Expression In Hepatic Stellate Cells Research Articles

IL-17a promotes hepatocellular carcinoma by increasing FAP expression in hepatic stellate cells via activation of the STAT3 signaling pathway

Studies have shown that hepatic stellate cells (HSCs) and interleukin-17a (IL-17a) play important roles in liver tumorigenesis. In addition, fibroblast activation protein-α (FAP) has been shown to be a key regulator of hepatic stellate cell activation. In this study, in vivo and in vitro experiments were performed to verify the promoting effects of IL-17a administration, IL-17a overexpression, and FAP upregulation in HSCs on liver fibrosis and liver tumorigenesis. The cleavage under targets & release using nuclease (CUT&RUN) technique was used to verify the binding status of STAT3 to the FAP promoter. The in vitro studies showed that IL-17a activated HSCs and promoted HCC development and progression. FAP and IL-17a overexpression also activated HSCs, promoted HCC cell proliferation and migration, and inhibited HCC cell apoptosis. The in vivo studies suggested that IL-17a and FAP overexpression in HSCs facilitated liver tumor development and progression. The CUT&RUN results indicated that FAP expression was regulated by STAT3, which could bind to the FAP promoter region and regulate its transcription status. We concluded that IL-17a promoted HCC by increasing FAP expression in HSCs via activation of the STAT3 signaling pathway.

Yohimbine ameliorates liver inflammation and fibrosis by regulating oxidative stress and Wnt/β-catenin pathway

Background and purposeChronic liver injury, caused by various aetiologies, causes recurrent tissue damage, culminating in decreased liver regenerative ability and resulting in fibrosis followed by cirrhosis. In this study, the anti-fibrotic activity of Yohimbine hydrochloride (YHC) was investigated using various in vitro models and in vivo models. MethodsTo assess the anti-inflammatory, antioxidant, and anti-fibrotic effects of YHC, lipopolysaccharide or TGF-β induced differentiation or lipid-induced oxidative-stress models were employed using HLECs, HSC-LX2, and HepG2 cells. Further, thioacetamide (TAA) induced hepatic inflammation/fibrosis models were utilized to validate the YHC's anti-fibrotic activity in rats. ResultsInflammation/differentiation experiments in HLECs and HSC-LX2 revealed that YHC treatment significantly (p < 0.001) mitigated the lipopolysaccharide or TGF-β induced upregulation of inflammatory and fibrotic markers expression respectively. In addition, YHC dose-dependently reduced the TGF-β induced migration and palmitic acid-induced oxidative stress in HepG2 cells. Further, TAA administration (5 weeks) in vivo rat model showed increased inflammatory marker levels/expression, oxidative stress, and pathological abnormalities. Additionally, TAA administration (9 weeks) elevated the fibrotic marker expression, collagen deposition in liver tissues, and shortened longevity in rats. Treatment with YHC dose-dependently mitigated the TAA-induced abnormalities in both inflammation and fibrosis models and improved the survival of the rats. Further mechanistic approaches revealed that TAA administration elevated the JNK, Wnt components and β-catenin expression in hepatic stellate cells and animal tissues. Further treatment with YHC significantly modulated the JNK/Wnt/β-catenin signaling. Moreover, the β-catenin nuclear translocation results showed that β-catenin levels were significantly elevated in the nuclear fraction of TAA control samples and reduced in YHC-treated samples. ConclusionYohimbine treatment significantly improved inflammation and fibrosis by inhibiting differentiation, oxidative stress, and collagen deposition by partly modulating the JNK/Wnt/β-catenin pathway. These results might serve as a foundation for proposing yohimbine as a potential lead compound for liver fibrosis.

Regulation of High-fat Diet-induced Liver Fibrosis by SOCS1 Expression in Hepatic Stellate Cells

Regulation of High-fat Diet-induced Liver Fibrosis by SOCS1 Expression in Hepatic Stellate Cells

Mediator subunit MED1 deficiency prevents carbon tetrachloride-induced hepatic fibrosis in mice.

