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的研

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的

研究＊

张琰1 ，宋志刚2，张会如1（272067，山东济宁，济宁医学院医学影像系1；250012，山东济南，山东大学药学院在读博士研究生2）

摘要目的探讨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，观察内质网应激相关因子Caspase-12、Caspase-3 的表达变化，探讨TSG产生脑保护作用的可能机制。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，研究氧糖剥夺（OGD）对不同组AS的影响；通过钙离子敏感的荧光探针Fura-2/AM负载细胞，荧光双波长分光光度计检测细胞内钙离子浓度的变化；免疫细胞化学染色和RT-PCR方法观测Caspase-12和Caspase-3在OGD前后的变化。结果与正常对照组相比，缺氧缺糖损伤可使细胞内游离钙离子浓度显著增高，TSG预处理后可明显降低钙离子浓度，调节钙稳态失衡。RT-PCR和免疫细胞化学染色结果显示，缺氧缺糖可使AS内Caspase-12、Caspase-3的表达增加，TSG预处理后可显著抑制两者的表达。结论凋亡因子Caspase-12、Caspase-3在缺氧缺糖处理后表达明显增加，提示内质网应激机制在氧糖剥夺所致AS细胞凋亡的发生发展中具有重要作用。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。

关键词星形胶质细胞；二苯乙烯苷；氧糖剥夺；细胞凋亡；内质网应激

Effects and mechanisms of

2,3,5,4’-tetrahydroxystibene-2-O-β-D-glucoside(TSG) against oxygen- glucose deprivation indused apoptosis in rat astrocytes＊Zhang Yan1,Song Zhigang2,Zhang Huiru1(1Department of Medical Imaging, Jining Medical University,Jining,272067;2School of Pharmaceutical Sciences Shandong

University,Jinan, 250012)

Abstract：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,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 microscope,and the detection of apoptotic cells was determined by the flow cytometry. Intracellular calcium concentration was monitored using the fluorescent Ca2+indicator fura-2/AM by fluorescence duel wavelength spectrophotometer. The expressions of apoptosis-related proteins,Caspase-12 and Caspase-3, were observed by Immunocytochemistry and the 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 can decrease it. RT-PCR and immunocytochemistry analysis demonstrated that the level of Caspase-12,Caspase-3 mRNA and protein increased by OGD, and the application of TSG can inhibit these expression.Conclusion The expressions 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, its mechanisms might be associated with the inhibition of the endoplasmic reticulum stress．

Key words: Astrocytes, TSG, Oxygen-Glucose Deprivation, Cell apoptosis, Endoplasmic reticulum stress

Supported by the Foundation of the Young Scholars of Jining Medical University

TSG是从何首乌中提取分离得到的一种多酚结构的水溶性成分，是何首乌的主要有效成分。大量研究表明，TSG具有抗炎、抗氧化、清除自由基、抑制动脉粥样硬化等多种作用[1]。随着TSG研究的不断深入，发现其在神经细胞损伤中具有显著