Primary Hepatic Stellate Cells Research Articles

Glial cell line-derived neurotrophic factor contributes to alcoholic-induced liver injury by regulating the NF-κB pathway.

Alcoholic liver disease (ALD) is associated with high morbidity and mortality worldwide. The pathogenesis of ALD is not completely understood. Although accumulating evidence suggests an important role of glial cell line-derived neurotrophic factor (GDNF) in several diseases, there are no data concerning its role in ALD. This study compared patients with ALD with control subjects and used a mouse model and a cell culture model to investigate the function of GDNF in ALD and its mechanism of action in hepatocyte injury. Serum levels of GDNF were measured in 25 patients with ALD and 25 healthy control subjects. A 4-week Lieber-DeCarli ethanol (EtOH) liquid diet combined with the Gao-Binge model was used in the mouse study. Mouse primary hepatocytes and Huh-7 cells were used for cell experiments. The parameters of liver injury, inflammatory cytokines, and lipid metabolism were measured. Patients with alcoholic hepatitis had higher serum GDNF than control subjects. Expression of GDNF mRNA and protein was markedly increased in mice in the chronic-plus-binge ALD mouse model. The level of GDNF mRNA was upregulated in primary hepatic stellate cells isolated from ethanol-fed mouse liver. Ethanol induced GDNF expression in LX2 cells. The levels of inflammatory cytokines (tumor necrosis factor α, interleukin 1β, and monocyte chemotactic protein 1) were significantly increased after GDNF stimulation in primary hepatocytes and Huh-7 cells. After GDNF stimulation, levels of both p-AKT and p-NF-κB were significantly increased in primary hepatocytes and Huh-7 cells. The NF-κB activity induced by GDNF was significantly decreased by an NF-κB inhibitor, which limited hepatocyte injury and inflammation. The concentration of GDNF is increased in the circulation of ALD patients. GDNF promotes alcohol-induced liver injury and inflammation via the activation of NF-κB, which mediates hepatocyte injury and inflammatory cytokine expression. Based on these findings, GDNF is a potential therapeutic target for preventing or ameliorating liver injury in ALD.

Decursin ameliorates carbon-tetrachloride-induced liver fibrosis by facilitating ferroptosis of hepatic stellate cells.

Decursin possesses the potential to alleviate transforming growth factor (TGF)-β-induced hepatic stellate cells (HSCs) activation. However, the mechanisms by which decursin alleviates hepatic fibrosis remain not fully understood. Our aim is to explore the function of decursin on regulating HSCs' activation and hepatic fibrosis. The anti-fibrotic effect of decursin was evaluated by Masson and Sirius red staining, and immunohistochemical (IHC) and quantitative real-time PCR (qRT-PCR) analyses for alpha-smooth muscle actin (α-SMA) and collagen type I(Col1α1) expression. Ferroptosis was assessed by measuring iron concentration, glutathioneperoxidase4 (Gpx4), and prostaglandin endoperoxide synthase 2 (Ptgs2) expression, glutathione (GSH) level, lipid peroxidation, and reactive oxygen species (ROS) level. We found that decursin treatment decreased carbon tetrachloride (CCl4)-induced liver fibrosis. The primary HSCs isolated from decursin-treated group showed an increased Fe2+, lipid ROS level, and decreased Gpx4 and GSH levels compared with HSCs from the model group. Moreover, decursin promoted ferroptosis in activated HSCs in vitro, as evidenced by declined Gpx4 and GSH levels, increased Fe2+, ROS, and Ptgs2 levels compared with control. More important, ferroptosis inhibitor destroyed the anti-fibrosis effect of decursin on HSCs. In summary, these data suggest that decursin has potential to treat hepatic fibrosis.

MiR-654-5p Contributes to the Activation and Proliferation of Hepatic Stellate Cells by Targeting RXRα.

