Gene expression profile of quiescent and activated rat hepatic stellate cells implicates Wnt signaling pathway in activation

BACKGROUND/AIMS: Liver fibrosis is characterized by accumulation of extracellular matrix proteins synthesized by activated hepatic stellate cells (HSCs). To understand molecular mechanisms of HSCs activation a comprehensive comparison of gene expression between quiescent and activated HSCs is needed. METHODS: Using DNA microarrays we compared expression of 31,100 genes between quiescent rat HSCs and culture activated rat HSCs. Expression of the components of Wnt signaling was analyzed in HSCs and fibrotic livers by RT-PCR. Activation of beta-catenin was analyzed by Western blot. RESULTS: Nine hundred genes were upregulated more than 4.6-fold and 500 genes were downregulated more than 5.7-fold in activated HSCs. The upregulated genes included Wnt receptor frizzled 2, ligands Wnt4 and Wnt5, which was confirmed in fibrotic livers. Expression of the target genes of Wnt signaling was increased from 5- to 70-fold. Phosphorylation and nuclear translocation of beta-catenin were unchanged, indicating activation of the noncanonical Wnt pathway. CONCLUSIONS: Highly upregulated expression of Wnt5a and its receptor frizzled 2 implicates this pathway in differentiation of quiescent HSCs into myofibroblasts. Activation of Wnt signaling pathway in HSCs and in animal models of liver fibrosis has not been described previously, suggesting an important role of Wnt signaling in development of liver fibrosis.     

Oxymatrine liposome attenuates hepatic fibrosis via targeting hepatic stellate cells

AIM: To investigate the potential mechanism of Arg-Gly-Asp (RGD) peptide-labeled liposome loading oxymatrine (OM) therapy in CCl₄-induced hepatic fibrosis in rats.METHODS: We constructed a rat model of CCl₄-induced hepatic fibrosis and treated the rats with different formulations of OM. To evaluate the antifibrotic effect of OM, we detected levels of alkaline phosphatase, hepatic histopathology (hematoxylin and eosin stain and Masson staining) and fibrosis-related gene expression of matrix metallopeptidase (MMP)-2, tissue inhibitor of metalloproteinase (TIMP)-1 as well as type I procollagen via quantitative real-time polymerase chain reaction. To detect cell viability and apoptosis of hepatic stellate cells (HSCs), we performed 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay and flow cytometry. To reinforce the combination of oxymatrine with HSCs, we constructed fluorescein-isothiocyanate-conjugated Arg-Gly-Asp peptide-labeled liposomes loading OM, and its targeting of HSCs was examined by fluorescent microscopy.RESULTS: OM attenuated CCl₄-induced hepatic fibrosis, as defined by reducing serum alkaline phosphatase (344.47 ± 27.52 U/L vs 550.69 ± 43.78 U/L, P < 0.05), attenuating liver injury and improving collagen deposits (2.36% ± 0.09% vs 7.70% ± 0.60%, P < 0.05) and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen (P < 0.05). OM inhibited cell viability and induced apoptosis of HSCs in vitro. RGD promoted OM targeting of HSCs and enhanced the therapeutic effect of OM in terms of serum alkaline phosphatase (272.51 ± 19.55 U/L vs 344.47 ± 27.52 U/L, P < 0.05), liver injury, collagen deposits (0.26% ± 0.09% vs 2.36% ± 0.09%, P < 0.05) and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen (P < 0.05). Moreover, in vitro assay demonstrated that RGD enhanced the effect of OM on HSC viability and apoptosis.CONCLUSION: OM attenuated hepatic fibrosis by inhibiting viability and inducing apoptosis of HSCs. The RGD-labeled formulation enhanced the targeting efficiency for HSCs and the therapeutic effect.     

Hydronidone ameliorates liver fibrosis by inhibiting activation of hepatic stellate cells via Smad7-mediated degradation of TGFβRI

Background and Purpose

Liver fibrosis is a wound-healing reaction that eventually leads to cirrhosis. Hydronidone is a new pyridine derivative with the potential to treat liver fibrosis. In this study, we explored the antifibrotic effects of hydronidone and its potential mode of action.

