Mild hypothermia alleviates oxygen-glucose deprivation/reperfusion-induced apoptosis by inhibiting ROS generation, improving mitochondrial dysfunction and regulating DNA damage repair pathway in PC12

活性氧 细胞凋亡 线粒体通透性转换孔 活力测定 细胞色素c 线粒体 细胞生物学 生物 乳酸脱氢酶 分子生物学 化学 程序性细胞死亡 生物化学
作者
Tianen Zhou,Jierong Mo,Weigan Xu,Qiaohua Hu,Hongfeng Liu,Yue Fu,Jun Jiang
出处
期刊:Research Square - Research Square
标识
DOI:10.21203/rs.3.rs-2159170/v1
摘要

Abstract The brain ischemia/reperfusion (I/R) injury have a great impact on human life and property safety, as far as we know, mild hypothermia (MH) is an effective measure which reduces neuronal injury. However, the precise mechanism is not extremely clear. The purpose of this study was to explore whether mild therapeutic hypothermia can play a protective role in nerve cells dealing with brain I/R injury and its specific mechanism in vitro. A flow cytometer, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) release assay were performed to detect apoptotic rate of cells, cell viability and cytotoxicity respectively, while reactive oxygen species (ROS) assay kit, JC-1 fluorescent methods, immunofluorescence, western blot were used to explore ROS, mitochondrial transmembrane potential (Δψm), mitochondrial permeability transition pore (MPTP), expression of proteins respectively. The result indicated that the activity was decreased, while the cytotoxicity and apoptosis rate were increased after treating with OGD/R in PC12, however, MH could antagonize this phenomenon. Strangely, treating with OGD/R increased the release of ROS and the transfer of Cytochrome C (Cyt-C) from mitochondria to cytoplasm, besides, it also upregulated the expression of γH2AX, Bax and Clv-caspase3 but downregulated the expression of PCNA, Rad51, Bcl-2 and inhibited the function of mitochondria in PC12, the opposite trend was observed after MH treatment. Therefore, our results suggest that MH alleviates PC12 against oxygen-glucose deprivation/ reoxygenation-induced injury with the mechanism of inhibiting cell apoptosis by reducing ROS production, improving mitochondrial function, reducing DNA damage, and enhancing DNA repair.
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