Liver fibrosis (LF) is a major disease that threatens human health. Hepatic stellate cells (HSCs) contribute directly to LF via extracellular matrix (ECM) secretion. Moreover, RXRα is an important nuclear receptor that plays a key regulatory role in HSC activation. Meanwhile, microRNAs (miRNAs) have been identified as significant regulators of LF development. In particular, miR-654-5p is involved in cellular migration and proliferation, and via bioinformatics analysis, has been identified as a potential factor that targets RXRα in humans and in mice. However, the precise relationship between miR-654-5p and RXRα in the context of LF, remains unknown and is the primary focus of the current study. To establish in vitro activated cell model human primary HSCs were cultured in vitro and LX-2 cells were stimulated with recombinant human TGF-β1. mRNA and protein levels of RXRα, miR-654-5p and fibrogenic genes were compared in quiescent and activated HSCs. Moreover, after transfected with miR-654-5p mimics, the expression changes of above related genes in LX-2 cells were estimated. Meanwhile, cell proliferation and apoptosis were detected in miR-654-5p overexpressed LX-2 cells. Simultaneously, the targeted binding between miR-654-5p and RXRα was verified in LX-2 cells. Carbon tetrachloride (CCl4)-induced mouse model with liver fibrosis was use to research the role of the miR-654-5p in vitro. Our results show that miR-654-5p expression levels increased in activated human HSCs and TGFβ-treated LX-2 cells. Moreover, miR-654-5p mimics markedly promoted LX-2 cell proliferation while inhibiting their apoptosis. Accordingly, the expression levels of RXRα are decreased in activated HSCs and LX-2 cells. Additionally, dual-luciferase reporter assay results reveal direct targeting of RXRα by miR-654-5p. Similarly, in vivo miR-654-5p overexpression aggravates LF in mice that are intraperitoneally injected with CCl4. Taken together, our findings elucidated a novel molecular mechanism with potential use for treatment of LF.

In vitro ballooned hepatocytes can be produced by primary human hepatocytes and hepatic stellate cell sheets

Despite the increasing prevalence of Nonalcoholic steatohepatitis (NASH) worldwide, there is no effective treatment available for this disease. “Ballooned hepatocyte” is a characteristic finding in NASH and is correlated with disease prognosis, but their mechanisms of action are poorly understood; furthermore, neither animal nor in vitro models of NASH have been able to adequately represent ballooned hepatocytes. Herein, we engineered cell sheets to develop a new in vitro model of ballooned hepatocytes. Primary human hepatocytes (PHH) and Hepatic stellate cells (HSC) were co-cultured to produce cell sheets, which were cultured in glucose and lipid containing medium, following which histological and functional analyses were performed. Histological findings showed hepatocyte ballooning, accumulation of fat droplets, abnormal cytokeratin arrangement, and the presence of Mallory–Denk bodies and abnormal organelles. These findings are similar to those of ballooned hepatocytes in human NASH. Functional analysis showed elevated levels of TGFβ-1, SHH, and p62, but not TNF-α, IL-8. Exposure of PHH/HSC sheets to a glucolipotoxicity environment induces ballooned hepatocyte without inflammation. Moreover, fibrosis is an important mechanism underlying ballooned hepatocytes and could be the basis for the development of a new in vitro NASH model with ballooned hepatocytes.

MicroRNA-122 inhibits hepatic stellate cell proliferation and activation in vitro and represses carbon tetrachloride-induced liver cirrhosis in mice

ObjectiveThis study aimed to determine the roles of microRNA (miR)-122 in the activation of hepatic stellate cells (HSCs) and liver cirrhosis. MethodsRat primary HSCs were incubated with transforming growth factor-beta (TGF-β), during which miR-122 and EphB2 expression was measured. miR-122 mimic and/or pcDNA3.1 EphB2 was transfected into TGF-β-induced HSCs. A mouse model of liver cirrhosis was established via an intraperitoneal injection of carbon tetrachloride (CCl4), followed by the injection of miR-122 agomir. Levels of serum alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured. Fibronectin (FN), alpha smooth muscle actin (α-SMA), Collagen I, miR-122, and EphB2 expression was evaluated in liver tissues and HSCs. Cell proliferation was measured using CCK-8 assay. Interactions between miR-122 and EphB2 were assessed using dual luciferase reporter assay. ResultsmiR-122 (0.15-fold) was downregulated and EphB2 (mRNA: 5.06-fold; protein: 2.35-fold) was upregulated after TGF-β induction of HSCs. Overexpressed miR-122 decreased proliferation and EphB2 (mRNA: 0.46-fold; protein: 0.62-fold), FN (mRNA: 0.45-fold; protein: 0.64-fold), α-SMA (mRNA: 0.48-fold; protein: 0.51-fold), and Collagen I (mRNA: 0.44-fold; protein: 0.51-fold) expression in HSCs, which was abrogated by EphB2 upregulation. miR-122 expression was reduced by 0.21-fold and serum ALT and AST levels were enhanced in mice following 8-week CCl4 induction along with increased expression of FN, α-SMA, and Collagen I in liver tissues, which was blocked by miR-122 overexpression. Moreover, EphB2 was a target gene of miR-122. ConclusionmiR-122 curtails HSC proliferation and activation by targeting EphB2 and suppresses liver cirrhosis in mice.

