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Background/Aims: Autoimmune hepatitis (AIH) is a chronic necroinflammatory disease of the liver whose pathogenic mechanisms have not yet been elucidated. Moreover, the current treatment used for the vast majority of AIH patients is largely dependent on immunosuppressant administration and liver transplantation. However, research on the pathogenesis of AIH and effective new treatments for AIH have been hampered by a lack of animal models that accurately reproduce the human condition. Methods: AIH models created by concanavalin A (ConA) injections at different times and doses. The levels of ALT, AST, LDH and inflammatory cytokines were examined at various times after 20 mg/kg ConA was administered by ELISA using commercially available kits. Moreover, liver pathological changes were observed by flow cytometry (FCM) and H&E staining. Results: Our experiments demonstrated that the levels of ALT, AST, LDH and several inflammatory cytokines, including TNF-α, IFN-γ, and IL-6, were higher in the 20 mg/kg 12 h ConA group than in the other groups. Importantly, the numbers of activated CD4+ and CD8+ T lymphocytes in the blood, spleen and liver were calculated. These results showed that ConA (20 mg/kg for 12 h)-induced hepatitis was similar to that in clinical AIH patients. Furthermore, we found that the number of MDSCs in the blood was significantly increased in the ConA (20 mg/kg for 12 h) group compared with controls. Our findings indicated that ConA (20 mg/kg for 12 h)-induced hepatitis could be used as an experimental murine model that mirrors most of the pathogenic properties of human type I AIH. Conclusion: This model [ConA (20 mg/kg for 12 h)] provides a valuable tool for studying AIH immunopathogenesis and rapidly assessing novel therapeutic approaches.

Background: The activation of hepatic stellate cells (HSCs) participates in liver fibrosis, and emerging evidences indicate that microRNAs (miRNAs) are abnormally expressed during HSC activation. However, the potential roles of miRNAs in liver fibrosis still remain elusive. Methods: The differential expression of miRNAs between the quiescent and activated HSCs was measured by miRNA microarray analysis, which was further validated by RT-qPCR in animal models and patients. The effect of miR-199a-3p on CAV2 expression was examined by Western blotting using miRNA mimics or inhibitor. Induction of miR-199a-3p expression by TWIST1 was explored by the luciferase assay and RT-qPCR. The role of miR-199a-3p in liver fibrosis was systematically investigated in vitro and in vivo. Findings: miR-199a-3p expression was dramatically upregulated during HSC activation in vitro, and during liver fibrogenesis in CCl4-treated rats, and was increased in livers from high-fat-diet-induced mice or the patients with cirrhosis. The expression of miR-199a-3p was driven by Twist1 which could be further induced by TGF-β treatment. Functional studies showed that inhibition of miR-199a-3p in both human LX2 cells and rat HSCs significantly decreased the expression of fibrotic markers, whereas the forced expression of miR-199a-3p exhibited opposite effects, demonstrating the role of miR-199a-3p in promoting HSCs activation. Mechanistically, miR-199a-3p plays an important role in TGF-β signalling pathway activation through targeting CAV2 that negatively regulates the expression of transforming growth factor-beta receptor type I (TGFβRI). Importantly, administration of antagomiR-199a-3p in the CCl4 treated mice significantly ameliorated hepatic fibrosis. Interpretation: Twist1-induced miR-199a-3p mediates the activation of HSCs by suppressing CAV2 expression and subsequently increasing TGFβRI expression to promote TGF-β pathway. Our findings highlight the therapeutic potential of miR-199a-3p for hepatic fibrosis. Funding Statement: This study was supported by National Natural Science Foundation of China (81570554 and 81770568 to LY; 81572739 to YP), and by National Key R&D Program of China (2016YFA0502204 to YP). TS is supported by a joint PhD studentship by King’s College London, UK and China Scholarship Council, China. Declaration of Interests: None of the authors has any potential conflict of interests to disclose. Ethics Approval Statement: All animal studies were approved by the Medical Ethics Committee of the West China Hospital of Sichuan University. All specimens were collected upon obtaining informed consent from all patients, and the study was approved by the Human Ethics Committee of Sichuan University.

