毛蕊异黄酮对氧糖剥夺再灌注BV2小胶质细胞极化的影响

Effects of Calycosin on Microglia Polarization in BV2 Cells After Oxygen-Glucose Deprivation and Reperfusion

目的探讨毛蕊异黄酮对氧糖剥夺再灌注BV2小胶质细胞极化的影响。方法将对数生长期BV2细胞随机分为6组:正常组、模型组、尼莫地平组(5μg·mL^(-1))、毛蕊异黄酮高剂量组(20μg·mL^(-1))、毛蕊异黄酮中剂量组(10μg·mL^(-1))、毛蕊异黄酮低剂量组(5μg·mL^(-1))。氧糖剥夺3 h后,各给药组换成含药的完全培养基,复氧6 h。采用CCK-8法检测细胞活力;测定细胞上清液中的一氧化氮(NO)、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-10含量;免疫荧光双染法检测BV2细胞极化;WesternBlot法检测BV2细胞的iNOS、CD206蛋白表达水平。结果与正常组比较,模型组的BV2细胞活力显著降低(P<0.01),NO、TNF-α、IL-1β水平明显升高(P<0.05),IL-10水平明显降低(P<0.05);免疫荧光双染检测中M1型小胶质细胞标记物iNOS表达显著增强(P<0.01);Western Blot检测中iNOS蛋白表达明显上调(P<0.05),M2型细胞标志物CD206蛋白表达明显下调(P<0.05)。与模型组比较,毛蕊异黄酮低、中、高剂量组及尼莫地平组的BV2细胞活力显著提高(P<0.01),NO、TNF-α、IL-1β水平明显降低(P<0.05),IL-10水平明显升高(P<0.05);免疫荧光双染检测中毛蕊异黄酮(10μg·mL^(-1))组BV2细胞的iNOS表达显著减弱(P<0.01);Western Blot检测中毛蕊异黄酮(10μg·mL^(-1))组BV2细胞的iNOS蛋白表达明显下调(P<0.05),CD206蛋白表达明显上调(P<0.05)。结论毛蕊异黄酮能促进活化的BV2小胶质细胞向M2型极化,抑制其向M1型极化,减少炎性介质的产生,降低氧化损伤,对缺血后脑组织损伤具有保护作用。

To research the effect of calycosin on the polarization of BV2 microglia cells after oxygenglucose deprivation and reperfusion.Methods The BV2 cells in logarithmic growth phase were randomly divided into 6 groups:normal group,model group,Nimodipine group(5μg·mL^(-1)),calycosin high-dose group(20μg·mL^(-1)),calycosin medium-dose group(10μg·mL^(-1)),calycosin low-dose group(5μg·mL^(-1)).After oxygen-glucose deprivation for 3 hours,the drug-containing complete medium was replaced in each administration group,and reoxygenation for 6 hours.CCK-8 was used to detect cell viability;the contents of NO,tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and IL-10 in the cell supernatant were measured.BV2 cell polarization was detected by Western Blot.Results Compared with the normal group,the activity of BV2 cells in the model group was significantly decreased(P<0.01),the levels of NO,TNF-αand IL-1βwere significantly increased(P<0.05),and the level of IL-10 was significantly decreased(P<0.05).The expression of iNOS,a marker of M1 microglia,was significantly increased in immunofluorescence double staining(P<0.01).The protein expression of iNOS was significantly up-regulated in the detection of Western Blot(P<0.05),and the protein expression of M2 cell marker CD206 was significantly down-regulated in the detection of Wenstern Blot(P<0.05).Compared with the model group,the viability of BV2 cells in the low-,medium-and high-dose calycosin groups and Nimodipine group was significantly increased(P<0.01),the levels of NO,TNF-αand IL-1βwere significantly decreased(P<0.05),and the level of IL-10 was significantly increased(P<0.05).The protein expression of iNOS in BV2 cells of calycosin(10μg·mL^(-1))group was significantly decreased by immunofluorescence double staining(P<0.01).Western Blot showed that the protein expression of iNOS in BV2 cells of calycosin(10μg·mL^(-1))group was significantly down-regulated(P<0.05),and the protein expression of CD206 was significantly up-regulated(P<0.05).Conclusion Calycosin can promote the polarization of activated BV2 microglia to M2 type,inhibit its polarization to M1 type,reduce the production of inflammatory mediators and oxidative damage,and protect brain tissue damage after ischemia.