线粒体转录因子A和细胞色素c氧化酶途径对肿瘤坏死因子受体相关蛋白1调节大鼠缺氧心肌细胞能量生成的作用及机制

Effects and mechanism of mitochondrial transcription factor A and cytochrome c oxidase pathway in the energy production of hypoxic cardiomyocytes of rats regulated by tumor necrosis factor receptor associated protein 1

目的探讨线粒体转录因子A(TFAM)和细胞色素c氧化酶(COX)途径在肿瘤坏死因子受体相关蛋白1(TRAP1)调节大鼠缺氧心肌细胞能量生成中的作用及机制。方法取135只1~3 d龄SD大鼠乳鼠,分离培养心肌细胞。(1)取细胞,按随机数字表法(分组方法下同)分为常氧空白对照组、缺氧空白对照组、缺氧+TRAP1过表达对照组、缺氧+TRAP1过表达组,每组1瓶。常氧空白对照组细胞常规培养;缺氧空白对照组细胞常规培养后缺氧6 h;缺氧+TRAP1过表达对照组、缺氧+TRAP1过表达组细胞常规培养后,分别加入TRAP1过表达空病毒载体、TRAP1过表达腺病毒载体病毒悬液转染48 h后缺氧6 h。蛋白质印迹法检测各组细胞TFAM蛋白表达。(2)取细胞,分为常氧空白对照组、缺氧空白对照组、缺氧+TRAP1过表达对照组、缺氧+TRAP1过表达组、缺氧+TRAP1过表达+TFAM干扰对照组、缺氧+TRAP1过表达+TFAM干扰组,每组1孔,前4组细胞处理同实验(1)对应组别。缺氧+TRAP1过表达+TFAM干扰对照组、缺氧+TRAP1过表达+TFAM干扰组细胞分别加入TFAM干扰空病毒载体、TFAM干扰腺病毒载体病毒悬液转染48 h,其他处理同缺氧+TRAP1过表达组。采用ATP检测试剂盒及酶标仪检测细胞内ATP含量,采用COX活性检测试剂盒及酶标仪检测细胞内COX活性。(3)取细胞,分为常氧空白对照组、常氧+叠氮化钠组、缺氧空白对照组、缺氧+叠氮化钠组,每组1孔,常氧空白对照组和缺氧空白对照组细胞处理同实验(1)对应组别。常氧+叠氮化钠组细胞实验前30 min加入32 nmol叠氮化钠,缺氧+叠氮化钠组细胞缺氧前30 min加入32 nmol叠氮化钠后缺氧6 h,同前检测ATP含量。以上实验均重复3次。对数据行单因素方差分析、LSD检验。结果(1)与常氧空白对照组比较,缺氧空白对照组细胞TFAM蛋白表达量明显下降(P<0.01)。与缺氧空白对照组和缺氧+TRAP1过表达对照组比较,缺氧+TRAP1过表达组细胞TFAM蛋白表达量明显升高(P<0.01)。(2)与常氧空白对照组(0.552±0.041、1.99±0.15)比较,缺氧空白对照组细胞COX活性(0.270±0.044)和ATP含量(1.09±0.11)均明显降低(P<0.01)。与缺氧空白对照组和缺氧+TRAP1过表达对照组(0.269±0.042、1.17±0.12)比较,缺氧+TRAP1过表达组和缺氧+TRAP1过表达+TFAM干扰对照组细胞COX活性(0.412±0.032、0.404±0.016)和ATP含量(1.75±0.06、1.69±0.07)均明显升高(P<0.01)。与缺氧+TRAP1过表达组和缺氧+TRAP1过表达+TFAM干扰对照组比较,缺氧+TRAP1过表达+TFAM干扰组细胞COX活性(0.261±0.036)和ATP含量(1.23±0.07)均明显降低(P<0.01)。(3)与常氧空白对照组比较,常氧+叠氮化钠组和缺氧空白对照组细胞ATP含量明显降低(P<0.01)。与缺氧空白对照组比较,缺氧+叠氮化钠组细胞ATP含量明显降低(P<0.01)。结论TRAP1能够通过上调TFAM表达来提高COX活性,最终减轻缺氧导致的大鼠心肌细胞能量生成受损。

