Tianhao Zhou

The functional role of microRNAs in alcoholic liver injury

The function of microRNAs (miRNAs) during alcoholic liver disease (ALD) has recently become of great interest in biological research. Studies have shown that ALD associated miRNAs play a crucial role in the regulation of liver‐inflammatory agents such as tumour necrosis factor‐alpha (TNF‐α), one of the key inflammatory agents responsible for liver fibrosis (liver scarring) and the critical contributor of alcoholic liver disease. Lipopolysaccharide (LPS), a component of the cell wall of gram‐negative bacteria, is responsible for TNF‐α release by Kupffer cells. miRNAs are the critical mediators of LPS signalling in Kupffer cells, hepatocytes and hepatic stellate cells. Certain miRNAs, in particular miR‐155 and miR‐21, show a positive correlation in up‐regulation of LPS signalling when they are exposed to ethanol. ALD is related to enhanced gut permeability that allows the levels of LPS to increase, leads to increased secretion of TNF‐α by the Kupffer cells and subsequently promotes alcoholic liver injury through specific miRNAs. Meanwhile, two of the most frequently dysregulated miRNAs in steatohepatitis, miR‐122 and miR‐34a are the critical mediators in ethanol/LPS activated survival signalling during ALD. In this review, we summarize recent findings regarding the experimental and clinical aspects of functions of specific microRNAs, focusing mainly on inflammation and cell survival after ethanol/LPS treatment, and advances on the role of circulating miRNAs in human alcoholic disorders.

Regulation of the Extrinsic Apoptotic Pathway by MicroRNA-21 in Alcoholic Liver Injury

Background: miR-21 is an anti-apoptotic microRNA, and its role in alcoholic liver disease (ALD) is unknown. Results: miR-21, increased in ALD and regulated by IL-6/Stat3, is essential for transformation, survival, and liver fibrosis. Conclusion: miR-21 plays a protective role against alcoholic hepatitis through the anti-extrinsic apoptotic pathway. Significance: Understanding the recovery function of miR-21 may have important implications for patients with ALD. IL-6/Stat3 is associated with the regulation of transcription of key cellular regulatory genes (microRNAs) during different types of liver injury. This study evaluated the role of IL-6/Stat3 in regulating miRNA and miR-21 in alcoholic liver disease. By microarray, we identified that ethanol feeding significantly up-regulated 0.8% of known microRNAs in mouse liver compared with controls, including miR-21. Similarly, the treatment of normal human hepatocytes (N-Heps) and hepatic stellate cells (HSCs) with ethanol and IL-6 significantly increased miR-21 expression. Overexpression of miR-21 decreased ethanol-induced apoptosis in both N-Heps and HSCs. The expression level of miR-21 was significantly increased after Stat3 activation in N-Heps and HSCs, in support of the concept that the 5′-promoter region of miR-21 is regulated by Stat3. Using real time PCR, we confirmed that miR-21 activation is associated with ethanol-linked Stat3 binding of the miR-21 promoter. A combination of bioinformatics, PCR array, dual-luciferase reporter assay, and Western blot analysis revealed that Fas ligand (TNF superfamily, member 6) (FASLG) and death receptor 5 (DR5) are the direct targets of miR-21. Furthermore, inhibition of miR-21 by specific Vivo-Morpholino and knock-out of IL-6 in ethanol-treated mice also increased the expression of DR5 and FASLG in vivo during alcoholic liver injury. The identification of miR-21 as an important regulator of hepatic cell survival, transformation, and remodeling in vitro, as well as its upstream modulators and downstream targets, will provide insight into the involvement of altered miRNA expression in contributing to alcoholic liver disease progression and testing novel therapeutic approaches for human alcoholic liver diseases.

