Effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on carbon tetrachloride‐induced hepatic cirrhosis in rats

Aim: The present study was undertaken to evaluate the effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a synthesized vitamin E derivative, on carbon tetrachloride (CCl₄)‐induced cirrhosis. Methods: Rats were treated with hypodermic injections of CCl₄ twice a week to induce the hepatic cirrhosis, and given drinking water containing HTHQ or solvent. Primary cultures of rat hepatocytes were performed to evaluate the effects of HTHQ on the expression of inducible nitric oxide synthase (iNOS). Results: Masson's staining of rat livers showed fibrosis around pseudo‐lobules in the CCl₄ group, the lesions being reduced in the CCl₄ HTHQ group. Increases in liver tissue hydroxyproline and α₁(I) collagen, α‐smooth muscle actin and iNOS induced by CCl₄, were also markedly diminished by HTHQ. Furthermore, both HTHQ and vitamin E attenuated interleukin‐1β‐induced iNOS protein expression in cultured hepatocytes, the potency of HTHQ being 10‐times higher than that of vitamin E. Conclusion: HTHQ may inhibit development of hepatic cirrhosis in rats, more potently than vitamin E, by inhibiting the iNOS expression in hepatocytes. Because vitamin E has a radical scavenging action, roles of NO and peroxynitrite will be discussed in the effects of HTHQ on the fibrosis.     

Endoglin in human liver disease and murine models of liver fibrosis—A protective factor against liver fibrosis

Background & Aims

Liver fibrosis is the outcome of chronic liver injury. Transforming growth factor‐β (TGF ‐β) is a major profibrogenic cytokine modulating hepatic stellate cell (HSC ) activation and extracellular matrix homeostasis. This study analyses the effect of Endoglin (Eng), a TGF ‐β type III auxiliary receptor, on fibrogenesis in two models of liver injury by HSC ‐specific endoglin deletion.

Methods

Eng expression was measured in human and murine samples of liver injury. After generating GFAP^Cre(+)Eng^ΔHSC mice, the impact of Endoglin deletion on chronic liver fibrosis was analysed. For in vitro analysis, Eng^flox/flox HSC s were infected with Cre‐expressing virus to deplete Endoglin and fibrogenic responses were analysed.

Results

Endoglin is upregulated in human liver injury. The receptor is expressed in liver tissues and mesenchymal liver cells with much higher abundance of the L‐Eng splice variant. Comparing GFAP^{C re(?)}Eng^f/f to GFAP^{C re(+)}Eng^ΔHSC mice in toxic liver injury, livers of GFAP^{C re(+)}Eng^ΔHSC mice showed 39.9% (P  < .01) higher Hydroxyproline content compared to GFAP^{C re(?)}Eng^f/f littermates. Sirius Red staining underlined these findings, showing 58.8% (P  < .05) more Collagen deposition in livers of GFAP^{C re(+)}Eng^ΔHSC mice. Similar results were obtained in mice subjected to cholestatic injury.

Conclusion

Endoglin isoforms are differentially upregulated in liver samples of patients with chronic and acute liver injury. Endoglin deficiency in HSC significantly aggravates fibrosis in response to injury in two different murine models of liver fibrosis and increases α‐SMA and fibronectin expression in vitro. This suggests that Endoglin protects against fibrotic injury, likely through modulation of TGF ‐β signalling.