Mediator subunit mediator 1 (MED1) mediates ligand-dependent binding of the mediator coactivator complex to various nuclear receptors and plays a critical role in embryonic development, lipid and glucose metabolism, liver regeneration, and tumorigenesis. However, the precise role of MED1 in the development of liver fibrosis has been unclear. Here, we showed that MED1 expression was increased in livers from nonalcoholic steatohepatitis (NASH) patients and mice and positively correlated with transforming growth factor β (TGF-β) signaling and profibrotic factors. Upon treatment with carbon tetrachloride (CCl4), hepatic fibrosis was much less in liver-specific MED1 deletion (MED1ΔLiv) mice than in MED1fl/fl littermates. TGF-β/Smad2/3 signaling pathway was inhibited, and gene expression of fibrotic markers, including α-smooth muscle actin (α-SMA), collagen type 1 α 1 (Col1a1), matrix metalloproteinase-2 (Mmp2), and metallopeptidase inhibitor 1 (Timp1) were decreased in livers of MED1ΔLiv mice with CCl4 injection. Transcriptomic analysis revealed that the differentially expressed genes in livers of CCl4-administered MED1ΔLiv mice were enriched in the pathway of oxidoreductase activity, followed by robustly reduced oxidoreductase activity-related genes, such as Gm4756, Txnrd3, and Etfbkmt. More importantly, we found that the reduction of reactive oxygen species (ROS) in MED1 knockdown hepatocytes blocked the activation of TGF-β/Smad2/3 pathway and the expression of fibrotic genes in LX2 cells. These results indicate that MED1 is a positive regulator for hepatic fibrogenesis, and MED1 may be considered as a potential therapeutic target for the regression of liver fibrosis.NEW & NOTEWORTHY In this study, we present the first evidence that liver mediator 1 (MED1) deficiency attenuated carbon tetrachloride-induced hepatic fibrosis in mouse. The underlying mechanism is that MED1 deficiency reduces reactive oxygen species (ROS) production in hepatocytes, thus restricts the activation of TGF-β/Smad2/3 signaling pathway and fibrogenic genes expression in hepatic stellate cells (HSCs). These data suggest that MED1 is an essential regulator for hepatic fibrogenesis, and MED1 may be considered as a potential therapeutic target for liver fibrosis.

FAT10 Silencing Prevents Liver Fibrosis through Regulating SIRT1 Expression in Hepatic Stellate Cells.

Background and objectives: Hepatic stellate cell (HSC) activation is the cardinal factor due to the accumulation of extracellular matrix proteins during the development of liver fibrosis. The aim of the present study was to find new targets for developing drugs to treat liver fibrosis, by screening the key genes involved in the activation of hepatic stellate cells. Methods: Differentially expressed genes were identified through TCGA database. RT-PCR, immunohistochemistry (IHC) assay, western blot, and ELISA were performed to evaluate the expression levels of FAT10 and fibrotic molecules. In vitro experiments were conducted to investigate the signaling pathways and biological functions of FAT10 in LX-2 cell lines. Results: In the present study, expression profiles obtained from the Gene Expression Omnibus (GEO) were used to explore the different genes expression between HSCs treated with or without carbon tetrachloride (CCl4). Human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10) was selected for further investigations. In animal model of carbon tetrachloride-induced liver fibrosis, the expression of FAT10 on activated HSCs is upregulated. In vitro, silencing FAT10 reduced TGF-β1-induced ECM activation and accumulation in LX-2 cells, and also suppressed the inflammatory response of LX-2 cells. Further Transwell results suggested that knockdown of FAT10 could inhibit TGF-β1-induced LX-2 cell migration and invasion. Mechanistically, FAT10 promotes its fibrotic activity through regulating sirtuin 1 (SIRT1), with a concomitant activation of ECM. Conclusions: These findings indicated an unexpected role of FAT10 in liver fibrosis development, suggesting that silencing FAT10 might represent a new strategy for the treatment of fibrotic liver diseases.

