【摘要】 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、1 000μmol/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1 000μmol/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),1 000μmol/L METH组细胞活力显著下降(P<0.05)。与1 000μmol/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、1 000μmol/L METH组CD11b表达显著增加(均P<0.05),且呈浓度依赖性;300、600、1 000μmol/L METH组TLR4表达及1 000μmol/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,1 000μmol/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1 000μmol/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,1 000μmol/L METH组BV2细胞TLR4、p-NF-κB表达显著增加(均P<0.05)。与1 000μmol/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。更多还原

【Abstract】 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 1 000 μmol/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 1 000 μmol/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, 960 μmol/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, 120 μmol/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-1β and IL-6 were also detected by ELISA. Results Compared with that in control group, BV2 cell activity in 30,60,120,240,480,960 μmol/L GB + METH group had no statistical significance( all P > 0. 05),and BV2 cell activity in 1 000 μmol/L METH group was significantly decreased( P < 0. 05). Compared with that in1 000 μmol/L METH group,BV2 cell activity in 120,240 μmol/L GB + METH groups were significantly increased( P < 0. 05-0. 01). Compared with that in 0 μmol/L METH group,the expression of CD11 b in 300 μmol/L METH group had no statistical significance( P > 0. 05),the expression of CD11 b in 600,1 000 μmol/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 300,600,1000 μmol/L METH groups and the expression of p-NF-κB in 1 000 μmol/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 1 000 μmol/L METH group was significantly increased( P < 0. 05),the expression of CD11 b in 120 μmol/L GB group and 120 μmol/L GB +METH group had no statistical significance( all P > 0. 05). Compared with that in 1 000 μmol/L METH group,the expression of CD11 b in 120 μmol/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 1 000 μmol/L METH group were significantly increased( all P < 0. 05).Compared with those in 1 000 μmol/L METH group,the expression of TLR4,p-NF-κB in 120 μmol/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.更多还原