The Role of Casr Inhibition-Mediated M2 Microglial Transformation in Ischemic Preconditioning Against Stroke

Abstract

Objective

Stroke is a main cause of disability and mortality worldwide. It has been reported that ischemic preconditioning (IP) has neuroprotective effects against stroke. This study aimed to verify the mechanism by which calcium-sensing receptor (Casr) inhibition-mediated M2 microglial transformation in the IP protects against stroke, which will provide a potential therapeutic target for stroke.

Methods

Middle cerebral artery occlusion (MCAO) rats and oxygen-glucose deprivation (OGD) neurons were used in this study. IP was induced via the transient MCAO and OGD methods. RNA sequencing (RNA-Seq) was used to explore the underlying key molecules. Western blotting and immunohistochemistry were performed to detect the expression of Casr and the M1 and M2 microglial markers. CCK8 was used to detect cell viability. The calcium concentration was detected via the use of Fluo-4 AM, a fluorescence probe. The Casr inhibitor NPS2143 and the Casr activator R568 were used to explore the role of Casr in M2 microglial transformation and neuroprotection.

Results

We first revealed that IP induced M2 microglial transformation in ischemic injury. In addition, MCAO injury increased Casr expression and the calcium concentration, which was inhibited by IP. Furthermore, Casr activation inhibited the M2 microglial transformation induced by IP. Finally, we found that Casr inhibition improved the survival rate, alleviated neurological deficits, and reduced the infarct volume induced by MCAO.

Conclusions

We confirmed that Casr-related neuroprotection induced by IP is associated with the transformation of M2 microglia. These findings can be used to understand the protective mechanisms of IP against ischemic stroke.

Keywords

Stroke / Transient ischemic attacks / Calcium-sensing receptor / Microglial transformation / Ischemic preconditioning

© The Author(s), under exclusive licence to Huazhong University of Science and Technology 2025

Acknowledgements None.

Authors’ Contributions Yun-feng Yang designed the study. Zhi- hao Zhai conducted the experiments, analyzed the data, and prepared the manuscript and images. Zuo-yu Huang, Kai-xun Huang, Yuan- qiang Zhong and En-xiang Tao helped prepare the manuscript and images, and collected and analyzed the data and literature. Yun- feng Yang revised the manuscript. All authors read and approved the final version of the manuscript.

Funding This work was supported by the National Natural Science Foundation of China (grant numbers 81371440, 82271454 and 81971195).

Availability of Data and Materials Data will be made available on reasonable request.

Declarations

Conflict of interest The authors declare that there is no potential conflict of interest.

Ethics Approval and Consent to participate All experimental procedures were approved by the Ethics Committee of The Eighth Affiliated Hospital, Sun Yat-sen University(No. 2024-309-01) in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals.

Consent for Publication All authors have agreed the submission of the transfer statement.

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