Suppression of MT1 and Melatonin Treatment Improves Liver Phenotypes in Mdr2‐/‐ mice

Background &amp; AimPrimary Sclerosing Cholangitis (PSC) is a cholestatic liver disease characterized by hepatic fibrosis and portal inflammation. Melatonin is synthesized by arylalkylamine N‐acetyltransferase (AANAT) in the pineal gland, as well as extrapineal sites such as cholangiocytes. We previously found that: (i) the MT1 receptor is primarily expressed in cholangiocytes with low and absent expression in hepatic stellate cells and hepatocytes, respectively; (ii) melatonin reduces biliary proliferation via MT1 receptor signaling; and (iii) melatonin treatment for 1 wk decreases biliary proliferation and liver fibrosis in bile duct ligated rats by downregulation of MT1 and clock genes (PER1, CRY1, CLOCK and BMAL1). We aimed to evaluate the beneficial effects of long‐term melatonin treatment and MT1 signaling on biliary phenotypes, liver fibrosis and portal inflammation in Mdr2‐/‐ mice (a model of PSC).MethodsMale FVB/NJ and Mdr2‐/‐ mice had access ad libitum to drinking water with/without melatonin for 3 months. We evaluated: (i) ductular reaction (DR) by immunohistochemistry (IHC) for CK19, (ii) liver fibrosis by Sirius Red staining and (iii) portal inflammation by IHC for F4/80. MT1, AANAT, and clock genes immunoreactivity was evaluated by immunofluorescence (IF) co‐stained with CK19 and qPCR in isolated cholangiocytes. Male C3H‐Hej (WT for MT1‐/‐), FVB/NJ (WT for Mdr2‐/‐), MT1‐/‐, Mdr2‐/‐ mice and MT1‐/‐/Mdr2‐/‐ (DKO) mice were euthanized at 12 wk of age. We analyzed: (i) MT1 expression by IF and mRNA expression in total liver by qPCR to validate our model; (ii) liver damage by H&amp;E, DR by IHC for CK19 and liver fibrosis by Sirius Red staining and (iii) portal inflammation by IHC for F4/80 and expression of the inflammatory markers by qPCR in total liver.ResultsLong‐term melatonin treatment reduces DR, liver fibrosis and portal inflammation in Mdr2‐/‐mice. Prolonged administration of melatonin in Mdr2‐/‐mice improves liver phenotype by decreasing the immunoreactivity of MT1, AANAT and clock genes in Mdr2‐/‐ mice, which suggests the chronobiotic action of melatonin on biliary circadian rhythm. DKO mice have no MT1 expression and display ameliorated liver phenotype compared to Mdr2‐/‐ mice.ConclusionWe demonstrated that chronic melatonin treatment improves liver histology and restores the biliary circadian rhythm by interaction with MT1. Suppression of MT1 receptor in Mdr2‐/‐mice ameliorates biliary/liver phenotypes through changes in clock genes and AANAT. Restoration of the circadian rhythm by modulation of melatonin/MT1 signaling may be key for the management of cholangiopathies.

MAPK p38/Ulk1 pathway inhibits autophagy and induces IL-1β expression in hepatic stellate cells.

In the past, hepatic stellate cells (HSCs) were considered to be noninflammatory cells and to contribute to liver fibrosis by producing extracellular matrix. Recently, it was found that HSCs can also secrete cytokines and chemokines and therefore participate in hepatic inflammation. Autophagy participates in many immune response processes in immune cells. It is unclear whether autophagy is involved in inflammatory cytokine induction in HSCs. MAPK p38, Ulk1 phosphorylation, and the Ulk1-Atg13 complex were analyzed in HSC-T6 cells after LPS treatment. The relationship between autophagy inhibition and inflammation was investigated in primary rat HSCs. We discovered that LPS inhibited autophagy through MAPK p38. The activation of MAPK p38 induced Ulk1 phosphorylation, which disrupted the Ulk1-Atg13 complex and therefore inhibited autophagy. Furthermore, in primary rat HSCs, we demonstrated that autophagy inhibition regulated IL-1β induction, which depended on the MAPK p38/Ulk1 pathway. Our results reveal a continuous signaling pathway, MAPK p38-Ulk1 phosphorylation-Ulk1-Atg13 disruption, which inhibits autophagy and induces IL-1β expression in HSCs.NEW & NOTEWORTHY LPS inhibits autophagy in a concentration- and dose-dependent manner in HSC-T6 cells. MAPK p38 induces phosphorylation of Ulk1, which disrupts the Ulk1-Atg13 complex and is therefore required for the inhibition of autophagy by LPS. LPS induces IL-1β expression via the MAPK p38/Ulk1 pathway in HSCs.

Cluster of Differentiation 44 Promotes Liver Fibrosis and Serves as a Biomarker in Congestive Hepatopathy.

