摘要
目的:探究敲低丙酮酸脱氢酶激酶4(PDK4)对D-半乳糖(D-gal)诱导的心肌细胞衰老的影响及对线粒体-内质网偶联结构(MAMs)的调节作用。方法:通过将si-PDK4重组质粒及阴性对照si-NC重组质粒转染至大鼠心肌细胞H9c2中以敲低PDK4表达,实时荧光定量聚合酶链式反应(qRT-PCR)和蛋白质免疫印迹(Western Blot)测定转染效果。将H9c2细胞分为对照组、D-gal组、si-NC+D-gal组、si-PDK4+D-gal组。对照组正常培养,D-gal组给予10 g/L D-gal诱导48 h,si-NC+D-gal组与si-PDK4+D-gal组分别转染si-NC、si-PDK4后再给予10 g/L D-gal诱导48 h,结束后收集各组H9c2细胞,细胞活性检测(CCK-8)法检测各组细胞活性,β-半乳糖苷酶活性(SAβ-gal)染色判定各组细胞衰老状态,2′,7′-二氯荧光黄双乙酸盐(DCFH-DA)荧光探针法检测各组细胞活性氧(ROS)水平,细胞荧光染色检测各组细胞MAMs,Western Blot测定各组细胞中线粒体融合蛋白-2(MFN2)蛋白表达水平,Fluo-3 AM荧光探针法检测各组细胞钙离子(Ca^(2+))水平。结果:转染si-PDK4的H9c2细胞中PDK4 mRNA相对表达量与蛋白相对表达量均低于转染si-NC的H9c2细胞与正常H9c2细胞(P<0.05),提示成功敲低PDK4表达。与对照组比较,D-gal组H9c2细胞存活率下降,SAβ-gal标记的衰老细胞比例增加,ROS水平升高,MAMs共定位减少,MFN2蛋白相对表达量下调,细胞内Ca^(2+)水平增加(P<0.05)。与D-gal组和si-NC+D-gal组比较,si-PDK4+D-gal组H9c2细胞存活率升高,SAβ-gal标记的衰老细胞比例减少,ROS水平下降,MAMs共定位增加,MFN2蛋白相对表达量上调,细胞内Ca^(2+)水平减少(P<0.05)。结论:敲低PDK4能够延缓D-gal诱导的心肌细胞衰老,其作用机制可能与增加MAMs形成以促进细胞内Ca^(2+)运转有关。
Objective:To explore the effect of knockdown pyruvate dehydrogenase kinase 4(PDK4)on D-galactose(D-gal)-induced cardiomyocyte senescence and its regulatory effect on mitochondria-associated endoplasmic reticulum membranes(MAMs).Methods:si-PDK4 recombinant plasmid and negative control si-NC recombinant plasmid were transfected into rat cardiomyocytes H9c2 to knock down the expression of PDK4.Real-time quantitative fluorescent polymerase chain reaction(qRT-PCR)and Western Blot were used to determine the transfection effect.H9c2 cells were divided into control group,D-gal group,si-NC+D-gal group,and si-PDK4+D-gal group.The control group was cultured normally,the D-gal group was given 10 g/L D-gal induction for 48 h,and the si-NC+D-gal group and the si-PDK4+D-gal group were transfected with si-NC and si-PDK4 respectively,and then given 10 g/L D-gal for 48 h.H9c2 cells were collected from each group after completion.Cell activity assay(CCK-8)was used to detect cell activity in each group.Cell senescence status was determined by β-galactosidase activity(SAβ-gal)staining.The levels of reactive oxygen species(ROS)were detected by 2′,7′-dichlorofluorescein yellow diacetate(DCFH-DA)fluorescence probe.Cell fluorescence staining was used to detect MAMs in each group.The expression level of mitochondrial fusion protein-2(MFN2)protein in each group was determined by Western Blot.Fluo-3 AM fluorescence probe was used to detect calcium ion(Ca^(2+))levels in each group.Results:The relative expression of PDK4 mRNA and protein in H9c2 cells transfected with si-PDK4 was significantly lower than that in H9c2 cells transfected with si-NC and normal H9c2 cells(P<0.05),indicating that PDK4 expression was successfully knocked down.Compared with the control group,the survival rate of H9c2 cells in D-gal group significantly decreased,the proportion of SAβ-gal labeled senile cells significantly increased,ROS level significantly increased,and MAMs co-localization significantly decreased,the relative expression of MFN2 protein significantly down-regulated,and the intracellular Ca^(2+)level significantly increased(P<0.05).Compared with D-gal group and si-NC+D-gal group,the survival rate of H9c2 cells in si-PDK4+D-gal group significantly increased,the proportion of senile cells labeled with SAβ-gal significantly decreased,and the ROS level significantly decreased,MAMs colocalization significantly increased,MFN2 protein relative expression significantly up-regulated,and intracellular Ca^(2+)level significantly decreased(P<0.05).Conclusion:Knockdown of PDK4 can delay D-gal induced cardiomyocyte senescence,and its mechanism may be related to increasing the formation of MAMs to promote intracellular Ca^(2+)operation.
作者
郑扬
黄玉冰
黄文霞
李海涛
ZHENG Yang;HUANG Yubing;HUANG Wenxia;LI Haitao(Hainan General Hospital,Haikou 570311,Hainan,China)
出处
《中西医结合心脑血管病杂志》
2025年第1期61-69,共9页
Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease
基金
海南省卫生健康行业科研项目(No.22A200204)。
关键词
心肌细胞
衰老
丙酮酸脱氢酶激酶4
线粒体-内质网偶联结构
D-半乳糖
cardiomyocyte
aging
pyruvate dehydrogenase kinase 4
mitochondria-associated endoplasmic reticulum membranes
D-galactose
