蛋白酶活化受体1调控Mcl-1和Bax表达介导热打击致人脐静脉内皮细胞早期凋亡的研究

Early apoptosis of human umbilical vein endothelial cells resulting from heat stress mediated by proteaseactivated receptor 1 through regulating the expressions of Mcl-1 and Bax

目的:观察热打击人脐静脉内皮细胞(HUVECs)后蛋白酶活化受体1(PAR1)对凋亡相关蛋白Mcl-1、Bax水平表达调控及细胞早期凋亡的影响,明确PAR1在热打击致HUVECs凋亡中的作用。方法 :采用PAR1抑制剂SCH79797(SCH)、PAR1激动剂TFLLR-NH2(TF)、PAR1si RNA、PAR1腺病毒过表达预处理HUVECs,给予42℃热打击2 h,后于8 h提取各处理组总蛋白,western blot检测Mcl-1、Bax、Caspase 3及CleavedCaspase 3蛋白表达,流式细胞计数仪检测各处理组细胞早期凋亡。结果 :与正常对照组比较,热打击组Mcl-1、Bax、Caspase 3表达增加及Cleaved-Caspase 3活化程度增加(P<0.05);与热打击组比较,PAR1抑制剂或PAR1si RNA联合热打击组Mcl-1表达增加(P<0.05),Bax减少(P<0.05),Cleaved-Caspase 3活化程度降低,细胞早期凋亡数目减少(P<0.05,P<0.01),PAR1激动剂或PAR1腺病毒联合热打击组Bax表达增加(P<0.05),Mcl-1表达减少(P<0.05),Cleaved-Caspase 3活化程度增加,且细胞早期凋亡数目增加(P<0.05或P<0.01)。结论 :热打击HUVECs可发生细胞凋亡,Mcl-1、Bax蛋白表达增加;PAR1通过调控Mcl-1及Bax的表达最终起到促凋亡作用。

Objective： To observe the effect of proteinase-activated receptor 1 （PAR1） on regulation of Mcl-1 andBax expression and early apoptosis of human umbilical vein endothelial cells （HUVECs） after heat stress for makingclear the role of PAR1 in the apoptosis of HUVECs induced by heat stress. Methods： HUVECs were pretreatedwith PAR1 inhibitor SCH, PAR1 agonist TFLLR-NH2（TF）, PRA1siRNA and PAR1 adenovirus respectively andthen received heat stress（42℃）for 2 hours. After 8 hours, the total protein was extracted , the protein expressionsof Mcl-1, Bax, caspase 3 and cleaved-caspase 3 were detected, and early apoptosis of the cells was determined byflow cytometry for all the groups. Results： Compared to the normal control group, expressions of Mcl-1, Bax andcaspase 3 increased after heat stress, with activation of cleaved-caspase 3 （P〈0.05）. Compard to the heat stress group,expression of Mcl-1 was up-regulated and Bax down-regulated in the groups of PAR1 inhibitor or PAR1siRNAcombined with heat stress （P〈0.05）, while the activation of cleaved-caspase 3 weakened and the number of earlyapoptotic cells reduced （P〈0.05 or P〈0.01）. Otherwise, increasing PAR1 expression and PAR1 adenovirus-pretreatedcombined with heat stress lead to the increase of Bax expression （P〈0.05）, decrease of Mcl-1 expression （P〈0.05）,activation of cleaved-caspase-3 and increase in number of apoptotic cells （P〈0.05 or P〈0.01）. Conclusions： Heatstress induces apoptosis of HUVECs, at the same time the expression of apoptosis-related protein Mcl-1 and Baxcould increase. PAR1 plays an important role in promoting apoptosis by regulating expressions of Mcl-1 and Bax. Study on the mechanisms of PAR1 mediating endothelial cell apoptosis contributes to further revealing of thepathological process of endothelial cells in heat stress and also provides a theoretical basis for clinical prevention andtreatment of heatstroke.