熊果酸对TGF-β_1诱导肝细胞凋亡的抑制作用及其机制

Inhibiting effect of ursolic acid on hepatocyte apoptosis induced by TGF-β_1 and its mechanism

目的研究熊果酸(UA)对转化生长因子β_1(TGF-β_1)诱导肝细胞凋亡的干预作用及其机制。方法采用原位灌注法分离健康SD大鼠原代肝细胞,随机分成空白对照组、UA自身对照组(UA 25μmol/L)、TGF-β_1组(TGF-β_12.5ng/ml)、UA干预组(UA 25μmol/L+TGF-β_1 2.5ng/ml)、DPI干预组(DPI 0.5μmol/L+TGF-β_1 2.5ng/ml)。药物作用相应时间后,采用流式细胞术检测肝细胞增殖及凋亡情况,荧光定量PCR法检测肝细胞内CD95(Fas)mRNA的表达,Western blotting检测肝细胞内CD95蛋白的表达以及NADPH氧化酶(NOX)亚基p47phox移位至胞膜的蛋白量,并采用活性氧(ROS)检测试剂盒检测肝细胞内ROS水平。结果在TGF-β_1刺激肝细胞前30min加入UA进行干预可明显降低TGF-β_1诱导的肝细胞凋亡率(80.53%±1.56%vs 63.97%±3.19%,P<0.01),肝细胞增殖指数明显增加(10.83%±2.03%vs18.67%±1.60%,P<0.01),细胞内CD95 mRNA及蛋白表达均明显下调(2.40±0.25 vs 1.28±0.15,P<0.01;1.37±0.18vs 1.05±0.15,P<0.05),p47^(phox)向膜移位明显减少(1.76±0.22 vs 1.13±0.12,P<0.01),肝细胞中ROS水平明显降低(3.23±0.53 vs 2.12±0.45,P<0.01)。结论 UA可能通过抑制肝细胞内NOX激活,减少ROS产生,下调CD95的表达来抑制TGF-β_1诱导的肝细胞凋亡。

Objective To study the effect of ursolic acid （UA） intervention on hepatocyte apoptosis induced by TGF-β1 and its potential mechanism. Methods Primary hepatocytes were extracted from healthy SD rats by in situ perfusion, cultured for 12-24h, then randomly divided into the following groups： blank control group, UA control group （UA 25μmol/L）, TGF-β1 group （TGF-β1 2.5ng/ml）, UA intervention group （UA 25μmol/L and TGF-β1 2.5ng/ml）, DPI intervention group （DPI 0.5μmol/L and TGF-β1 2.5ng/ml）. Each group was treated with drugs for corresponding time and their proliferation and apoptosis were detected by flow cytometry, the expression of CD95 （Fas） mRNA was analyzed by RT-qPCR, the expression of protein CD95 and membrane translocation of NADPH oxidase （NOX） subunit p47phox were analyzed by Western blotting, and the reactive oxygen species （ROS） generation in primary hepatocytes was analyzed with reactive oxygen detection kit. Results UA intervention at 30min before TGF-β1 stimulating hepatocytes markedly reduced hepatocyte apoptosis （63.97 ± 3.19 vs 80.53 ± 1.56, P〈0.01） and promoted hepatocyte proliferation （18.67 ± 1.60 vs 10.83 ± 2.03, P〈0.01）. UA intervention notably down-regulated the expressions of CD95 mRNA and protein （1.28 ± 0.15 vs 2.40 ± 0.25, P〈0.01; 1.05 ± 0.15 vs 1.37 ± 0.18, P〈0.05）, restrained membrane translocation of p47phox （1.13 ± 0.12 vs 1.76 ± 0.22, P〈0.01）, and decreased ROS level in primary hepatocytes induced by TGF-β1 （2.12 ± 0.45 vs 3.23 ± 0.53, P〈0.01）. Conclusion The mechanism of UA inhibiting hepatocyte apoptosis induced by TGF-β1 is likely to be that UA intervention reduced hepatocyte apoptosis by inhibiting NOX activation and decrease generation of ROS so as to down-regulate expression of CD95 in hepatocytes.