蛋白酶抑制剂H89对神经元细胞氧糖剥夺再灌注后高尔基体形态及细胞凋亡的影响

Effect of protease inhibitor H89 on morphology of the Golgi apparatus and apoptosis after oxygen-glucose deprivation followed by reperfusion in neurons

目的探讨蛋白酶抑制剂H89对神经元细胞经氧糖剥夺再灌注损伤后高尔基体形态和细胞凋亡的影响。方法将体外常规培养的小鼠海马神经元细胞HT22分为对照组、模型组和H89干预组。模型组与H89干预组按再灌注时间点分6h、12h和24h三个亚组。采用MTT法检测细胞存活率;Hoechest33258荧光染色法评估细胞凋亡;细胞免疫荧光技术观察高尔基体形态;Western blot技术检测GM130与GAAP蛋白表达。结果 H89干预组较模型组细胞活力有所上升,其中12h时间点(OD值0.1467±0.0090)与同期模型组比较,差异有统计学意义(P<0.05)。H89干预组较模型组细胞凋亡率有所下降,但差异均无统计学意义(P>0.05)。H89干预组在6h和12h时间点高尔基体碎裂程度较同期模型组稍有减轻。H89干预组GM130表达水平较模型组无显著升高(P>0.05)。GAAP表达水平仅在24h时间点(灰度比值0.4066±0.0288)显著升高(P<0.05)。结论 H89干预不能减轻神经元细胞经氧糖剥夺再灌注损伤后的高尔基体碎裂和细胞凋亡。

Objective To investigate the effect of protease inhibitor H89 on the morphology of the Golgi apparatus and apoptosis after oxygen-glucose deprivation followed by reperfusion （OGD/R） in neurons. Methods HT22 hippocampal neurons from mice cultured in vitro were divided in to control group, model group, and H89 intervention group. The cells in the model group and the H89 intervention group were further divided into 6-hour, 12-hour, and 24-hour subgroups according to the time point of reperfusion. MTT assay was used to measure cell viability; Hoechest33258 fluorescence staining was used to evaluate cell apoptosis; cell immunofluorescence was used to observe the morphology of the Golgi apparatus ; Western blot was used to measure the expression of GM130 and GAAP proteins. Results Compared with the model group, the H89 intervention group had a significant increase in the viability of HT22 cells （P 〈 0.05）, and there was a significant difference between the 12-hour subgroups of the H89 intervention group and the model group （OD = 0. 1467 ± 0.0090, P 〈 0.05）. Compared with the model group, the H89 intervention group had a reduction in cell apoptosis rate （P 〉 0. 05）. At 6 and 12 hours, the H89 intervention group had a slight reduction in the degree of fragmentation of the Golgi apparatus compared with the model group. The H89 intervention group had similar expression of GM130 as the model group （P 〉 0.05）, and compared with the model group, the H89 intervention group a significant increase in the expression of GAAP at 24 hours （ gray level ratio =0.4066 ± 0.0288, P 〈 0.05）. Conclusions H89 intervention cannot alleviate Golgi fragmentation and cell apoptosis after OGD/R.