Antibody-drug conjugates targeting CD248 inhibits liver fibrosis through specific killing on myofibroblasts

BackgroundChronic liver injury induces pathological repair, resulting in fibrosis, during which hepatic stellate cells (HSCs) are activated and transform into myofibroblasts. CD248 is mainly expressed on myofibroblasts and was considered as a promising target to treat fibrosis. The primary aim of this study was to generate a CD248 specific antibody-drug conjugate (ADC) and evaluate its therapeutic efficacy for liver fibrosis and its safety in vivo.MethodsCD248 expression was examined in patients with liver cirrhosis and in mice with CCl4-induced liver fibrosis. The ADC IgG78-DM1, which targets CD248, was prepared and its bioactivity on activated primary HSCs was studied. The anti-fibrotic effects of IgG78-DM1 on liver fibrosis were evaluated in CCl4-induced mice. The reproductive safety and biosafety of IgG78-DM1 were also evaluated in vivo.ResultsCD248 expression was upregulated in patients with liver cirrhosis and in CCl4-induced mice, and was mainly expressed on alpha smooth muscle actin (α-SMA)+ myofibroblasts. IgG78-DM1 was successfully generated, which could effectively bind with and kill CD248+ activated HSCs in vitro and inhibit liver fibrosis in vivo. In addition, IgG78-DM1 was demonstrated to have qualified biosafety and reproductive safety in vivo.ConclusionsOur study demonstrated that CD248 could be an ideal target for myofibroblasts in liver fibrosis, and CD248-targeting IgG78-DM1 had excellent anti-fibrotic effects in mice with liver fibrosis. Our study provided a novel strategy to treat liver fibrosis and expanded the application of ADCs beyond tumors.Graphic

Rab31 promotes activation of hepatic stellate cells by accelerating TGF-β receptor II complex endocytosis.

Hepatic stellate cells activation is the key process of liver fibrosis, revealing the molecular mechanism of which is helpful to provide an effective target for inhibiting liver fibrosis. Rab31, a small GTPase, regulates the specificity of intracellular vesicular transport system, and is crucial for signal transduction. However, whether Rab31 is involved in hepatic stellate cells activation is unknown. Analysis of the differences in gene expression between human healthy and fibrotic liver tissues by sequencing revealed that Rab31 was significantly upregulated in fibrotic tissues. Immunohistochemistry and immunofluorescence analysis confirmed that Rab31 positively correlated with hepatic fibrosis. Next, mouse primary hepatic stellate cells were prepared, and their continuous activation was accompanied by Rab31 expression increased. Interestingly, knockdown of Rab31 by lentivirus can significantly restrict those cell activation. Subsequently, the vary of signal transduction after Rab31 knockdown was detected, its presented that the TGF-β/Smads signaling was obviously affected. Following experiments identified that Rab31 knockdown significantly inhibited the TGF-β activation and led to the failure of hepatic stellate cells activation. Importantly, we revealed that Rab31 knockdown could inhibit TGF-β receptor II complex endocytosis, a prerequisite for the activation of TGF-β signaling. Finally, in a mouse CCl4 fibrosis model, we proved that Rab31 knockdown markedly inhibited hepatic fibrosis. Our study demonstrated that Rab31 could promote hepatic stellate cells activation by accelerating TGF-β Receptor II complex endocytosis, suggesting that interfering with Rab31 could be an effectively strategy to inhibit hepatic fibrosis progression.

Astaxanthin Attenuates the Changes in the Expression of MicroRNAs Involved in the Activation of Hepatic Stellate Cells.

