Hepatitis C virus NS5A and core protein induce fibrosis-related genes regulation on Huh7 cells through activation of LX2 cells.

INTRODUCTION AND OBJECTIVES: Liver fibrosis remains a complication derived from a chronic Hepatitis C Virus (HCV) infection even when it is resolved, and no liver antifibrotic drug has been approved. Molecular mechanisms on hepatocytes and activation of hepatic stellate cells (HSCs) play a central role in liver fibrogenesis. To elucidate molecular mechanisms, it is important to analyze pathway regulation during HSC activation and HCV infection. MATERIALS AND METHODS: We evaluate the fibrosis-associated molecular mechanisms during a co-culture of human HSCs (LX2), with human hepatocytes (Huh7) that express HCV NS5A or Core protein. We evaluated LX2 activation induced by HCV NS5A or Core expression in Huh7 cells during co-culture. We determined a fibrosis-associated gene expression profile in Huh7 that expresses NS5A or Core proteins during the co-culture with LX2. RESULTS: We observed that NS5A induced 8.3-, 6.7- and 4-fold changes and that Core induced 6.5-, 1.8-, and 6.2-fold changes in the collagen1, TGFß1, and timp1 gene expression, respectively, in LX2 co-cultured with transfected Huh7. In addition, NS5A induced the expression of 30 genes while Core induced 41 genes and reduced the expression of 30 genes related to fibrosis in Huh7 cells during the co-culture with LX2, compared to control. The molecular pathways enriched from the gene expression profile were involved in TGFB signaling and the organization of extracellular matrix. CONCLUSIONS: We demonstrated that HCV NS5A and Core protein expression regulate LX2 activation. NS5A and Core-induced LX2 activation, in turn, regulates diverse fibrosis-related gene expression at different levels in Huh7, which can be further analyzed as potential antifibrotic targets during HCV infection.

Identification of (E)-1-((1H-indol-3-yl)methylene)-4-substitute-thiosemicarbazones as potential anti-hepatic fibrosis agents.

Liver fibrosis remains a global health challenge due to its rapidly rising prevalence and limited treatment options. The orphan nuclear receptor Nur77 has been implicated in regulation of autophagy and liver fibrosis. Targeting Nur77-mediated autophagic flux may thus be a new promising strategy against hepatic fibrosis. In this study, we synthesized four types of Nur77-based thiourea derivatives to determine their anti-hepatic fibrosis activity. Among the synthesized thiourea derivatives, 9e was the most potent inhibitor of hepatic stellate cells (HSCs) proliferation and activation. This compound could directly bind to Nur77 and inhibit TGF-ß1-induced α-SMA and COLA1 expression in a Nur77-dependent manner. In vivo, 9e significantly reduced CCl4-mediated hepatic inflammation response and extracellular matrix (ECM) production, revealing that 9e is capable of blocking the progression of hepatic fibrosis. Mechanistically, 9e induced Nur77 expression and enhanced autophagic flux by inhibiting the mTORC1 signaling pathway in vitro and in vivo. Thus, the Nur77-targeted lead 9e may serve as a promising candidate for treatment of chronic liver fibrosis.

S100a16 deficiency prevents hepatic stellate cells activation and liver fibrosis via inhibiting CXCR4 expression.

INTRODUCTION: Liver fibrosis caused by hepatic stellate cells (HSCs) activation is implicated in the pathogenesis of liver diseases. To date, there has been no effective intervention means for this process. S100 proteins are calcium-binding proteins that regulate cell growth and differentiation. This study aimed to investigate whether S100A16 induces HSCs activation and participates in liver fibrosis progression. METHODS: HSCs were isolated, and the relationship between S100A16 expression and HSCs activation was studied. S100a16 knockdown and transgenic mice were generated and subjected to HSCs activation and liver fibrosis stimulated by different models. Clinical samples were collected for further confirmation. Alterations in gene expression in HSCs were investigated, using transcriptome sequencing to determine the underlying mechanisms. RESULTS: We observed increased S100A16 levels during HSCs activation. Genetic silencing of S100a16 prevented HSCs activation in vitro. Furthermore, S100a16 silencing exhibited obvious protective effects against HSCs activation and fibrosis progression in mice. In contrast, S100a16 transgenic mice exhibited spontaneous liver fibrosis. S100A16 was also upregulated in the HSCs of patients with fibrotic liver diseases. RNA sequencing revealed that C-X-C motif chemokine receptor 4 (Cxcr4) gene was a crucial regulator of S100A16 induction during HSCs activation. Mechanistically, S100A16 bound to P53 to induce its degradation; this augmented CXCR4 expression to activate ERK 1/2 and AKT signaling, which then promoted HSCs activation and liver fibrosis. CONCLUSIONS: These data indicate that S100a16 deficiency prevents liver fibrosis by inhibiting Cxcr4 expression. Targeting S100A16 may provide insight into the pathogenesis of liver fibrosis and pave way for the design of novel clinical therapeutic strategies.

