二苯乙烯苷对氧糖剥夺星形胶质细胞凋亡的影响及其机制

Effects and mechanisms of 2,3,5,4'-tetrahydroxystibene-2-O-β-D-glucoside(TSG)against oxygen-glucose deprivation induced apoptosis in rat astrocytes

目的观察2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷(TSG)对大鼠大脑皮层星形胶质细胞(AS)缺氧、缺糖损伤所致凋亡的影响,以及内质网应激相关因子Caspase-12、Caspase-3的表达变化,探讨TSG脑保护作用的作用机制。方法原代培养Sprague-Dawley大鼠大脑皮层AS,建立缺氧、缺糖损伤模型,以流式细胞术检测细胞凋亡率,以透射电镜观察细胞超微结构尤其是凋亡小体的变化,研究缺氧缺糖对各组AS的影响;以钙离子敏感的荧光探针Fura-2/AM负载细胞检测细胞内游离钙离子浓度,研究氧糖剥夺(OGD)对不同组AS的影响;以免疫细胞化学染色方法和反转录-聚合酶链反应(RT-PCR)方法观测内质网应激相关因子Caspase-12和Caspase-3各组的表达。结果与正常对照组相比,缺氧缺糖处理后AS内游离钙离子浓度增高,TSG预处理可明显降低细胞内游离钙离子,调节钙稳态失衡。免疫细胞化学染色和RT-PCR结果显示,缺氧缺糖损伤后细胞内Caspase-12、Caspase-3表达增加,TSG预处理可显著抑制两者的表达。结论 TSG可抑制OGD诱导的AS凋亡,其机制可能通过抑制内质网应激发挥作用。

Objective To investigate the effects of TSG on the apoptosis of rat cerebral cortical astrocytes induced by oxygen-glucose deprivation. To observe the endoplasmic reticulum stress related factors caspase-12 and caspase-3 expression in AS and explore the possible mechanism of brain protection by TSG. Methods Primary cultured cerebral cortical astrocytes were prepared from Sprague-Dawley rats, and a cell damage model was induced by oxygen-glucose deprivation （OGD）. The change of cell morphology was observed by transmission electron mi- croscope, and the detection of apoptotic cells was determined by the flow cytometry. Intracellular calcium concen- tration was monitored using the fluorescent Ca2＋ indicator fura-2/AM by fluorescence duel wavelength spectropho- tometer. The expression of apoptosis-related proteins, caspase-12 and caspase-3, was observed by immunocyto- chemistry and caspase-12 and caspase-3 mRNA were evaluated by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR）. Results The concentration of intracellular Ca2＋ was increased by OGD, and the application of TSG could decrease it. RT-PCR and immunocytochemistry analysis demonstrated that the levels of caspase-12, caspase-3 mRNA and protein increased by OGD, and the application of TSG could inhibit these expression. Conclusion The expression of caspase-12 and caspase-3 increases dramatically after OGD, which indicates that the endoplasmic reticulum stress could contribute to the occurrence and development of cell apoptosis induced by OGD in AS. TSG could inhibit the cell apoptosis induced by OGD in AS, and its mechanisms might be associated with the inhibition of the endoplasmic reticulum stress.