We previously demonstrated that astaxanthin (ASTX), a xanthophyll carotenoid, has an antifibrogenic effect in hepatic stellate cells (HSC), primarily responsible for the accumulation of extracellular matrix protein during the development of liver fibrosis. Studies have shown that microRNAs (miRNAs) are involved in HSC activation. Therefore, we analyzed the expression of 84 miRNAs using miRNA arrays in primary mouse quiescent HSC (qHSC) and activated HSC (aHSC) treated with/without ASTX during their activation. Compared with qHSC, the expression of 14 miRNAs and 23 miRNAs was increased and decreased by more than 2-fold, respectively, in aHSC. Among the 14 miRNAs increased in aHSC, the expression of miR-192-5p, miR-382-5p, and miR-874-3p was reduced by ASTX. In addition, ASTX increased the expression of miR-19a-3p, miR-19b-3p, and miR-101a-3p among 23 miRNAs decreased in aHSC. Moreover, we confirmed miR-382-5p expression was ~15-fold higher in aHSC than qHSC, and ASTX markedly inhibited the induction measured by quantitative real-time PCR. We identified that the expression of Baz1a and Zfp462 from the predicted miR-382-5p target genes was significantly reduced in aHSC while increased by ASTX treatment similar to the levels in qHSC. The roles of Baz1a and Zfp462 in HSC activation and the antifibrogenic effect of ASTX need to be further investigated.

Mulberrin confers protection against hepatic fibrosis by Trim31/Nrf2 signaling

Mulberrin (Mul) is a key component of the traditional Chinese medicine Romulus Mori with various biological functions. However, the effects of Mul on liver fibrosis have not been addressed, and thus were investigated in our present study, as well as the underlying mechanisms. Here, we found that Mul administration significantly ameliorated carbon tetrachloride (CCl4)-induced liver injury and dysfunction in mice. Furthermore, CCl4-triggerd collagen deposition and liver fibrosis were remarkably attenuated in mice with Mul supplementation through suppressing transforming growth factor β1 (TGF-β1)/SMAD2/3 signaling pathway. Additionally, Mul treatments strongly restrained the hepatic inflammation in CCl4-challenged mice via blocking nuclear factor-κB (NF-κB) signaling. Importantly, we found that Mul markedly increased liver TRIM31 expression in CCl4-treated mice, accompanied with the inactivation of NOD-like receptor protein 3 (NLRP3) inflammasome. CCl4-triggered hepatic oxidative stress was also efficiently mitigated by Mul consumption via improving nuclear factor E2-related factor 2 (Nrf2) activation. Our in vitro studies confirmed that Mul reduced the activation of human and mouse primary hepatic stellate cells (HSCs) stimulated by TGF-β1. Consistently, Mul remarkably retarded the inflammatory response and reactive oxygen species (ROS) accumulation both in human and murine hepatocytes. More importantly, by using hepatocyte-specific TRIM31 knockout mice (TRIM31Hep-cKO) and mouse primary hepatocytes with Nrf2-knockout (Nrf2KO), we identified that the anti-fibrotic and hepatic protective effects of Mul were TRIM31/Nrf2 signaling-dependent, relieving HSCs activation and liver fibrosis. Therefore, Mul-ameliorated hepatocyte injury contributed to the suppression of HSCs activation by improving TRIM31/Nrf2 axis, thus providing a novel therapeutic strategy for hepatic fibrosis treatment.

Inhibition of carnitine palmitoyltransferase 1A in hepatic stellate cells protects against fibrosis

The pathogenesis of liver fibrosis requires activation of hepatic stellate cells (HSCs); once activated, HSCs lose intracellular fatty acids but the role of fatty acid oxidation and carnitine palmitoyltransferase 1A (CPT1A) in this process remains largely unexplored. CPT1A was found in HSCs of patients with fibrosis. Pharmacological and genetic manipulation of CPT1A were performed in human HSC cell lines and primary HCSs. Finally, we induced fibrosis in mice lacking CPT1A specifically in HSCs. Herein, we show that CPT1A expression is elevated in HSCs of patients with non-alcoholic steatohepatitis, showing a positive correlation with the fibrosis score. This was corroborated in rodents with fibrosis, as well as in primary human HSCs and LX-2 cells activated by transforming growth factor β1 (TGFβ1) and fetal bovine serum (FBS). Furthermore, both pharmacological and genetic silencing of CPT1A prevent TGFβ1- and FBS-induced HSC activation by reducing mitochondrial activity. The overexpression of CPT1A, induced by saturated fatty acids and reactive oxygen species, triggers mitochondrial activity and the expression of fibrogenic markers. Finally, mice lacking CPT1A specifically in HSCs are protected against fibrosis induced by a choline-deficient high-fat diet, a methionine- and choline-deficient diet, or treatment with carbon tetrachloride. These results indicate that CPT1A plays a critical role in the activation of HSCs and is implicated in the development of liver fibrosis, making it a potentially actionable target for fibrosis treatment. We show that the enzyme carnitine palmitoyltransferase 1A (CPT1A) is elevated in hepatic stellate cells (HSCs) in patients with fibrosis and mouse models of fibrosis, and that CPT1A induces the activation of these cells. Inhibition of CPT1A ameliorates fibrosis by preventing the activation of HSCs.

