mink lung epithelial cells (Mv1Lu) and attenuated TGF-β1-induced Smad2/3 phosphorylation, α-SMA, collagen type 1 alpha 2 (COL1A2), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in the rat hepatic stellate cell (HSC) line, HSC-T6. Vaccination against TGF-β1 with the kinoids significantly suppressed CCl₄-induced collagen deposition and the expression of α-SMA and desmin, attenuated hepatocyte apoptosis and accelerated hepatocyte proliferation in BALB/c mice. These results demonstrated that immunization with the TGF-β1 kinoids efficiently attenuated CCl₄-induced hepatic fibrosis and liver injury. Our study suggests that vaccination against TGF-β1 might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases.     

Traditional Chinese Medicine Ganshuang Granules Attenuate CCl4-Induced Hepatic Fibrosis by Modulating Gut Microbiota

Gut dysbiosis contributes to hepatic fibrosis. Emerging evidence revealed the major role of traditional Chinese medicine (TCM) in gut microbiota homeostasis. Here, we aimed to investigate the anti-fibrotic activity and underlying mechanism of ganshuang granules (GS), particularly regarding gut microbiota homeostasis. CCl₄-induced hepatic fibrosis models were allocated into 4 groups receiving normal saline (model), 1.0, 2.0, or 4.0 g/kg GS for 5 weeks. As result, GS treatment alleviated liver injury in CCl₄-induced hepatic fibrosis, presenting as decreases of the liver index, alanine aminotransferase, and aspartate transaminase. Histological staining and expression revealed that the enhanced oxidative stress, inflammatory and hepatic fibrosis in CCl₄-induced models were attenuated by GS. Immunohistochemical staining showed that tight junction-associated proteins in intestinal mucosa were up-regulated by GS. 16S rRNA sequencing showed that GS rebalanced the gut dysbiosis manifested as improving alpha and beta diversity of gut microbiota, reducing the ratio of Firmicutes to Bacteroidetes, and regulating the relative abundance of various bacteria. In summary, GS decreased the intestinal permeability and rebalanced the gut microbiota to reduce the oxidative stress and inflammation, eventually attenuating CCl₄-induced hepatic fibrosis.     

Effects of blocking chemokine receptor CCR1 with BX471 in two models of fibrosis prevention and rescue in mice

BackgroundThe induction, progression and resolution of liver fibrosis are influenced by multiple chemokines. The inhibition of CCR1 signalling by a specific non-peptide inhibitor (BX471) reduces kidney fibrosis after unilateral ureteral obstruction via suppression of leukocyte recruitment in mice. However, it remains unclear whether selective CCR1 inhibition also affects hepatic fibrogenesis. Therefore we aimed to study the effect of this intervention on liver fibrosis in prevention (CCl₄ administration) and rescue (ABCB4-deficient mice) mouse models.MethodsIn the prevention model, hepatic fibrosis was induced by repeated injections of CCl₄. Additionally, the verum group was treated with subcutaneous injections of BX471, while controls received vehicle only. ABCB4 deficient mice (on the BALB/c-background) with sclerosing cholangitis and biliary fibrosis received BX471 or vehicle, respectively (rescue model). Liver histopathology was assessed after Sirius red staining of collagen, and hepatic collagen contents were measured. In addition, we performed gene expression analyses of fibrosis-related genes.ResultsBX471 injections were tolerated moderately well by all mice, and all mice developed hepatic fibrosis. Significant differences were neither observed in serum aminotransferase activities after 6 weeks of treatment between the two groups in the prevention nor in the rescue model. Interestingly, hepatic collagen contents were significantly higher in mice treated with BX471 in the prevention model as compared to controls but histological stages of liver sections did not differ. Of note, we observed only moderate effects on liver fibrosis in the ABCB4 knock-out model.ConclusionsOur data indicate that BX471 treatment did neither affect serum and tissue markers of liver injury and fibrosis in the CCl₄ model and only moderately in the Abcb4^-/- model of biliary fibrosis. The animal models indicate that treatment with BX471 alone is unlikely to exert major beneficial effects in chronic liver disease.     

