| Background:Liver fibrosis is a reversible wound-healing response to virtually all forms of chronic liver injury. The eventual result of uncontrolled liver fibrosis is cirrhosis. Thus, efforts to prevent and reverse fibrosis have considerable clinical implications. Recent years, some studies indicated that ROS derived from NOX can act as a second messenger for various profibrogenic factors signal transduction in HSC,place NOX in the center of the fibrogenic signaling response in HSC. Therefore, regulation of NOX enzymatic activity and expression may inhibit ROS generation and interfere with various profibrogenic factors signal transduction in HSC, which would play a potential role as a pharmacological target for antifibrotic therapies. Ursolic acid is a triterpenoid that exists in nature and is the major component of some traditional medicinal herbs. Our pre-project works discovered that Ursolic acid could significantly inhibit HSC proliferation and induce apoptosis in vitro, and can inhibit DMN-induced liver fibrosis in rats in vivo. But it is not clear that whether ursolic acid has effects on NADPH oxidase in HSC and its mechanisms.Objective:To determine the cellular source of ROS generated by HSC in response to leptin, and the effect of ursolic acid on the ROS generation, and to investigate the effect of Ursolic acid on NADPH oxidase subunit Rac1mRNA and P22PhoxmRNA expression and its enzymatic activity in HSC and explore the possible mechanisms of Ursolic acid inhibiting ROS generation in HSC.Materials and Methods:1. To determine the cellular source of ROS generated by HSC in response to leptin, and the effect of ursolic acid on the ROS generation, exponential phase of growth hepatic stellate cells(HSC-T6) were randomly divided into thirteen groups: 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, Rotenone(20uM) pretreat group, Metyrapone(250 uM) pretreat group, Allopurinol(100uM) pretreat group , Indomethacin(100uM) pretreat group, Rosup(5ug/ml)positive control group, normal control group and blank control group. According to empirical procedure, to choose different medicine treat HSC for 1hour,12 hour or 24 hour. Intracellular ROS was measured by using flow cytometry to detect DCF fluorescence.2. To investigate the effect of Ursolic acid on NADPH oxidase subunit Rac1mRNA and P22PhoxmRNA expression and its enzymatic activity in HSC, exponential phase of growth hepatic stellate cells(HSC-T6) were randomly divided into seven groups: 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, NADPH oxidase subunit Rac1,P22PhoxmRNA expression levels were measured by RT-PCR; Uniformly, HSC were treated for 6 hour, 12 hour or 24 hour, then incubation with 250μmol/l NADPH, NADPH oxidase activity was analyzed by using spectrophotometer to calculate the amount of NADPH consumed.Results:1. In normal control group , the DCF fluorescence were higher than blank control group, but lower than positive control group (P<0.01); In leptin group, the DCF fluorescence increased significantly compared with normal control group (P<0.01), had no difference compared with positive control group(P>0.05);In DPI pretreat group, the DCF fluorescence were lower than leptin group and positive control group (P<0.01),and had no difference compared with positive control group(P>0.05);In Rotenone, Metyrapone, Allopurinol and Indomethacin pretreat groups, the DCF fluorescence increased significantly compared with normal control group (P<0.01), and had no difference compared with leptin group and positive control group(P>0.05); In AG490 pretreat group, the DCF fluorescence were lower than leptin group and positive control group (P<0.01),but higher than DPI pretreat group and normal control group(P<0.05,P<0.01).2. In UA pretreat group and UA own control group, the DCF fluorescence were lower than leptin group and positive control group in1hour (P<0.01), but higher than normal control group (P<0.01).However, the DCF fluorescence of UA pretreat group were lower than leptin group in 12 and 24 hours, furthermore, the DCF fluorescence in12 hour were lower than 1hour (P<0.01),and in 24 hour were lower than 12hour (P<0.01),in 24 hour, its DCF fluorescence had no difference compared with DPI pretreat group(P>0.05).In NAC pretreat group, the DCF fluorescence were lower than leptin group and positive control group (P<0.01), and the DCF fluorescence in 12 hour and 24 hour were higher than in 1 hour (P<0.01).3. The Rac1 mRNA expression levels of leptin group in 6 hour,12 hour and 24 hour increased significantly compared with normal control group (P<0.01), and the highest expression levels was in 12 hour; In UA pretreat group and UA own control group, the Rac1 mRNA expression levels were lower 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 NAC pretreat group, DPI pretreat group and AG490 pretreat group, the Rac1 mRNA expression levels had no difference compared with leptin group in 6 hour,12 hour and 24 hour(P>0.05); Furthermore, the Rac1 mRNA expression levels of NAC pretreat group, DPI pretreat group and AG490 pretreat group were higher than normal control group in 12 hour (P<0.01), and DPI pretreat group and AG490 pretreat group were higher than normal control group in 24 hour (P<0.01,P<0.05).4. The p22Phox mRNA expression levels of leptin group in 6 hour,12 hour and 24 hour increased significantly compared with normal control group (P<0.01); In UA pretreat group and UA own control group, the p22Phox mRNA expression levels were lower 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 NAC pretreat group, p22Phox mRNA expression levels were higher than normal control group in 6 hour (P<0.01),but lower than leptin group in 24 hour (P<0.01); In DPI pretreat group, the p22Phox mRNA expression levels were lower than leptin group in 6 hour and 24 hour (P<0.01, P<0.05); In AG490 pretreat group, the p22Phox mRNA expression levels were higher than normal control group in 6 hour and 12 hour (P<0.01, P<0.05).5. In leptin group, the NADPH oxidase activity of HSC increased significantly compared with normal control group in 6 hour,12 hour and 24 hour (P<0.01); In DPI and AG490 pretreat groups, the NADPH oxidase activity were lower than leptin group in 6hour,12 hour and 24 hour (P<0.01); In UA pretreat group, the NADPH oxidase activity of HSC were lower than leptin group in 6 hour,12hour and 24 hour (P<0.01), and had no difference compared with DPI pretreat group(P>0.05); In NAC pretreat group, the NADPH oxidase activity were higher than normal control group(P<0.01),had no difference compared with leptin group in 6hour,12 hour and 24 hour(P>0.05).Conclusions:1. Leptin can stimulate HSC to generate ROS. The mechanism is probably that leptin activate NOX through JAK signal transduction, and (or) induce the expression of NADPH oxidase subunit.2. Pretreatment with UA can inhibit ROS production induced by leptin in HSC, and inhibit the expression of NADPH oxidase subunit Rac1 and p22Phox mRNA and its enzymatic activity. We presume that the mechanism of Ursolic acid inhibiting ROS generation in HSC is probably related to depressing the expression of NADPH oxidase subunit and the NOX activity.
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