Transcriptional and Epigenetic Alterations in the Progression of Non-Alcoholic Fatty Liver Disease and Biomarkers Helping to Diagnose Non-Alcoholic Steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) encompasses a broad spectrum of conditions from simple steatosis (non-alcoholic fatty liver (NAFL)) to non-alcoholic steatohepatitis (NASH), and its global prevalence continues to rise. NASH, the progressive form of NAFLD, has higher risks of liver and non-liver related adverse outcomes compared with those patients with NAFL alone. Therefore, the present study aimed to explore the mechanisms in the progression of NAFLD and to develop a model to diagnose NASH based on the transcriptome and epigenome. Differentially expressed genes (DEGs) and differentially methylated genes (DMGs) among the three groups (normal, NAFL, and NASH) were identified, and the functional analysis revealed that the development of NAFLD was primarily related to the oxidoreductase-related activity, PPAR signaling pathway, tight junction, and pathogenic Escherichia coli infection. The logistic regression (LR) model, consisting of ApoF, THOP1, and BICC1, outperformed the other five models. With the highest AUC (0.8819, 95%CI: 0.8128-0.9511) and a sensitivity of 97.87%, as well as a specificity of 64.71%, the LR model was determined as the diagnostic model, which can differentiate NASH from NAFL. In conclusion, several potential mechanisms were screened out based on the transcriptome and epigenome, and a diagnostic model was built to help patient stratification for NAFLD populations.

Lnc-AIFM2-1 promotes HBV immune escape by acting as a ceRNA for miR-330-3p to regulate CD244 expression.

Chronic hepatitis B (CHB) virus infection is a major risk factor for cirrhosis and hepatocellular carcinoma (HCC). Hepatitis B virus (HBV) immune escape is regulated by the exhaustion of virus-specific CD8+ T cells, which is associated with abnormal expression of negative regulatory molecule CD244. However, the underlying mechanisms are unclear. To investigate the important roles of non-coding RNAs play in CD244 regulating HBV immune escape, we performed microarray analysis to determine the differential expression profiles of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs in patients with CHB and patients with spontaneous clearance of HBV. Competing endogenous RNA (ceRNA) was analyzed by bioinformatics methods and confirmed by the dual-luciferase reporter assay. Furthermore, gene silencing and overexpression experiments were used to further identify the roles of lncRNA and miRNA in HBV immune escape through CD244 regulation. The results showed that the expression of CD244 on the surface of CD8+ T cells was significantly increased in CHB patients and in the co-culture system of T cells and HBV-infected HepAD38 cells, which was accompanied by the reduction of miR-330-3p and the elevation of lnc-AIFM2-1. The down-regulated miR-330-3p induced the apoptosis of T cells by lifting the inhibition of CD244, which was reversed by miR-330-3p mimic or CD244-siRNA. Lnc-AIFM2-1 promotes the accumulation of CD244, which is mediated by decreased miR-330-3p, and then reduced the clearance ability of CD8+ T cells to HBV through regulated CD244 expression. And the injury in the ability of CD8+ T cells to clear HBV can be reversed by lnc-AIFM2-1-siRNA, miR-330-3p mimic, or CD244-siRNA. Collectively, our findings indicate that lnc-AIFM2-1 on CD244 by acting as a ceRNA of miR-330-3p contributes to HBV immune escape, which may provide novel insights into the roles of interaction networks among lncRNA, miRNA, and mRNA in HBV immune escape, highlighting potential applications of lnc-AIFM2-1 and CD244 for diagnosis and treatment in CHB.

Selective suppression of M1 macrophages is involved in zinc inhibition of liver fibrosis in mice.

Zinc deficiency is common in the liver of patients with chronic liver disease. Zinc supplementation suppresses the progression of liver fibrosis induced by bile duct ligation (BDL) in mice. The present study was undertaken to specifically investigate a possible mechanism by which zinc plays this role in the liver. Kunming mice were subjected to BDL for 4 weeks to induce liver fibrosis, and concomitantly treated with zinc sulfite or saline as control by gavage once a day. The results showed that zinc supplementation significantly suppressed liver fibrosis and inflammation along with inhibition of hepatic stellate cells activation induced by BDL. These inhibitory effects were accompanied by the reduction of collagen deposition and a significant reduction of macrophage infiltration affected livers. Importantly, zinc selectively inhibited M1 macrophage polarization and M1-related inflammatory cytokines. This inhibitory effect was further confirmed by the reduction of relevant biomarkers of M1 macrophages including inducible NO synthase (iNOS), monocyte chemotactic protein-1 (MCP-1/CCL2), and tumor necrosis factor-α in the zinc supplemented BDL livers. In addition, zinc inhibition of M1 macrophages was associated with a decrease of Notch1 expression. Taken together, these data indicated that zinc supplementation inhibited liver inflammation and fibrosis in BDL mice through selective suppression of M1 macrophages, which is associated with inhibition of Notch1 pathway in M1 macrophage polarization.

Comparison of serological assessments in the diagnosis of liver fibrosis in bile duct ligation mice.

Liver fibrosis assessment is essential to make a prognosis and to determine the appropriate anti-fibrosis treatment. Non-invasive serum markers are widely studied in patients to assess liver fibrosis due to the limitations of liver biopsy. When using animal models to study the mechanism and intervention of hepatic fibrosis, serum markers might be useful for the continuous assessment of liver fibrosis in individual animals, which could avoid the influence of biological differences between individuals. However, it is unclear whether serum markers can assess hepatic fibrosis in the animal model. In the present study, we evaluated and compared the ability of four serum markers to assess liver fibrosis in bile duct ligation mice. According to the stages of liver fibrosis assessed by pathological changes, mice in this study were divided into five groups (F0, F1, F2, F3, and F4). Subsequently, four serum markers, aspartate aminotransferase-to-alanine aminotransferase ratio (AAR), aspartate aminotransferase-to-platelet ratio index (APRI), fibrosis index based on the 4 factors (FIB-4), and Forns Index, were calculated for each group. Furthermore, the correlations between serum markers and pathological stages and the ability of serological markers to evaluate liver fibrosis were analyzed. AAR, APRI, FIB-4, and Forns Index could significantly distinguish F0-2 from F3-4 mice. APRI, FIB-4, and Forns Index could detect F0-3 from F4 mice. Among these four markers, FIB-4 was the best able to distinguish ≥F2 and ≥F3, with area under the curve values of 0.882 and 0.92, respectively. Forns Index was best for diagnosing F4 with area under the curve value of 0.879. These results demonstrated that serum markers could be used for assessing liver fibrosis in bile duct ligation mice, and therefore, these markers might lead to more accurate diagnostic and therapeutic studies through continuous monitoring in individual animals. Impact statement The assessment of liver fibrosis is essential for making a prognosis and determining the appropriate anti-fibrosis treatment. In studies focusing on the mechanism and treatment of liver fibrosis using animal models, it would be more accurate to continuously evaluate liver fibrosis in a single animal to avoid individual biological differences. Unfortunately, it is difficult to perform continuous assessment through liver biopsy in the most commonly used rodent models. It is unclear whether serum markers, which have been used in hepatic fibrosis patients, could be used in animal models. Our results demonstrate that serum markers could be used for assessing liver fibrosis in bile duct ligation mice. This study might contribute to more accurate diagnostic and therapeutic studies through continuous monitoring in individual animals.