Congestive hepatopathy (CH) with chronic passive congestion is characterized by the progression of liver fibrosis without prominent inflammation and hepatocellular damage. Currently, the lack of reliable biomarkers for liver fibrosis in CH often precludes the clinical management of patients with CH. To explore fibrosis biomarkers, we performed proteome analysis on serum exosomes isolated from patients with CH after the Fontan procedure. Exosomal cluster of differentiation (CD)44 levels were increased in patients with CH compared to healthy volunteers and was accompanied by increases in serum levels of soluble CD44 and CD44 expression in the liver. To address the roles of CD44 in CH, we established a mouse model of chronic liver congestion by partial inferior vena cava ligation (pIVCL) that mimics CH by fibrosis progression with less inflammation and cellular damage. In the pIVCL mice, enhanced CD44 expression in hepatic stellate cells (HSCs) and deposition of its ligand hyaluronan were observed in the liver. Blood levels of soluble CD44 were correlated with liver fibrosis. The blockade of CD44 with specific antibody inhibited liver fibrosis in pIVCL mice and was accompanied by a reduction in S100 calcium‐binding protein A4 expression following activation of HSCs. Conclusion: Chronic liver congestion promotes fibrosis through CD44. This identifies CD44 as a novel biomarker and therapeutic target of liver fibrosis in patients with CH.

Prophylactical Low Dose Whole-Liver Irradiation Inhibited Colorectal Liver Metastasis by Regulating Hepatic Niche in Mice.

BackgroundThe liver is the most common target for metastatic colorectal cancer. Changes of the local hepatic niche due to hepatic diseases such as cirrhosis decrease the incidence of colorectal cancer liver metastasis. Hepatic niche heterogeneity could influence the risk of hepatic metastasis.Materials and MethodsWe simulated changes of the hepatic niche via prophylactical liver irradiation with a safe dose of 6 Gy. GEO dataset and GO analysis revealed a difference in the expression of matrix metalloproteinase 1 (MMP1) in primary colorectal cancer versus liver metastasis, as well as synchronous versus metachronous liver metastasis. Western blotting, Immunofluorescence and qRT-PCR were conducted to measure protein expressions, location and RNA expressions. Colony formation, wound-healing, transwell assays experiments were performed to determine the malignant biological properties of colorectal cancer cells. shRNA transfection was used to conduct stable transfected cell lines.ResultsTissue inhibitor of metalloproteinases 1 (TIMP1) expression was significantly higher in metastases lesions than primary tumors. In vivo, TIMP1 expression in the hepatic niche increased after a safe dose of 6 Gy irradiation, along with MMP1 decreased, leading to collagen fiber deposition and impairment of hepatic microcirculation. In vitro, irradiated hepatic stellate cells-conditioned media reduced the migration and clone formation ability of colon cancer cells SW480 and HCT116. Low TIMP1 expression in hepatic stellate cells reduced tumor cell invasion and migration.ConclusionProphylactical 6 Gy whole-liver irradiation could inhibit colorectal cancer liver metastasis by regulating TIMP1/MMP1 balance in the hepatic niche before liver metastatic lesion formed.

CCL20 induced by visfatin in macrophages via the NF-\u03baB and MKK3/6-p38 signaling pathways contributes to hepatic stellate cell activation

Chemokines interact with hepatic resident cells during inflammation and fibrosis. CC chemokine ligand (CCL) 20 has been reported to be important in inflammation and fibrosis in the liver. We hypothesized that visfatin, an adipocytokine, could play a role in hepatic fibrosis via CCL20. We investigated the effect of visfatin on CCL20 in THP-1 human promonocytic cells and examined the molecular mechanisms involved. Following treatment of THP-1 cells with visfatin, CCL20 expression and secretion were assessed. We assessed the intracellular signaling molecules IKK/NF-κB, JAK2/STAT3, MAPKs, and MKK3/6 by western blotting. We treated THP-1 cells with visfatin and signaling inhibitors, and examined CCL20 mRNA and protein levels. To investigate the effect of visfatin-induced CCL20 expression in hepatic stellate cells (HSCs), LX-2 cells were co-cultured with the culture supernatant of THP-1 cells with or without anti-CCL20 neutralizing antibodies, and fibrosis markers were examined by RT-PCR and immunoblotting. In THP-1 cells, visfatin increased the CCL20 mRNA and protein levels. visfatin increased the activities of the NF-κB, p38, and MLK3/6 signaling pathways but not those of the JAK2/STAT3 and ERK pathways. Visfatin treatment together with an NF-κB, p38, or MLK3 inhibitor reduced the mRNA and protein levels of CCL20. The visfatin-induced CCL20 increased the expression of fibrosis markers and CCR6 in HSCs. Following neutralization of CCL20, the levels of fibrosis markers and CCR6 were decreased. Visfatin increases the expression of CCL20 via the NF-κB and MKK3/6-p38 signaling pathways in macrophages, and visfatin-induced CCL20 expression promotes the fibrosis markers in HSCs.

