Mouse Hepatic Stellate Cells Research Articles

Role of WISP1 in Stellate Cell Migration and Liver Fibrosis.

The mechanisms underlying the remarkable capacity of the liver to regenerate are still not completely understood. Particularly, the cross-talk between cytokines and cellular components of the process is of utmost importance because they represent potential avenues for diagnostics and therapeutics. WNT1-inducible-signaling pathway protein 1 (WISP1) is a cytokine member of the CCN family, a family of proteins that play many different roles in liver pathophysiology. WISP1 also belongs to the earliest and strongest upregulated genes in mouse livers after CCl4 intoxication and has recently been shown to be secreted by tumor cells and to bind to type 1 collagen to cause its linearization in vitro and in tumor tissue in vivo. We show that WISP1 expression is strongly induced by TGFβ, a critical cytokine in wound healing processes. Additionally, secretion of WISP1 protein by hepatic stellate is increased in cells upon TGFβ stimulation (~seven-fold increase). Furthermore, WISP1 facilitates the migration of mouse hepatic stellate cells through collagen in vitro. However, in WISP1 knockout mice, no difference in stellate cell accumulation in damaged liver tissue and no influence on fibrosis was obtained, probably because the knockout of WISP1 was compensated by other factors in vivo.

Activin A plays an essential role in migration and proliferation of hepatic stellate cells via Smad3 and calcium signaling

Activin A and hepatic stellate cells (HSCs) are involved in tissue repair and fibrosis in liver injury. This study investigated the impact of activin A on HSC activation and migration. A microfluidic D4-chip was used for examining the cell migration of mouse hepatic stellate cell line MHSteC. The analysis of differentially expressed genes revealed that activin βA (Inhba), activin receptor type 1A (Acvr1a) and type 2A (Acvr2a) mRNAs were more significantly expressed in human HSCs than in the hepatocytes. Moreover, activin A promoted MHSteC proliferation and induced MHSteC migration. Furthermore, the MHSteCs treated with activin A exhibited increased levels of migration-related proteins, N-cadherin, Vimentin, α-SMA, MMP2 and MMP9, but a decreased level of E-cadherin. Additionally, activin A treatment significantly increased the p-Smad3 levels and p-Smad3/Smad3 ratio in the MHSteCs, and the Smad3 inhibitor SIS3 attenuated activin A-induced MHSteC proliferation and migration. Simultaneously, activin A increased the calcium levels in the MHSteCs, and the migratory effects of activin A on MHSteCs were weakened by the intracellular calcium ion-chelating agent BAPTA-AM. These data indicate that activin A can promote MHSteC activation and migration through the canonical Smad3 signaling and calcium signaling.

FoxD1 expression identifies a distinct subset of hepatic stellate cells involved in liver fibrosis

Hepatic stellate cells (HSCs) are pericytes of the liver responsible for liver fibrosis and cirrhosis, which are the end stages of chronic liver diseases. TGF-β activates HSCs, leading to the differentiation of myofibroblasts in the process of liver fibrosis. While the heterogeneity of HSCs is appreciated in the fibrotic liver, it remains elusive which HSC subsets mainly contribute to fibrosis. Here, we show that the expression of the pericyte marker FoxD1 specifically marks a subset of HSCs in FoxD1-fate tracer mice. HSCs fate-mapped by FoxD1 were preferentially localized in the portal and peripheral areas of both the homeostatic and fibrotic liver induced by carbon tetrachloride. Furthermore, the deletion of Cbfβ, which is necessary for TGF-β signaling, in FoxD1-expressing cells ameliorated liver fibrosis. Thus, we identified an HSC subset that preferentially responds to liver injuries.

Association of IL-9 Cytokines with Hepatic Injury in Echinococcus granulosus Infection.