MicroRNAs (miRNAs) have been demonstrated to modulate cellular processes in the liver. However, the role of miRNAs in liver fibrosis is poorly understood. Because the activation of hepatic stellate cells (HSCs) is a pivotal event in the initiation and progression of hepatic fibrosis, we investigate the differential expression of miRNAs in activated and quiescent rat HSCs by microarray analysis and find that miR-214 (miR-214-3p) is significantly upregulated during HSC activation. Moreover, the robust induction of miR-214 is correlated with liver fibrogenesis in carbon tetrachloride (CCl4)-treated rats and mice, high-fat diet-induced non-alcoholic steatohepatitis in mice, and cirrhosis in humans. We identify that miR-214 expression is driven by the helix–loop–helix transcription factor Twist1 via the E-box element. The increased miR-214 inhibits the expression of suppressor-of-fused homolog (Sufu), a negative regulator of the Hedgehog signaling pathway, thereby contributing to HSC activation to promote the accumulation of fibrous extracellular matrix and the expression of profibrotic genes in HSCs and LX2 cells. Furthermore, miR-214 expression is inversely correlated with the expression of Sufu in clinical cirrhosis samples. To explore the clinical potential of miR-214, we inject antagomiR-214 oligos into mice to induce hepatic fibrosis. The knockdown of miR-214 in vivo enhances Sufu expression and reduces fibrosis marker expression, which ameliorates liver fibrosis in mice. In conclusions, the Twist1-regulated miR-214 promotes the activation of HSC cells through targeting Sufu involved in the Hedgehog pathway and participates in the development of hepatic fibrosis. Hence, the knockdown of miR-214 expression may be a promising therapeutic strategy for liver fibrosis.

Aim. To explore the efficacy and safety of immunosuppressive therapy for the treatment of primary biliary cirrhosis-autoimmune hepatitis (PBC-AIH) overlap syndrome accompanied by decompensated cirrhosis. Methods. A cohort study was performed to evaluate the usefulness of immunosuppressive therapy in this unique group. This cohort study was performed between October 2013 and June 2017 and included 28 biopsy-proven patients diagnosed according to the Paris criteria. The therapies included ursodeoxycholic acid (UDCA) alone (N=14) or in combination with immunosuppression (IS) therapy (N=14). The primary endpoints were biochemical remission, liver-related adverse events, transplant-free survival, and drug side-effects. Results. The frequency of biochemical remission for the AIH features was significantly higher in the UDCA+IS group than in the UDCA-only group (60.0 versus 9.1%, P=0.024) after 12 months of therapy but not after 3 and 6 months (28.6 versus 0%, P=0.165; 35.7 versus 7.1%, P=0.098). The rates of liver-related adverse events were lower in the combined group (2/14 versus 9/14, P=0.018). The Kaplan-Meier estimate showed that the transplant-free survival was distinct between the two groups (P=0.019). In the UDCA+IS group, mild and transient leukopenia occurred in two patients receiving azathioprine (AZA), and an infection was observed in one patient receiving mycophenolate mofetil (MMF). Conclusions. PBC-AIH patients with decompensated cirrhosis receiving a combination of UDCA and immunosuppressors presented with higher biochemical remission rates and experienced fewer liver-related adverse events, implying that the combined treatment might be a better therapeutic option for strictly defined decompensated PBC-AIH overlap syndrome.

