Bezafibrate prevents hepatic stellate cell activation and fibrogenesis in a murine steatohepatitis model,and suppresses fibrogenic response induced by transforming growth factor‐β1 in a cultured stellate cell line

Aim: The aim of this study was to investigate the preventive actions of bezafibrate against non‐alcoholic steatohepatitis (NASH), the activation of hepatic stellate cells (HSC), and fibrogenesis by using a model of NASH and an in vitro model. Methods: Male KK‐A^y/TaJcl (KK‐A^y) mice were fed a methionine and choline‐deficient (MCD) diet or a MCD diet containing bezafibrate or pioglitazone for 7 weeks, after which biochemical parameters, pathological changes, and hepatic mRNA levels were assessed. An in vitro HSC model was designed by using a previously described RI‐T cell line stimulated by transforming growth factor‐β1 (TGF‐β1). Results: MCD diet‐fed KK‐A^y mice developed hepatic steatosis, oxidative stress, inflammation, and hepatic fibrosis. Bezafibrate markedly decreased the hepatic content of triglyceride accumulation of fatty droplets within hepatocytes, and increased the expression of hepatic fatty acid β‐oxidative genes in MCD diet‐fed KK‐A^y mice. Bezafibrate markedly inhibited the increases in the plasma alanine aminotransferase level and hepatic content of thiobarbituric acid‐reactive substances in this model. Moreover, it dramatically reduced hepatic inflammatory changes and fibrosis concomitantly with marked reductions in the mRNA levels for inflammatory cytokine, chemokine, and profibrogenic genes. Importantly, both bezafibrate and pioglitazone markedly reduced the mRNA levels of profibrogenic and fibrogenic genes in TGF‐β1‐stimulated cells. Conclusion: Bezafibrate improved hepatic steatosis and potently prevented inflammation, oxidative stress, HSC activation, and fibrogenesis in the liver. Moreover, this study was the first to demonstrate that bezafibrate directly inhibits hepatic fibrogenic response induced by TGF‐β1 in vitro. Hence bezafibrate may be a new therapeutic strategy against NASH and hepatic fibrosis.     

Ursodeoxycholic acid dose‐dependently improves liver injury in rats fed a methionine‐ and choline‐deficient diet

Aim: The data on the beneficial effect of ursodeoxycholic acid (UDCA) in non‐alcoholic steatohepatitis (NASH) are controversial. The difference of opinion is connected with UDCA dosage to be used. Therefore, we evaluated the dose‐dependent efficacy of UDCA in experimental NASH. Methods: Male Wistar rats were fed the methionine‐ and choline‐deficient (MCD) diet for 10 weeks. Rats were administrated UDCA (10, 20, 40 and 80 mg/kg bodyweight intragastrically) after 6 weeks of the MCD diet. Results: Animals fed the MCD diet developed severe steatohepatitis. Treatment with UDCA dose‐dependently decreased liver damage, but only high‐dose UDCA (80 mg/kg) significantly diminished ultrastructural changes in addition to preventing steatosis, ballooning and inflammatory changes in the liver. The activities of serum marker enzymes and the content of liver triglyceride and blood glucose were increased in MCD diet‐fed rats, but decreased in all the UDCA‐treated groups. Serum insulin concentration was decreased whereas the quantitative insulin sensitivity check index did not changed in MCD diet‐fed groups. Serum tumor necrosis factor‐α content was strongly increased after MCD diet and normalized in the UDCA‐treated rats, with the most pronounced effect in the highest dose groups, 40 and 80 mg/kg. The contents of endogenous ethanol in blood and intestinal mucus were increased in MCD diet‐fed rats which were significantly lowered by UDCA (40 and 80 mg/kg per day). Conclusion: The present data demonstrate a beneficial effect of UDCA that manifested by the decrease of liver steatosis, inflammatory signs and serum tumor necrosis factor‐α content especially of the highest 40 and 80 mg/kg day doses.     

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.     

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.     

Effect of the regulation of retinoid X receptor‐α gene expression on rat hepatic fibrosis

Aim: To study the effect of retinoid X receptor‐α (RXR‐α) expression on rat hepatic fibrosis. Methods: Rat hepatic fibrosis was induced by CCl₄, and the rats were randomly divided into an early‐phase hepatic fibrosis group (2 weeks) and a sustained hepatic fibrosis group (8 weeks). They were then divided into four groups (normal control, hepatic fibrosis, negative control and RXR‐α groups). A recombinant lentiviral expression vector carrying the rat RXR‐α gene was injected into the rats to induce RXR‐α expression by intraportal infusion, hepatic tissue pathological examination was performed, and hydroxyproline content was detected. Hepatic stellate cells (HSC) were cultured in vitro, an RXR‐α lentivirus vector was used to activate HSC, and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) activation was assayed to detect HSC proliferation. Results: In vivo experiments indicated that in the sustained hepatic fibrosis group, there were significant differences in the hydroxyproline content, and expression of RXR‐α, α‐smooth muscle actin (α‐SMA) and type I collagen (P < 0.01). However, in the early‐phase hepatic fibrosis group, hydroxyproline content and the protein level of RXR‐α showed no significant difference compared with the normal control group (P > 0.05). In vitro studies revealed that expression of RXR‐α significantly inhibited expression of α‐SMA and type I collagen in activated HSC (P < 0.01), as well as HSC proliferation (P < 0.01). Conclusion: The increased RXR‐α gene expression inhibited HSC activation and proliferation and the degree of hepatic fibrosis.     