Long noncoding RNA XIST enhances ethanol-induced hepatic stellate cells autophagy and activation via miR-29b/HMGB1 axis.

Activation of hepatic stellate cells (HSCs) is a prominent driver of liver fibrogenesis, including alcoholic liver fibrosis (ALF). Furthermore, autophagy contributes to HSCs activation. This study aims to investigate the role and the mechanisms of long noncoding RNA XIST in regulating HSCs autophagy and activation. Human HSC cells (LX-2) were treated with 100 mmol/L ethanol to mimic HSCs activation. The HSCs activation was evaluated by determining cell viability and protein levels of fibrosis markers α-smooth muscle actin (α-SMA) and collagen type 1 α1 (CoL1A1). The autophagy was evaluated by measuring autophagy markers Beclin-1 and LC3-II. The interaction among XIST, miR-29b, and high-mobility group box-1 (HMGB1) were analyzed using luciferase reporter assay, qRT-PCR, and western blot. Lentiviruses targeting sh-XIST (LV-sh-XIST) were injected into ALF model mice via tail vein to elucidate the in vivo role of XIST in ALF injury. XIST was upregulated in ethanol-activated LX-2 cells. Furthermore, XIST served as a competitive endogenous RNA of miR-29b to facilitate HMGB1 expression, and thus enhanced ethanol-induced HSCs autophagy and activation. Further in vivo assay showed that downregulation of XIST by LV-sh-XIST alleviated ALF injury in ALF model mice. Collectively, XIST enhances ethanol-induced HSCs autophagy and activation via miR-29b/HMGB1 axis.

Exogenous expression of caveolin-1 is sufficient for hepatic stellate cell activation.

Caveolin-1 (Cav-1) expression is increased in hepatic stellate cells (HSC) upon liver cirrhosis and it functions as an integral membrane protein of lipid rafts and caveolae that regulates and integrates multiple signals as a platform. This study aimed to evaluate the role of Cav-1 in HSC. Thus, the effects of exogenous expression of Cav-1 in GRX cells, a model of activated HSC, were determined. Here, we demonstrated through evaluating well-known HSC activation markers - such as α-smooth muscle actin, collagen I, and glial fibrillary acidic protein - that up regulation of Cav-1 induced GRX to a more activated phenotype. GRXEGFP-Cav1 presented an increased migration, an altered adhesion pattern, a reorganization f-actin cytoskeleton, an arrested cell cycle, a modified cellular ultrastructure, and a raised endocytic flux. Based on this, GRX EGFP-Cav1 represents a new cellular model that can be an important tool for understanding of events related to HSC activation. Furthermore, our results reinforce the role of Cav-1 as a molecular marker of HSC activation.

DNMT1 controls LncRNA H19/ERK signal pathway in hepatic stellate cell activation and fibrosis.

Hepatic stellate cells (HSCs) activation is considered as a pivotal event in liver fibrosis. In HSCs activation and fibrosis, epigenetic events are important. Although HSCs activation alters DNA methylation, it is unknown, whether it also affects other epigenetic processes, including LncRNA and its recognition. The aim of this study was to identify the mechanism of DNA methyltransferase 1 (DNMT1) expression and its role in regulating LncRNA H19 during HSCs activation and fibrosis. Expression of DNMT1 and LncRNA H19 were determined in activated HSCs and CCl4-induced rat liver fibrosis tissue. The relationship between the LncRNA H19 and DNMT1 expression was examined in vitro. LncRNA H19 expression was reduced in activated HSCs and rat liver fibrosis tissue, whereas DNMT1 expression and methylation of the LncRNA H19 promoter were increased. Treatment of HSCs of DNMT1-siRNA blocked cell proliferation. Knockdown of DNMT1 elevated H19 expression in activated HSCs, and over-expression of DNMT1 inhibited H19 expression in activated HSCs. Moreover, we investigated the effect of H19 on ERK signal pathway. Treatment HSCs with H19-siRNA increased the expression of p-ERK1/2 in HSCs. Treatment with 5'-aza-2'-deoxycytidine in activated HSCs model reduced fibrosis gene and DNMT1 expression, enhanced H19 expression, and attenuated HSCs activation. These data connect HSCs activation with a DNMT1-LncRNA H19 epigenetic pathway that is important for liver fibrosis.