Cystic echinococcosis (CE) is a zoonotic disease caused by the parasite Echinococcus granulosus (E. granulosus), which can lead to the formation of liver lesions. Research indicates that E. granulosus releases both Toll-like receptor 2 (TLR2) and Interleukin-9 (IL-9), which can potentially impair the body's innate immune defenses and compromise the liver's ability to fight against diseases. To investigate the role of TLR2 and IL-9 in liver damage caused by E. granulosus infection, samples were initially collected from individuals diagnosed with CE. Subsequently, BALB/c mice were infected with E. granulosus at multiple time points (4 weeks, 12 weeks, 32 weeks) and the expression levels of these markers was then assessed at each of these phases. Furthermore, a BALB/c mouse model was generated and administered anti-IL-9 antibody via intraperitoneal injection. The subsequent analysis focused on the TLR2/MyD88/NF-κB signaling pathway and the expression of IL-9 in E. granulosus was examined. A co-culture experiment was conducted using mouse mononuclear macrophage cells (RAW264.7) and hepatic stellate cells (HSCs) in the presence of E. granulosus Protein (EgP). The findings indicated elevated levels of IL-9 and TLR2 in patients with CE, with the activation of the signaling pathway significantly increased as the duration of infection progressed. Administration of anti-IL-9 in mice reduced the activation of the TLR2/MyD88/NF-κB signaling pathway, exacerbating liver injury. Moreover, EgP stimulates the TLR2/MyD88/NF-κB signaling pathway, resulting in the synthesis of α-SMA and Collagen I. The data suggest that infection with E. granulosus may stimulate the production of IL-9 through the activation of the TLR2/MyD88/NF-κB signaling pathway, which is mediated by TLR2. This activation stimulates RAW264.7 and HSCs, exacerbating liver injury and fibrosis.

A20 in hepatic stellate cells suppresses chronic hepatitis by inhibiting DCLK1-JNK pathway-dependent chemokines.

Hepatic stellate cells (HSCs) are responsible for liver fibrosis accompanied by its activation into myofibroblasts and the abundant production of extracellular matrix. However, the HSC contribution to progression of liver inflammation has been less known. We aimed to elucidate the mechanism in HSCs underlying the inflammatory response and the function of tumor necrosis factor α-related protein A20 (TNFAIP3). We established A20 conditional knockout (KO) mice crossing Twist2-Cre and A20 floxed mice. Using these mice, the effect of A20 was analyzed in mouse liver and HSCs. The human HSC line LX-2 was also used to examine the role and underlying molecular mechanism of A20. In this KO model, A20 was deficient in >80% of HSCs. Spontaneous inflammation with mild fibrosis was found in the liver of the mouse model without any exogenous agents, suggesting that A20 in HSCs suppresses chronic hepatitis. Comprehensive RNA sequence analysis revealed that A20-deficient HSCs exhibited an inflammatory phenotype and abnormally expressed chemokines. A20 suppressed JNK pathway activation in HSCs. Loss of A20 function in LX-2 cells also induced excessive chemokine expression, mimicking A20-deficient HSCs. A20 overexpression suppressed chemokine expression in LX-2. In addition, we identified DCLK1 in the genes regulated by A20. DCLK1 activated the JNK pathway and upregulates chemokine expression. DCLK1 inhibition significantly decreased chemokine induction by A20-silencing, suggesting that A20 controlled chemokine expression in HSCs via the DCLK1-JNK pathway. In conclusion, A20 suppresses chemokine induction dependent on the DCLK1-JNK signaling pathway. These findings demonstrate the therapeutic potential of A20 and the DCLK1-JNK pathway for the regulation of inflammation in chronic hepatitis.

Tricetin protects against liver fibrosis through promoting autophagy and Nrf2 signaling in hepatic stellate cells

AimsThe study aims to investigate the role and underlying mechanisms of tricetin in regulating hepatic stellate cells (HSCs) activation. Main methodsWe treated human hepatic stellate cells line LX-2 and freshly isolated primary mouse hepatic stellate cells (mHSCs) with tricetin, pharmacological inhibitors and siRNAs, western blot, immunofluorescence, quantitative PCR were used to evaluate the expression of fibrotic markers, autophagy levels and Nrf2 (nuclear factor E2-related factor 2) signaling. Key findingsHerein, we demonstrated that tricetin strongly attenuated the proliferation, migration, lipid droplets (LDs) loss and fibrotic markers Col 1a1 (type I α 1 collagen) and α-SMA (α-smooth muscle actin) expression in LX-2 cells. Moreover, tricetin time- and dose-dependently provoked autophagic formation in LX-2 cells. Autophagy inhibition by pharmacological intervention or genetic ATG5 (autophagy related 5) silencing facilitated tricetin-induced downregulation of profibrotic markers in LX-2 cells. Additionally, tricetin treatment reduced reactive oxygen species (ROS) accumulation, promoted Nrf2 signaling in LX-2 cells and pretreatment with ROS scavenger NAC partially reversed tricetin-induced autophagy and enhanced tricetin-mediated HSCs inactivation. Nrf2 silencing partially reversed tricetin-mediated inhibition of α-SMA expression. Finally, utilizing primary mouse hepatic stellate cells (mHSCs), we demonstrated that tricetin also induced autophagy activation, repressed TGF-β1-induced LDs loss and fibrotic marker expression and pretreatment with CQ further sensitized these effects. SignificanceOur study indicates that tricetin's actions may represent an effective strategy to treat liver fibrosis and help identify novel therapeutic targets, especially in combination with autophagy inhibitors.

