熊果酸对血管紧张素Ⅱ诱导肝星状细胞内NADPH氧化酶的活化及下游信号通路的影响

Effects of ursolic acid on activation of NADPH oxidase and downstream signaling pathways of hepatic stellate cells induced by angiotensinⅡ

目的分析熊果酸(UA)对血管紧张素Ⅱ(AngⅡ)引起肝星状细胞(HSC)NADPH氧化酶(NOX)激活及其下游的PI3K/Akt、P38MAPK信号通路的影响。方法将培养激活的HSC-T6细胞株分组:对照组,不加任何药物;AngⅡ组,给予AngⅡ(1μmol/L)刺激细胞;各干预组分别给予UA(50μmol/L)、NOX抑制剂DPI(20μmol/L)、PI3K/Akt信号通路抑制剂LY294002(10μmol/L)、P38MAPK信号通路抑制剂SB203580(10μmol/L)预处理30 min,再加入AngⅡ处理不同时间。通过Western blotting检测细胞膜上NOX亚基p47Phox蛋白、细胞总PI3K蛋白和磷酸化的Akt、P38MAPK蛋白的表达;采用RT-PCR法检测UA对AngⅡ诱导的Ⅰ型胶原表达,CCK-8比色法检测HSC-T6的增殖率。结果用AngⅡ处理15 min后,细胞膜上p47phox蛋白的表达明显高于对照组(P<0.05);分别给予UA及DPI进行干预后,细胞膜上p47phox蛋白的表达较AngⅡ组明显降低(P<0.05)。用AngⅡ处理30 min后,PI3K、p-Akt的蛋白表达明显高于对照组(P<0.05);而分别给予UA、LY294002、DPI进行干预后,PI3K、p-Akt蛋白的表达较AngⅡ组均降低(P<0.05)。用AngⅡ处理30 min后,p-P38MAPK蛋白表达明显高于对照组(P<0.05);分别给予UA、SB203580、DPI干预后,p-P38MAPK蛋白的表达较AngⅡ组明显降低(P<0.05)。AngⅡ处理12 h后,Ⅰ型胶原的mRNA的表达明显高于对照组(P<0.05);分别给予UA、DPI、SB203580、LY294002干预后,Ⅰ型胶原的mRNA表达较AngⅡ组均明显降低(P<0.05)。AngⅡ刺激12、24、48 h后,细胞增殖率明显升高;分别给予UA、DPI、SB203580、LY294002干预后,细胞增殖率均低于AngⅡ组(P<0.05)。结论 UA能够通过抑制NOX活化阻断AngⅡ在肝星状细胞内的信号转导,从而抑制肝星状细胞的增殖及Ⅰ型胶原基因的表达。

Objective To analyze the effects of ursolic acid （UA） on the activation of NADPH oxidase （NOX） and the downstream signaling pathways of PI3K/Akt and P38MAPK of hepatic steUate cells （HSC） induced by angiotensin Ⅱ （Ang Ⅱ）. Methods Culture-activated HSC-T6 cells were divided into the control group （ received no medicines）, Ang Ⅱ group （ received Ang Ⅱ of 1μmol/L）, and 4 intervention groups, which were pretreated with UA of 50 μmol/L, NOX inhibitor DPI of 20 μmol/L, PI3K/Akt inhibitor LY294002 of 10 pmol/L, and P38MAPK inhibitor SB203580 of 10 μmol/L for 30 min and then treated with AngⅡ for different periods of time. Expressions of NOX snbunit p47^phox, total PI3K, phosphorylated p-Akt, and p-P38MAPK of the membrane were detected by the Western blotting. Collagen Ⅰ mRNA expressions induced by Ang Ⅱ were detected by the RT-PCR and the proliferation rate of HSC-T6 was measured by the CCK-8. Results After being treated with Ang Ⅱ for 15 min, the expression of p47^phox of membrane was significantly higher than that of the control group （P〈0.05）. After being intervened by DPI and UA, the expression of p47^phox of membrane was significantly lower than that of the Ang Ⅱ group （P〈0.05）. After being treated with Ang Ⅱfor 30 min, the expressions of PI3K and p-Akt were significantly higher than those of the control group （P〈0.05）. After being intervened by UA, DPI, and LY294002, the expressions of PI3K and p-Akt were lower than those of the Ang Ⅱ group （P〈0.05）. After being treated with Ang Ⅱ for 30 min, the expression of p-P38MAPK was significantly higher than that of the control group （ P 〈 0.05）. After being intervened by SB203580, UA, and DPI, the expression of p-P38MAPK was significantly lower than that of the Ang 1I group （P〈0.05）. After being treated with AngⅡ for 12 h, the mRNA expression of collagen I was significantly higher than that of the control group （P〈0.05）. After being intervened by SB203580, LY294002, UA, and DPI, the mRNA expression of collagen I was significantly lower than that of the AngⅡ group （P〈0.05）. After being treated with AngⅡ for 12, 24, and 48 h, the proliferation rate of HSC-T6 significantly increased. After being intervened by LY294002, SB203580, UA, and DPI, the proliferation rate HSC-T6 was lower than that of the AngⅡ group （P〈0.05）. Conclusion UA can block the signal transduction of Ang Ⅱ in HSC by inhibiting the activation of NOX subunit p47^phox, therefore inhibit the proliferation of HSC and the mRNA expression of Collagen I .