Polaprezinc attenuates liver fibrosis in a mouse model of non-alcoholic steatohepatitis

Background and Aim: The effect of polaprezinc, a zinc‐carnosine chelate compound, on the development of non‐alcoholic steatohepatitis (NASH) was investigated in dietary methionine and choline deficient (MCD) mice. Methods: Mice were fed the MCD diet with or without polaprezinc (2.2 g/kg diet) for 10 weeks. Liver histopathology, triglyceride and lipid peroxide levels, and the expression of genes linked to fibrosis were then assessed. Results: MCD mice developed steatohepatitis accompanied by mild fibrosis with an increase in lipid peroxidation, hepatic stellate cell (HSC) activation, and the augmented mRNA expression of tumor necrosis factor‐α, transforming growth factor‐β1 and procollagen α1(I). The mRNA expression levels of matrix metalloproteinase (MMP)‐2 and tissue inhibitors of metalloproteinase (TIMP)‐1 and TIMP‐2 were also enhanced. Histopathologically, polaprezinc supplementation did not influence the development of steatosis but it apparently attenuated fibrosis. Polaprezinc slightly reduced lipid peroxidation and suppressed HSC activation as well as the mRNA expression of pro‐inflammatory cytokines. Polaprezinc affected the MCD diet‐enhanced expression of TIMP‐1 even when administered relatively late. Conclusion: These results suggest that polaprezinc attenuates fibrosis in NASH by reducing inflammation and lipid peroxidation and, during a later phase, promoting fibrolysis via the inhibition of TIMP expression in the liver. Further investigation is required to clarify the clinical efficacy of polaprezinc in patients with NASH.     

Transforming growth factor‐α attenuates hepatic fibrosis: possible involvement of matrix metalloproteinase‐1

Background: The effect of transforming growth factor (TGF)‐α on fibrosis varies between cell types and the role of TGF‐α in hepatic fibrosis has not been fully elucidated. Methods: We examined the effect of TGF‐α on hepatic fibrosis using TGF‐α‐expressing transgenic mice fed a methionine‐ and choline‐deficient (MCD) diet and human hepatic stellate cells (HSCs) line LX‐2, rat and human primary HSCs. Results: Although the expression levels of the tissue inhibitor of metalloproteinases‐1 and α1(I) collagen mRNA were unchanged, feeding the TGF‐α transgenic mice the MCD diet resulted in greater expression of the murine functional analogue of matrix metalloproteinase‐1 (MMP‐1), MMP‐13 mRNA and protein and attenuated hepatic fibrosis compared with wild‐type mice. TGF‐α overexpression did not affect the extent of the steatosis, oxidative stress and hepatic inflammation in the MCD diet‐fed mice. The effect of TGF‐α on the fibrogenic and anti‐fibrogenic gene expressions varied between cell types in vitro. TGF‐α increased MMP‐1 mRNA expressions that were completely blocked by gefitinib in LX‐2 cells. The extracellular signal‐regulated kinase (ERK) 1/2, c‐Jun N‐terminal kinase and p38 pathways were involved in MMP‐1 mRNA expression in LX‐2 cells. Although TGF‐α increased the phosphorylation of p38, the p38 inhibitor activated the RAS‐ERK pathway and increased TGF‐α‐induced MMP‐1 mRNA expression, which suggested that there may be a crosstalk between the RAS‐ERK and the p38 pathways in LX‐2 cells. Conclusions: The TGF‐α may attenuate hepatic fibrosis in part because of upregulation of the expression of MMP‐1. The balance between fibrogenic and anti‐fibrogenic gene expression and between the activity of the RAS‐ERK and the p38 pathways may be crucial for the fibrotic process.     

Melatonin suppresses activation of hepatic stellate cells through RORα‐mediated inhibition of 5‐lipoxygenase

Liver fibrosis is scar tissue resulting from an uncontrolled wound‐healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture‐activated rat HSC. Melatonin dose‐dependently suppressed the expression of HSC activation markers Col1a1 and alpha‐smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor‐related orphan receptor‐alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5‐lipoxygenase (5‐LO). The pharmacological 5‐LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα‐mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis.     