Objective To investigate the effects and mechanism of mitochondrial transcription factor A(TFAM)and cytochrome c oxidase(COX)pathway in the energy production of hypoxic cardiomyocytes of rats regulated by tumor necrosis factor receptor associated protein 1(TRAP1).Methods The cardiomyocytes were isolated from 135 neonatal Sprague-Dawley rats(aged 1-3 d)and cultured for the following experiments.(1)Cells were collected and divided into normoxia blank control(NBC)group,hypoxia blank control(HBC)group,hypoxia+TRAP1 over-expression control(HTOC)group,and hypoxia+TRAP1 over-expression(HTO)group according to the random number table(the same grouping method below),with 1 bottle in each group.Cells in NBC group were cultured routinely,cells in HBC group were cultured in hypoxic condition for 6 hours after routine culture,cells in HTOC and HTO groups were respectively added with TRAP1 over-expression empty virus vector and TRAP1 over-expression adenovirus vector virus suspension for transfection for 48 hours after routine culture and then cultured in hypoxic condition for 6 hours.The protein expression of TFAM of cells in each group was detected by Western blotting.(2)Cells were collected and divided into NBC,HBC,HTOC,HTO,HTO+TFAM interference control(HTOTIC),and HTO+TFAM interference(HTOTI)groups,with 1 well in each group.Cells in the former 4 groups were dealt with the same methods as the corresponding groups in experiment(1).Cells in HTOTIC and HTOTI groups were respectively added with TFAM interference empty virus vector and TFAM interference adenovirus vector virus suspension for transfection for 48 hours,and the other processing methods were the same as those in HTO group.The content of ATP of cells in each group was determined by ATP determination kit and microplate reader,and the COX activity of cells in each group was determined by COX activity assay kit and microplate reader.(3)Cells were collected and divided into NBC group,normoxia+sodium azide(NSA)group,HBC group,and hypoxia+sodium azide(HSA)group,with 1 well in each group.Cells in NBC and HBC groups were respectively dealt with the same methods as the corresponding groups in experiment(1).Cells in NSA and HSA groups were respectively added with 32 nmol sodium azide at 30 min before experiment or hypoxia,and then cells in HSA group were cultured in hypoxic condition for 6 hours.The content of ATP was determined by the same method as above.The above three experiments were repeated for three times.Data were statistically analyzed with one-way analysis of variance and least significant difference test.Results(1)Compared with that in NBC group,the protein expression of TFAM of cells in HBC group was significantly decreased(P<0.01).Compared with that in HBC group or HTOC group,the protein expression of TFAM of cells in HTO group was significantly increased(P<0.01).(2)Compared with 0.552±0.041 and 1.99±0.15 in NBC group,the COX activity(0.270±0.044)and ATP content(1.09±0.11)of cells in HBC group were significantly decreased(P<0.01).Compared with 0.269±0.042 and 1.17±0.12 in HBC group and those in HTOC group,the COX activity(0.412±0.032 and 0.404±0.016)and ATP content(1.75±0.06 and 1.69±0.07)of cells in HTO and HTOTIC groups were significantly increased(P<0.01).Compared with those in HTO and HTOTIC groups,the COX activity(0.261±0.036)and ATP content(1.23±0.07)of cells in HTOTI group were significantly decreased(P<0.01).(3)Compared with that in NBC group,the ATP content of cells in NSA and NBC groups was significantly decreased(P<0.01).Compared with that in HBC group,the ATP content of cells in HSA group was significantly decreased(P<0.01).Conclusions TRAP1 can increase the COX activity of cardiomyocytes by raising the expression of TFAM,and finally alleviate the impairment in energy production of cardiomyocytes caused by hypoxia.