丹参酚酸B盐抑制大鼠肝星状细胞中内皮素-1激活的RhoA/ROCK Ⅱ信号通路

Salvianolic acid B inhibits ET-1-activated RhoA/ROCK Ⅱ signaling pathway in rat HSC

目的探讨丹参酚酸B盐(Sal B)对大鼠肝星状细胞(HSC)中内皮素-1(ET-1)激活的RhoA/ROCK信号通路的影响。方法采用肝脏原位灌流酶消化、Nycodenz密度梯度离心法分离大鼠HSC。免疫蛋白印迹法检测肌球蛋白磷酸酶靶亚基1(MYPT1)磷酸化。特异性抗体沉淀ROCK,进行体外磷酸化反应,以磷酸化底物Thr696-MYPT1(654-880)的含量反映ROCK活性。GST下拉实验检测RhoA活性。结果在大鼠HSC中,ET-1刺激后,RhoA和ROCK Ⅱ活性显著增加,ROCK I活性无明显变化,MYPT1 Thr696和Thr850磷酸化水平均显著增加。ET-1刺激1 min和10 min时,RhoA活性分别是基础状态下的1.95倍(P<0.05)和5.84倍(P<0.01)。ET-1刺激2.5 min和15 min时,ROCK Ⅱ活性分别是基础状态下的3.49倍和4.83倍,均P<0.01,差异有统计学意义。ET-1刺激2.5 min时,MYPT1 Thr696磷酸化水平是基础状态下的3.86倍;刺激15 min时,Thr696磷酸化达到高峰,是基础状态下的5.17倍。Thr850磷酸化亦在ET-1刺激15 min达到高峰,是基础状态下的3.33倍,均P<0.01,差异有统计学意义。在ET-1刺激前给予10-5 mol/L Sal B预处理,则使ET-1诱导的RhoA和ROCK Ⅱ活性分别下降66.84%和76.79%,ET-1诱导的MYPT1 Thr696磷酸化下降80.09%,对Thr850磷酸化水平无影响。结论 Sal B能显著抑制大鼠HSC中ET-1诱导的RhoA和ROCK Ⅱ活化,抑制MYPT1Thr696磷酸化。

Objective To investigate the effect of salvianolic acid B （Sal B）on ET-1-activated RhoA/ROCK signaling pathway in rat hepatic stellate cells （HSC）. Methods HSC from Sprague-Dawley rats were isolated by perfusion with pronase E in situ and density-gradient centrifugation with Nycodenz. Myosin phosphotase targeting subunit-1 （MYPT1 ） phosphorylation was determined by western blot. The content of active GTP-bound RhoA was determined by Rhotekin RBD binding assay. Followed by immunoprecipitation of ROCK with specific antibody,phosphorylation was performed in vitro. Phosphorylated Thr696-MYPT1 （654-880）represented the activation of ROCK.Results Stimulated rat HSC by ET-1 ,the activities of RhoA and ROCK II were increased significantly, ROCK I activity was not varied, Thr696 and Thr850 phosphorylation of MYPT1 were increased significantly. After ET-1 stimulated for 1 min （P〈0.05 ）and 10 min （P〈0.01）,RhoA activity was increased up to nearly 1.95-fold and 5.84-fold than that of control,respectively. After ET-1 stimulated for 2.5 min and 15 min,ROCK II activity was increased significantly up to 3.49-fold and 4.83-fold than that of control ,respectively. Thr^696 phosphorylation was also increased up to 3 .86-fold than that of control when stimulated by ET-1 for 2.5 min,and reaching a maximal level of 5.17-fold for 15 min. Peak Thr^850 phosphorylation was observed after ET-1 stimulated for 15 min,reaching 3.3-fold compared to control. ET-1-induced RhoA and ROCK II activation were decreased by 66.84% and 76.79% when pre-treated with 10-5 mol/L Sal B,respectively,and Thr696 phosphorylation of MYPT1 was inhibited by 80.09% ,but Thr850 phosphorylation was not varied. Conclusion Sal B effectively inhibits ET-1-induced RhoA/ROCK II activation and MYPT1 phosphorylation at Thr^696 in rat HSC.