Objective. To examine Nogo-B in liver tissues and plasma of patients with liver cirrhosis and associate them with various clinical parameters.Materials and Methods. Nogo-B protein expression was examined by immunohistochemistry in 24 human fibrotic/cirrhotic liver specimens and 10 healthy controls. We determined plasma Nogo-B levels by enzyme-linked immunosorbent assay in 301 patients with liver cirrhosis and 153 healthy controls, and then analyzed various clinical parameters.Results. Nogo-B was mainly expressed in nonparenchymal cells in the liver and was marked increased in liver with significant fibrosis/cirrhosis compared to controls. Moreover, Metavir F4 showed a higher level of expression than F2. Plasma Nogo-B levels were significantly higher in cirrhotic patients than in healthy controls and were the highest in Child-Pugh class C patients. Plasma Nogo-B levels were positively correlated with Child-Pugh scores. However, there was no relationship between plasma Nogo-B levels and etiology of liver diseases, ALT, AST, platelet counts, and the severity of esophagogastric varices.Conclusions. Nogo-B is mainly expressed in hepatic nonparenchymal cells and is present in plasma. Abnormally high plasma levels of Nogo-B are associated with hepatic cirrhosis and Child-Pugh score, but not correlated with the grade of liver inflammation or portal hypertension. Plasma Nogo-B may be a novel surrogate marker to reflect liver function reserve.

Aim To understand the contribution of miR-142-3p in the activation of hepatic stellate cells (HSCs) and liver fibrosis, and the underlying mechanism. Materials and methods We detected microRNAs expression profiles in quiescent and activated HSCs by microRNA-array, and performed qRT-PCR to validate these data in HSCs and plasma of cirrhosis patients. In vitro, the 3rd–5th passage HSCs was transfected with mir-142-3p mimics or stimulated with TGF β. The markers of HSCs activation (i.e. FN and α-SMA) were examined by qRT-PCR and western blotting, and cell viability was detected by MTT, colony formation assays respectively. Key finding In our study, we identified miR-142-3p as a novel regulator of HSCs activation and indicator of hepatic cirrhosis. We found that miR-142-3p was significantly reduced in activated HSCs, while TGFβRI was distinctly up-regulated in activated HSCs. Ectopic expression of miR-142-3p in activated HSCs inhibited cell viability as well as cell growth, and blocked HSCs activation, concomitant with decreased transdifferentiation markers (i.e. FN and α-SMA). Further, we confirmed that miR-142-3p was reduced upon TGF-β exposure, while diminishing TGF-β-Smad signaling pathway in turn by reducing TGFβRI expression in HSCs. Besides, the plasma level of miR-142-3p declined significantly in patients with hepatic cirrhosis. Significance In conclusion, we demonstrated that miR-142-3p repressed TGF-β-Smad signaling pathway to prevent HSCs activation through directly targeting TGFβRI in HSCs.

Krüppel-like Factor 4 (KLF4), a target gene of miR-145, can negatively regulate lung fibrosis. However, the potential role of KLF4 and miR-145 in hepatic stellate cells (HSCs) activation or in hepatic fibrosis keeps unclear. This study aims to characterize miR-145 and KLF4 in activated HSCs and liver cirrhotic, and the underlying molecular basis. miR-145 was significantly up-regulated, while KLF4 was dramatically down-regulated during the activation of rat primary HSCs and TGF-βtreated HSCs. Furthermore, miR-145 mimics induced and inhibition of miR-145 reduced α-SMA and COL-I expression in primary HSCs. Additionally, the mRNA and protein levels of KLF4 in the liver of cirrhotic patients and rats were significantly down-regulated. α-SMA and COL-I were increased after inhibition of KLF4 by specific shRNA in primary HSCs. Forced KLF4 expression led to a reduction of α-SMA and COL-I expression in HSCs. miR-145 promotes HSC activation and liver fibrosis by targeting KLF4.