Expression of Carboxypeptidase X M14 Family Member 2 Accelerates the Progression of Hepatocellular Carcinoma via Regulation of the gp130/JAK2/Stat1 Pathway.

BackgroundCarboxypeptidase X, M14 family member 2 (CPXM2) has been reported to be involved with several human malignancies. However, the impact of CPXM2 on human hepatocellular carcinoma (HCC) tumorigenesis has not been studied.Materials and MethodsUsing immunohistochemistry, the detailed CPXM2 expression patterns were examined in HCC cell lines and tissues. Additionally, a hepatic stellate cell line overexpressing CPXM2 and an HCC CPXM2-knockdown cell line were established by lipofection of an expression plasmid or short hairpin RNA, respectively. The transfection efficiencies were confirmed by reverse transcription-quantitative PCR, Western blotting and immunofluorescence. Moreover, Western blotting was conducted to determine the phosphorylation levels of the tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (Stat1) pathway. Furthermore, gp130-specific hairpin RNA was used to knockdown gp130 expression in hepatic stellate cells overexpressing CPXM2. The malignant phenotype of cultured HCC cells was assessed by a Cell Counting Kit-8 (CCK8) assay, plate cloning assay, Matrigel invasion assay and wound-healing assay in vitro.ResultsIt was demonstrated that CPXM2 was upregulated in HCC, and its upregulation predicted a poor prognosis. Besides, the upregulation of CPXM2 markedly enhanced the metastatic potential of HCC via the gp130/JAK2/Stat1 signaling pathway in vitro.ConclusionIn summary, this evidence suggests a positive role for CPXM2 in HCC progression via modulation of the gp130/JAK2/Stat1 signaling pathway in HCC.

Leptin promotes methionine adenosyltransferase 2A expression in hepatic stellate cells by the downregulation of E2F-4 via the β-catenin pathway.

Most obese patients develop hyperleptinaemia. Leptin, mainly produced by adipocytes, demonstrates a promotional role in liver fibrosis. Hepatic stellate cell (HSC) activation, a key step in liver fibrogenesis, requires global reprogramming of gene expression. The remodeling of DNA methylation is a mechanism of the epigenetic regulation of gene expression. The biosynthesis of S-adenosylmethionine, a principle biological methyl donor, is catalyzed by methionine adenosyltransferase (MAT) such as MATⅡ which has been shown to promote HSC activation in vitro. This study was mainly aimed to determine the effect of leptin on MAT2A expression (the catalytic subunit of MATⅡ) in HSCs. Results showed that MAT2A knockdown reduced leptin-induced HSC activation and liver fibrosis in the leptin-deficient mouse model. Leptin promoted MAT2A expression in HSCs and increased MAT2A promoter activity. The axis of the β-catenin pathway/E2F-4 mediated the effect of leptin on MAT2A expression. Leptin-induced β-catenin signaling reduced E2F-4 expression and thus abated E2F-4 binding to MAT2A promoter at a site around -2779bp, leading to an increase in the MAT2A promoter activity. These data might shed more light on the mechanisms responsible for liver fibrogenesis in obese patients with hyperleptinaemia.

