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-论文.pdf

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1、肝脏2014年4月第19卷第4期·261··论著·丹参酚酸B盐抑制大鼠肝星状细胞中内皮素一1激活的RhoA／ROCK11信号通路徐虹平键卢超周扬徐列明【摘要】目的探讨丹参酚酸B盐(SalB)对大鼠肝星状细胞(HSC)中内皮素一1(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。G

2、ST下拉实验检测RhoA活性。结果在大鼠HSC中，ET一1刺激后，RhoA和ROCKII活性显著增加，ROCKI活性无明显变化，MYPT1Thr和Thr。磷酸化水平均显著增加。ET一1刺激1min和10min时，RhoA活性分别是基础状态下的1．95倍(P

3、化达到高峰，是基础状态下的5．17倍。Thr”磷酸化亦在ET-1刺激I5min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ET1刺激前给予10mol／LSalB预处理，则使ET一1诱导的RhoA和ROCKII活性分别下降66．84和76．79％，ET1诱导的MYPT1Thr磷酸化下降8O．09，对Thr“磷酸化水平无影响。结论SalB能显著抑制大鼠HSC中ET一1诱导的RhoA和ROCKII活化，抑制MYPT1Thr磷酸化。【关键词】肝星状细胞；内皮素一1；丹参酚酸B；RhoA／ROCK信号通路Salvia

4、nolieacidBinhibitsET-1。activatedRhoA／ROCKIIsignalingpathwayinratHSCXUHong。PINGJian．LUChao，ZHOUYang，XULieruing，ShuguangHospitalAffiliatedtoShanghaiUniversity0／TraditionalChineseMedicine，InstituteofLiverDiseases，ShanghaiUniversityofTraditionalChineseMedicine，Shanghai20120

5、3，ChinaC0rrsogauthor：XULie—ruing，Email：xuliemingr@shutcm．edu．cn[Abstract]ObjectiveToinvestigatetheeffectofsalvianolicacidB(SalB)onET一1一activatedRhoA／ROCKsignalingpathwayinrathepaticstellatecells(HSC)．MethodsHSCfromSprague—DawleyratswereisolatedbyperfusionwithpronaseEins

6、ituanddensity—gradientcentrifugationwithNycodenz．Myosinphosphotasetargetingsubunit一1(MYPT1)phosphorylationwasdeterminedbywesternblot．ThecontentofactiveGTP—boundRhoAwasdeterminedbyRhotekinRBDbindingassay．FollowedbyimmunoprecipitationofROCKwithspecificantibody，phosphory1a

7、tionwasperformedinvitro．PhosphorylatedThr6。一MYPT1(654—880)representedtheactivationofROCK．ResultsStimulatedratHSCbyET一1，theactivitiesofRhoAandROCKI1wereincreasedsignificantly．ROCKIactivitywasnotvaried，ThandThr”phosphorylationofMYPT1wereincreasedsignificantly．AfterET一1sti

8、mulatedfor1min(P<0．05)and10rain(P<0．01)，RhoAactivitywasincreaseduptonearly1．95一foldand5．84一foldthanthatofcontr