Nicotine aggravates liver fibrosis via α7 nicotinic acetylcholine receptor expressed on activated hepatic stellate cells in mice.

Smoking is a risk factor for liver cirrhosis; however, the underlying mechanisms remain largely unexplored. The α7 nicotinic acetylcholine receptor (α7nAChR) has recently been detected in nonimmune cells possessing immunoregulatory functions. We aimed to verify whether nicotine promotes liver fibrosis via α7nAChR. We used osmotic pumps to administer nicotine and carbon tetrachloride to induce liver fibrosis in wild-type and α7nAChR-deficient mice. The severity of fibrosis was evaluated using Masson trichrome staining, hydroxyproline assays, and real-time PCR for profibrotic genes. Furthermore, we evaluated the cell proliferative capacity and COL1A1 mRNA expression in human HSCs line LX-2 and primary rat HSCs treated with nicotine and an α7nAChR antagonist, methyllycaconitine citrate. Nicotine exacerbated carbon tetrachloride-induced liver fibrosis in mice (+42.4% in hydroxyproline assay). This effect of nicotine was abolished in α7nAChR-deficient mice, indicating nicotine promotes liver fibrosis via α7nAChR. To confirm the direct involvement of α7nAChRs in liver fibrosis, we investigated the effects of genetic suppression of α7nAChR expression on carbon tetrachloride-induced liver fibrosis without nicotine treatment. Profibrotic gene expression at 1.5 weeks was significantly suppressed in α7nAChR-deficient mice (-83.8% in Acta2, -80.6% in Col1a1, -66.8% in Tgfb1), and collagen content was decreased at 4 weeks (-22.3% in hydroxyproline assay). The in vitro analysis showed α7nAChR expression in activated but not in quiescent HSCs. Treatment of LX-2 cells with nicotine increased COL1A1 expression (+116%) and cell proliferation (+10.9%). These effects were attenuated by methyllycaconitine citrate, indicating the profibrotic effects of nicotine via α7nAChR. Nicotine aggravates liver fibrosis induced by other factors by activating α7nAChR on HSCs, thereby increasing their collagen-producing capacity. We suggest the profibrotic effect of nicotine is mediated through α7nAChRs.

LncRNA Snhg12/IGFBP3 axis is involved in liver fibrosis by promoting the proliferation and activation of mouse hepatic stellate cells.

Liver fibrosis is a persistent damage repair response triggered by various injury factors, which leads to an abnormal accumulation of extracellular matrix within liver tissue samples. The current clinical treatment of liver fibrosis is currently ineffective; therefore, elucidating the mechanism of liver fibrogenesis is of significant importance. Herein, the function and related mechanisms of lncRNA Snhg12 within hepatic fibrosis were investigated. Snhg12 expression was shown to be increased in mouse hepatic fibrotic tissue samples, and Snhg12 knockdown suppressed hepatic pathological injury and down-regulated the expression levels of fibrosis-associated proteins. Mechanistically, Snhg12 played a role in the early activation of mouse hepatic stellate cells (mHSCs) based on bioinformatics analysis, and Snhg12 was positively correlated with Igfbp3 expression. Further experimental results demonstrated that Snhg12 knockdown impeded mHSCs proliferation and activation and also downregulated the protein expression of Igfbp3. Snhg12 could interact with IGFBP3 and boost its protein stability, and overexpression of Igfbp3 partially reversed the inhibition of mHSCsproliferation and activation by the knockdown of Snhg12. In conclusion, LncRNA Snhg12 mediates liver fibrosis by targeting IGFBP3 and promoting its protein stability, thereby promoting mHSC proliferation and activation. Snhg12 has been identified as an underlying target for treating liver fibrosis.

