丹参酮ⅡA通过NLRP3炎症体信号通路对小胶质细胞糖氧剥夺/再灌注损伤的保护作用

The Protective Effect of TanshinoneⅡA on Oxygen-glucose Deprivation and Reperfusion Injury of Microglia Through the NLRP3 Inflammatory Signaling Pathway

目的探讨丹参酮ⅡA对小胶质细胞BV-2糖氧剥夺/再灌注（oxygen-glucose deprivation and reperfusion,OGD/R）损伤的保护作用及其机制。方法 取对数生长期的BV-2细胞OGD 3h后再灌注4~24h,采用Western blot筛选细胞内Nod受体蛋白3（NLPR3）表达水平最高的再灌注时间。将实验组细胞OGD处理3h后分别加入终质量浓度为0~2.5μg/mL的丹参酮ⅡA,CCK8法检测细胞存活率,筛选丹参酮ⅡA的最大有效质量浓度。对BV-2细胞OGD 3h后分别加入0、0.5、1.0、2.0μg/mL丹参酮ⅡA,再灌注12h,Western blot检测BV-2细胞内NLRP3和凋亡蛋白caspase-1的表达水平,ELISA检测BV2细胞白介素（IL）-1β和IL-18的质量浓度。结果 OGD 3h后,再灌注12h时NLPR3蛋白表达水平最高。CCK8法显示丹参酮ⅡA最大有效质量浓度为2.0μg/mL。NLRP3、caspase-1、IL-1β和IL-18表达均随着丹参酮ⅡA质量浓度的升高而降低。结论 丹参酮ⅡA能抑制OGD/R处理后BV-2细胞内NLRP3炎症体信号通路分子的表达,这可能是其保护OGD/R细胞的分子机制之一。

Objective To investigate the protective effect of Tanshinone lI A （TSA） on oxygen-glucose deprivation and reperfusion （OGD/R） injury of BV-2 cell and its NLRP3 inflammatory signaling pathway. Methods The highest expression level of NLPR3 in BV-2 cells was detected by Western blot after oxygen-glucose deprivation （OGD） for 3 h and reperfusion for different time, to determine the most suitable reperfusion time. Cell viability of TSA （0-2.5 μg/mL） treatment was detected by CCK8 assay to determine the maximum effect concentration of TSA. In TSA 0 （also called OGD group）, 0.5, 1.0, 2.0μg/mL groups, expression levels of NLRP3 and caspase- 1 were detected by Western blot, while IL-1β and IL-18 in culture medium of those groups were detected by ELISA assay. Results The highest expression level of NLRP3 came to 12 h of reperfusion. The maximum effective concentration of TSA was 2.0 〉g/mL. The expression levels of NLRP3, caspase-1, IL-1β and IL-18 decreased with the increase of TSA concentration. Conclusion TSA can inhibit the expression of protein and cytokines of NLRP3 inflammatory signaling pathway in OGD/R BV-2 cells, which may be one of the molecular mechanisms of the protective effect of TSA on OGD/R cells.