TDP-43对氧糖剥夺诱导的小鼠HL-1心房肌细胞凋亡的影响及其机制
Effect and mechanism of TDP-43 on oxygen-glucose deprivation-induced apoptosis in mouse HL-1 atrial myocytes cells
- 解放军医学杂志 2023年48卷第11期 页码：1305-1313
- 作者机构：
1.西南交通大学医学院，四川成都 610031
2.解放军西部战区总医院心内科，四川成都 610083
4.南方医科大学第五附属医院泌尿外科，广东广州 510900
3.解放军西部战区总医院卫勤训练中心，四川成都 610083
5.四川大学国家生物医学工程研究中心，四川成都 610064
- 作者简介：
岳娇，硕士研究生，主要从事心肌损伤与心肌重构方面的研究
裴海峰，E-mail：web2010@foxmail.com
- 基金信息：国家自然科学基金(81970241);西部战区总医院院管重点项目(2021-XZYG-A03)
DOI：10.11855/j.issn.0577-7402.2656.2023.0911
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岳娇, 龚美婷, 徐伍, 等. TDP-43对氧糖剥夺诱导的小鼠HL-1心房肌细胞凋亡的影响及其机制[J]. 解放军医学杂志, 2023, 48(11): 1305-1313.
Yue Jiao,Gong Mei-Ting,Xu Wu,et al.Effect and mechanism of TDP-43 on oxygen-glucose deprivation-induced apoptosis in mouse HL-1 atrial myocytes cells[J].Medical Journal of Chinese People′s Liberation Army,2023,48(11):1305-1313.
岳娇, 龚美婷, 徐伍, 等. TDP-43对氧糖剥夺诱导的小鼠HL-1心房肌细胞凋亡的影响及其机制[J]. 解放军医学杂志, 2023, 48(11): 1305-1313. DOI： 10.11855/j.issn.0577-7402.2656.2023.0911.
Yue Jiao,Gong Mei-Ting,Xu Wu,et al.Effect and mechanism of TDP-43 on oxygen-glucose deprivation-induced apoptosis in mouse HL-1 atrial myocytes cells[J].Medical Journal of Chinese People′s Liberation Army,2023,48(11):1305-1313. DOI： 10.11855/j.issn.0577-7402.2656.2023.0911.
摘要
目的,2,探讨TAR DNA结合蛋白43(TDP-43)对氧糖剥夺(OGD)诱导小鼠HL-1心房肌细胞凋亡的影响及其机制。,方法,2,将体外培养的小鼠HL-1心房肌细胞分为：(1)对照组及不同OGD处理时间(2、4、8、16 h)组，采用CCK-8法检测细胞活力，Western blotting检测TDP-43蛋白表达水平，以此确定OGD诱导时间点用于后续研究；(2)对照组与OGD组，采用流式细胞术检测细胞凋亡率，JC-1染色法检测线粒体膜电位，化学发光法检测三磷酸腺苷(ATP)相对含量，微板法检测丙二醛(MDA)含量，WST-1法检测超氧化物歧化酶(SOD)含量。将转染慢病毒的小鼠HL-1心房肌细胞分为：(1)阴性对照慢病毒干预组(NC-shRNA)、TDP-43敲低慢病毒干预组(TDP-43-shRNA1、TDP-43-shRNA2、TDP-43-shRNA3)，Western blotting检测TDP-43蛋白表达水平，选择慢病毒敲低效率最高的TDP-43-shRNA用于后续试验；(2)NC-shRNA组、TDP-43-shRNA组、OGD+NC-shRNA组、OGD+TDP-43-shRNA组，在常氧条件和OGD条件下，采用流式细胞术检测细胞凋亡率，MitoTracker染色法检测线粒体形态，JC-1染色法检测线粒体膜电位，化学发光法检测ATP含量，流式细胞术检测活性氧(ROS)荧光强度，微板法检测MDA含量，WST-1法检测SOD含量。,结果,2,CCK-8法检测结果显示，随OGD时间的延长，小鼠HL-1心房肌细胞的活力逐渐下降；Western blotting检测结果显示，TDP-43蛋白表达水平逐渐升高，两者均呈现较强的时间依赖性。与对照组比较，在OGD 16 h时小鼠HL-1心房肌细胞活力最低(,P,<,0.05)、TDP-43蛋白表达量最高(,P,<,0.05)，据此后续实验选择OGD 16 h为诱导时间点。与对照组比较，OGD组细胞凋亡率、线粒体膜电位荧光强度比值、MDA含量升高，ATP相对含量、SOD含量降低，差异均有统计学意义(,P,<,0.05)。Western blotting检测结果显示，与NC-shRNA组比较，TDP-43-shRNA2组TDP-43蛋白表达水平降低最为明显(,P,<,0.05)，敲低效率最高，因此选择TDP-43-shRNA2进行后续实验。流式细胞术检测结果显示，在常氧条件下，与NC-shRNA组比较，TDP-43-shRNA组细胞凋亡率无明显变化(,P,>,0.05)；在OGD条件下，与OGD+NC-shRNA组比较，OGD+TDP-43-shRNA组细胞凋亡率降低(,P,<,0.05)。MitoTracker染色结果显示，与NC-shRNA组比较，TDP-43-shRNA组线粒体形态完整，无明显变化；OGD+NC-shRNA组线粒体数量增多，多数为碎片状，分布散乱；与OGD+NC-shRNA组比较，OGD+TDP-43-shRNA组线粒体形态有所恢复。在常氧条件下，与NC-shRNA组比较，TDP-43-shRNA组线粒体膜电位荧光强度比值、ATP相对含量、ROS荧光强度、MDA含量、SOD含量均无明显变化(,P,>,0.05)；但在 OGD条件下，与OGD+NC-shRNA组比较，OGD+TDP-43-shRNA组细胞线粒体膜电位荧光强度比值、ROS荧光强度、MDA含量降低，ATP相对含量、SOD含量升高，差异均有统计学意义(,P,<,0.05)。,结论,2,TDP-43通过调控心肌细胞凋亡加重OGD诱导的线粒体功能障碍，因此，敲低TDP-43的表达有望成为缺血性心肌病的潜在治疗策略。
Abstract
Objective,2,To investigate the effects and mechanisms of TAR DNA-binding protein 43 (TDP-43) on oxygen-glucose deprivation (OGD)-induced apoptosis in mouse atrial myocytes (HL-1 cells).,Methods,2,The ,in vitro, cultured mouse atrial myocytes (HL-1 cells) were divided into: (1) control group and groups with different OGD treatment times (2, 4, 8, 16 h), and cell viability was detected by CCK-8 assay, and TDP-43 protein expression level was detected by Western blotting, which was used to determine the time point of OGD induction for the subsequent study; (2) control and OGD groups, flow cytometry was used to detect apoptosis, JC-1 staining to detect mitochondrial membrane potential, chemiluminescence to detect adenosine triphosphate (ATP) relative content, microplate method to detect malondialdehyde (MDA) content, and WST-1 method to detect superoxide dismutase (SOD) content. Mouse atrial myocytes (HL-1 