Sphingosine-1-phosphate promotes liver fibrosis in metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatohepatitis (MASH) is one of the most prevalent liver diseases and is characterized by steatosis and the accumulation of bioactive lipids. This study aims to understand the specific lipid species responsible for the progression of liver fibrosis in MASH. Changes in bioactive lipid levels were examined in the livers of MASH mice fed a choline-deficient diet (CDD). Additionally, sphingosine kinase (SphK)1 mRNA, which generates sphingosine 1 phosphate (S1P), was examined in the livers of patients with MASH. CDD induced MASH and liver fibrosis were accompanied by elevated levels of S1P and increased expression of SphK1 in capillarized liver sinusoidal endothelial cells (LSECs) in mice. SphK1 mRNA also increased in the livers of patients with MASH. Treatment of primary cultured mouse hepatic stellate cells (HSCs) with S1P stimulated their activation, which was mitigated by the S1P receptor (S1PR)2 inhibitor, JTE013. The inhibition of S1PR2 or its knockout in mice suppressed liver fibrosis without reducing steatosis or hepatocellular damage. S1P level is increased in MASH livers and contributes to liver fibrosis via S1PR2.

Protective Effects of Resveratrol on Cytotoxicity of Mouse Hepatic Stellate Cells Induced by PM2.5

The atmosphere’s fine particulate matter (PM2.5) can enter the liver through the circulatory system, leading to hepatic inflammation and fibrosis. As a non-flavonoid polyphenolic compound, resveratrol (RES) has anti-oxidant, anti-inflammatory and hepatoprotective effects, but the molecular mechanisms of liver fibrosis induced by PM2.5 exposure are still limited. In this study, we established an in vitro cell model to investigate the intervention effect of RES with different concentrations (5 and 20 μmol/mL) on mouse hepatic stellate cells (mHSCs) injury induced by PM2.5 (100 μg/mL). We determined the cell viability in mHSCs after treatment with PM2.5 or/and RES for 24 h. We investigated the intracellular oxidative stress by detecting the changes in reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and lactate dehydrogenase (LDH) levels. We also measured the protein expressions of fibrosis-related genes (α-SMA, Collagen I and Collagen III) and key genes (SIRT1, NF-κB, NLRP3, Cleaved-Caspase1, IL-1β) in the NLRP3 pathway in mHSCs exposed to PM2.5 with or without RES. The results showed that (1) PM2.5 has cytotoxic effects on mHSCs, whereas RES (5 μmol/L and 20 μmol/L) inhibited PM2.5-induced cytotoxicity and LDH leakage; (2) RES effectively reduces ROS and MDA production caused by PM2.5 while concurrently enhancing SOD levels, thereby improving cellular anti-oxidant capacity; (3) the expression of α-SMA, Collagen I and Collagen III were notably downregulated in the PM2.5 plus RES treatment group compared to the PM2.5-exposed group; (4) RES significantly increased SIRT1 expression and decreased the expression of NF-κB, NLRP3, Cleaved-Caspase1 and IL-1β in mHSCs exposure to PM2.5 compared to the PM2.5 group. These results demonstrate that RES can up-regulate SIRT1 and mitigate PM2.5-induced fibrosis by suppressing oxidative stress in mHSCs and the SIRT1/NF–κB/NLRP3 pathway activated by PM2.5.

Loss of SREBP-1c ameliorates iron-induced liver fibrosis by decreasing lipocalin-2

Sterol regulatory element-binding protein (SREBP)-1c is involved in cellular lipid homeostasis and cholesterol biosynthesis and is highly increased in nonalcoholic steatohepatitis (NASH). However, the molecular mechanism by which SREBP-1c regulates hepatic stellate cells (HSCs) activation in NASH animal models and patients have not been fully elucidated. In this study, we examined the role of SREBP-1c in NASH and the regulation of LCN2 gene expression. Wild-type and SREBP-1c knockout (1cKO) mice were fed a high-fat/high-sucrose diet, treated with carbon tetrachloride (CCl4), and subjected to lipocalin-2 (LCN2) overexpression. The role of LCN2 in NASH progression was assessed using mouse primary hepatocytes, Kupffer cells, and HSCs. LCN2 expression was examined in samples from normal patients and those with NASH. LCN2 gene expression and secretion increased in CCl4-induced liver fibrosis mice model, and SREBP-1c regulated LCN2 gene transcription. Moreover, treatment with holo-LCN2 stimulated intracellular iron accumulation and fibrosis-related gene expression in mouse primary HSCs, but these effects were not observed in 1cKO HSCs, indicating that SREBP-1c-induced LCN2 expression and secretion could stimulate HSCs activation through iron accumulation. Furthermore, LCN2 expression was strongly correlated with inflammation and fibrosis in patients with NASH. Our findings indicate that SREBP-1c regulates Lcn2 gene expression, contributing to diet-induced NASH. Reduced Lcn2 expression in 1cKO mice protects against NASH development. Therefore, the activation of Lcn2 by SREBP-1c establishes a new connection between iron and lipid metabolism, affecting inflammation and HSCs activation. These findings may lead to new therapeutic strategies for NASH.

