| Background:Hepatic cirrhosis is inconvertible. Hepatic stellate cell (HSC) infected by activation and transformation of various cytokines is the key factors in incidence and development of hepatic fibrosis. So inhibiting activation and proliferation of HSC,and inducing apoptosis of activated HSC are important method in converting hepatic fibrosis. Leptin which recently founded to be an important factor in the development of hepatic fibrosis, as it can cause hepatic fibrosis by JAK-STAT signal transduction pathway which can activate HSC, promote the proliferation of HSC and depress its apoptosis, regulate the expression of extracellular matrix(ECM), MMP-1 and TIMP-1.Our pre-project works discovered that Ursolic acid had a more significant effect in inhibiting HSC proliferation and inducing apoptosis in vitro, and reducing incidence of DMN-induced liver fibrosis in rats in vivo. Recently , we found that UA can inhibit the expression of NADPH oxidase subunit Rac1 and p22Phox mRNA and inhibit ROS production induced by leptin in HSC, and depressing the expression of NF-κB, and then induces HSC apoptosis in vitro.This study based on prior research aimed at observing the influence of Ursolic acid on JAK-STAT signal transduction pathway of leptin in HSC and expression of MMP-1 and TIMP-1 and discussing the possible molecular mechanism of Ursolic acid in hepatic fibrosis.Objective : To determine the effect of Ursolic acid on TIMP-1mRNA and MMP-1mRNA expression and the ROS generation; and to investigate the effect of Ursolic acid on p-JAK2 protein and p-STAT3 protein expression and explore the possible mechanisms of Ursolic acid on the pathway of leptin signal transduction of HSC.Materials and Methods: Randomization of exponential phase of growth hepatic stellate cells(HSC-T6) were : leptin(100 ng/ml) group, UA(50uM)pretreat group, NAC(10mM)pretreat group, DPI(20uM)pretreat group, AG490(50uM)pretreat group, UA(50uM)own control group and normal control group. After HSC be treated with medicine for 6 hour,12 hour or 24 hour, extracted total RNA,TIMP-1, MMP-1mRNA expression levels were measured by RT-PCR; Intracellular ROS was measured by using fluorescence microscope to detect DCF fluorescence.the nuclear translocation of p-STAT3 was evaluated by immunocytochemical staining assay after the HCS-T6 was treated within 24 hours.Results:1. The effect of Ursolic acid on TIMP-1mRNA expressionThe TIMP-1 mRNA expression levels of Leptin group had no difference compared with normal control group in 6 hour(P>0.05),but increased significantly compared with normal control group in 12 hour and 24 hour (P<0.05);In UA pretreatment group, the TIMP-1 mRNA expression levels were lower than Leptin group in 12 hour and 24 hour (P<0.05), but had no difference compared with normal control group(P>0.05); In NAC Pretreatment group, DPI Pretreatment group and AG490 Pretreatment group, the TIMP-1 mRNA expression levels were lower than Leptin group in 12 hour and 24 hour (P<0.01 or P<0.05); In UA pretreatment group, the TIMP-1 mRNA expression levels had no difference compared with NAC Pretreatment group, DPI Pretreatment group and AG490 Pretreatment group in 6 hour,12 hour and 24 hou(rP>0.05); In UA own control group, the TIMP-1 mRNA expression levels had no difference compared with normal control group in 6 hour,12 hour and 24 hour(P>0.05).2. The effect of Ursolic acid on MMP-1mRNA expressionThe MMP-1 mRNA expression levels of Leptin group in 6 hour,12 hour and 24 hour decreased significantly compared with normal control group (P<0.05 or P<0.01); In UA Pretreatment group, the MMP-1 mRNA expression levels were higher than Leptin group in 6 hour ,12 hour and 24 hour (P<0.01), and had no difference compared with normal control group(P>0.05); In UA Pretreatment group, the MMP-1 mRNA expression levels were higher than NAC Pretreatment group in 24 hour (P<0.05), and had no difference compared with DPI Pretreatment group and AG490 Pretreatment group in 6 hour,12 hour and 24 hour(P>0.05); In UA own control group, the TIMP-1 mRNA expression levels had no difference compared with normal control group in 6 hour,12 hour and 24 hour(P>0.05).3. The effect of ursolic acid on the ROS generationIn Leptin group, the DCF fluorescence increased significantly compared with normal control group (P<0.01) ; In UA Pretreatment group, the DCF fluorescence were lower than Leptin group (P<0.01); In NAC Pretreatment group, DPI Pretreatment group and AG490 Pretreatment group, the DCF fluorescence were lower than Leptin group (P<0.01); In UA Pretreatment group, the DCF fluorescence were lower than NAC Pretreatment group (P<0.01),but had no difference compared with DPI Pretreatment group and AG490 Pretreatment group(P>0.05); In UA own control group , the DCF fluorescence were higher than normal control group(P<0.05).4. The effect of ursolic acid on p-JAK2 protein expressionThe analysis of immunocytochemistry techniques suggested that compared with normal group, p-JAK2 in HSC-T6 were increasingly activated when HSC-T6 had been treated by Leptin after 24 hours,and the difference was considered significant(P<0.01). In UA Pretreatment group, NAC Pretreatment group and DPI Pretreatment group, the protein expression of p-JAK2 were lower than the Leptin group (P<0.05 or P<0.01), but higher than normal control group(P<0.01); In AG490 Pretreatment group, the protein expression of p-JAK2 were lower than the Leptin group (P<0.01), and lower than normal control group(P<0.05); In UA own control group, the protein expression of p-JAK2 were lower than normal control group(qP<0.01).5. The effect of ursolic acid on p-STAT3 protein expressionThe analysis of immunocytochemistry techniques suggested that compared with normal group, p-STAT3 in HSC-T6 were increasingly activated when HSC-T6 had been treated by Leptin after 24 hours,and the difference was considered significant(P<0.01); In UA Pretreatment group, the protein expression of p-STAT3 were lower than the Leptin group(P<0.01), had no difference compared with normal control group(P>0.05); In NAC Pretreatment group and DPI Pretreatment group, the protein expression of p-STAT3 were lower than the Leptin group(P<0.01),but higher than normal control group(P<0.01); In AG490 Pretreatment group, the protein expression of p-STAT3 were lower than the Leptin group(P<0.01), but had no difference compared with normal control group ( q=1.80, P>0.05 ) ; In UA Pretreatment group, the protein expression of p-STAT3 were lower than NAC Pretreatment group and DPI Pretreatment group(P<0.05), but had no difference compared with AG490 Pretreatment group(P>0.05); In UA own control group, the protein expression of p-STAT3 had no difference compared with normal control group(P>0.05).Conclusions:1. UA can inhibit the expressions of TIMP-1 mRNA, and increase the expression of MMP-1 mRNA.2. Leptin can induce ROS production in HSC-T6 cells, and ROS probably participate in leptin–induced JAK2 -STAT3 activation, while pretreatment with UA can inhibit leptin induced ROS generation.3. Leptin can activate JAK2-STAT3 signal transduction in HSC-T6 cells, while pretreatment with UA can inhibit leptin induced JAK2 -STAT3 activation.4. The mechanism of UA regulating the expressions of TIMP-1 mRNA and MMP-1 mRNA is probably related to inhibiting the production of ROS, then inhibiting the JAK-STAT signal transduction.
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