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 CoCl(2) for 8 h. Cells were divided into the control, CoCl(2) (hypoxia-induced model), and CoCl(2)+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 CoCl(2) 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 CoCl(2) group increased compared with that of the control group. Therefore, hypoxia could enhance Kir2.1 function. The apoptosis rate of the CoCl(2) group was significantly higher than that of the control group. Further, the ML133 group had a significantly lower apoptosis rate than the CoCl(2) group. The expression of apoptotic proteins Bax and cleaved caspase-3 increased in the CoCl(2) group, and that of the antiapoptotic protein Bcl-2 decreased. The expression of apoptotic proteins Bax and cleaved caspase-3 reduced in the CoCl(2)+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.