ClC-3氯通道调节氧糖剥夺所诱导的小胶质细胞的表型转化

ClC-3 chloride channel regulated microglial phenotypic transformation induced by oxygen-glucose deprivation

目的 :研究ClC-3氯通道在氧糖剥夺所致小胶质细胞表型转化中的作用。方法:应用小胶质细胞株(BV-2)制备氧糖剥夺模型,分别给予氯通道阻断剂DIDS和NPPB预处理BV-2细胞。通过MTT活性测定确定BV-2细胞损伤及药物的有效浓度;通过实时荧光定量PCR法测定细胞表型相关分子,如M1型相关分子[肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素-1β(interleukin 1β,IL-1β)、CD86]和M2型相关分子[转化生长因子β(transforming growth factorβ,TGF-β)、CD206]m RNA水平的表达;通过RNA干扰的方法下调ClC-3的表达,再给予氯通道阻断剂DIDS和NPPB干预,进一步检测细胞的MTT活性,观察药物作用效果。结果:预先给予DIDS(1和10μmol/L)和NPPB(1μmol/L)能够改善氧糖剥夺所诱导的BV-2细胞MTT活性下降,抑制M1型相关分子如TNF-α、IL-1β和CD86的表达并促进M2型相关分子(TGF-β和CD206)的表达;通过RNA干扰下调ClC-3的表达,能够消除DIDS和NPPB的作用。结论:ClC-3氯通道参与调控氧糖剥夺所致小胶质细胞的表型转化,阻断ClC-3氯通道抑制氧糖剥夺诱导小胶质细胞向M1型转化。

Objective：To study the roles of ClC-3 chloride channel played in the microglial phenotypic transformation induced by oxygen-glucose deprivation. Methods：Microglia（BV-2 cell line） was applied to establish the oxygen-glucose deprivation（OGD）model,and then respectively pretreated with different concentrations of chloride channel blockers,including DIDS and NPPB. Cell damage and the effective concentration of drugs were determined by MTT activity. The m RNA level of cell phenotypic factors,such as M1 markers including tumor necrosis factor α（TNF-α）,interleukin 1β（IL-1β）and CD86,M2 markers containing transforming growth factor β（TGF-β） and cell surface molecule CD206 were detected by real-time fluorescent quantitative PCR. Expression of ClC-3 was downregulated by small RNA interference,and then pretreated with chloride channels blockers-DIDS and NPPB. Finally,the effects of blockers were observed by the MTT activity. Results：Pretreatment with DIDS（1,10 μmol / L） and NPPB（1 μmol / L） could partially reverse the decrease of BV-2 cells viability induced by OGD. DIDS（1 μmol / L） and NPPB（1 μmol / L） pretreatment could reduce the m RNA level of TNF-α,IL-1β and CD86,they could also promote expression of TGF-β m RNA. However,the reversal effect of DIDS and NPPB could be abolished by the down-regulation of ClC-3 expression. Conclusion：ClC-3 chloride channel is essential for the phenotypic transformation of microglia caused by OGD. Blocking ClC-3 chloride channel could inhibit its transformation into M1 phenotype.