氧葡萄糖剥夺－再恢复后抑制小胶质细胞TLR9激活对神经元的保护作用

摘要

AIM:To observe the Toll-like receptor 9 (TLR9) activation in microglia BV-2 cells after oxygen-glucose deprivation and reoxygenation ( OGDR) , and its effects on neuronal apoptosis.METHODS:The BV-2 cell super-natants were collected after the corresponding treatment and added to mouse primary cortical neurons after OGDR for 4 h, followed by normal culture for 24 h.The cells were divided into normal BV-2 group, NC-siRNA group, TLR9-siRNA group, OGDR group, OGDR+NC-siRNA group, OGDR+TLR9-siRNA group and control group (without adding BV-2 cell supernatant) .The changes of the neuronal morphology were observed under an inverted phase-contrast microscope, and the neuronal apoptosis was detected by TUNEL.The protein expression of cleaved caspase-3 was detected by Western blot-ting.RESULTS:After OGDR, the axon turned thin, twisted and broken, and neuronal swelling, decrease in refraction and vacuolar degeneration were observed.The green-stained apoptotic bodies in the neurons in all groups were positive. Compared with control group, the caspase-3 protein levels in other groups were increased.Compared with the normal BV-2 group, the caspase-3 protein in OGDR group and TLR9-siRNA group was increased.Compared with OGDR+TLR9-siRNA group, the caspase-3 protein in TLR9-siRNA group and OGDR group was decreased.CONCLUSION: After OGDR, TLR9 activation in BV-2 cells induces neuronal apoptosis with the increase in caspase-3 protein level.Inhibition of TLR9 expression reduces neuronal damage.%目的：观察氧葡萄糖剥夺－再恢复（OGDR）后小胶质细胞BV-2 Toll 样受体9（TLR9）激活对神经元凋亡的影响。方法：对BV-2细胞或TLR9-siRNA转染的BV-2细胞进行OGDR处理4 h后，将细胞上清添加至OGDR处理4 h的小鼠原代皮层神经元中，继续正常培养24 h后，倒置显微镜下观察神经元形态变化，TUNEL染色检测神经元凋亡，Western blotting检测神经元caspase-3蛋白的表达。实验分为正常BV-2组、negative control-siRNA组、TLR9-siRNA组、OGDR组、OGDR＋NC-siRNA组、OGDR＋TLR9-siRNA组和对照组（神经元OGDR后不添加BV-2细胞上清）。结果：OGDR后神经元胞体肿胀，折光性下降，出现空泡样变，轴突变细、扭曲、断裂。 TUNEL染色各组均可见绿染凋亡小体。与对照组比较，其它组的caspase-3蛋白表达升高（ P＜0.05）；与正常BV-2组比较，OGDR组和TLR9-siRNA组的caspase-3蛋白表达升高（ P＜0.05）；OGDR＋TLR9-siRNA转染组与TLR9-siRNA转染组和OGDR组比较， caspase-3蛋白表达下降（ P＜0.05）。结论： OGDR后 BV-2细胞TLR9激活致神经元凋亡增多， caspase-3蛋白表达升高；抑制TLR9表达后，神经元损伤减轻。