| Liver.fibrosis results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix(ECM) proteins,which is a characteristic of most types of chronic liver diseases.Advanced liver fibrosis results in cirrhosis,liver failure,and portal hypertension and often requires liver transplantation.Extensive and persistent hepatic fibrosis has for a long time been considered irreversible.However, accumulating evidence suggests that liver fibrosis is reversible and recovery from cirrhosis may be possible.Hepatic stellate cells(HSCs) are major sources of the excess ECM deposited in fibrotic liver.Transforming growth factorβ1(TGF-β1) is considered to play a generic role in the development of tissue fibrosis,as it stimulates the synthesis and deposition.Smad pathways are central mediators of signals from the receptors for TGF-β1 to the nucleus.However,growing biochemical and developmental evidence supports that PGE2-EP2-cAMP signal transduction also participates in TGF-β1 signalling.Despite efforts to develop antifibrotic agents,no drugs have been approved as antifibrotic agents in humans.Radix Astragali is one of the valuable medical herbs that have been widely used in East Asia for a long time.Astragalosides is the major active component extracted from the root of Astragalus membranaceus.The anti-inflammatory,anti-aging, anti-oxidative,anti-myocardial injury and immunoregulatory activities without evident toxic or side effects of astragalosides have been extensively proved for many years.Previous studies from our laboratory demonstrated that crude astragalosides fraction can significantly inhibit the progression of hepatic fibrosis induced by Carbon tetrachloride(CCl4).AstragalosideⅣ(AGS-Ⅳ),a new glycoside of cycloartane-type triterpene, 3-O-β-Dxylopyranosyl-6-O-β-D-glucopyranosylcycloastragenol was purified from the Chinese medical herb Astragalus membranaceus(Fisch) Bge.Up to now,whether AGS-Ⅳhas an exact antifibrotic effect in immunological hepatic fibrosis and the mechanism are not known.In the present study,we examined the in vivo effect of AGS-Ⅳon liver fibrosis induced by administration of porcine serum(PS) and observed the changes of transforming growth factor-β1 in serum and liver tissue in rats treated with AGS-Ⅳ.In vitro effects of AGS-Ⅳon primary rat HSCs proliferation,apoptosis and collagen synthesis were detected,the effects of ASG.-Ⅳon the expressions of Smad3 and Smad7 were investigated.Further more The relationship between the PGE2-EP2-cAMP signal transduction and HSCs fibrogenesis and the effects of AGS-Ⅳwere also evaluated.AIMTo investigate the anti-fibrositic effects of AGS-Ⅳon immunological hepatic fibrosis in rats induced by PS;to investigate the in vitro effects of AGS-Ⅳon the proliferation,apoptosis and collagen synthesis in HSCs and the effects on the expressions of Smad3 and Smad7;to investigate the role of PGE2-EP2-cAMP signal transduction in fibrogenesis and the effects of AGS-Ⅳ.METHODSLiver fibrosis was induced by PS injection(0.5 ml,twice a week) for 8 weeks in Wistar rats.At the same time,AGS-Ⅳ(2.0,4.0mg·kg-1) was administered intragastrically.Histopathology was examined by hematoxylin-eosin(HE) and Masson's staining.Serum marker and TGF-β1 levels were detected by radioimmunoassay and enzyme-linked immunosorbent assay(ELISA),respectively. The expression of TGFβ1 in liver tissue was evaluated by immunohistochemistry. Effect of AGS-Ⅳon platelet-derived growth factor-B subunit homodimer(PDGF-BB) stimulated proliferation was detected by[3H]-thymidine incorporation assays. Flow-cytometric analysis was performed to examine the apoptosis-inducing effect. Collagen synthesis stimulated by TGF-β1 was detected by[3H]-proline incorporation assays.Western blotting analysis was performed to detect the expressions of Smads protein.COX-2 inhibitor,celecoxib 15 mg·kg-1 body weight daily were given by gastric gavage to evaluated the changes and the effects of PGE2 and EP2 on PS induced liver fibrosis.Detected the effects of PGE2(5,500nM) and EP2 agonist ONO-AE1-259-01(1,100 nM) on TGF-β1 stimulated collagen synthesis and the changes of intracellular cAMP level.RESULTS1.Protective effect of AGS-Ⅳon immunological hepatic fibrosis in rats induced by porcine-serumAGS-Ⅳat two dose of 2.0 and 4.0 mg·kg-1 had obvious protective effects on PS-induced immunological hepatic fibrosis.Serum levels of hyaluronic acid(HA), laminin(LN),procollagen typeⅢ(PCⅢ) and collagenⅣ(CⅣ),the surrogate markers of liver fibrogenesis,increased significantly in hepatic fibrotic rats in model group and were suppressed by treatment with AGS-Ⅳ.Hyp contents in liver tissue were carried out as an index of liver fibrosis,the increased was effectively reduced by the administration with AGS-Ⅳ(2.0 and 4.0mg·kg-1).Histological results showed that hepatic fibrosis caused by PS was significantly inhibited by AGS-Ⅳ.AGS-Ⅳimproved