Active matrix metalloproteinase‐2 promotes apoptosis of hepatic stellate cells via the cleavage of cellular N‐cadherin

Background and Aims: Hepatic stellate cells (HSC) are known to synthesise excess matrix that characterises liver fibrosis and cirrhosis. Activated HSC express the matrix‐degrading matrix metalloproteinase enzymes (MMPs) and their tissue inhibitors (TIMPs). During spontaneous recovery from experimental liver fibrosis, the expression of TIMP‐1 declines and hepatic collagenolytic activity increases. This is accompanied by HSC apoptosis. In this study, we examine a potential mechanism whereby MMP activity might induce HSC apoptosis by cleaving N‐cadherin at the cell surface. Results: N‐cadherin expression was upregulated in human HSC during activation in culture. Addition of function‐blocking antibodies or a peptide targeting the extracellular domain of N‐cadherin, to cultured HSC, promoted apoptosis. During apoptosis, there was cleavage of N‐cadherin into 20–100 kDa fragments. MMP‐2 became activated early during HSC apoptosis and directly cleaved N‐cadherin in vitro. Addition of activated MMP‐2 to HSCs in culture resulted in enhanced apoptosis and loss of N‐cadherin. Conclusions: Together, these studies identify a role for both N‐cadherin and MMP‐2 in mediating HSC apoptosis, where N‐cadherin works to provide a cell survival stimulus and MMP‐2 promotes HSC apoptosis concomitant with N‐cadherin degradation.     

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.     

Experience with anti‐tumor necrosis factor‐α therapy in India

Aims: To review the Indian experience with anti‐tumor necrosis factor (TNF)‐α therapy. Methods: ‘PubMed’ and ‘IndMED’ were searched for Indian studies on anti‐TNF‐α therapy. Data were compiled and analysed. Results: Data on infliximab from 176 patients from five different series were collated. One hundred and forty‐seven had ankylosing spondylitis (AS), nine had polyarticular juvenile idiopathic arthritis (JIA), 12 had rheumatoid arthritis (RA), six had undifferentiated spondyloarthropathy, one had inflammatory bowel disease‐related spondyloarthritis and one had psoriatic arthritis. Thus, 155/176. (88%) had spondyloarthropathy (SpA). No screening for latent tuberculosis was done in any of the studies. One series comprising 108 cases of AS, used 3 mg/kg infliximab infusions (instead of 5 mg/kg) at 8‐weekly intervals with omission of the 2‐week and 6‐week doses. All others with SpA (n = 47) followed the standard protocol: 171/176 patients had a significant improvement. Reactivation tuberculosis developed in 5/47 (10.6%) SpA patients treated with standard doses of infliximab. This amounted to 56 times increased risk compared to baseline (0.187%). None of the 129 patients treated with 3 mg/kg infusions of infliximab developed reactivation tuberculosis (AS ?108, RA ?12, JIA ?9). The lone study on etanercept showed good efficacy in 40 patients with RA. However, seven serious adverse events occurred. Conclusions: Infliximab showed expected efficacy in SpA, RA and JIA. Reactivation tuberculosis developed in 10.6% of the SpA group treated with standard regimen. Patients treated with lower doses of infliximab which included a large subgroup of SpA patients and those with RA or JIA did not develop tuberculosis.     

Appearance of α-smooth-muscle-actin-positive cells in hepatic fibrosis

ABSTRACT— The appearance of α-smooth-muscle-actin (α-smA)-positive cells during hepatic fibrosis was studied immunohistochemically in rat and human livers. In the normal rat liver, α-smA was observed only in vascular smooth muscle cells. With the progression of fibrosis induced by CCl₄ injection, α-smA-positive cells appeared in the perisinusoidal space and the fibrous septa, and ultimately surrounded regenerative nodules. An increase of desmin-positive cells was recognized in the fibrotic areas and the perisinusoidal area. In the human liver, α-smA-positive cells appeared in the fibrotic area, whereas no desmin-positive cells were observed, except in vascular walls of the central vein and the portal tract. α-smA is a good marker for the detection of myofibroblast-like cells, and the appearance of α-smA in liver mesenchymal cells seems closely related to the process of hepatic fibrosis in both rat and man.