Methods

The anti-hepatic fibrosis effects of hydronidone were studied in carbon tetrachloride (CCl₄)- and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)- induced animal liver fibrosis. The antifibrotic mechanisms of hydronidone were investigated in hepatic stellate cells (HSCs). The antifibrotic effect of hydronidone was further tested after Smad7 knockdown in HSCs in mouse models of fibrosis.

Results

In animal models, hydronidone attenuated liver damage and collagen accumulation, and reduced the expression of fibrosis-related genes. Hydronidone decreased the expression of fibrotic genes in HSCs. Impressively, hydronidone significantly upregulated Smad7 expression and promoted the degradation of transforming growth factor β receptor I (TGFβRI) in HSCs and thus inhibited the TGFβ-Smad signalling pathway. Specific knockdown of Smad7 in HSCs in vivo blocked the antifibrotic effect of hydronidone.

Conclusion

Hydronidone ameliorates liver fibrosis by inhibiting HSCs activation via Smad7-mediated TGFβRI degradation. Hydronidone is a potential drug candidate for the treatment of liver fibrosis.     

Involvement of 90-kuD ribosomal S6 kinase in collagen type I expression in rat hepatic fibrosis

AIM： To investigate the relationship between 90-kuD ribosomal $6 kinase （pg0RSK） and collagen type I expression during the development of hepatic fibrosis in vivo and in vitro.METHODS： Rat hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. The protein expression and cell location of p90RSK and their relationship with collagen type I were determined by co-immunofluoresence and confocal microscopy.Subsequently, RNAi strategy was employed to silence p90RSK mRNA expression in HSC-T6, an activated hepatic stellate cell （HSC） line. The expression of collagen type I in HSC-T6 cells was assessed by Western blotting and real-time polymerase chainreaction. Furthermore, HSCs were transfected with expression vectors or RNAi constructs of p90RSK to increase or decrease the p90RSK expression, thencollagen type I promoter activity in the transfected HSCs was examined by reporter assay. Lastly HSC-T6 cells transfected with p90RSK siRNA was treated withor without platelet-derived growth factor （PDGF）-BB at a final concentration of 20μg/L and the cell growthwas determined by MTS conversion.RESULTS： In fibrotic liver tissues, p90RSK was over-expressed in activated HSCs and had a significantpositive correlation with collagen type I levels.In HSC-T6 cells transfected with RNAi targeted top90RSK, the expression of collagen type I was down-regulated （61.8% in mRNA, P 〈 0.01, 89.1% inprotein, P 〈 0.01）. However, collagen type ] promoteractivity was not increased with over-expression of p90RSK and not decreased with low expression either,compared with controls in the same cell line （P = 0.076）.Furthermore, p90RSK siRNA exerted the inhibitionof HSC proliferation, and also abolished the effect of PDGF on the HSC proliferation.CONCLUSION： p90RSK is over-expressed in activatedHSCs and involved in regulating the abnormalexpression of collagen type I through initiating theproliferation of HSCs.     

New role and molecular mechanism of Gadd45a in hepatic ?brosis

AIM: To investigate the role of Gadd45a in hepatic fibrosis and the transforming growth factor (TGF)-β/Smad signaling pathway.METHODS: Wild-type male BALB/c mice were treated with CCl₄ to induce a model of chronic liver injury. Hepatic stellate cells (HSCs) were isolated from the liver of BALB/c mice and were treated with small interfering RNAs (siRNAs) targeting Gadd45a or the pcDNA3.1-Gadd45a recombinant plasmid. Cellular α-smooth muscle actin (α-SMA), β-actin, type I collagen, phospho-Smad2, phospho-Smad3, Smad2, Smad3, and Smad4 were detected by Western blots. The mRNA levels of α-SMA, β-actin, and type I collagen were determined by quantitative real-time (qRT)-PCR analyses. Reactive oxygen species production was monitored by flow cytometry using 2,7-dichlorodihydrofluorescein diacetate. Gadd45a, Gadd45b, anti-Gadd45g, type I collagen, and SMA local expression in liver tissue were measured by histologic and immunohistochemical analyses.RESULTS: Significant downregulation of Gadd45a, but not Gadd45b or Gadd45g, accompanied by activation of the TGF-β/Smad signaling pathways was detected in fibrotic liver tissues of mice and isolated HSCs with chronic liver injury induced by CCl₄ treatment. Overexpression of Gadd45a reduced the expression of extracellular matrix proteins and α-SMA in HSCs, whereas transient knockdown of Gadd45a with siRNA reversed this process. Gadd45a inhibited the activity of a plasminogen activator inhibitor-1 promoter construct and (CAGA)₉ MLP-Luc, an artificial Smad3/4-specific reporter, as well as reduced the phosphorylation and nuclear translocation of Smad3. Gadd45a showed protective effects by scavenging reactive oxygen species and upregulating antioxidant enzymes.CONCLUSION: Gadd45a may counteract hepatic fibrosis by regulating the activation of HSCs via the inhibition of TGF-β/Smad signaling.     