     

Pentoxifylline prevents pig serum‐induced rat liver fibrosis by inhibiting interleukin‐6 production

Background/Aim: Pig serum‐induced rat liver fibrosis is a model of liver fibrosis in the absence of obvious hepatocyte injury. Penoxifylline (PTX), a xanthine derivative, which is a well‐known suppressor of tumor necrosis factor‐α (TNF‐α) production from inflammatory cells, has also been shown to inhibit the growth of hepatic stellate cells and to inhibit collagen synthesis in these cells in vitro. We investigated the effect of PTX on pig serum‐induced liver fibrosis in vivo, and assessed the mechanisms of prevention of fibrogenesis by this drug. Methods: Male Wistar rats were given intraperitoneal injections of 0.5 ml normal pig serum twice a week for 10 weeks with or without concomitant oral administration of PTX (20 mg/kg). Results: Rats that received pig serum showed significant liver fibrosis, and their serum interleukin‐6 (IL‐6) and hyaluronic acid levels were significantly increased. The serum levels of IL‐6 were well correlated with the serum levels of hyaluronic acid, and increased as the liver fibrosis progressed. Penoxifylline prevented the development of fibrosis in this animal model and reduced the serum levels of IL‐6 in a dose‐dependent manner. In vitro, by the addition of PTX to the culture medium of the rat hepatic stellate cells (HSCs), the proliferation of the HSCs was significantly inhibited and IL‐6 in the culture supernatant was also reduced significantly. Exogenous addition of IL‐6 partially restored the proliferation. Conclusion: Penoxifylline prevents pig serum‐induced rat liver fibrosis by inhibiting the proliferation of HSCs and by inhibiting the production of IL‐6 from HSCs.     

Overexpression of NK2 promotes liver fibrosis in carbon tetrachloride‐induced chronic liver injury

Background/Aims: Hepatocyte growth factor (HGF) inhibits liver fibrosis induced by carbon tetrachloride (CCl₄) in animal models. NK2 is a natural splice variant of HGF, but its in vivo function remains to be elucidated. We investigated the in vivo effects of NK2 on CCl₄‐induced liver fibrosis. Methods: NK2 transgenic mice and wild‐type (WT) mice were injected intraperitoneally with CCl₄ twice a week. The extent of hepatic fibrosis was evaluated by Azan–Mallory staining. Expression levels of mRNAs of transforming growth factor‐β1 (TGF‐β1) and matrix metalloproteinase‐13 (MMP‐13) were examined by real‐time polymerase chain reaction. The protein levels of α‐smooth muscle actin (α‐SMA), c‐Met and its phosphorylation were determined by Western blot analysis. Results: Liver fibrosis was significantly more severe in NK2 transgenic mice than in WT mice. CCl₄ administration increased the expression levels of TGF‐β1 mRNA and α‐SMA protein, and decreased the expression of MMP‐13 mRNA in livers of NK2 transgenic mice compared with those of WT mice. c‐Met protein expression in the liver was compatible with the degree of fibrosis. As for c‐Met activation, no difference was found between NK2 and WT livers. Conclusion: Overexpression of NK2 acts as an antagonist of HGF and promotes liver fibrosis in CCl₄‐induced chronic liver injury.     

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.     

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.     

Conditional knockout of heparin‐binding epidermal growth factor‐like growth factor in the liver accelerates carbon tetrachloride‐induced liver injury in mice

Aim: We previously demonstrated that heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) is induced in response to several liver injuries. Because the HB‐EGF knockout (KO) mice die in utero or immediately after birth due to cardiac defects, the loss of function study in vivo is limited. Here, we generated liver‐specific HB‐EGF conditional knockout mice using the interferon‐inducible Mx‐1 promoter driven cre recombinase transgene and investigated its role during acute liver injury. Methods: We induced acute liver injury by a single i.p. injection of carbon tetrachloride (CCl₄) in HB‐EGF KO mice and wild‐type mice and liver damage was assessed by biochemical and immunohistochemical analysis. We also used AML12 mouse hepatocyte cell lines to examine the molecular mechanism of HB‐EGF‐dependent anti‐apoptosis and wound‐healing process of the liver in vitro. Results: HB‐EGF KO mice exhibited a significant increase of alanine aminotransferase level and also showed a significant increase in the number of apoptotic hepatocytes assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining at 24 h after CCl₄ injection. We also demonstrated that HB‐EGF treatment inhibited tumor necrosis factor‐α‐induced apoptosis of AML12 mouse hepatocytes and promoted the wound‐healing response of these cells. Conclusion: This study showed that HB‐EGF plays a protective role during acute liver injury.     