STUB1 increases adiponectin expression by inducing ubiquitination and degradation of NR2F2, thereby reducing hepatic stellate cell activation and alleviating non-alcoholic fatty liver disease

BackgroundAdiponectin (APN) has exhibited ameliorating effects on non-alcoholic fatty liver disease (NAFLD). This study investigates the roles of APN and its regulatory molecules in hepatic stellate cell (HSC) activation and the progression of NAFLD. MethodsMice were subjected to a high-fat diet (HFD) to establish NAFLD models. Liver tissue was examined for lipid metabolism, fibrosis, and inflammation. Mouse 3T3-L1 adipocytes were exposed to palmitic acid (PA) to mimic a high-fat environment. The conditioned medium (CM) from adipocytes was collected for the culture of isolated mouse HSCs. Gain- or loss-of-function studies of APN, nuclear receptor subfamily 2 group F member 2 (NR2F2), and STIP1 homology and U-box containing protein 1 (STUB1) were performed to analyze their roles in NAFLD and HSC activation in vivo and in vitro. ResultsAPN expression was poorly expressed in HFD-fed mice and PA-treated 3T3-L1 adipocytes, which was attributed to the transcription inhibition mediated by NR2F2. Silencing of NR2F2 restored the APN expression, ameliorating liver steatosis, fibrosis, and inflammatory cytokine infiltration in mouse livers and reducing HSC activation. Similarly, the NR2F2 silencing condition reduced HSC activation in vitro. However, these effects were counteracted by artificial APN silencing. STUB1 facilitated the ubiquitination and protein degradation of NR2F2, and its upregulation mitigated NAFLD-like symptoms in mice and HSC activation, effects reversed by the NR2F2 overexpression. ConclusionThis study highlights the role of STUB1 in reducing HSC activation and alleviating NAFLD by attenuating NR2F2-mediated transcriptional repression of APN.

Phillygenin Inhibits TGF-β1-induced Hepatic Stellate Cell Activation and Inflammation: Regulation of the Bax/Bcl-2 and Wnt/β-catenin Pathways.

Hepatic fibrosis (HF), a precursor to cirrhosis and hepatocellular carcinoma, is caused by abnormal proliferation of connective tissue and excessive accumulation of extracellular matrix in the liver. Notably, activation of hepatic stellate cells (HSCs) is a key link in the development of HF. Phillygenin (PHI, C21H24O6) is a lignan component extracted from the traditional Chinese medicine Forsythiae Fructus, which has various pharmacological activities such as anti-inflammatory, antioxidant and anti-tumour effects. However, whether PHI can directly inhibit HSC activation and ameliorate the mechanism of action of HF has not been fully elucidated. Therefore, the aim of the present study was to investigate the in vitro anti-HF effects of PHI and the underlying molecular mechanisms. Transforming growth factor-β1 (TGF-β1)-activated mouse HSCs (mHSCs) and human HSCs (LX-2 cells) were used as an in vitro model of HF and treated with different concentrations of PHI for 24h. Subsequently, cell morphological changes were observed under the microscope, cell viability was analyzed by MTT assay, cell cycle and apoptosis were detected by flow cytometry, and the mechanism of anti-fibrotic effect of PHI was explored by immunofluorescence, ELISA, RT-qPCR and western blot. The results showed that PHI suppressed the proliferation of TGF-β1-activated mHSCs and LX-2 cells, arrested the cell cycle at the G0/G1 phase, decreased the levels of α-SMA, Collagen I, TIMP1 and MMP2 genes and proteins, and promoted apoptosis in activated mHSCs and LX-2 cells. Besides, PHI reduced the expression of inflammatory factors in activated mHSCs and LX-2 cells, suggesting a potential anti-inflammatory effect. Mechanically, PHI inhibited TGF-β1-induced HSC activation and inflammation, at least in part through modulation of the Bax/Bcl-2 and Wnt/β-catenin pathways. Overall, PHI has significant anti-HF effects and may be a promising agent for the treatment of HF.