Glycyrrhizic acid inhibits apoptosis and fibrosis in carbontetrachloride-induced rat liver injury

AIM:To investigate anti-apoptotic effects of glycyrrhizic acid(GA) against fibrosis in carbon tetrachloride(CCl4)-induced liver injury and its contributing factors.METHODS:Liver fibrosis was induced by administration of CCl4 for 8 wk.Pathological changes in the liver of rats were examined by hematoxylin-eosin staining.Collagen fibers were detected by Sirius red staining.Hepatocyte apoptosis was determined by TUNEL assay and the expression levels of cleaved caspase-3,Bax,α-SMA,connective tissue growth factor(CTGF),matrix metalloproteinase(MMP) 2 and MMP9 proteins were evaluated by western blot analysis,and α-SMA m RNA,collagen type Ⅰ and Ⅲ m RNA were estimated by real-time PCR.RESULTS:Treatment with GA significantly improved the pathological changes in the liver and markedly decreased the positive area of Sirius red compared with rats in the CCl4-treated group.TUNEL assay showed that GA significantly reduced the number of TUNEL-positive cells compared with the CCl4-treated group.The expression levels of cleaved caspase-3,Bax,α-SMA,CTGF,MMP2 and MMP9 proteins,and α-SMA m RNA,collagen type Ⅰ and Ⅲ m RNA were also significantly reduced by GA compared with the CCl4-treated group(P 0.05).CONCLUSION:GA treatment can ameliorate CCl4-induced liver fibrosis by inhibiting hepatocyte apoptosis and hepatic stellate cell activation.     

Ubiquitin specific peptidase 1 promotes hepatic fibrosis through positive regulation of CXCL1 by deubiquitinating SNAIL

BackgroundHepatic fibrosis is attributed to an imbalance of extracellular matrix production and lysis. Human hepatic stellate cells (HSCs) have been uncovered to converge through complex interactions with hepatocytes and immune cells, causing scarring in liver damage.AimsWe aimed to investigate the expression status of ubiquitin specific peptidase 1 (USP1) and its potential mechanisms on HSCs and hepatic fibrosis.MethodsHepatic fibrosis animal and cell models were generated using mice with carbon tetrachloride (CCl4) treatment and HSCs LX-2 with TGF-β1 treatment. Relationships among USP1, SNAIL, and CXCL1 were identified via dual-luciferase reporter gene assay, co-immunoprecipitation, and chromatin immunoprecipitation. With gain- and loss-of-experiments, CCK-8 and flow cytometry assays were employed for cell proliferation and apoptosis.ResultsUSP1 upregulated SNAIL expression through deubiquitination to increase CXCL1 expression. USP1 downregulation decreased expressions of fibrosis-related genes, suppressed proliferation, and promoted apoptosis in TGF-β1-induced LX-2 cells, which were reversed by SNAIL overexpression. The pro-fibrosis role caused by SNAIL upregulation was abolished by CXCL1 reduction. Promotive function of USP1/SNAIL/CXCL1 axis in hepatic fibrosis was further confirmed in vivo.ConclusionThese data supported siRNA-mediated silencing of USP1 improved hepatic fibrosis through inhibition of SNAIL and CXCL1, which yields a new therapeutic target for hepatic fibrosis treatment.     