β‐Glucuronidase inhibitor tectorigenin isolated from the flower of Pueraria thunbergiana protects carbon tetrachloride‐induced liver injury

Background/Aim: To understand the relationship between the fluctuation in serum β‐glucuronidase and hepatotoxicity, an inhibitor of β‐glucuronidase was isolated from the flowers of Pueraria thunbergiana and its hepatoprotective activity was measured. Method: Tectorigenin was isolated from the flowers of pueria thunbergiana as an inhibitor of β‐glucuronidase, and serum ALT, AST and biological parameters as markers for its hepatoprotective activity were measured on CCl₄‐induced liver injury in mice. The relationship between serum β‐glucuronidase and hepatoprotective activities in mice was measured. Results: When tectorigenin at a dose of 100 mg/kg was intraperitoneally administered on CCl₄‐induced liver injury in mice, it significantly inhibited the increase of plasma ALT, AST and LDH activities. The inhibitory effect of tectorigenin is much more potent than that of dimethyl diphenyl bicarboxylate (DDB), which has been used as a commercial hepatoprotective agent. When tectoridin transformed to tectorigenin by intestinal bacteria was orally administered to mice, it showed hepatoprotective activity. However, when tectoridin was intraperitoneally administrated to mice, it did not exhibit hepatoprotective activity. Moreover, orally administered tectoridin not only inhibited β‐glucuronidase but also increased GSH content and GST activity on CCl₄‐induced hepatotoxicity of mice. Conclusion: We insist that an inhibitor of β‐glucuronidase tectorigenin may be hepatoprotective and tectoridin should be a prodrug transformed to tectorigenin.     

Hydrodynamic-based in vivo transfection of retinoic X receptor-alpha gene can enhance vitamin A-induced attenuation of liver fibrosis in mice.

BACKGROUND/AIM: In hepatic stellate cells isolated from rat fibrotic livers, the amount of retinoid X receptor-alpha (RXR-alpha) mRNA is greatly reduced. However, the effectiveness of retinoids in the treatment of liver fibrosis is controversial. We hypothesized that increasing the expression levels of RXR-alpha in livers will improve the response of liver fibrosis to retinoids treatment. METHODS: pTracer-CMV2 vector harboring both green fluorescent protein and RXR-alpha genes was given to mice with carbon tetrachloride (CCl(4))-induced liver fibrosis, by hydrodynamic-based in vivo transfection. Vitamin A was simultaneously administered to the mice. Sirius red staining and measurement of hydroxyproline content were performed to evaluate liver fibrosis. The incorporation of 5-bromo-2-deoxyribouridine (BrdU) was carried out to determine liver cell proliferation. RESULTS: Successful transfection and expression of exogenous RXR-alpha gene in the liver was determined by observance of green fluorescence under a confocal microscope, and detection of RXR-alpha protein by immunohistochemistry. Hepatic fibrosis, evaluated by both Sirius red staining with image analysis and quantity of hydroxyproline in livers of RXR-alpha-transfected group, tapered off remarkably. The hydroxyproline content and Sirius red-positive staining area on liver sections from RXR-alpha-transfected mice decreased by 34.3% and 54.63%, respectively, compared with the control group receiving empty vector. The labeling index of BrdU in non-parenchymal cells was much lower in livers from the RXR-alpha-transfected group than that of empty vector-transfected group. CONCLUSIONS: Hydrodynamic-based in vivo transfection of the RXR-alpha gene can enhance the vitamin A-induced attenuation of liver fibrosis in mice. One of the possible mechanisms of action for this gene treatment is inhibition of non-parenchymal cell proliferation mainly composed of hepatic stellate cells in fibrotic livers.     

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.