Functional and structural features of cholangiocytes in health and disease

Cholangiocytes are the epithelial cells that line the bile ducts. Along the biliary tree, two different kinds of cholangiocytes exist: small and large cholangiocytes. Each type has important differences in their biological role in physiologic and pathologic conditions. In response to injury, cholangiocytes become reactive and acquire a neuroendocrine-like phenotype with the secretion of a number of peptides. These molecules act in an autocrine/paracrine fashion to modulate cholangiocyte biology and determine the evolution of biliary damage. The failure of such mechanisms is believed to influence the progression of cholangiopathies, a group of diseases that selectively target biliary cells. Therefore, the understanding of mechanisms regulating cholangiocyte response to injury is expected to foster the development of new therapeutic options to treat biliary diseases. In this review, we discuss the most recent findings in the mechanisms driving cholangiocyte adaptation to damage, with particular emphasis on molecular pathways that are susceptible of therapeutic intervention. Morphogenic pathways (Hippo, Notch, Hedgehog), which have been recently shown to regulate biliary ontogenesis and response to injury, are also reviewed as well as the results of ongoing clinical trials evaluating new drugs for the treatment of cholangiopathies.

Knockout of microRNA-21 reduces biliary hyperplasia and liver fibrosis in cholestatic bile duct ligated mice

Cholestasis is a condition that leads to chronic hepatobiliary inflammation, fibrosis, and eventually cirrhosis. Many microRNAs (miRs) are known to have a role in fibrosis progression; however, the role of miR-21 during cholestasis remains unknown. Therefore, the aim of this study was to elucidate the role of miR-21 during cholestasis-induced biliary hyperplasia and hepatic fibrosis. Wild-type (WT) and miR-21−/− mice underwent Sham or bile duct ligation (BDL) for 1 week, before evaluating liver histology, biliary proliferation, hepatic stellate cell (HSC) activation, fibrotic response, and small mothers against decapentaplegic 7 (Smad-7) expression. In vitro, immortalized murine biliary cell lines (IMCLs) and human hepatic stellate cell line (hHSC) were treated with either miR-21 inhibitor or control before analyzing proliferation, apoptosis, and fibrotic responses. In vivo, the levels of miR-21 were increased in total liver and cholangiocytes after BDL, and loss of miR-21 decreased the amount of BDL-induced biliary proliferation and intrahepatic biliary mass. In addition, loss of miR-21 decreased BDL-induced HSC activation, collagen deposition, and expression of the fibrotic markers transforming growth factor-β1 and α-smooth muscle actin. In vitro, IMCL and hHSCs treated with miR-21 inhibitor displayed decreased proliferation and expression of fibrotic markers and enhanced apoptosis when compared with control treated cells. Furthermore, mice lacking miR-21 show increased Smad-7 expression, which may be driving the decrease in biliary hyperplasia and hepatic fibrosis. During cholestatic injury, miR-21 is increased and leads to increased biliary proliferation and hepatic fibrosis. Local modulation of miR-21 may be a therapeutic option for patients with cholestasis.

Functional role of cellular senescence in biliary injury

Cellular senescence is a state of irreversible cell cycle arrest that has been involved in many gastrointestinal diseases, including human cholestatic liver disorders. Senescence may play a role in biliary atresia, primary sclerosing cholangitis, cellular rejection, and primary biliary cirrhosis, four liver diseases affecting cholangiocytes and the biliary system. In this review, we examine proposed mechanisms of senescence-related biliary diseases, including hypotheses associated with the senescence-associated phenotype, induction of senescence in nearby cells, and the depletion of stem cell subpopulations. Current evidence for the molecular mechanisms of senescence in the previously mentioned diseases is discussed in detail, with attention to recent advances on the role of pathways associated with senescence-associated phenotype, stress-induced senescence, telomere dysfunction, and autophagy.

Substance P increases liver fibrosis by differential changes in senescence of cholangiocytes and hepatic stellate cells.