Overexpression of miRNA-25-3p inhibits Notch1 signaling and TGF-\u03b2-induced collagen expression in hepatic stellate cells

During chronic liver injury hepatic stellate cells (HSCs), the principal source of extracellular matrix in the fibrotic liver, transdifferentiate into pro-fibrotic myofibroblast-like cells - a process potentially regulated by microRNAs (miRNAs). Recently, we found serum miRNA-25-3p (miR-25) levels were upregulated in children with Cystic Fibrosis (CF) without liver disease, compared to children with CF-associated liver disease and healthy individuals. Here we examine the role of miR-25 in HSC biology. MiR-25 was detected in the human HSC cell line LX-2 and in primary murine HSCs, and increased with culture-induced activation. Transient overexpression of miR-25 inhibited TGF-β and its type 1 receptor (TGFBR1) mRNA expression, TGF-β-induced Smad2 phosphorylation and subsequent collagen1α1 induction in LX-2 cells. Pull-down experiments with biotinylated miR-25 revealed Notch signaling (co-)activators ADAM-17 and FKBP14 as miR-25 targets in HSCs. NanoString analysis confirmed miR-25 regulation of Notch- and Wnt-signaling pathways. Expression of Notch signaling pathway components and endogenous Notch1 signaling was downregulated in miR-25 overexpressing LX-2 cells, as were components of Wnt signaling such as Wnt5a. We propose that miR-25 acts as a negative feedback anti-fibrotic control during HSC activation by reducing the reactivity of HSCs to TGF-β-induced collagen expression and modulating the cross-talk between Notch, Wnt and TGF-β signaling.

RSJYB1 inhibits collagen type I protein expression in hepatic stellate cells via down-regulating activity of collagen α1 (I) promoter.

YB1 is a negative regulator in liver fibrosis. We wondered whether SJYB1, a homologous protein of YB1 from Schistosoma japonicum, has an effect on liver fibrosis in vitro. Recombinant SJYB1 (rSJYB1) protein was expressed in a bacterial system and purified by Ni‐NTA His·Bind Resin. A human hepatic stellate cell line, the LX‐2 cell line, was cultured and treated with rSJYB1. The role of rSJYB1 on LX‐2 cells was then analysed by Western blot and luciferase assay. We succeeded in expressing and purifying SJYB1 in a bacterial system and the purified rSJYB1 could be recognized by S japonicum‐infected rabbit sera. Western bolt analysis showed that rSJYB1 inhibited the expression of collagen type I, but had little effect on α‐smooth muscle actin (α‐SMA). Further analysis revealed that rSJYB1 inhibited the activity of collagen α1 (I) (COL1A1) promoter and functioned at −1592/−1176 region of COL1A1 promoter. Our data demonstrate that rSJYB1‐mediated anti‐fibrotic activity involves inhibiting the activity of COL1A1 promoter and subsequently suppressing the expression of collagen type I in hepatic stellate cells.

A variant in the MICA gene is associated with liver fibrosis progression in chronic hepatitis C through TGF-\u03b21 dependent mechanisms

Hepatocarcinogenesis is tightly linked to liver fibrosis. Recently, two GWAS variants, MICA rs2596542 and DEPDC5 rs1012068 were identified as being associated with the development of HCV-induced hepatocellular carcinoma (HCC) in Japanese patients. The role of these variants on hepatic inflammation and fibrosis that are closely associated with HCC development is not known, nor are the biological mechanisms underlying their impact on the liver. Here, we demonstrate in 1689 patients with chronic hepatitis C (CHC) (1,501 with CHC and 188 with HCV-related HCC), that the MICA (T) allele, despite not being associated with HCC susceptibility, is associated with increased fibrosis stage (OR: 1.47, 95% CI: 1.05–2.06, p = 0.02) and fibrosis progression rate (hazards ratio: 1.41, 95% CI: 1.04–1.90, p = 0.02). The DEPDC5 variant was not associated with any of these phenotypes. MICA expression was down-regulated in advanced fibrosis stages. Further, (T) allele carriage was associated with lower MICA expression in liver and serum. Transforming growth factor-β1 (TGF-β1) expression suppresses MICA expression in hepatic stellate cells. Our findings suggest a novel mechanism linking susceptibility to advanced fibrosis and subsequently indirectly to HCC, to the level of MICA expression through TGF-β1-dependent mechanisms.