cells) transfected with lentivirus were divided into: (1) negative control lentiviral intervention group (NC-shRNA), TDP-43 knockdown lentiviral intervention group (TDP-43-shRNA1, TDP-43-shRNA2, TDP-43-shRNA3), and Western blotting was used to detect the TDP-43 protein expression level, and the group with the highest lentiviral knockdown efficiency was selected as the TDP-43-shRNA for subsequent experiments; (2) NC-shRNA group, TDP-43-shRNA group, OGD+NC-shRNA group, OGD+TDP-43-shRNA group, under normoxic and OGD conditions, flow cytometry was used to detect the apoptosis rate, MitoTracker staining to detect mitochondrial morphology, JC-1 staining to detect mitochondrial membrane potential, chemiluminescence to detect the relative content of ATP, flow cytometry to detect the fluorescence intensity of reactive oxygen species (ROS), microplate to detect the content of MDA, and WST-1 to detect the content of SOD.,Results,2,CCK-8 method showed that, with the prolongation of OGD time, the viability of mouse atrial myocytes (HL-1 cells) gradually decreased; Western blotting assay showed that the expression level of TDP-43 protein gradually increased, and both of them showed a strong time-dependence. Compared with control group, mouse atrial myocytes (HL-1 cells) viability was the lowest (,P,<,0.05) and TDP-43 protein expression was the highest (,P,<,0.05) at 16 h of OGD, accordingly, OGD 16 h was chosen as the induction time point for subsequent experiments. Compared with control group, the apoptosis rate, the fluorescence intensity ratio of mitochondrial membrane potential and the content of MDA increased, the relative content of ATP and SOD decreased in OGD group, and the differences were all statistically significant (,P,<,0.05). Western blotting detection showed that compared with NC-shRNA group, the TDP-43-shRNA2 group had the most obvious reduction in TDP-43 protein expression level (,P,<,0.05) and the highest knockdown efficiency, so the TDP-43-shRNA2 group was selected for subsequent experiments. The results of flow cytometry showed that under normoxic conditions, there was no significant change in the apoptosis rate in TDP-43-shRNA group compared with NC-shRNA group (,P,>,0.05); and under OGD conditions, the apoptosis rate in OGD+TDP-43-shRNA group reduced when compared with OGD+NC-shRNA group (,P,<,0.05). MitoTracker staining results showed that the mitochondrial morphology of TDP-43-shRNA group was intact without significant changes compared with NC-shRNA group; the mitochondria of OGD+NC-shRNA group increased in number, most of which were fragmented and scattered in distribution; compared with OGD+NC-shRNA group, the mitochondrial morphology of OGD+TDP-43-shRNA group was restored. Under normoxic conditions, there were no significant changes in mitochondrial membrane potential, relative ATP content, ROS fluorescence intensity, MDA content, and SOD content in TDP-43-shRNA group compared with NC-shRNA group (,P,>,0.05); however, under OGD conditions, the ratio of fluorescence intensity of mitochondrial membrane potential of cells the fluorescence intensity of ROS, and the content of MDA decreased, and the relative content of ATP and the content of SOD increased in OGD+TDP-43-shRNA group compared with that of OGD+NC-shRNA group, and all of these differences was statistically significant (,P,<,0.05).,Conclusion,2,TDP-43 exacerbates OGD-induced mitochondrial dysfunction by regulating cardiomyocyte apoptosis; therefore, knockdown of TDP-43 expression is expected to be a potential therapeutic strategy for ischemic cardiomyopathy.
关键词
TAR DNA结合蛋白43氧糖剥夺细胞凋亡氧化应激
Keywords
TAR DNA-binding protein 43oxygen-glucose deprivationapoptosisoxidative stress
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