Substance P (SP) is involved in the proliferation of cholangiocytes in bile duct–ligated (BDL) mice and human cholangiocarcinoma growth by interacting with the neurokinin‐1 receptor (NK‐1R). To identify whether SP regulates liver fibrosis during cholestasis, wild‐type or NK‐1R knockout (NK‐1R–/–) mice that received BDL or sham surgery and multidrug resistance protein 2 knockout (Mdr2–/–) mice treated with either an NK‐1R antagonist (L‐733,060) or saline were used. Additionally, wild‐type mice were treated with SP or saline intraperitoneally. In vivo, there was increased expression of tachykinin precursor 1 (coding SP) and NK‐1R in both BDL and Mdr2–/– mice compared to wild‐type mice. Expression of tachykinin precursor 1 and NK‐1R was significantly higher in liver samples from primary sclerosing cholangitis patients compared to healthy controls. Knockout of NK‐1R decreased BDL‐induced liver fibrosis, and treatment with L‐733,060 resulted in decreased liver fibrosis in Mdr2–/– mice, which was shown by decreased sirius red staining, fibrosis gene and protein expression, and reduced transforming growth factor‐β1 levels in serum and cholangiocyte supernatants. Furthermore, we observed that reduced liver fibrosis in NK‐1R–/– mice with BDL surgery or Mdr2–/– mice treated with L‐733,060 was associated with enhanced cellular senescence of hepatic stellate cells and decreased senescence of cholangiocytes. In vitro, L‐733,060 inhibited SP‐induced expression of fibrotic genes in hepatic stellate cells and cholangiocytes; treatment with L‐733,060 partially reversed the SP‐induced decrease of senescence gene expression in cultured hepatic stellate cells and the SP‐induced increase of senescence‐related gene expression in cultured cholangiocytes. Conclusion: Collectively, our results demonstrate the regulatory effects of the SP/NK‐1R axis on liver fibrosis through changes in cellular senescence during cholestatic liver injury. (Hepatology 2017;66:528–541).

Regulators of Cholangiocyte Proliferation.

Cholangiocytes, a small population of cells within the normal liver, have been the focus of a significant amount of research over the past two decades because of their involvement in cholangiopathies such as primary sclerosing cholangitis and primary biliary cholangitis. This article summarizes landmark studies in the field of cholangiocyte physiology and aims to provide an updated review of biliary pathogenesis. The historical approach of rodent extrahepatic bile duct ligation and the relatively recent utilization of transgenic mice have led to significant discoveries in cholangiocyte pathophysiology. Cholangiocyte physiology is a complex system based on heterogeneity within the biliary tree and a number of signaling pathways that serve to regulate bile composition. Studies have expanded the list of neuropeptides, neurotransmitters, and hormones that have been shown to be key regulators of proliferation and biliary damage. The peptide histamine and hormones, such as melatonin and angiotensin, angiotensin, as well as numerous sex hormones, have been implicated in cholangiocyte proliferation during cholestasis. Numerous pathways promote cholangiocyte proliferation during cholestasis, and there is growing evidence to suggest that cholangiocyte proliferation may promote hepatic fibrosis. These pathways may represent significant therapeutic potential for a subset of cholestatic liver diseases that currently lack effective therapies.

Forkhead box A2 regulated biliary heterogeneity and senescence during cholestatic liver injury in mice