Fibrogenic Gene Expression in Hepatic Stellate Cells Induced by HCV and HIV Replication in a Three Cell Co-Culture Model System

Retrospective studies indicate that co-infection of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) accelerates hepatic fibrosis progression. We have developed a co-culture system (MLH) comprising primary macrophages, hepatic stellate cells (HSC, LX-2), and hepatocytes (Huh-7), permissive for active replication of HCV and HIV, and assessed the effect of these viral infections on the phenotypic changes and fibrogenic gene expression in LX-2 cells. We detected distinct morphological changes in LX-2 cells within 24 hr post-infection with HCV, HIV or HCV/HIV in MLH co-cultures, with migration enhancement phenotypes. Human fibrosis microarrays conducted using LX-2 cell RNA derived from MLH co-culture conditions, with or without HCV and HIV infection, revealed novel insights regarding the roles of these viral infections on fibrogenic gene expression in LX-2 cells. We found that HIV mono-infection in MLH co-culture had no impact on fibrogenic gene expression in LX-2 cells. HCV infection of MLH co-culture resulted in upregulation (>1.9x) of five fibrogenic genes including CCL2, IL1A, IL1B, IL13RA2 and MMP1. These genes were upregulated by HCV/HIV co-infection but in a greater magnitude. Conclusion: Our results indicate that HIV-infected macrophages accelerate hepatic fibrosis during HCV/HIV co-infection by amplifying the expression of HCV-dependent fibrogenic genes in HSC.

High-Throughput Sequencing Strategy for miR-146b-regulated circRNA Expression in Hepatic Stellate Cells.

BackgroundThis study was designed to detect and analyze miR-146b-mediated circular RNA (circRNA) expression in hepatic stellate cells.Material/MethodsThe experiment was divided into a control group and a siRNA-miR-146b group. The interference efficiency of siRNA-miR-146b was confirmed by real-time quantitative reverse transcription PCR (qRT-PCR) and the cells were collected, and total RNA was collected for high flux sequencing. The miRNA-targeted carcass were predicted. Finally, the expression of 5 circRNAs was verified by qRT-PCR.ResultsmiR-146b expression in the siRNA-miR-146b group was significantly lower than that in the control group. The quality of the original sequencing data and the processed data satisfied with the analysis, and the expression of circRNAs was modulated after the reduction of miR-146b. Among them, 18 circRNAs were upregulated, while 77 circRNAs were downregulated in the miR-146b group compared with the control group. The gene prediction showed that hsa_circ1887 was the largest contact point in miRNA and circRNA regulatory networks. qRT-PCR showed that rno-circRNA-469, rno-circRNA-1138, rno-circRNA-2168 and rno-circRAN-1907 were significantly reduced, while circRNA-1984 was significantly promoted in the siRNA-miR-146b group compared with the control group, which were consistent with the measurements by high-throughput sequencing technique.ConclusionsmiR-146b could regulate the expression of circRNAs in HSCs, which might take part in the formation and development of hepatic fibrosis.

Tyrosine kinase SYK is a potential therapeutic target for liver fibrosis.

Spleen tyrosine kinase (SYK) plays a critical role in immune cell signaling pathways and has been reported as a biomarker for human hepatocellular carcinoma (HCC). We sought to investigate the mechanism by which SYK promotes liver fibrosis and to evaluate SYK as a therapeutic target for liver fibrosis. We evaluated the cellular localization of SYK and the association between SYK expression and liver fibrogenesis in normal, hepatitis B virus (HBV)-infected, hepatitis C virus (HCV)-infected and non-alcoholic steatohepatitis (NASH) liver tissue (n=36, 127, 22 and 30, respectively). A polymerase chain reaction (PCR) array was used to detect the changes in transcription factor (TF) expression in hepatic stellate cells (HSCs) with SYK knockdown. The effects of SYK antagonism on liver fibrogenesis were studied in LX-2 cells, TWNT-4 cells, primary human HSCs, and three progressive fibrosis/cirrhosis animal models, including a CCL4 mouse model, and diethylnitrosamine (DEN) and bile duct ligation (BDL) rat models. We found that SYK protein in HSCs and hepatocytes correlated positively with liver fibrosis stage in human liver tissue. HBV or HCV infection significantly increased SYK and cytokine expression in hepatocytes. Increasing cytokine production further induced SYK expression and fibrosis-related gene transcription in HSCs. Up-regulated SYK in HSCs promoted HSC activation by increasing the expression of specific TFs related to activation of HSCs. SYK antagonism effectively suppressed liver fibrosis via inhibition of HSC activation, and decreased obstructive jaundice and reduced HCC development in animal models. Conclusion: SYK promotes liver fibrosis via activation of HSCs and is an attractive potential therapeutic target for liver fibrosis and prevention of HCC development. (Hepatology 2018).