PD-L1 promotes myofibroblastic activation of hepatic stellate cells by distinct mechanisms selective for TGF-β receptor I versus II.

SUMMARYIntrahepatic cholangiocarcinoma (ICC) contains abundant myofibroblasts derived from hepatic stellate cells (HSCs) through an activation process mediated by TGF-β. To determine the role of programmed death-ligand 1 (PD-L1) in myofibroblastic activation of HSCs, we disrupted PD-L1 of HSCs by shRNA or anti-PD-L1 antibody. We find that PD-L1, produced by HSCs, is required for HSC activation by stabilizing TGF-β receptors I (TβRI) and II (TβRII). While the extracellular domain of PD-L1 (amino acids 19–238) targets TβRII protein to the plasma membrane and protects it from lysosomal degradation, a C-terminal 260-RLRKGR-265 motif on PD-L1 protects TβRI mRNA from degradation by the RNA exosome complex. PD-L1 is required for HSC expression of tumor-promoting factors, and targeting HSC PD-L1 by shRNA or Cre/loxP recombination suppresses HSC activation and ICC growth in mice. Thus, myofibroblast PD-L1 can modulate the tumor microenvironment and tumor growth by a mechanism independent of immune suppression.

Hepatitis B and Hepatitis C Virus Infection Promote Liver Fibrogenesis through a TGF-β1-Induced OCT4/Nanog Pathway.

Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-β1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide-transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-β1-induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-β1 mRNA levels in sodium taurocholate cotransporting polypeptide-Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-β1-induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-β1-induced OCT4/Nanog-dependent pathway.

The Invasive Potential of Hepatoma Cells Induced by Radiotherapy is Related to the Activation of Hepatic Stellate Cells and Could be Inhibited by EGCG Through the TLR4 Signaling Pathway.

Post-radiotherapy recurrence and metastasis of liver cancer were thought to arise from the invasion and metastasis of residual hepatocellular carcinoma cells, but it has now been shown to be closely related to the increased metastatic potential of residual liver cancer cells mediated by radiotherapy. The changes of liver microenvironment after radiotherapy also provide a favorable condition for promoting the metastatic potential of hepatocellular carcinoma. Studies have shown that radiation-induced activation of hepatic stellate cells (HSCs) is one of the main changes in the microenvironment of hepatocellular carcinoma. Therefore, we hypothesized that activated HSCs are involved in regulating the metastatic capacity of residual cancer cells after radiotherapy. The present study observed that 48 h co-culture of three human hepatoma cell lines (MHCC97-L, Hep-3B, LM3) with a irradiated human HSC line (LX-2) in a transwell chamber could significantly improve the invasion of the human hepatoma cells; and the culture supernatant of activated HSCs could also enhance the invasion of the hepatoma cells. In contrast, co-culture with irradiated hepatoma cells enhanced the invasion of LX-2 cells. In vitro, irradiation enhanced the activation phenotype and the toll like receptor 4 (TLR4) signaling pathway of LX-2 cells or primary mouse HSCs, which upregulated intercellular cell adhesion molecule-1 (ICAM1), laminin receptor (67 LR), Interleukin- 6 (IL-6), and CX3C chemokine ligand 1 (CX3CL1) and downregulated toll-interacting proteins. The compound (-)-epigallocatechin-3-gallate (EGCG) inhibited signal transduction of activated TLR4 and radiation-induced invasion of LX-2 cells by binding to 67 LR. These observations indicated that the enhancement of the metastatic potential of hepatoma cells after irradiation was relevant to the activation of HSCs, and the activation of TLR4 signaling pathway was involved in this process, which was inhibited by EGCG. Our results will help enhance the therapeutic efficacy of liver cancer stereotactic body radiation therapy to prevent and decrease the risks of post-radiotherapy recurrence and metastasis.

