Prevention Of Combining Na～+/H～+ Exchanger Inhibitor With Antioxidants On Liver Fibrosis In Rats

Objective To investigate the preventive effects of combination of amiloride used as a Na+/H+ exchanger inhibitor with antioxidants including curcumin , tanshinone II A and vitamin E on experimental liver fibrosis in vivo and on rat active hepatic stellate cells in vitro.Methods In vivo.the rat liver fibrosis was induced by dimethhiitrosamine(DMN),malotilate was emjoyed as a postive control, liver/weight index.spleen/weight index,serum and tissue biochemical indexes,liver hydroxyproline(HYP) content,serum hyaluronic acid (HA)and laminin(LN) content were detected after treating by amiloride,curcumin, tanshinone II A,vitamin E and the histological changes were observed with light microscope. In vitro, hepatic stellate cell was used as a target cell,cellular model of liver fibrosis in rat was induced by naked plastic incubated dish.HSC proliferation and extracellular matrix secretion were measured after treating by amiloride,curcumin, tanshinone II A by MTT colorimetry, ELISA and Radioimmunoassay respectively. Model of oxidative stress in rat hepatic stellate cells was induced by FeNTA .Collagen type I accummulation in the culture media was measured by ELISA. Intracellular malonildialdehyde(MDA),superoxide dismutase(SOD), reduced glutathione (GSH) and glutathione peroxidase (GSH-PX) levels in the culture media were measured by their Kits.Results In the model of liver fibrosis induced by DMN,serum ALT and AKP were decreased in all preventive groups,while serum A/G were increased in all preventive groups,liver function was ameliorative in preventive groups compared with those of DMN group except amiloridegroup(P<0.05).Serum HA,LN and liver HYP content were declined in all preventive groups compared with those of DMN group(P<0.05).Liver SOD,GSH-PX were higher in preventive groups than those in the model group,while MDA was reduced.Pathological examination suggested that all preventive groups had therapeutical effect on expertimental liver fibrosis in different degrees.Combining antioxidant with Na'TtF exchanger inhibitor had better effects on relieving experimental liver fibrosis induced by DMN than amiloride or curcumin or tanshinone II A,vitaminE,especially,combining curcumin with amiloride.In vitro, adding l~20umol/L amiloride,5~50jAg/ml tanshinone II A and l~20|Amol/L curcumin into culture medium could significantly inhibite HSC proliferation,and also inhibit collagen type I ,hyaluronic Acid and laminin secretion in different degrees. Combining antioxidant with Na+/H exchanger inhibitor had better effects on inhibiting HSC proliferation and extracellular matrix secretion,especially,combining curcumin with amiloride.HSC incubation with FeNTA resulted in a significant production of intracellular MDA and GSH,associated with decrease SOD and GSH-PX activity.Exposure of HSC to FeNTA significantly enhanced the number of proliferating HSC and collagen type I levels in the culture medium. All these effects were reversed by the curcumin or tanshinone IIA or amiloride.Combining curcumin with amiloride had better effects on the fibrosis of rat hepatic stellate cells induced by oxidative stress than amiloride or curcumin.Conclusions Both antioxidants and Na+/H exchanger inhibitor show prophylactic effects on DMN induced rat liver fibrosis.inhibitory effects on HSC proliferation and extracellular matrix secretion and inhibitory effect on the fibrosis of rat hepatic stellate cells induced by oxidative stress.The coeffects on rat liver fibrosis were observed in combination of antioxidants with Na+/H" exchanger inhibitor in different degrees,especially,combining curcumin with amiloride.