银杏内酯B对甲基苯丙胺引发的小胶质细胞活化及炎症反应的影响及机制研究

Effects and mechanism research of Ginkgolide B on microglia activation and inflammatory mechanisms induced by methamphetamine

目的研究银杏内酯B(GB)对甲基苯丙胺(METH)引发的小胶质细胞活化及炎症反应的影响及潜在机制。方法根据METH浓度不同分为0、300、600、1000μmol/L METH组,采用相应浓度METH刺激BV2细胞24 h,选择最佳METH浓度。根据METH处理时间不同分为0、12、24、48 h METH组,采用1000μmol/L METH(最佳浓度)分别刺激BV2细胞相应时间,选择最佳METH刺激时间。检测30、60、120、240、480、960μmol/L GB+METH组以及对照组的BV2细胞活力,选择最佳GB浓度。Western Blotting检测对照组、120μmol/L GB+METH组、阴性对照组(NC组)、METH+NC组、TLR4siRNA组及TLR4siRNA+METH组CD11b、Toll样受体4(TLR4)、磷酸化的核因子κB(p-NF-κB)的表达,ELISA法检测TNF-α、IL-1β和IL-6水平。结果与对照组比较,30、60、120、240、480、960μmol/L GB组细胞活力差异无统计学意义(均P>0.05),1000μmol/L METH组细胞活力显著下降(P<0.05)。与1000μmol/L METH组比较,120、240μmol/L GB+METH组细胞活力显著升高(P<0.05~0.01)。与0μmol/L METH组相比,300μmol/L METH组CD11b表达差异无统计学意义(P>0.05),600、1000μmol/L METH组CD11b表达显著增加(均P<0.05),且呈浓度依赖性;300、600、1000μmol/L METH组TLR4表达及1000μmol/L METH组p-NF-κB表达显著升高(P<0.05~0.01)。与0 h METH组相比,12 h、24 h及48 h METH组CD11b、TLR4及p-NF-κB表达均显著增加(P<0.05~0.001)。与对照组比较,1000μmol/L METH组CD11b表达显著升高(P<0.05),120μmol/L GB组及120μmol/L GB+METH组CD11b表达差异无统计学意义(均P>0.05)。与1000μmol/L METH组相比,120μmol/L GB+METH组CD11b表达显著降低(P<0.05)。与NC组比较,METH+NC组TLR4、p-NF-κB、TNF-α、IL-1β、IL-6表达水平均显著升高(P<0.05~0.01)。与METH+NC组比较,TLR4siRNA组TLR4、p-NF-κB表达显著降低(均P<0.01),TLR4siRNA+METH组TLR4、TNF-α、IL-1β、IL-6表达显著降低(P<0.05~0.01)。与对照组比较,1000μmol/L METH组BV2细胞TLR4、p-NF-κB表达显著增加(均P<0.05)。与1000μmol/L METH组相比,120μmol/L GB+METH组BV2细胞TLR4、p-NF-κB表达显著降低(均P<0.05)。结论GB可能通过抑制TLR4-NF-κB通路,发挥对METH引发的小胶质细胞活化及炎症反应的抑制作用。

Objective To investigate the effect of Ginkgolide B(GB)on microglia activation and inflammatory response induced by methamphetamine(METH)and its potential mechanism.Methods BV2 cells were divided into 0,300,600 and 1000μmol/L METH groups according to different METH concentrations and stimulated by METH at corresponding concentrations for 24 h to select the best METH concentration.According to METH treatment time,BV2 cells were divided into 0,12,24 and 48 h METH groups and stimulated with 1000μmol/L METH(the optimal concentration)for corresponding time respectively to select the optimal METH stimulation time.To detect BV2 cell activity in 30,60,120,240,480,960μmol/L GB+METH group and control group,and the optimal GB concentration was selected.Western Blotting was used to detect CD11 b,toll-like receptor 4(TLR4)and phosphorylated nuclear factor B(p-NF-κB)expression in the control group,120μmol/L GB+METH group,negative control(NC)group,METH+NC group,TLR4 siRNA group and TLR4 siRNA+METH group,and levels of TNF-α,IL-1βand IL-6 were also detected by ELISA.Results Compared with that in control group,BV2 cell activity in 30,60,120,240,480,960μmol/L GB+METH group had no statistical significance(all P>0.05),and BV2 cell activity in 1000μmol/L METH group was significantly decreased(P<0.05).Compared with that in1000μmol/L METH group,BV2 cell activity in 120,240μmol/L GB+METH groups were significantly increased(P<0.05-0.01).Compared with that in 0μmol/L METH group,the expression of CD11 b in 300μmol/L METH group had no statistical significance(P>0.05),the expression of CD11 b in 600,1000μmol/L METH groups were significantly increased(P<0.05-0.01),and it was in a dose-dependent manner.Compared with those in 0μmol/L METH group,the expression of TLR4 in 300,600,1000μmol/L METH groups and the expression of p-NF-κB in 1000μmol/L METH group were significantly increased(P<0.05-0.01).Compared with those in 0 h METH group,the expression of CD11 b,TLR4,p-NF-κB in 12,24,48 h METH groups were significantly increased(P<0.05-0.01).Compared with that in control group,the expression of CD11 b in 1000μmol/L METH group was significantly increased(P<0.05),the expression of CD11 b in 120μmol/L GB group and 120μmol/L GB+METH group had no statistical significance(all P>0.05).Compared with that in 1000μmol/L METH group,the expression of CD11 b in 120μmol/L GB+METH group was significantly decreased(P<0.05).Compared with those in NC group,the expression of TLR4,p-NF-κB,TNF-α,IL-1β,IL-6 in METH+NC group were significantly increased(P<0.05-0.01).Compared with those in METH+NC group,the expression of TLR4,p-NF-κB in TLR4 siRNA group were significantly decreased(all P<0.01),the expression of TLR4,TNF-α,IL-1β,IL-6 in TLR4 siRNA+METH group were significantly decreased(P<0.05-0.01).Compared with those in control group,the expression of TLR4,p-NF-κB in 1000μmol/L METH group were significantly increased(all P<0.05).Compared with those in 1000μmol/L METH group,the expression of TLR4,p-NF-κB in 120μmol/L GB+METH group were significantly decreased(all P<0.05).Conclusion GB may play an anti-inflammatory role in METHinduced microglia activation by inhibiting the TLR4-NF-κB pathway.