Nonmuscle myosin II regulates migration but not contraction in rat hepatic stellate cells

AIM: To identify and characterize the function of non-mu-scle myosin Ⅱ (NMM Ⅱ) isoforms in primary rat hepatic stellate cells (HSCs).METHODS: Primary HSCs were isolated from male Spra-gue-Dawley rats by pronase/collagenase digestion. Total RNA and protein were harvested from quiescent and culture-activated HSCs. NMM Ⅱ isoform (Ⅱ-A, Ⅱ-B and Ⅱ-C) gene and protein expression were measured by RealTime polymerase chain reaction and Western blot analyses respectively. NMM Ⅱ protein localization was visualized in vitro using immunocytochemical analysis. For in vivo assessment, liver tissue was harvested from bile duct-ligated (BDL) rats and NMM Ⅱisoform expression determined by immunohistochemistry. Using a selective myosin Ⅱ inhibitor and siRNA-mediated knockdown of each isoform, NMM Ⅱ functionality inprimary rat HSCs was determined by contraction and migration assays.RESULTS: NMM Ⅱ-A and Ⅱ-B mRNA expression was increased in culture-activated HSCs (Day 14) with sig-niflicant increases seen in all pairwise comparisons (Ⅱ-A: 12.67 ± 0.99 (quiescent) vs 17.36 ± 0.78 (Day 14), P < 0.05; Ⅱ-B: 4.94 ± 0.62 (quiescent) vs 13.90 ±0.85 (Day 14), P < 0.001). Protein expression exhibited similar expression patterns (Ⅱ-A: 1.87 ± 2.50 (quiescent) vs 58.64 ± 8.76 (Day 14), P < 0.05; Ⅱ-B: 1.17 ± 1.93 (quiescent) vs 103.71 ± 21.73 (Day 14), P < 0.05). No signif icant differences were observed in NMM Ⅱ-C mRNA and protein expression between quiescent and activated HSCs. In culture-activated HSCs, NMM Ⅱ-A and Ⅱ-B merged with F-actin at the cellular periphery and throughout cytoplasm respectively. In vitro stud-ies showed increased expression of NMM Ⅱ-B in HSCs activated by BDL compared to sham-operated animals. There were no apparent increases of NMM Ⅱ-A and Ⅱ-C protein expression in HSCs during hepatic BDL injury. To determine the contribution of NMM Ⅱ-A and Ⅱ-B to migration and contraction, NMM Ⅱ-A and Ⅱ-B expres-sion were downregulated with siRNA. NMM Ⅱ-A and/or Ⅱ-B siRNA inhibited HSC migration by approximately 25% compared to scramble siRNA-treated cells. Conversely, siRNA-mediated NMM Ⅱ-A and Ⅱ-B inhibition had no signif icant effect on HSC contraction; however, contraction was inhibited with the myosin Ⅱ inhibitor, blebbistatin (38.7% ± 1.9%).CONCLUSION: Increased expression of NMM Ⅱ-A and Ⅱ-B regulates HSC migration, while other myosin Ⅱclasses likely modulate contraction, contributing to development and severity of liver f ibrosis.     

LncRNA DIRC1 is a novel prognostic biomarker and correlated with immune infiltrates in stomach adenocarcinoma

The potential application value of Long non-coding RNA disrupted in renal carcinoma 1 (DIRC1) has not yet been explored, the purpose of this study was to explore the relationship between DIRC1 and stomach adenocarcinoma (STAD) based on the cancer genome atlas database. Wilcoxon rank sum test, Chi-square test, Fisher test and logistic regression were used to evaluate relationships between clinical-pathologic features and DIRC1 expression. Receiver operating characteristic (ROC) curves were used to describe binary classifier value of DIRC1 using area under curve (AUC) score. Kaplan-Meier method was used to assess the impact of DIRC1 on prognosis and the impact of DIRC1-related hub genes on prognosis. Gene oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were used to predict the function of differentially expressed genes associated with DIRC1. Gene set enrichment analysis (GSEA) was used to predict biological states or processes associated with DIRC1. Immune infiltration analysis was performed to identify the significantly involved functions of DIRC1. Protein–protein interaction (PPI) networks were established and 10 hub genes identified with Cytoscape software. Real time-polymerase chain reaction (RT-PCR) was used to detect the expression of DIRC1 in Gastric Cancer patients and healthy people. Increased DIRC1 expression in STAD was associated with T stage (P = .004), race (P = .045), histologic grade (P = .029) and anatomic neoplasm subdivision (P = .034). ROC curve suggested the significant diagnostic ability of DIRC1 (AUC = 0.779). High DIRC1 expression predicted a poorer Overall survival (P = .004, hazard ratio: 1.63; 95% confidence interval: 1.17‐2.27; P = .034). GO and KEGG analysis demonstrated that DIRC1 is related to epidermis, collagen-containing extracellular matrix, receptor-ligand activity, protein digestion and absorption, etc. GSEA demonstrated that E2F target, G2M checkpoint, Myc target, interferon γ reaction were differentially enriched in the high DIRC1 expression phenotype. SsGSEA and Spearman correlation revealed the relationships between DIRC1 and macrophages, dendritic cells, and Th1 cells were the strongest. Coregulatory proteins were included in the PPI network, higher expressions of 4 hub genes were associated with worse prognosis in STAD. RT-PCR showed that the expression of DIRC1 in the serum of Gastric Cancer patients was higher than healthy people (P = .027). DIRC1 expression was significantly correlated with poor survival and immune infiltrations in STAD, and it may be a promising prognostic biomarker in STAD.     