RNA-seq combined network pharmacology reveals that Fu-Gan-Wan (FGW) inhibits liver fibrosis via NF-κB/CCL2/CCR2 and lipid peroxidation via Nrf2/HMOX1 signaling pathway

Ethnopharmacological relevanceLiver fibrosis is a serious complication of liver disease characterized by excessive collagen deposition, without effective therapeutic agents in the clinic. Fu-Gan-Wan (FGW) is an empirical formula used for the clinical treatment of hepatitis and cirrhosis. It has been shown to reverse experimental liver fibrosis. However, its corresponding mechanisms remain unclear. Aim of the reviewThis study aimed to elucidate the key pathways and target genes of FGW in attenuating liver fibrosis. Materials and methodsThe therapeutic effects of different doses of FGW on liver fibrosis were investigated using a 2 mL/kg 15% CCl4-induced mouse model. Then, RNA-seq combined with network pharmacology was used to analyze the key biological processes and signaling pathways underlying the anti-liver fibrosis exertion of FGW. These findings were validated in a TGF-β1-induced model of activation and proliferation of mouse hepatic stellate cell line JS-1. Finally, the key signaling pathways and molecular targets were validated using animal tissues, and the effect of FGW on tissue lipid peroxidation was additionally observed. ResultsWe found that 19.5 g/kg FGW significantly down-regulated CCl4-induced elevation of hepatic ALT and AST, decreased collagen deposition, and inhibited the expression of pro-fibrotic factors α-SMA, COL1α1, CTGF, TIMP-1, as well as pro-inflammatory factor TGF-β1. Additionally, FGW at doses of 62.5, 125, and 250 μg/mL dose-dependently blocked JS-1 proliferation, migration, and activation. Furthermore, RNA-seq identified the NF-κB signaling pathway as a key target molecular pathway for FGW against liver fibrosis, and network pharmacology combined with RNA-seq focused on 11 key genes. Significant changes were identified in CCL2 and HMOX1 by tissue RT-PCR, Western blot, and immunohistochemistry. We further demonstrated that FGW significantly attenuated CCl4-induced increases in p-p65, CCL2, CCR2, and HMOX1, while significantly elevating Nrf2. Finally, FGW significantly suppressed the accumulation of lipid peroxidation products MDA and 4-HNE and reconfigured the oxidation-reduction balance, including promoting the increase of antioxidants GPx, GSH, and SOD, and the decrease of peroxidation products ROS and GSSG. ConclusionsThis study demonstrated that FGW exhibits potential in mitigating CCl4-induced hepatic fibrosis, lipid peroxidation, and iron metabolism disorders in mice. This effect may be mediated through the NF-κB/CCL2/CCR2 and Nrf2/HMOX1 pathways.

Orphan receptor GPR176 in hepatic stellate cells exerts a profibrotic role in chronic liver disease

Background & AimsChronic liver disease (CLD) remains a global health issue associated with a significant disease burden. Liver fibrosis, a hallmark of CLD, is characterised by the activation of hepatic stellate cells (HSCs) that gain profibrotic characteristics including increased production of extracellular matrix protein. Currently, no antifibrotic therapies are available clinically, in part because of the lack of HSC-specific drug targets. Here, we aimed to identify HSC-specific membrane proteins that can serve as targets for antifibrotic drug development. MethodsSmall interfering RNA-mediated knockdown of GPR176 was used to assess the in vitro function of GPR176 in HSCs and in precision cut liver slices (PCLS). The in vivo role of GPR176 was assessed using the carbon tetrachloride (CCl4) and common bile duct ligation (BDL) models in wild-type and GPR176 knockout mice. GPR176 in human CLD was assessed by immunohistochemistry of diseased human livers and RNA expression analysis in human primary HSCs and transcriptomic data sets. ResultsWe identified Gpr176, an orphan G-protein coupled receptor, as an HSC-enriched activation associated gene. In vitro, Gpr176 is strongly induced upon culture-induced and hepatocyte-damage-induced activation of primary HSCs. Knockdown of GPR176 in primary mouse HSCs or PCLS cultures resulted in reduced fibrogenic characteristics. Absence of GPR176 did not influence liver homeostasis, but Gpr176-/- mice developed less severe fibrosis in CCl4 and BDL fibrosis models. In humans, GPR176 expression was correlated with in vitro HSC activation and with fibrosis stage in patients with CLD. ConclusionsGPR176 is a functional protein during liver fibrosis and reducing its activity attenuates fibrogenesis. These results highlight the potential of GPR176 as an HSC-specific antifibrotic candidate to treat CLD. Impact and implicationsThe lack of effective antifibrotic drugs is partly attributed to the insufficient knowledge about the mechanisms involved in the development of liver fibrosis. We demonstrate that the G-protein coupled receptor GPR176 contributes to fibrosis development. Since GPR176 is specifically expressed on the membrane of activated hepatic stellate cells and is linked with fibrosis progression in humans, it opens new avenues for the development of targeted interventions.

