熊果酸对活化型肝星状细胞NADPH氧化酶亚基p47^(Phox)表达及ERK1/2信号通路活化的影响

Effects of ursolic acid on NADPH oxidase subunit p47^(Phox) expression and ERK1/2 pathway activation in rat hepatic stellate cells

目的研究熊果酸(ursolic acid,UA)对瘦素诱导的大鼠肝星状细胞(HSC-T6)NADPH氧化酶(NOX)亚基p47Phox表达及ERK1/2信号通路活化的影响,并观察Ⅰ型胶原合成及细胞增殖情况。方法将培养激活的HSC-T6细胞株分为6组:正常对照组,不加任何药物;瘦素组,给予重组大鼠瘦素(100 ng/ml)刺激细胞;各干预组分别给予UA(50μmol/L)、JAK抑制剂AG490(50μmol/L)、NOX抑制剂DPI(20μmol/L)、ERK抑制剂PD98059(30μmol/L)预处理30 min,再加入瘦素刺激不同时间。采用蛋白质印迹分析检测细胞膜移位的p47Phox蛋白、细胞总p47Phox蛋白和磷酸化的ERK1/2(p-ERK1/2)蛋白表达;采用RT-PCR法检测Ⅰ型胶原mRNA的表达;采用MTT法检测细胞增殖。结果瘦素刺激HSC-T6细胞30 min后细胞膜p47Phox蛋白表达较正常对照组增高(P<0.01),细胞内p-ERK1/2蛋白表达也随之增高(P<0.05);UA、AG490、DPI、PD98059干预后抑制了p47Phox蛋白向细胞膜移位以及细胞内ERK1/2蛋白磷酸化。瘦素刺激HSC-T6细胞12 h后Ⅰ型胶原的mRNA表达较正常对照组升高(P<0.01),UA、AG490、DPI及PD98059干预组Ⅰ型胶原mRNA的表达均低于瘦素组(P均<0.01)。瘦素刺激HSC-T6细胞12、24、48 h后细胞增殖率高于正常对照组(P均<0.01);UA、AG490、DPI及PD98059干预不同时间点的细胞增殖率均低于瘦素组(P均<0.01),UA的抑制细胞增殖作用弱于DPI(P<0.01)。结论 UA能抑制瘦素诱导的HSC-T6细胞增殖及Ⅰ型胶原表达,机制可能与抑制NOX亚基p47Phox向细胞膜移位及下游信号通路ERK1/2的激活有关。

Objective To investigate the effects of ursolic acid（UA） on leptin-induced NADPH oxidase（NOX） subunits p47Phox expression and ERK1/2 pathway activation of rat hepatic stellate cells（HSC-T6）,and to observe the cells proliferation and collagen Ⅰ synthesis.Methods Culture-activated HSC-T6 cells were divided into six groups： normal control group received no treatment;leptin group received recombinant rat leptin（100 ng/ml）;the four intervention groups were pretreated with UA（50 μmol/L）,JAK inhibitor AG490（50 μmol/L）,NOX inhibitor DPI（20 μmol/L）,and ERK inhibitor PD98059（30 μmol/L） for 30 min,and then stimulated with leptin for different time periods.Western blotting analysis was used to examined the expression of p47Phox protein translocation to the cell membrane,total cellular p47Phox protein and phosphorylated ERK1/2（p-ERK1/2） protein.Collagen Ⅰ mRNA expression was detected by RT-PCR and cell proliferation was examined by MTT assay.Results Expression of p47Phox membrane protein was significantly increased compared with the normal control 30 min after leptin stumulation（P〈0.01）,and p-ERK1/2 protein expression was also significantly increased correspondingly（P〈0.05）.UA,AG490,DPI and PD98059 inhibited the p47Phox protein translocation to membrane and ERK1/2 protein phosphorylation in HSC-T6 cells.Compared with normal control group,leptin stimulation for 12 h significantly up-regulated collagen Ⅰ mRNA expression in HSC-T6 cells（P〈0.01）;UA,AG490,DPI and PD98059 treatment inhibited collagen Ⅰ mRNA expression in HSC-T6 cells compared with the leptin group（P〈0.01）.Proliferation rates of HSC-T6 cells were significantly higher at 12,24 and 48 h after leptin stimulation compared with the control group（all P〈0.01）;UA,AG490,DPI and PD98059 treatment inhibited leptin-induced cell proliferation at different time points（all P〈0.01）,with the inhibitory effect of UA being significantly weaker than that of DPI（P〈0.01）.Conclusion UA can inhibit leptin-induced proliferation of HSC-T6 cells and collagen Ⅰexpression,which might be associated with the inhibition of NOX subunit p47Phox translocation to the cell membrane and the ERK1/2 pathway activation.