Wnt5a inhibits canonical Wnt signaling in hematopoietic stem cells and enhances repopulation

The mechanisms that regulate hematopoietic stem cell (HSC) fate decisions between proliferation and multilineage differentiation are unclear. Members of the Wnt family of ligands that activate the canonical Wnt signaling pathway, which utilizes beta-catenin to relay the signal, have been demonstrated to regulate HSC function. In this study, we examined the role of noncanonical Wnt signaling in regulating HSC fate. We observed that noncanonical Wnt5a inhibited Wnt3a-mediated canonical Wnt signaling in HSCs and suppressed Wnt3a-mediated alterations in gene expression associated with HSC differentiation, such as increased expression of myc. Wnt5a increased short- and long-term HSC repopulation by maintaining HSCs in a quiescent G(0) state. From these data, we propose that Wnt5a regulates hematopoiesis by the antagonism of the canonical Wnt pathway, resulting in a pool of quiescent HSCs.     

Inhibitory effects of microRNA 19b in hepatic stellate cell-mediated fibrogenesis

Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGF-β). MicroRNAs (miRs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGF-β signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Because miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGF-β signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGF-β signaling components demonstrated by decreased TGF-β receptor II (TGF-βRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3' untranslated region of TGF-βRII was validated by luciferase reporter assay. Inhibition of TGF-β signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGF-β-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased smooth muscle α-actin expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared with normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared with normal human livers. CONCLUSION: miR 19b is a novel regulator of TGF-β signaling in HSCs, suggesting a potential therapeutic approach for hepatic fibrosis.     

Notch3 regulates the activation of hepatic stellate cells

AIM: To investigate whether Notch signaling is involved in liver fibrosis by regulating the activation of hepatic stellate cells (HSCs).METHODS: Immunohistochemistry was used to detect the expression of Notch3 in fibrotic liver tissues of patients with chronic active hepatitis. The expression of Notch3 in HSC-T6 cells treated or not with transforming growth factor (TGF)-β1 was analyzed by immunofluorescence staining. The expression of Notch3 and myofibroblastic marker α-smooth muscle actin (α-SMA) and collagen I in HSC-T6 cells transfected with pcDNA3.1-N3ICD or control vector were detected by Western blotting and immunofluorescence staining. Moreover, effects of Notch3 knockdown in HSC-T6 by Notch3 siRNA were investigated by Western blotting and immunofluorescence staining.RESULTS: The expression of Notch3 was significantly up-regulated in fibrotic liver tissues of patients with chronic active hepatitis, but not detected in normal liver tissues. Active Notch signaling was found in HSC-T6 cells. TGF-β1 treatment led to up-regulation of Notch3 expression in HSC-T6 cells, and over-expression of Notch3 increased the expression of α-SMA and collagen I in HSC-T6 without TGF-β1 treatment. Interestingly, transient knockdown of Notch3 decreased the expression of myofibroblastic marker and antagonized TGF-β1-induced expression of α-SMA and collagen I in HSC-T6.CONCLUSION: Notch3 may regulate the activation of HSCs, and the selective interruption of Notch3 may provide an anti-fibrotic strategy in hepatic fibrosis.