The Protective Effect of Esculentoside A on Experimental Acute Liver Injury in Mice

Inflammatory response and oxidative stress are considered to play an important role in the development of acute liver injury induced by carbon tetrachloride (CCl₄) and galactosamine (GalN)/lipopolysaccharides (LPS). Esculentoside A (EsA), isolated from the Chinese herb phytolacca esculenta, has the effect of modulating immune response, cell proliferation and apoptosis as well as anti-inflammatory effects. The present study is to evaluate the protective effect of EsA on CCl₄ and GalN/LPS-induced acute liver injury. In vitro, CCK-8 assays showed that EsA had no cytotoxicity, while it significantly reduced levels of TNF-α and cell death rate challenged by CCl₄. Moreover, EsA treatment up-regulated PPAR-γ expression of LO2 cells and reduced levels of reactive oxygen species (ROS) challenged by CCl₄. In vivo, EsA prevented mice from CCl₄-induced liver histopathological damage. In addition, levels of AST and ALT were significantly decreased by EsA treatment. Furthermore, the mice treated with EsA had a lower level of TNF-α, Interleukin (IL)-1β and IL-6 in mRNA expression. EsA prevented MDA release and increased GSH-Px activity in liver tissues. Immunohistochemical staining showed that over-expression of F4/80 and CD11b were markedly inhibited by EsA. The western bolt results showed that EsA significantly inhibited CCl₄-induced phosphonated IkBalpha (P-IκB) and ERK. Furthermore, EsA treatment also alleviated GalN/LPS-induced acute liver injury on liver enzyme and histopathological damage. Unfortunately, our results exhibited that EsA had no effects on CCl₄-induced hepatocyte apoptosis which were showed by TUNEL staining and Bax, Caspase-3 and cleaved Caspase-3 expression. Our results proved that EsA treatment attenuated CCl₄ and GalN/LPS-induced acute liver injury in mice and its protective effects might be involved in inhibiting inflammatory response and oxidative stress, but not apoptosis with its underlying mechanism associated with PPAR-γ, NF-κB and ERK signal pathways.     

Glycoprotein Nonmetastatic Melanoma B (Gpnmb)-Positive Macrophages Contribute to the Balance between Fibrosis and Fibrolysis during the Repair of Acute Liver Injury in Mice

Background and aims

Glycoprotein nonmetastatic melanoma B (Gpnmb), a transmembrane glycoprotein that is expressed in macrophages, negatively regulates inflammation. We have reported that Gpnmb is strongly expressed in the livers of rats fed a choline-deficient, L-amino acid-defined (CDAA) diet. However, the role of macrophage-expressed Gpnmb in liver injury is still unknown. This study aimed to clarify the characteristics of infiltrating macrophages that express Gpnmb, and the involvement of Gpnmb in the repair process in response to liver injury.

Methods

C57BL/6J, DBA/2J [DBA] and DBA/2J-Gpnmb⁺ [DBA-g+] mice were treated with a single intraperitoneal injection of carbon tetrachloride (CCl₄) at a dose of 1.0 mL/kg body weight. Mice were sacrificed at predetermined time points, followed by measurement of serum alanine aminotransferase (ALT) levels and histological examination. Expression of Gpnmb, pro-/anti-inflammatory cytokines, and profibrotic/antifibrotic factors were examined by quantitative RT-PCR and/or Western blotting. Immunohistochemistry, fluorescent immunostaining and flow cytometry were used to determine the expression of Gpnmb, CD68, CD11b and α-SMA, phagocytic activity, and the presence of apoptotic bodies. We used quantitative RT-PCR and ELISA to examine TGF-β and MMP-13 expression and the concentrations and supernatants of isolated infiltrating hepatic macrophages transfected with siGpnmb.

Results

In C57BL/6J mice, serum ALT levels increased at two days after CCl₄ injection and decreased at four days. Gpnmb expression in the liver was stimulated four days after CCl₄ injection. Histological examination and flow cytometry showed that Gpnmb-positive cells were almost positive for CD68-positive macrophages, contained engulfed apoptotic bodies and exhibited enhanced phagocytic activity. Isolated infiltrating hepatic macrophages transfected with siGpnmb showed high MMP-13 secretion. There was no significant difference in the magnitude of CCl₄-induced liver injury between DBA-g+ and DBA mice. However, hepatic MMP-13 expression, as well as α-SMA expression and collagen production, increased significantly in DBA-g+ compared with DBA mice.

Conclusions

Gpnmb-positive macrophages infiltrate the liver during the recovery phase of CCl₄–induced acute liver injury and contribute to the balance between fibrosis and fibrolysis in the repair process following acute liver injury.     

