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Hypoxia Induces Apoptosis of Microglia BV2 by Upregulating Kir2.1 to Activate Mitochondrial-Related Apoptotic

Yu-Fang Xie^1,2,3, Yan Wang^1,2, Yi Rong^1,2,3

¹Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Ministry of Education, Shihezi University School of Medicine, Shihezi, China.

Disease Markers

|March 28, 2022

Abstract

Aim:

To explore the role of Kir2.1 in hypoxia-induced microglial apoptosis.

Methods:

BV2 microglial cell lines were cultured and treated with ML133 hydrochloride, a Kir2.1 channel blocker, for 23 h and with 500 μmol/L of CoCl₂ for 8 h. Cells were divided into the control, CoCl₂ (hypoxia-induced model), and CoCl₂+ML133 (hypoxia-induced model established after ML133 pretreatment) groups. Cell activity was assessed using the CCK-8 technique. The membrane potential and Kir2.1 current of BV2 were evaluated with the whole-cell patch-clamp technique. The protein levels and mRNA levels of Kir2.1, apoptotic proteins Bax and caspase-3, and antiapoptotic protein Bcl-2 in BV2 cells were evaluated via immunofluorescence, Western blot analysis, and real-time quantitative reverse transcription. The apoptosis rate of BV2 cells was detected via flow cytometry.

Results:

CCK-8 analysis showed that the cell activity of each group increased initially and then decreased. The 2 h intervention group had the highest cell activity, and that of the 8 h group was >90%. Hence, there was a significant difference in the results (P < 0.05). Western blot analysis revealed that the expression of cleaved caspase-3 significantly increased in the 8 h group compared with the 0 h group. Compared with the control group, the expression of Kir2.1 and mRNA in the CoCl₂ group increased. Thus, hypoxia could upregulate the expression of Kir2.1. The whole-cell patch-clamp results showed that the Kir2.1 channel current amplitude of the CoCl₂ group increased compared with that of the control group. Therefore, hypoxia could enhance Kir2.1 function. The apoptosis rate of the CoCl₂ group was significantly higher than that of the control group. Further, the ML133 group had a significantly lower apoptosis rate than the CoCl₂ group. The expression of apoptotic proteins Bax and cleaved caspase-3 increased in the CoCl₂ group, and that of the antiapoptotic protein Bcl-2 decreased. The expression of apoptotic proteins Bax and cleaved caspase-3 reduced in the CoCl₂+ML133 group, whereas that of the antiapoptotic protein Bcl-2 increased.

Conclusion:

Hypoxia can induce microglia BV2 apoptosis accompanied by the upregulation of Kir2.1 and mRNA expression levels and an increase in the Kir2.1 current. Moreover, ML133 can inhibit hypoxia-induced BV2 cell apoptosis. Hence, Kir2.1 may be involved in the process of hypoxia-induced BV2 cell apoptosis.

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