Aim To evaluate plasma Nogo-B levels in liver cirrhotic patients and declare a novel molecular basis by which Nogo-B modulates hepatic stellate cell (HSC) activation. Methods Plasma Nogo-B levels from liver cirrhotic patients were detected by enzyme-linked immunosorbent assay. Rat primary HSC were culture activated or stimulated with transforming growth factor (TGF)-β. Activated HSC were transfected for 48 h with Nogo-B shRNA to inhibit Nogo-B expression. Gene expressions of Nogo-B, α-smooth muscle actin (SMA), collagen type I, TGF-β, endoplasmic reticulum (ER) stress key molecules, including C/EBP homologous protein (CHOP), glucose-regulated protein 78 (GRP78), activating transcription factor (ATF)4, ATF6, X-box binding protein 1 (Xbp-1) and calnexin, and the marker of autophagy beclin 1, were detected by quantitative reverse transcription polymerase chain reaction. The protein expressions of Nogo-B, α-SMA, collagen type I, CHOP, GRP78 and the marker of autophagy LC3B were evaluated by western blot. Results Liver cirrhotic patients showed a much higher level of plasma Nogo-B compared with the healthy controls. Nogo-B expression and ER stress could be induced during the process of cultured HSC activation. TGF-β treatment increased Nogo-B expression time- and dose-dependently. Knockdown of Nogo-B in HSC reduced the activation of HSC. After Nogo-B gene knockdown, there was a decline of expression of ER stress markers and autophagic markers. Agonist or antagonist of ER stress could regulate autophagy level. Conclusion Circulating Nogo-B may be an effective indicator for liver cirrhosis. Nogo-B inhibition could diminish HSC activation, in which alleviating ER stress may be one of the mechanisms, suggesting a potential approach to interference Nogo-B in liver fibrosis.

Aims Proliferation is a ‘multiplier’ for extracellular matrix production and contraction of activated hepatic stellate cells (HSC) in fibrotic liver. Transient receptor potential melastatin-like 7 channels (TRPM7) are implicated in the survival and proliferation of several kinds of cells. This study was aimed to investigate the effect of TRPM7 blocker 2-APB on survival and proliferation of HSC and the underlying mechanisms. Main methods Rat HSC were stimulated by 2-APB for 24 h and then collected for further use. Cell viability was detected by MTT, and apoptosis was determined by AnnexinV/PI staining and TUNEL assay. Gene expressions of TRPM7, α-SMA, bcl-2, bax, and endoplasmic reticulum (ER) stress key members CHOP, caspase-12, ATF4, ATF6, Xbp1, GRP78 and calnexin were evaluated with quantitative RT-PCR. Quantifications of α-SMA, TRPM7, CHOP and GRP78 proteins were carried out by Western blot. Transmission electron microscopy and Xbp1 mRNA splicing analysis were also used for detection of ER stress. Key findings 2-APB decreased TRPM7 and α-SMA expressions in primary HSC, and inhibited proliferation of activated HSC in a dose-dependent manner. 2-APB also decreased total count of activated HSC and increased the number of apoptotic cells. 2-APB increased expressions of bax and ER stress key factors CHOP, caspase-12, ATF4, ATF6, Xbp1, GRP78 and calnexin. Meanwhile, ultra-structural ER changes and spliced Xbp1 mRNA were also observed in 2-APB treated HSC. Significance Blockage of TRPM7 could inhibit activation and proliferation of primary HSC and induce apoptotic death of activated cells, in which ER stress was identified as one of possible underlying molecular bases.

Aims This study aimed to determine the role of miR-30c in the process of hepatic stellate cells (HSCs) activation and liver fibrosis/cirrhosis and to explore the underlying mechanism. Main methods A microarray analysis of miRNAs in HSCs was performed, and quantitative RT-PCR analyses were conducted to validate the results in HSCs, cirrhotic liver tissues and plasma. Rat HSCs were stimulated with angiotensin II (AngII) and transfected with miR-30c mimics/inhibitor to elucidate the underlying mechanism. Key findings miR-30c was down-regulated during HSCs activation and in cirrhotic liver tissues and plasma. This miRNA was found to be involved in HSCs activation by repressing the expression of one of its target genes—plasminogen activator inhibitor-1 (PAI-1), and AngII stimulation decreased the expression of miR-30c. However, the up-regulation of miR-30c by specific mimics could down-regulate the mRNA and protein expression of α-SMA, a marker of HSCs activation, in AngII-stimulated HSCs and attenuate the AngII-induced activation of HSCs. Significance miR-30c is involved in HSCs activation and might be a novel biomarker of liver fibrosis/cirrhosis.