Expression patterns of STAT3, ERK and estrogen-receptor \u03b1 are associated with development and histologic severity of hepatic steatosis: a retrospective study

BackgroundHepatic steatosis renders hepatocytes vulnerable to injury, resulting in the progression of preexisting liver disease. Previous animal and cell culture studies implicated mammalian target of rapamycin (mTOR), signal transducer and activator of transcription-3 (STAT3), extracellular signal-regulated kinase (ERK) and estrogen-receptor α in the pathogenesis of hepatic steatosis and disease progression. However, to date there have been few studies performed using human liver tissue to study hepatic steatosis. We examined the expression patterns of mTOR, STAT3, ERK and estrogen-receptor α in liver tissues from patients diagnosed with hepatic steatosis.MethodsWe reviewed the clinical and histomorphological features of 29 patients diagnosed with hepatic steatosis: 18 with non-alcoholic fatty liver disease (NAFLD), 11 with alcoholic fatty acid disease (AFLD), and a control group (16 biliary cysts and 22 hepatolithiasis). Immunohistochemistry was performed on liver tissue using an automated immunostainer. The histologic severity of hepatic steatosis was evaluated by assessing four key histomorphologic parameters common to NAFLD and AFLD: steatosis, lobular inflammation, ballooning degeneration and fibrosis.ResultsmTOR, phosphorylated STAT3, phosphorylated pERK, estrogen-receptor α were found to be more frequently expressed in the hepatic steatosis group than in the control group. Specifically, mTOR was expressed in 78% of hepatocytes, and ERK in 100% of hepatic stellate cells, respectively, in patients with NAFLD. Interestingly, estrogen-receptor α was diffusely expressed in hepatocytes in all NALFD cases. Phosphorylated (active) STAT3 was expressed in 73% of hepatocytes and 45% of hepatic stellate cells in patients with AFLD, and phosphorylated (active) ERK was expressed in hepatic stellate cells in all AFLD cases. Estrogen-receptor α was expressed in all AFLD cases (focally in 64% of AFLD cases, and diffusely in 36%). Phosphorylated STAT3 expression in hepatocytes and hepatic stellate cells correlated with severe lobular inflammation, severe ballooning degeneration and advanced fibrosis, whereas diffusely expressed estrogen-receptor α correlated with a mild stage of fibrosis.ConclusionsOur data indicate ERK activation and estrogen-receptor α may be relevant in the development of hepatic steatosis. However, diffuse expression of estrogen-receptor α would appear to impede disease progression, including hepatic fibrosis. Finally, phosphorylated STAT3 may also contribute to disease progression.

Delta-like homolog1/GATA binding protein 2 axis mediates leptin inhibition of PPARγ2 expression in hepatic stellate cells in vitro

AimsPeroxisome-proliferator activated receptor γ (PPARγ) plays a pivotal role in inhibition of hepatic stellate cell (HSC) activation, a key step for liver fibrogenesis. Adipocyte-derived hormone leptin has been shown to promote liver fibrosis in murine and human. PPARγ includes two subtypes, PPARγ1 and PPARγ2. Our previous study indicated that leptin down-regulated PPARγ1 expression in HSCs. The aim of this study was to investigate the effect of leptin on PPARγ2 expression and the underlying mechanisms in HSCs. Main methodsReal-time PCR and western blot analyses were used to examine gene expression. The promoter activities were detected by luciferase assay. Key findingsLeptin reduced PPARγ2 expressions at promoter level, mRNA level, and protein level in HSCs, which required β-catenin, p38 mitogen-activated protein kinase, and delta-like homolog1 (DLK1) signaling pathways. Leptin induced GATA binding protein 2 (GATA2) expression through DLK1 pathway and GATA2 reduced PPARγ2 expression. Ectopic expression of PPARγ2 reduced the protein levels of α-smooth muscle actin and α1(I)collagen in HSCs. SignificanceSince obese patients, often accompanied by hyperleptinemia, are more prone to liver fibrosis, the data from this study might have potential implications for clarifying the mechanisms for liver fibrogenesis in obese patients with hyperleptinemia.