氧葡萄糖剥夺-再恢复后小胶质细胞Toll样受体9信号通路的激活

Activation of toll-like receptor 9 signaling pathway in mouse microglia after oxygen-glucose deprivation/recovery

目的观察氧葡萄糖剥夺-再恢复(OGDR)后小胶质细胞(BV-2细胞)Toll样受体9(TLR9)及其下游信号分子的表达变化。方法对BV-2细胞进行OGDR处理,模拟脑缺血再灌注的体内过程。以常氧培养的BV-2细胞作为对照。细胞免疫化学方法检测BV-2细胞的激活。在OGDR后0h、6h、12h、24h、48h、72h,荧光定量PCR检测细胞内TLR9mRNA表达,Western Blot检测细胞内NF-κB(P65)、磷酸化NF-κB(P-P65)、IRF7、磷酸化IRF7(P-IRF7)蛋白表达,ELISA检测细胞上清TNF-α、IL-1β和IFN-β的含量。结果 OGDR后BV-2细胞由原来静止的分枝状变成激活的阿米巴状,CD68染色阳性。除0h外,其余时间点OGDR组TLR9mRNA的相对表达量与对照组相应时间点比较均有上调,表达高峰位于24h。OGDR后各时间点P65和IRF7表达无明显变化。P-P65 0h、6h、12h明显增高,24h、48h、72h表达无明显增高。P-IRF7在0h、6h无明显增高,12h-72h明显升高。OGDR后TNF-α和IL-1β表达增多,TNF-α表达高峰于24-48h,IL-1β表达高峰位于48h。IFN-β表达无明显变化。结论 OGDR后小胶质细胞TLR9信号通路的活化以促炎途径为主。

Objective To explore the activation of BV-2microglia and the expression of toll-like receptor 9(TLR9)signaling pathway after oxygen-glucose deprivation/recovery(OGDR).Methods The OGDR model was established by placing BV-2 microglia into a tri-gas incubator containing 1% oxygen and glucose-free medium for four hours and then recovering normal gas and glucose in order to mimic brain ischemia-reperfusion injury.Cells receiving no stimulation were taken as control.The activation of BV-2microglia was measured by immunocytochemistry after the executions.Fluorogenic quantitative PCR was utilized to detect TLR9 expression at 0h,6h,12 h,24h,48 hand 72hafter OGDR.Western Blot was used to detect the expression of NF-κB(P65),phosphorated NF-κB(P-P65),IRF7 and phosphorated IRF7(PIRF7).Enzyme linked immunosorbent assay(ELISA)was used to determined the concentrations of TNF-α,IL-1βand IFN-βin the supernatant.Results OGDR changed BV-2microglial cells to the activated ameboid state from resting highly branched ramified state,which showed positive staining of CD68.TLR9 mRNA level of the OGDR group was statistically higher than that of the control group excepted at 0h,with an expressing peak at 24 h.Relative protein expressing level of phosphorated NF-κB(P-P65)was statistically higher at 0h,6hand 12 h,while the level of phosphorated IRF7 elevated after 12 h.Relative protein expressing level of NF-κB(P65)and IRF7 showed no obvious change.The level of TNF-αincreased gradually after OGDR and reached a peak at 24-48 hrs before decrease,while that of IL-1βreached a peak at 48 h.No statistical difference was detected between groups in the levels of IFN-β.Conclusion BV-2microglial cells and its TLR9 signaling pathways are activated after OGDR,mainly playing aproinflammatory role.