An RORα agonist, ODH-08, inhibits fibrogenic activation of hepatic stellate cells via suppression of SMAD3

AimsHepatic fibrosis is a dynamic process characterized by the net accumulation of an extracellular matrix resulting from chronic liver injury such as nonalcoholic steatohepatitis. Activation of hepatic stellate cells (HSCs) plays a role in transdifferentiation of quiescent cells into fibrogenic myofibroblasts. We aimed to examine the function of retinoic acid receptor-related orphan receptor alpha (RORα) and its novel agonistic ligand, 1-(4-benzyloxybenzyl)-3-(2-dimethylaminoethyl)-thiourea (ODH-08) against activation of HSCs using hepatic fibrosis mouse models. Main methodsChemical synthesis, a reporter gene assay, surface plasmon resonance analysis, and a docking study were performed to evaluate ODH-08 as a ligand of RORα. In vivo experiments with mice fed a Western diet were performed to evaluate the effect of ODH-08. The human HSC line, Lx-2, and primary mouse HSCs were employed to identify the molecular mechanisms underlying the antifibrogenic effect of ODH-08. Key findingsA novel RORα-selective ligand, ODH-08, was developed based on modification of JC1-40, an analog of N-methylthiourea. Administration of ODH-08 to the Western diet-fed mice reduced hepatic collagen deposition and expression levels of fibrogenic markers such as α-smooth muscle actin and collagen type I alpha 1 chain. Activation of RORα—either by transient overexpression of RORα or treatment with ODH-08—suppressed the expression of fibrogenic proteins in HSCs. The activation of RORα suppressed the activity of SMAD2 and 3, which are the primary downstream proteins of transforming growth factor β. SignificanceRORα and its agonist ODH-08 have a potent antifibrotic effect, which could provide a novel antifibrotic strategy against hepatic fibrosis.

Ginkgetin exhibits antifibrotic effects by inducing hepatic stellate cell apoptosis via STAT1 activation.

Liver fibrosis affects approximately 800 million patients worldwide, with over 2 million deaths each year. Nevertheless, there are no approved medications for treating liver fibrosis. In this study, we investigated the impacts of ginkgetin on liver fibrosis and the underlying mechanisms. The impacts of ginkgetin on liver fibrosis were assessed in mouse models induced by thioacetamide or bile duct ligation. Experiments on human LX-2 cells and primary mouse hepatic stellate cells (HSCs) were performed to explore the underlying mechanisms, which were also validated in the mouse models. Ginkgetin significantly decreased hepatic extracellular matrix deposition and HSC activation in the fibrotic models induced by thioacetamide (TAA) and bile duct ligation (BDL). Beneficial effects also existed in inhibiting hepatic inflammation and improving liver function. In vitro experiments showed that ginkgetin markedly inhibited HSC viability and induced HSC apoptosis dose-dependently. Mechanistic studies revealed that the antifibrotic effects of ginkgetin depend on STAT1 activation, as the effects were abolished in vitro after STAT1 silencing and in vivo after inhibiting STAT1 activation by fludarabine. Moreover, we observed a meaningful cross-talk between HSCs and hepatocytes, in which IL-6, released by ginkgetin-induced apoptotic HSCs, enhanced hepatocyte proliferation by activating STAT3 signaling. Ginkgetin exhibits antifibrotic effects by inducing HSC apoptosis via STAT1 activation and enhances hepatocyte proliferation secondary to HSC apoptosis via the IL-6/STAT3 pathway.

Water extract of earthworms mitigates mouse liver fibrosis by potentiating hepatic LKB1/Nrf2 axis to inhibit HSC activation and hepatocyte death

