CAJ | 학술논문

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10-5mol/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 MYPT1 Thr696磷酸化。
목적：탐토단삼분산B염（Sal B）대대서간성상세포（HSC）중내피소-1（ET-1）격활적RhoA/ROCK 신호통로적영향。방법채용간장원위관류매소화、Nycodenz밀도제도리심법분리대서 HSC。면역단백인적법검측기구단백린산매파아기1（MYPT1）린산화。특이성항체침정ROCK，진행체외린산화반응，이린산화저물Thr696-MYPT1（654-880）적함량반영ROCK활성。GST하랍실험검측RhoA활성。결과재대서HSC중，ET-1자격후，RhoA화ROCK II활성현저증가，ROCK I활성무명현변화，MYPT1 Thr696화Thr850린산화수평균현저증가。ET-1자격1 min화10 min시，RhoA활성분별시기출상태하적1．95배（P＜0．05）화5．84배（P＜0．01）。ET-1자격2．5 min화15 min시，ROCK II 활성분별시기출상태하적3．49배화4．83배，균P＜0．01，차이유통계학의의。ET-1자격2．5 min시，MYPT1 Thr696린산화수평시기출상태하적3．86배；자격15 min 시，Thr696린산화체도고봉，시기출상태하적5．17배。Thr850린산화역재 ET-1자격15 min체도고봉，시기출상태하적3．33배，균P＜0．01，차이유통계학의의。재ET-1자격전급여10-5mol/L Sal B예처리，칙사ET-1유도적RhoA화ROCK II활성분별하강66．84％화76．79％，ET-1유도적MYPT1 Thr696린산화하강80．09％，대Thr850린산화수평무영향。결론 Sal B 능현저억제대서 HSC 중 ET-1유도적 RhoA 화 ROCK II 활화，억제 MYPT1 Thr696린산화。
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. Thr696 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 Thr850 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 Thr696 in rat HSC.