调控NF—κB信号通路对大鼠脑星形胶质细胞继发性死亡的影响

Effect of regulation of nuclear factor-κB signaling pathway on brain astrocyte secondary death in rats

目的观察干预细胞核因子-κB（NF-κB）信号通路对星形胶质细胞（AST）自噬、凋亡和坏死的影响。方法取纯化AST分成3组：（1）空白组：加入2μLPBS液1h后，再加入2μLPBS液；（2）激活组：加入2μLPBS液1h后，再加入2μLNF-κB激活剂PMA（终浓度1μmol／L）；（3）抑制组：先加入2μLNF．κB阻断剂PDTC（终浓度20μmol／L）预处理1h，再加入2μLPMA（终浓度1μmol／L）。各组细胞分别处理1、6、12、24、48h，应用免疫细胞化学荧光法测定NF-κB蛋白的表达，应用AnnexinV／PI染色法检测凋亡和坏死．应用Westernblotting检测自噬蛋白Beclin1表达情况。结果（1）随着作用时间增加，NF-κB蛋白表达量也逐渐增高，24h时空白组、激活组及抑制组表达量分别为0．27±0．46、3．13±0．35、2．00±0．53，差异有统计学意义（P〈0．05）。（2）各组细胞凋亡及坏死程度也随着作用时间增加而相应增高。激活组AST凋亡程度总体高于空白组与抑制组，12、24、48h时间点3组细胞两两比较差异均有统计学意义（P〈0．05）。激活组AST坏死程度总体高于空白组与抑制组，24h、48h时间点3组细胞两两比较差异均有统计学意义（P〈0．05）。（3）激活组Beclin1蛋白各时间点均较抑制组及空白组明显增加，6h时空白组、激活组及抑制组Beclin1蛋白表达量分别为0．17±0．01、0．59±0．03、0．51±0．03，两两比较差异均有统计学意义（P〈0．05）。结论PMA诱发的NF-κB活化能够引起AST不同程度的凋亡、坏死及自噬进程，早期以凋亡和自噬为主，晚期以坏死为主。而PDTC能够在一定程度上抑制NF-κB通路所诱导的这些死亡进程。

Objective To study the effect of nuclear factor-κb （NF-κB） signaling pathway on autophagy, apoptosis and necrosis of astrocytes （ASTs）. Methods Purified ASTs were divided into three groups： blank control group, adding 2μL PBS at one h after 2 μL PBS; activator group, adding 2 μL NF-κB activator PMA （final concentration 1 μmol/L） at one h after 2 μL PBS; blocker group, adding 21xL NF-κB activator PMA （final concentration 1 μmol/L） at one h after 2 μL NF-κB blocker PDTC （final concentration 20 μmol/L）; each treatment was given to the cells for one, 6, 12, 24 and 48 h. NF-κB protein expression was detected by immunocytochemistry assay, apoptosis and necrosis of the cells in each group were observed by Annexin V/PI staining, autophagy protein Beclin 1 expression was measured by Western blotting. Results （1） With time being prolonged, NF-κB protein expression gradually increased; its expression quantity at 24 h after treatment was 0.27±0.46, 3.13±0.35 and 2.00± 0.53 in the blank control group, activator group and blocker group, with significant differences （P〈0.05）. （2） With time being prolonged, apoptosis and necrosis of the cells gradually became much severe; ASTs apoptosis in the activator group was significantly severer than that in the other two groups （P〈0.05）, andat 12, 24 and 48 h after treatment, the ASTs apoptosis was significantly different between each two groups （P〈0.05）; ASTs necrosis in the activator group was significantly severer than that in the other two groups （P〈0.05）, and at 24 and 48 h after treatment, the ASTs necrosis was significantly different between each two groups （P〈0.05）. （3） Beclinl protein expression in the activator group was obviously increased as compared with that in the other two groups; its expression quantity at 6 h after treatment was 0.17± 0.01, 0.59±0.03 and 0.51±0.03 in the blank control group, activator group and blocker group, with significant differences （P〈0.05）. Conclusion NF-κB activation induced by PMA could cause apoptosis, necrosis and autophagy of ASTs at different levels correspondingly; apoptosis and autophagy appear mainly at early stage, and necrosis is mainly at late stage; PDTC could inhibit these programmed cell death process induced by NF-κB pathway to some extent.