To evaluate plasma Nogo-B levels in liver cirrhotic patients and declare a novel molecular basis by which Nogo-B modulates hepatic stellate cell (HSC) activation.Plasma Nogo-B levels from liver cirrhotic patients were detected by enzyme-linked immunosorbent assay. Rat primary HSC were culture activated or stimulated with transforming growth factor (TGF)-β. Activated HSC were transfected for 48 h with Nogo-B shRNA to inhibit Nogo-B expression. Gene expressions of Nogo-B, α-smooth muscle actin (SMA), collagen type I, TGF-β, endoplasmic reticulum (ER) stress key molecules, including C/EBP homologous protein (CHOP), glucose-regulated protein 78 (GRP78), activating transcription factor (ATF)4, ATF6, X-box binding protein 1 (Xbp-1) and calnexin, and the marker of autophagy beclin 1, were detected by quantitative reverse transcription polymerase chain reaction. The protein expressions of Nogo-B, α-SMA, collagen type I, CHOP, GRP78 and the marker of autophagy LC3B were evaluated by western blot.Liver cirrhotic patients showed a much higher level of plasma Nogo-B compared with the healthy controls. Nogo-B expression and ER stress could be induced during the process of cultured HSC activation. TGF-β treatment increased Nogo-B expression time- and dose-dependently. Knockdown of Nogo-B in HSC reduced the activation of HSC. After Nogo-B gene knockdown, there was a decline of expression of ER stress markers and autophagic markers. Agonist or antagonist of ER stress could regulate autophagy level.Circulating Nogo-B may be an effective indicator for liver cirrhosis. Nogo-B inhibition could diminish HSC activation, in which alleviating ER stress may be one of the mechanisms, suggesting a potential approach to interference Nogo-B in liver fibrosis.

The Child–Turcotte–Pugh (CTP) score is widely used worldwide to predict outcomes across a broad spectrum of liver diseases, mainly cirrhosis. Portal hypertension and variceal bleed are significant causes of morbidity and mortality in cirrhotic patients, although the variceal status is not incorporated into the classical CTP score. We sought to determine whether the inclusion of variceal status, specifically the Child–Turcotte–Pugh–Kumar (CTPK) score, would improve the utility of the classical CTP score to predict the clinical outcomes of cirrhotic patients in a single but high-volume center in China. We retrospectively analyzed the records of 253 patients from January 1, 2014 to December 31, 2014 and performed follow-up for at least 12 months. The CTPK score and the CTP score were obtained as soon as possible after the patient's admission. Telephone follow-up was performed to assess survival situations. At 3 and 12 months, the cumulative number of deaths was 9.1% (n = 23) and 13.8% (n = 35), respectively. In the multivariate Cox proportional hazards models, the CTPK score was independently associated with death within 3 and 12 months after adjusting for potential confounders. The predictive ability related to the 2 scores was evaluated by the area under the receiver operating characteristic curve (AUC-ROC) respectively. At 3 months of enrollment, the AUCs of CTPK and CTP were 0.814 and 0.838, respectively. At 12 months of enrollment, the AUCs of CTPK and CTP were 0.825 and 0.840, respectively. No significant difference between time points was observed. Both the CTPK score and the CTP score displayed prognostic value in cirrhotic patients, as the Kaplan–Meier analysis showed that the CTPK score could clearly discriminate patients in the intermediate term (P