Proanthocyanidin derived from the leaves of Vaccinium virgatum suppresses platelet‐derived growth factor‐induced proliferation of the human hepatic stellate cell line LI90

Aim: Hepatic stellate cell (HSC) proliferation plays a pivotal role in liver fibrogenesis, and agents that suppress HSC activation, including platelet‐derived growth factor (PDGF)‐induced HSC proliferation, are good candidates for antifibrogenic therapies. In this report, we use the LI90 HSC line to elucidate the antifibrogenic effects of proanthocyanidin derived from the leaves of Vaccinium virgatum. Methods: Proanthocyanidin (PAC) was extracted from the leaves of blueberry V. virgatum (BB‐PAC), grape seeds (GS‐PAC) and Croton lechleri (CL‐PAC). These extracts were examined for their effects on PDGF‐BB‐induced LI90 cell proliferation and DNA synthesis. Extracellular signal‐regulated kinase (ERK) and Akt phosphorylation and PDGF receptor‐β (PDGFR‐β) expression were evaluated by western blot analysis. Results: BB‐PAC potently suppressed PDGF‐BB‐induced proliferation and DNA synthesis of LI90 cells. BB‐PAC also suppressed PDGF‐BB‐induced DNA synthesis in primary cultured rat HSC. Moreover, GS‐PAC and CL‐PAC suppressed PDGF‐BB‐induced DNA synthesis in LI90 cells. In contrast, the monomeric PAC catechin and epicatechin and dimeric PAC procyanidin B2 only slightly suppressed PDGF‐BB‐induced DNA synthesis. Western blot analysis showed that BB‐PAC completely or partially inhibited PDGF‐BB‐induced ERK and Akt phosphorylation, respectively. In addition, BB‐PAC partially inhibited the PDGF‐BB‐induced degradation of PDGFR‐β. Conclusion: Our results suggest that BB‐PAC suppresses activated HSC by inhibiting the PDGF signaling pathway. In addition, these results provide novel findings that may facilitate the development of antifibrogenic agents.     

RNA interference targeting the platelet‐derived growth factor receptor β subunit ameliorates experimental hepatic fibrosis in rats

Background/Aims: Platelet‐derived growth factor (PDGF) is the strongest stimulator of the proliferation of hepatic stellate cells (HSCs). PDGF receptor β subunit (PDGFR‐β) is acquired on HSCs proliferation induced by PDGF. In this study, we aim to investigate the effect of PDGFR‐β small interference RNA (siRNA) on experimental hepatic fibrosis. Methods: We constructed a PDGFR‐β siRNA expression plasmid and investigated its effect on the activation of HSCs. Bromodeoxyuridine incorporation was performed to investigate the effect of PDGFR‐β siRNA on HSCs proliferation. A hydrodynamics‐based transfection method was used to deliver PDGFR‐β siRNA to rats with hepatic fibrosis. The distribution of transgenes in the liver was observed by immunofluorescence. The antifibrogenic effect of PDGFR‐β siRNA was investigated pathologically. Results: Platelet‐derived growth factor receptor‐β subunit siRNA could significantly downregulate PDGFR‐β expression, suppress HSCs activation, block the mitogen‐activated protein kinase signalling pathway and inhibit HSCs proliferation in vitro. PDGFR‐β siRNA expression plasmid could be delivered into activated HSCs by the hydrodynamics‐based transfection method, and remarkably improve the liver function of the rat model induced by dimethylnitrosamine and bile duct ligation. Furthermore, the progression of fibrosis in the liver was significantly suppressed by PDGFR‐β siRNA in both animal models. Conclusions: Platelet‐derived growth factor receptor‐β subunit siRNA may be presented as an effective antifibrogenic gene therapeutic method for hepatic fibrosis.