低氧诱导因子1α调控蛋白酶激活受体1在低氧诱导心肌细胞凋亡中的作用及机制

Role and Mechanism of PAR1 in Hypoxia-Induced Cardiomyocyte Apoptosis Regulated by HIF-1α

目的探讨低氧条件下低氧诱导因子1α(HIF-1α)调控蛋白酶激活受体1(PAR1)在低氧诱导心肌细胞凋亡中的作用及机制。方法将大鼠心肌细胞系H9c2细胞培养在95%O2及5%CO2环境中作为常氧组,培养在1%O2、94%N2、5%CO2环境中作为低氧组,并作不同时间梯度;采用四氮唑比色(MTT)法和原位末端转移酶标记(TUNEL)试剂盒法分别检测常氧组和低氧组中H9c2细胞活性和细胞凋亡情况;采用实时荧光定量聚合酶链式反应(qRT-PCR)和蛋白质印迹(WB)法分别从mRNA和蛋白水平上检测HIF-1α和PAR1的表达。通过细胞转染siNC与PAR1 siRNA质粒24 h处理H9c2细胞,采用MTT法和TUN试剂盒检测细胞活性和细胞凋亡情况;通过qRT-PCR和WB法检测常氧组(A组),常氧+NC siRNA组(B组),低氧+NC siRNA组(C组)和低氧+PAR1 siRNA组(D组)H9c2细胞中Bcl-2与Bax的表达;在细胞转染HIF-1αsiRNA后再低氧处理H9c2细胞,采用RNA印迹(NB)法和WB法检测PAR1蛋白表达水平。结果与A组比较,在低氧环境下,H9c2细胞活性降低并发生凋亡,HIF-1α和PAR1稳定高表达;与直接低氧处理组比较,在H9c2细胞中敲除PAR1后再低氧处理细胞,可明显缓解因低氧诱导的心肌细胞凋亡,提高H9c2细胞活力,在H9c2细胞中敲除PAR1后会使Bcl-2表达上调、Bax表达下调;细胞转染HIF-1αsiRNA后再低氧处理H9c2细胞时,PAR1的表达下降,表明PAR1受HIF-1α的正调控。结论低氧条件下诱导H9c2心肌细胞的凋亡可能与低氧诱导因子HIF-1α正调控PAR1的表达有关,在H9c2细胞中敲除PAR1对处于低氧环境下心肌细胞的凋亡有阻断作用,可能源于上调Bcl-2的表达及降低Bax的表达。

Objective To investigate the role and mechanism of hypoxiainducible factor-1α(HIF-1α)regulation of protease activated receptors1(PAR1)in hypoxia-induced apoptosis of cardiomyocytes.Methods The rat myocardial cell line H9 c2 cells were cultured as normal oxygen(Normoxia)group at 95%O2 and 5%CO2,and as Hypoxia group at 1%O2,94%N2 and 5%CO2,with different time gradients.Methyl thiazolyl tetrazolium(MTT)assay and TdT-mediated-d UTP nick end labeling(TUNEL)in situ assay kit were used to detect the changes of H9 c2 cell activity and apoptosis in normoxic group and hypoxic group,respectively.The expressions of HIF-1αand thrombin receptor PAR1 were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and Western Blot(WB)respectively at mRNA and protein levels.H9 c2 cells were transfected with siNC and PAR1 siRNA plasmid for 24 h,and changes in cell activity and apoptosis were detected by MTT assay and TUNEL in situ assay kit.The expressions of Bcl-2 and Bax in H9c2 cells of Normox group(group A),Normox+siNC group(group B),Hypoxia+siNC group(group C)and Hypoxia+PAR1 siRNA group(group D)were detected by qRT-PCR and WB.Finally,H9 c2 cells were treated with hypoxia after transfection of HIF-1αsiRNA,and PAR1 expression levels were analyzed by NB and WB.Results Compared with group A,H9c2 cell activity decreased,and apoptosis occurred in the hypoxic environment.Hypoxic inducer HIF-1αand thrombin receptor PAR1 were stable and highly expressed.In H9 c2 cells,thrombin receptor PAR1 was knocked out and hypoxia-treated cells were then hypoxia-treated.Compared with the direct hypoxia-treated H9 c2 cells,MTT and TUNEL in situ assay showed that hypoxia-induced apoptosis of cardiomyocytes was alleviated.Real-time fluorescence quantitative PCR and Western Blot analysis showed that after knocking out the thrombin receptor PAR1 in H9 c2 cells,the expression of Bcl-2 was up-regulated,the expression of Bax was down-regulated.Northern Blot and Western Blot analysis showed that the expression of thrombin receptor PAR1 decreased in H9 c2 cells after transfection with HIF-1αsiRNA,indicating that PAR1 was positively regulated by HIF-1α.Conclusion Induction of apoptosis of H9c2 cardiomyocytes under hypoxic conditions may be related to the regulation of expression of the thrombin receptor PAR1 by hypoxic inducible factor HIF-1αpositive in H9c2 cells.The knockdown of the thrombin receptor PAR1 in H9C2 cells may block the apoptosis of cardiomyocytes under hypoxic conditions,which may be due to the up-regulation of Bcl-2 expression and the decreased expression of Bax.