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

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Abstract

The brain ischemia/reperfusion (I/R) injury has a great impact on human life and property safety. As far as we know, mild hypothermia (MH) is an effective measure to reduce neuronal injury after I/R. However, the precise mechanism is not extremely clear. The purpose of this study was to investigate whether mild therapeutic hypothermia can play a protective role in nerve cells dealing with brain I/R injury and explore its specific mechanism in vitro. A flow cytometer, cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) release assay were performed to detect apoptotic rate of cells, cell viability and cytotoxicity, respectively, reactive oxygen species (ROS) assay kit, JC-1 fluorescent methods, immunofluorescence and western blot were used to explore ROS, mitochondrial transmembrane potential (Δψm), mitochondrial permeability transition pore (MPTP) and protein expression, respectively. The result indicated that the cell activity was decreased, while the cytotoxicity and apoptosis rate were increased after treating with oxygen–glucose deprivation/reperfusion (OGD/R) in PC12 cells. However, MH could antagonize this phenomenon. Interestingly, treating with OGD/R increased the release of ROS and the transfer of Cytochrome C (Cyt-C) from mitochondria to cytoplasm. In addition, it up-regulated the expression of γH2AX, Bax and Clv-caspase3, down-regulated the expression of PCNA, Rad51 and Bcl-2, and inhibited the function of mitochondria in PC12 cells. Excitingly, the opposite trend was observed after MH treatment. Therefore, our results suggest that MH protects PC12 cells against OGD/R-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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Data availability

The data that support the findings of this study are available.

Abbreviations

CCK-8:

Cell counting Kit-8

Cyt-C:

Cytochrome C

I/R:

Ischemia/reperfusion

LDH:

Lactate dehydrogenase

MH:

Mild hypothermia

MPTP:

Mitochondrial permeability transition pore

OGD/R:

Oxygen–glucose deprivation/reperfusion

ROS:

Reactive oxygen species

Δψm:

Mitochondrial transmembrane potential

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Acknowledgements

We are grateful to the anonymous reviewers for their critical insight into improving this manuscript.

Funding

This work was supported by The Medical Science and Technology Research Project of Foshan (No. 2016AB002111) (to JJ), The Basic and Applied Basic Research Fund of Guangdong (Regional Cooperation Fund Project, 2022) (to JJ) and The 14th Five-Year High-Level Medical Key Specialty Project of Foshan. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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  1. Tianen Zhou and Jierong Mo have contributed equally to this work.

Authors and Affiliations

  1. Department of Emergency, The First People’s Hospital of Foshan, Foshan, 528000, Guangdong, China

    Tianen Zhou, Jierong Mo, Weigan Xu, Qiaohua Hu, Hongfeng Liu & Jun Jiang

  2. Department of General Medicine, The First People’s Hospital of Foshan, Foshan, 528000, Guangdong, China

    Yue Fu

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  1. Tianen Zhou
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Contributions

TEZ, JJ and YF designed the experiments. JRM, QHH and HFL participated in the experiments. WGX finished statistical analyses. TEZ and JRM wrote the article and performed figures. JJ and YF provided the supervision and the financial support. All authors reviewed and approved the manuscript.

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Correspondence to Yue Fu or Jun Jiang.

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Zhou, T., Mo, J., Xu, W. et al. Mild hypothermia alleviates oxygen−glucose deprivation/reperfusion-induced apoptosis by inhibiting ROS generation, improving mitochondrial dysfunction and regulating DNA damage repair pathway in PC12 cells. Apoptosis 28, 447–457 (2023). https://doi.org/10.1007/s10495-022-01799-w

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