Focal adhesion kinase-related non-kinase ameliorates liver fibrosis by inhibiting aerobic glycolysis via the FAK/Ras/c-myc/ENO1 pathway.

BACKGROUNDHepatic stellate cell (HSC) hyperactivation is a central link in liver fibrosis development. HSCs perform aerobic glycolysis to provide energy for their activation. Focal adhesion kinase (FAK) promotes aerobic glycolysis in cancer cells or fibroblasts, while FAK-related non-kinase (FRNK) inhibits FAK phosphorylation and biological functions.AIMTo elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs.METHODSMouse liver fibrosis models were established by administering CCl4, and the effect of FRNK on the degree of liver fibrosis in the model was evaluated. Transforming growth factor-β1 was used to activate LX-2 cells. Tyrosine phosphorylation at position 397 (pY397-FAK) was detected to identify activated FAK, and the expression of the glycolysis-related proteins monocarboxylate transporter 1 (MCT-1) and enolase1 (ENO1) was assessed. Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region, which were validated with chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays.RESULTSThe pY397-FAK level was increased in human fibrotic liver tissue. FRNK knockout promoted liver fibrosis in mouse models. It also increased the activation, migration, proliferation and aerobic glycolysis of primary hepatic stellate cells (pHSCs) but inhibited pHSC apoptosis. Nevertheless, opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells. Mechanistically, the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis, which was inhibited by exogenous FRNK.CONCLUSIONFRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ENO1 pathway, thereby improving liver fibrosis. FRNK might be a potential target for liver fibrosis treatment.

Autophagy-Related Activation of Hepatic Stellate Cells Reduces Cellular miR-29a by Promoting Its Vesicular Secretion.

Background & AimsLiver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis.MethodsIntracellular and extracellular miRNA levels of primary and immortalized myofibroblastic HSC in response to profibrogenic stimulation by transforming growth factor β (TGFβ) or platelet-derived growth factor-BB (PDGF-BB) or upon inhibition of vesicular transport and autophagy processes were determined by quantitative polymerase chain reaction. Autophagy flux was studied by electron microscopy, flow cytometry, immunoblotting, and immunocytochemistry. Hepatic and serum miR-29a levels were quantified by using both liver tissue and serum samples from a cohort of chronic hepatitis C virus patients and a murine CCl4 induced liver fibrosis model.ResultsIn our study, we show that TGFβ and PDGF-BB resulted in decrease of intracellular miR-29a and a pronounced increase of vesicular miR-29a release into the supernatant. Strikingly, miR-29a vesicular release was accompanied by enhanced autophagic activity and up-regulation of the autophagy marker protein LC3. Moreover, autophagy inhibition strongly prevented miR-29a secretion and repressed its targets’ expression such as Col1A1. Consistently, hepatic miR-29a loss and increased LC3 expression in myofibroblastic HSC were associated with increased serum miR-29a levels in CCl4-treated murine liver fibrosis and specimens of hepatitis C virus patients with chronic liver disease.ConclusionsWe provide evidence that activation-associated autophagy in HSC induces release of miR-29a, whereas inhibition of autophagy represses fibrogenic gene expression in part through attenuated miR-29a secretion.

Non-heat-stressed Method to Isolate Hepatic Stellate Cells From Highly Steatotic Tumor-bearing Liver Using CD49a.

Non-heat-stressed Method to Isolate Hepatic Stellate Cells From Highly Steatotic Tumor-bearing Liver Using CD49a.

A Selective Adenosine A3 Receptor Antagonist, HL3501, Has Therapeutic Potential in Preclinical Liver and Renal Fibrosis Models.

Adenosine and 4 G-protein-associated membrane receptors (A1, A2A, A2B, and A3) and their derivatives regulate the central nervous, cardiovascular, peripheral, and immune system. We developed a novel selective A3 AR antagonist, HL3501, and examined its anti-fibrotic effects across various models. The anti-fibrotic activity of HL3501 was evaluated in three cell lines (HK2, LX2, and Primary hepatic stellate cell) and a methionine-choline-deficient (MCD) model including use of mouse pharmacokinetics (PK). HL3501 decreased alpha-smooth muscle actin (α-SMA) and collagen 1 in TGF-β1-induced pro-fibrotic activation in HK2 cells. HL3501 also inhibited TGF-β1-induced HSC activation, which resulted in reduction of α-SMA and fibronectin in LX2 and human primary HSCs. In the nonalcoholic fatty liver disease activity score (NAS) analysis, HL3501 showed improved anti-steatosis and anti-inflammatory activity. The mouse PK study revealed the oral bioavailability (%F) of HL3501 at 30 mg/kg and 60 mg/kg as 92.5 and 107.2%, respectively. HL3501 presents anti-fibrotic effects in in vitro and in vivo studies. We also demonstrated that HL3501 is orally available and has a good bioavailability (BA >90%) profile from in mouse PK. HL3501, therefore, has a therapeutic potential for various fibrotic diseases, including those of liver and kidney tissues.