Biliary‐committed progenitor cells (small mouse cholangiocytes; SMCCs) from small bile ducts are more resistant to hepatobiliary injury than large mouse cholangiocytes (LGCCs) from large bile ducts. The definitive endoderm marker, forkhead box A2 (FoxA2), is the key transcriptional factor that regulates cell differentiation and tissue regeneration. Our aim was to characterize the translational role of FoxA2 during cholestatic liver injury. Messenger RNA expression in SMCCs and LGCCs was assessed by polymerase chain reaction (PCR) array analysis. Liver tissues and hepatic stellate cells (HSCs) from primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) patients were tested by real‐time PCR for methylation, senescence, and fibrosis markers. Bile duct ligation (BDL) and multidrug resistance protein 2 (MDR2) knockout mice (MDR2–/–) were used as animal models of cholestatic liver injury with or without healthy transplanted large or small cholangiocytes. We demonstrated that FoxA2 was notably enhanced in murine liver progenitor cells and SMCCs and was silenced in human PSC and PBC liver tissues relative to respective controls that are correlated with the epigenetic methylation enzymes, DNA methyltransferase (DNMT) 1 and DNMT3B. Serum alanine aminotransferase and aspartate aminotransferase levels in nonobese diabetic/severe combined immunodeficiency mice engrafted with SMCCs post‐BDL showed significant changes compared to vehicle‐treated mice, along with improved liver fibrosis. Enhanced expression of FoxA2 was observed in BDL mouse liver after SMCC cell therapy. Furthermore, activation of fibrosis signaling pathways were observed in BDL/MDR2–/– mouse liver as well as in isolated HSCs by laser capture microdissection, and these signals were recovered along with reduced hepatic senescence and enhanced hepatic stellate cellular senescence after SMCC engraft. Conclusion: The definitive endoderm marker and the positive regulator of biliary development, FoxA2, mediates the therapeutic effect of biliary‐committed progenitor cells during cholestatic liver injury. (Hepatology 2017;65:544‐559).

The let-7/lin28 axis regulates activation of hepatic stellate cells in alcoholic liver injury

The let-7/Lin28 axis is associated with the regulation of key cellular regulatory genes known as microRNAs in various human disorders and cancer development. This study evaluated the role of the let-7/Lin28 axis in regulating a mesenchymal phenotype of hepatic stellate cells in alcoholic liver injury. We identified that ethanol feeding significantly down-regulated several members of the let-7 family in mouse liver, including let-7a and let-7b. Similarly, the treatment of human hepatic stellate cells (HSCs) with lipopolysaccharide (LPS) and transforming growth factor-β (TGF-β) significantly decreased the expressions of let-7a and let-7b. Conversely, overexpression of let-7a and let-7b suppressed the myofibroblastic activation of cultured human HSCs induced by LPS and TGF-β, as evidenced by repressed ACTA2 (α-actin 2), COL1A1 (collagen 1A1), TIMP1 (TIMP metallopeptidase inhibitor 1), and FN1 (fibronectin 1); this supports the notion that HSC activation is controlled by let-7. A combination of bioinformatics, dual-luciferase reporter assay, and Western blot analysis revealed that Lin28B and high-mobility group AT-hook (HMGA2) were the direct targets of let-7a and let-7b. Furthermore, Lin28B deficiency increased the expression of let-7a/let-7b as well as reduced HSC activation and liver fibrosis in mice with alcoholic liver injury. This feedback regulation of let-7 by Lin28B is verified in hepatic stellate cells isolated by laser capture microdissection from the model. The identification of the let-7/Lin28 axis as an important regulator of HSC activation as well as its upstream modulators and down-stream targets will provide insights into the involvement of altered microRNA expression in contributing to the pathogenesis of alcoholic liver fibrosis and novel therapeutic approaches for human alcoholic liver diseases.

Role of stem cells during diabetic liver injury

Diabetes mellitus is one of the most severe endocrine metabolic disorders in the world that has serious medical consequences with substantial impacts on the quality of life. Type 2 diabetes is one of the main causes of diabetic liver diseases with the most common being non‐alcoholic fatty liver disease. Several factors that may explain the mechanisms related to pathological and functional changes of diabetic liver injury include: insulin resistance, oxidative stress and endoplasmic reticulum stress. The realization that these factors are important in hepatocyte damage and lack of donor livers has led to studies concentrating on the role of stem cells (SCs) in the prevention and treatment of liver injury. Possible avenues that the application of SCs may improve liver injury include but are not limited to: the ability to differentiate into pancreatic β‐cells (insulin producing cells), the contribution for hepatocyte regeneration, regulation of lipogenesis, glucogenesis and anti‐inflammatory actions. Once further studies are performed to explore the underlying protective mechanisms of SCs and the advantages and disadvantages of its application, there will be a greater understand of the mechanism and therapeutic potential. In this review, we summarize the findings regarding the role of SCs in diabetic liver diseases.