Ethnopharmacological relevanceWhen left untreated, liver fibrosis (LF) causes various chronic liver diseases. Earthworms (Pheretima aspergillum) are widely used in traditional medicine because of their capacity to relieve hepatic diseases.Aim of the study: This study aimed to explore the anti-LF effects of water extract of earthworms (WEE) and the underlying molecular mechanisms. Materials and methodsA CCl4-induced mouse model of LF was used to study the impact of WEE on LF in vivo. The anti-LF activity of WEE in mice was compared with that of silybin, which can be clinically applied in LF intervention and was used as a positive control. Activation of LX-2 hepatic stellate cells (HSCs) and apoptosis and ferroptosis of AML-12 hepatocytes induced by TGFβ1 were used as in vitro models. ResultsWEE drastically improved LF in mice. WEE reduced markers of activated HSCs in mice and inhibited TGFβ1-induced activation of LX-2 HSCs in vitro. Additionally, WEE suppressed CCl4-induced apoptosis and ferroptosis in mouse hepatocytes. Mechanistically, WEE induced Nrf2 to enter the nuclei of the mouse liver cells, and the hepatic levels of Nrf2-downstream antioxidative factors increased. LKB1/AMPK/GSK3β is an upstream regulatory cascade of Nrf2. In the LF mouse model, WEE increased hepatic phosphorylated LKB1, AMPK, and GSK3β levels. Similar results were obtained for the LX-2 cells. In AML-12 hepatocytes and LX-2 HSCs, WEE elevated intracellular Nrf2 levels, promoted its nuclear translocation, and inhibited TGFβ1-induced ROS accumulation. Knocking down LKB1 abolished the impact of WEE on the AMPK/GSK3β/Nrf2 cascade and eliminated its protective effects against TGFβ1. ConclusionsOur findings reveal that WEE improves mouse LF triggered by CCl4 and supports its application as a promising hepatoprotective agent against LF. The potentiation of the hepatic antioxidative AMPK/GSK3β/Nrf2 cascade by activating LKB1 and the subsequent suppression of HSC activation and hepatocyte apoptosis and ferroptosis are implicated in WEE-mediated alleviation of LF.

Caveolin-1 depletion attenuates hepatic fibrosis via promoting SQSTM1-mediated PFKL degradation in HSCs.

The key glycolytic enzyme phosphofructokinase (PFK) is responsible for maintaining glycolytic stability and an important energy source for activating hepatic stellate cells (HSCs). However, its regulation in activated HSCs remains unclear. Caveolin-1 (Cav1), a major constituent of caveolae, has emerged as a key target for triggering glycolysis. However, the relationship between Cav1 and glycolysis during HSC activation is not well established. In this study, Cav1 was upregulated in mouse and human fibrotic liver tissues. We concluded that HSC-specific Cav1 knockdown markedly alleviates liver injury and fibrosis. Mechanistically, Cav1 was elevated during primary mouse HSC activation, competing with SQSTM1 for the regulatory subunit of PFK liver type and inhibiting the SQSTM1-mediated autophagy-independent lysosomal degradation pathway to sustain HSC activation. We also identified the heptapeptide alamandine as a promising therapeutic agent that downregulates Cav1 protein levels via proteasomal degradation and may impair glycolysis. Our study provides evidence of the crucial role and mechanism of Cav1 in the glucose metabolic network in HSCs and highlights Cav1 as a critical therapeutic target for the treatment of liver fibrosis.

GARP on hepatic stellate cells is essential for the development of liver fibrosis

Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-β docking molecule. While the immune regulatory properties of GARP on blood cells have been studied, the function of GARP on tissue stromal cells remains unclear. Here, we investigate the role of GARP expressed on hepatic stellate cells (HSCs) in the development of liver fibrosis. The function of GARP on HSCs was explored in toxin-induced and metabolic liver fibrosis models, using conditional GARP-deficient mice or a newly generated inducible system for HSC-specific gene ablation. Primary mouse and human HSCs were isolated to evaluate the contribution of GARP to the activation of latent TGF-β. Moreover, cell contraction of HSCs in the context of TGF-β activation was tested in a GARP-dependent fashion. Mice lacking GARP in HSCs were protected from developing liver fibrosis. Therapeutically deleting GARP on HSCs alleviated the fibrotic process in established disease. Furthermore, natural killer T cells exacerbated hepatic fibrosis by inducing GARP expression on HSCs through IL-4 production. Mechanistically, GARP facilitated fibrogenesis by activating TGF-β and enhancing endothelin-1-mediated HSC contraction. Functional GARP was expressed on human HSCs and significantly upregulated in the livers of patients with fibrosis. Lastly, deletion of GARP on HSCs did not augment inflammation or liver damage. GARP expressed on HSCs drives the development of liver fibrosis via cell contraction-mediated activation of latent TGF-β. Considering that systemic blockade of TGF-β has major side effects, we highlight a therapeutic niche provided by GARP and surface-mediated TGF-β activation. Thus, our findings suggest an important role of GARP on HSCs as a promising target for the treatment of liver fibrosis. Liver fibrosis represents a substantial and increasing public health burden globally, for which specific treatments are not available. Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-β docking molecule. Here, we show that GARP expressed on hepatic stellate cells drives the development of liver fibrosis. Our findings suggest GARP as a novel target for the treatment of fibrotic disease.