Isolation and Culture of Primary Rat Hepatic Stellate Cells

Isolation and Culture of Primary Rat Hepatic Stellate Cells

The miR-23b/27b/24-1 Cluster Inhibits Hepatic Fibrosis by Inactivating Hepatic Stellate Cells.

Background & AimsHepatic fibrosis is characterized by hepatic stellate cell (HSC) activation and transdifferentiation-mediated extracellular matrix (ECM) deposition, which both contribute to cirrhosis. However, no antifibrotic regimen is available in the clinic. microRNA-23b/27b/24-1 cluster inhibition of transforming growth factor-β (TGF-β) signaling during hepatic development prompted us to explore whether this cluster inhibits HSC activation and hepatic fibrosis.MethodsExperimental fibrosis was studied in carbon tetrachloride (CCl4)-treated C57BL/6 mice. After administration of miR-23b/27b/24-1 lentivirus or vehicle, animals were euthanized for liver histology. In primary rat HSC and HSC-T6, the anti-fibrotic effect of miR-23b/27b/24-1 cluster was furtherly investigated by RNA-sequencing, luciferase reporter assay, western blotting and bioinformatic means.ResultsIn this study, we showed that increasing the miR-23b/27b/24-1 level through intravenous delivery of miR-23b/27b/24-1 lentivirus ameliorated mouse hepatic fibrosis. Mechanistically, the miR-23b/27b/24-1 cluster directly targeted messenger RNAs, which reduced the protein expression of 5 secretory profibrotic genes (TGF-β2, Gremlin1, LOX, Itgα2, and Itgα5) in HSCs. Suppression of the TGF-β signaling pathway by down-regulation of TGF-β2, Itgα2, and Itgα5, and activation of the bone morphogenetic protein signaling pathway by inhibition of Gremlin1, decreased extracellular matrix secretion of HSCs. Furthermore, down-regulation of LOX expression softened the ECM. Moreover, a reduction in tissue inhibitors of metalloproteinase 1 expression owing to weakened TGF-β signaling increased ECM degradation.ConclusionsHepatic overexpression of the miR-23b/27b/24-1 cluster blocked hepatic fibrosis and may be a novel therapeutic regimen for patients with hepatic fibrosis.

Fisetin Ameliorates Alcohol-Induced Liver Injury through Regulating SIRT1 and SphK1 Pathway.

Alcoholic liver disease (ALD) often leads to hepatitis, hepatic cirrhosis, and even hepatocellular carcinoma. Fisetin has been shown to confer protection against liver injury. Herein, we investigated whether fisetin could prevent ethanol-induced hepatotoxicity. Mice were fed on 5% (v/v) Lieber-DeCarli ethanol diet. Human primary hepatic stellate cells (HSCs) co-cultured with ethanol were used to verify the therapeutic effect of fisetin. The results of alanine/aspartate aminotransferase (ALT/AST), Triglyceride (TG), total cholesterol (TC) in serum, Oil O Red and Masson staining revealed that fisetin (80[Formula: see text]mg/kg) ameliorated ethanol-induced mice liver injury and fibrosis. Besides, immunofluorescence results of [Formula: see text]-SMA revealed that fisetin suppressed HSCs activation. The suppression was dose-dependent. Furthermore, fisetin promoted SIRT1-mediated autophagy and inhibited Sphk1-mediated endoplasmic reticulum stress (ER stress) both in vitro and in vivo. Molecular docking results indicated potential interaction of fisetin with SIRT1 and SphK1. The inhibitory effect of fisetin on HSCs activation was reversed on co-culturing with EX-527, a specific inhibitor against STIR1 overexpression. Thus, fisetin has the potential to ameliorate alcohol-induced liver injury through suppression of HSCs activation, SIRT1-mediated autophagy and Sphk1-mediated ER stress.