Effect Of Ursolic Acid On The Level Of ROS Of Rats HSC And The Mechanism Of Apoptosis Of Rats HSC Induced By UA

Background:Hepatic fibrosis which is associated with the production and deposition of extracellular matrix is a pathological response to various chronic liver diseases. The perisinusoidal HSC is largely responsible for the increase in extracellular matrix(ECM) deposition. Inducing apoptosis of activated HSC is an important therapeutic approach to advanced hepatic fibrosis. Our pre-experiment had proved that Ursolic acid can inhibit the activation and proliferation of HSC and induce apoptosis of activated HSC effectively, and when the Concentration of UA is lower than 75μM , the proliferation of Liver cells(L02) is inhibited. When the Ursolic acid was used to the SD rat hepatic fibrosis model which had been treated by dimethy- nitrosamine(DMN), we found UA can improve the liver function, lessen hepatic cell necrosis and inhibit hyperplasia of fibrous tissue . Based on the newest researches on hepatic fibrosis and our prior research basis, we want to find what role the ROS plays in HSC apoptosis and how the UA induces HSC apoptosis.Objective:To observe the effect of Ursolic acid on the level of ROS of rats HSC and to explore the possible mechanisms of HSC apoptosis of induced by UA.Methods:Randomization of exponential phase of growth hepatic stellate cells(HSC -T6) were : leptin(100 ng/ml) group, UA(50uM),NAC(10mM)pretreatment groups, normal group. In pretreatment groups HSC will be pretreated by UA,NAC for 30min, then be treated by Leptin. Intracellular ROS was measured using flow cytometry to detect DCF fluorescence at 1h,12h,24h;the nuclear translocation of NF-κB subunit P65 was evaluated by immunocytochemical staining assay after the HCS-T6 was treated within 2 hours; the expressions of XIAP mRNA were determined by RT-PCR in 12h,24h; finally the rate of HSC-T6 apoptosis was identified by flow cytometry (FCM) after the HCS-T6 was treated within 48 hours.Results:1.The DCF fluorescence intensity of leptin group was higher than normal group (P<0.01);The DCF fluorescence intensity of leptin group at 24h was higher than 12h(P<0.05); The DCF fluorescence intensity of NAC pretreatment group,UA pretreatment group was lower than leptin group(P<0.001); The DCF fluorescence intensity of UA pretreatment group was reduced gradually from 1h to 24h.2.The analysis of immunocytochemistry techniques suggested that compared with normal group,NF-κB in HSC-T6 were increasingly activated when HSC-T6 had been treated by leptin after 2 hours,and the difference was considered significant(P<0.001).When the HSC-T6 was pretreated by UA or NAC within half an hour, then treated by leptin,and we found that the nuclear translocation of NF-κB in the first two group was lower than the leptin group(P<0.001),but the nuclear translocation of NF-κB had no significant different between them(P>0.05).3.When HSC-T6 was treated by leptin, the expressions of XIAP mRNA were significantly higher than normal group At 24h(P<0.01); at 12h,when HSC-T6 was pretreated by UA or NAC, the expressions of XIAP mRNA were both lower than the leptin group and normal group(P<0.001~0.05),and the expressions of XIAP mRNA of the first two group had no significant different between them(P>0.05). at 24h, the expressions of XIAP mRNA in the UA pretreatment group was lower than leptin group,normal group and the NAC pretreatment group(P<0.01); the expressions of XIAP mRNA of normal group and NAC pretreatment group had no significant different between 12h,24h(P>0.05); the expressions of XIAP mRNA of leptin group at 24h was higher than when it was at 12h(P<0.01); the expressions of XIAP mRNA of UA pretreatment group at 24h was lower than it was at 12h(P<0.01).4.After treating HSC-T6 with leptin for 48h, the apoptosis rate of HSC-T6 was lower than normal group(P<0.05);when HSC-T6 was pretreated by UA or NAC, the apoptosis rate of them was higher than leptin group(P<0.001-0.05),and the HSC-T6's apoptosis rate of UA pretreatment group was higher than NAC pretreatment group and normal group(P<0.05).Conclusions:1.leptin can inhibit the apoptosis of HSC-T6; but the rate of HSC-T6 apoptosis is significantly increased when HSC-T6 were pretreated by UA.2. leptin can increase the level of ROS in HSC-T6; but the level of ROS which was induced by leptin is significantly dicreased when HSC-T6 are pretreated by UA.3.Leptin can promote the activation of NF-κB and the expressions of XIAP mRNA,,but the activation of NF-κB and the expressions of XIAP mRNA which were induced by leptin are siginificantly decreased when HSC-T6 were pretreated by UA. 4.The mechanism of HSC-T6 apoptosis induced by Ursolic acid is probably related to decrease of ROS, down-regulate the expression of NF-κB and XIAP mRNA.