alterations in the liver structure.AGS-Ⅳalso ameliorated the oxidative stress state of hepatic fibrosis rats, decreased the production of malondiadehyde(MDA) and enhanced the activities of anti-oxidative enzyme including super oxide dismutase(SOD) and glutathione peroxidase(GSH-px).Because TGF-β1 plays an important role in hepatic fibrosis,we further examined whether the PS induced TGF-β1 expression was affected by treatment with AGS-Ⅳ.In rats treated with AGS-Ⅳ,the positive staining TGF-β1 in HSCs and in fibroblasts was markedly reduced than in model group,serum levels of TGF-β1 were increased markedly in model group and were significantly decreased by treatment with AGS-Ⅳ(2.0,4.0mg·kg-1).2.Effect of AGS-Ⅳon HSCs proliferation,apoptosis,collagen synthesis and on TGF-β1/SmadsThe in vitro study was carried out to investigate the effects of AGS-Ⅳon the proliferation,apoptosis and collagen synthesis in HSCs.Platelet-derived growth factor-B subunit homodimer(PDGF-BB) stimulated proliferation was detected by [3H]-thymidine incorporation assays.Flow-cytometric analysis was performed to examine the apoptosis-inducing effect of AGS-Ⅳon HSCs.TGF-β1 stimulated collagen synthesis was detected by[3H]-proline incorporation assays.Results revealed that AGS-Ⅳ(1.5,3.0,6.0,12.0,24.0 mg·L-1) suppressed the proliferation of HSCs in a concentration-dependent manner,and increased sub-G1 DNA content and increased the activity of caspase-3.Meanwhile,TGF-β1 stimulated collagen synthesis was significantly inhibited by AGS-Ⅳ.The results suggest that AGS-Ⅳsignificantly inhibited HSCs proliferation,induced apoptosis in HSCs via caspase-3 activation.To determine the effects of AGS-Ⅳon Smads expressions,western blotting analysis was applied to detect its protein levels,The level of Smad3 was increased stimulated by TGF-β1,while treated with AGS-Ⅳ,it declined gradually with an increase in the concentration from 6.0 to 24.0mg·L-1.Meanwhile,AGS-Ⅳfuther increased the expression of Smad7 stimulated by TGF-β1.3.Effects of AGS-Ⅳon PGE2-EP2-cAMP signal transduction in liver fibrosis in rat and in HSCs In order to examine the effects of decreased prostaglandin E2(PGE2) level on PS-induced hepatic fibrosis,rats were treated with selective cyclooxygenase-2 (COX-2) inhibitor,celecoxib.Histological results showed that fibrosis was significantly increased by co-administration of celecoxib.Most notably,besides hepatic fibrosis formation,livers in PS plus celecoxib treated group showed hepatocellular necrosis,no obvious hepatocellular necrosis was found in other three groups.Celecoxib alone without PS injection did not result in any hepatic fibrosis and hepatocellular necrosis.Plasma levels of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β),the principal pro-inflammatory cytokines were measured, neither PS nor celecoxib alone induced notable change in the serum levels of TNF-αand IL-1β.However,co-treatment with PS and celecoxib resulted in a marked increase in plasma TNF-αand IL-1βlevel.In rats treated with PS alone,plasma level of PGE2 was increased after 2 weeks PS injection followed by a slight decrease, at week 8,it was significantly lower than that in normal control group.At week 8,the level of PGE2 in plasma and EP2 expression in HSCs were significantly lower in all groups of rats receiving celecoxib,when compared with normal control.The plasma level of PGE2 and EP2 expression in rats treated with PS was further decreased by celecoxib administration at week 8.In vitro studies investigated the effects of PGE2 and EP2 agonist on collegen synthesis in HSCs,results shown that,PGE2(5,500nM) and EP2 agonist ONO-AE1-259-01(1,100 nM) could induce inhibitory effect on TGF-β1 stimulated HSCs collagen synthesis via increase the intracellular cAMP level. We investigated the effects of AGS-Ⅳon the expression of EP2 and intracellular cAMP level.Results showed that AGS-Ⅳ(6.0,12.0,24.0 mg·L-1) could significantly increased the expression of EP2 and cAMP level in HSCs.CONCLUSION1.AGS-Ⅳhas an anti-hepatic fibrosis effects in PS-induced rat models via ameliorated the oxidative stress,decreased the TGF-β1 levels in plasma and the expression in liver tissue; 2.AGS-Ⅳmay achieve its inhibit effects on HSCs by inhibiting the proliferation, inducing apoptosis in HSCs via caspase-3 activation,and modulating TGF-β1/Smads signaling response and consequently suppressing the collagen productions deposition;3.PGE2-EP2-cAMP signal pathway had important role in liver fibrosis induced by PS injection in rats and in collegen synthesis in HSCs.Increaseing EP2 expression and intracellular cAMP level may inhibit collagen synthesis in HSCs. 4.Anti-fibrosis effects of AGS-Ⅳmay be associated with its effects of increaseing PGE2 level in plasma in rats,increasing EP2 expression and cAMP level in HSCs.
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