Protective effects of L-carnosine on CCl4 -induced hepatic injury in rats

The present study was undertaken to investigate the possible protective effect of L-carnosine (CAR), an endogenous dipeptide of alanine and histidine, on carbon tetrachloride (CCl₄)-induced hepatic injury. Liver injury was induced in male Sprague-Dawley rats by intraperitoneal (i.p.) injections of CCl₄, twice weekly for six weeks. CAR was administered to rats daily, at dose of 250 mg/kg, i.p. At the end of six weeks, blood and liver tissue specimens were collected. Results show that CAR treatment attenuated the hepatic morphological changes, necroinflammation and fibrosis induced by CCl₄, as indicated by hepatic histopathology scoring. In addition, CAR treatment significantly reduced the CCl₄-induced elevation of liver-injury parameters in serum. CAR treatment also combated oxidative stress; possibly by restoring hepatic nuclear factor erythroid 2-related factor 2 (Nrf-2) levels. Moreover, CAR treatment prevented the activation of hepatic stellate cells (HSCs), as indicated by reduced α-smooth muscle actin (α-SMA) expression in the liver, and decreased hepatic inflammation as demonstrated by a reduction in hepatic tumor necrosis factor-α (TNF-α) and restoration of interleukin-10 (IL-10) levels. In conclusion, CCl₄-induced hepatic injury was alleviated by CAR treatment. The results suggest that these beneficial, protective effects are due, at least in part, to its anti-oxidant, anti-inflammatory and anti-fibrotic activities.     

MyD88 in hepatic stellate cells enhances liver fibrosis via promoting macrophage M1 polarization

During liver fibrosis, quiescent HSCs (qHSCs) are activated to become activated HSCs (aHSCs)/myofibroblasts. The signal adapter MyD88, an essential component of TLR signaling, plays an important role in liver fibrosis. However, far less is known about the specific effects of MyD88 signaling in both qHSCs and aHSCs in the progress of liver fibrosis. Here, we used a CCl₄-induced mouse fibrosis model in which MyD88 was selectively depleted in qHSCs (GFAP^MyD88−/− mice) or aHSCs (α-SMA^MyD88−/− mice). MyD88 deficiency in qHSCs or aHSCs attenuated liver fibrosis in mice and inhibited α-SMA-positive cell activation. Inhibition of MyD88 in HSCs decreased α-SMA and collagen I levels, inflammatory cell infiltration, and pro-inflammatory gene expression. Furthermore, MyD88 signaling in HSCs increased the secretion of CXCL10, which promoted macrophage M1 polarization through CXCR3, leading to activation of the JAK/STAT1 pathway. Inhibition of CXCL10 attenuated macrophage M1 polarization and reduced liver fibrosis. Thus, MyD88 signaling in HSCs crucially contributes to liver fibrosis and provides a promising therapeutic target for the prevention and treatment of liver fibrosis.Subject terms: Mechanisms of disease, Kupffer cells     

Inhibitory Effect of Gallic Acid on CCl4-Mediated Liver Fibrosis in Mice

The aim of this study was to investigate the effect of gallic acid (GA) on liver fibrosis induced by carbon tetrachloride (CCl₄). Male BALB/c mice were randomly divided into four groups: normal control group (group A), CCl₄-induced liver injury control group (group B), and CCl₄ induction with GA of low dose (5 mg/kg) and high dose (15 mg/kg) treatment group (group C and group D). GA was intra-gastric given for mice once a day after 2 weeks of CCl₄ induction. Animals were killed at the eighth week. Degrees of fibrosis and collagen percentage were measured. Hyaluronic acid (HA), type IV collagen (cIV), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γ-GT) were determined. Expression of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) mRNA levels were examined by RT-PCR. Western blotting was carried out to evaluate the changes of MMP-2 protein. HE and VG stainings showed GA in a dose-dependent manner improved significantly the fibrosis condition in CCl₄-injured mice (P < 0.05 or P < 0.01). Also, the concentrations of HA, cIV, and MDA, as well as the serum levels of ALT, AST, and γ-GT were markedly reduced by GA (P < 0.05 or P < 0.01), and decreases in MMP-2, TIMP-1 mRNA, and MMP-2 protein were observed as well (P < 0.05 or P < 0.01). GA could exert protective effect on liver injury and reduce liver fibrosis induced by CCl₄ in mice, which might be through the inhibition of hepatic stellate cell activity.