Fas/caspase-8/3和ERK信号通路在Fas siRNA减轻心肌细胞缺氧/复氧损伤中的作用  

Roles of Fas/caspase-8/3 and ERK signaling pathways in Fas siRNA induced reduction of hypoxia/reoxygenation injury to cardiomyocytes

作　　者：潘永露 何淑芳[1] 韩正怡 窦梦云 杨婉[1] 程洁[1] 陈志武 张野[1] PAN Yong-lu;HE Shu-fang;HAN Zheng-yi;DOU Meng-yun;YANG Wan;CHENG Jie;CHEN Zhi-wu;ZHANG Ye(Dept of Anesthesiology,the Second Hospital of Anhui Medical University,Hefei 230601,China;Dept of Pharmacology,Anhui Medical University,Hefei 230032,China)

机构地区：[1]安徽医科大学第二附属医院麻醉科,安徽合肥230601 [2]安徽医科大学基础医学院药理学教研室,安徽合肥230032

出　　处：《中国药理学通报》2018年第8期1150-1157,共8页Chinese Pharmacological Bulletin

基　　金：安徽省科技厅重点项目(No 1301043030);安徽省高校省级自然科学研究重大项目(No KJ2014ZD16)

摘　　要：目的评价Fas/caspase-8/3和细胞外信号调节激酶(extracellular signaling-regulated kinase,ERK)信号通路在Fas siRNA减轻心肌细胞缺氧/复氧(hypoxia/reoxygenation,H/R)损伤中的作用。方法培养H9c2心肌细胞,随机分为4组(n=3):(1)正常对照组:H9c2细胞置于DMEM/F12细胞培养液中培养;(2)H/R组:将H9c2细胞进行5 h缺氧和1 h复氧处理;(3)Fas siRNA组(H/R+Fas-siRNA):H9c2细胞转染Fas siRNA 24 h后进行H/R损伤;(4)siRNA阴性对照组(H/R+siRNA-NC):H9c2细胞转染Fas siRNA-NC 24 h后进行H/R损伤。处理结束后,采用CCK-8法检测细胞活力;微量酶标法测定培养液中乳酸脱氢酶(LDH)的活性;AnnexinV-FITC/PI双染法检测细胞凋亡;荧光定量RT-PCR法检测细胞Fas mRNA表达水平;Western blot法检测细胞内Fas、caspase-8/3、ERK和GSK-3β蛋白表达。结果与对照组相比,H/R组细胞活力降低,LDH活性和细胞凋亡增加,Fas mRNA及蛋白水平升高,cleaved caspase-8/3、p-ERK、p-GSK-3β蛋白表达升高(P<0.05);与H/R组相比,H/R+FassiRNA组细胞活力明显提高,LDH活性和细胞凋亡率降低,Fas mRNA及蛋白表达水平降低,cleaved caspase-8/3蛋白表达降低,p-ERK、p-GSK-3β蛋白表达增高(P<0.05),而H/R+siRNA-NC组上述指标差异无统计学意义。结论 Fas/caspase-8/3和ERK信号通路在Fas siRNA减轻心肌细胞H/R损伤中发挥着重要作用。Aim To evaluate the roles of Fas/caspase-8/3 and extracellular signaling-regulated kinase（ ERK） signaling pathways in Fas siRNA induced reduction of hypoxia/reoxygenation（ H/R） injury to cardiomyocytes. Methods H9c2 cardiomyocytes were cultured in DME/F-12 culture medium supplemented with 10% fetal bovine serum and randomly divided into four groups（ n = 3 each） using a random number table： control group,H/R group,Fas siRNA group（ H/R ＋ Fas-siRNA） and siRNA control group（ H/R ＋siRNA-NC）. H9 c2 cardiomyocytes were cultured in normal culture atmosphere in control group; treatment with 5 h hypoxia and 1 h reoxygenation was carried out to H9 c2 cells to establish H/R injury in group H/R.In addition,the H9c2 cells in groups H/R ＋ Fas siRNA and H/R ＋ siRNA-NC were transfected with Fas siRNA and Fas siRNA-NC for 24 h before H/R injury,respectively. Cell viability was determined using CCK-8 method,lactate dehydrogenase（ LDH） activity in the culture medium was detected using chemical colorimetry,and cell apoptosis was assessed using AnnexinV-FITC/PI flow cytometry in each group at the end of treatment. Relative expression of Fas mRNA was detected by fluorescent quantitative RT-PCR,while the levels of Fas, caspase-8/3, ERK, GSK-3β protein were observed by Western blot. Results Compared with control group,cell viability was significantly repressed,and LDH activity and apoptotic rate as well as the expression of Fas mRNA,protein, and cleaved caspase-8/3 protein and p-ERK,p-GSK-3β protein all increased in group H/R（ P〈0. 05）,while Fas siRNA alleviated H/R injury when compared with group H/R,as evidenced by the significantly increased cell viability,and further proved by the suppressed LDH activity and apoptotic rate as well as the expression of Fas mRNA,protein and cleaved caspase-8/3 protein. In addition, p-ERK, p-GSK-3β protein expression increased in group H/R ＋ Fas siRNA（ P〈0. 05）.There was no difference in the parameters mentioned above in group H/R ＋ siRNA-NC. Conclusi

关 键 词：FAS caspase 细胞外信号调节激酶 心肌细胞 缺氧/复氧损伤 凋亡 

分 类 号：R-332[医药卫生] R322.11