葡萄籽原花青素提取物对HT22细胞糖氧剥夺损伤的保护作用

目的:通过研究小鼠海马神经元细胞系HT22细胞糖氧剥夺(OGD)后凋亡/自噬相关蛋白表达的变化,探讨葡萄籽原花青素提取物(GSPE)预处理对OGD诱导的HT22细胞损伤的保护作用。方法:将HT22细胞分为对照组、OGD组、OGD+GSPE组。CCK8法检测OGD不同时间(4、6、8 h)干预后HT22细胞的活性,确定后续实验中OGD时间;CCK8法检测同一OGD时间下,不同GSPE浓度(20、40、100、200μmol/L)处理后HT22细胞的活性,确定GSPE最佳浓度;免疫荧光技术标记Tubulin蛋白,观察细胞形态的改变;AnnexinⅤ-FITC/PI染色和TUNEL染色检测细胞凋亡;免疫荧光技术检测Bax、Bcl-2、cleaved caspase-3、LC3Ⅱ和Beclin1等蛋白的表达量变化;caspase-3活性检测试剂盒检测caspase-3相对活性。结果:观察细胞形态结果表明:对照组细胞形态完整、胞体较大,OGD组中细胞大量皱缩成团、胞体变小,OGD+GSPE组细胞状态较OGD组明显改善;观察细胞凋亡情况结果表明:和OGD组相比,GSPE预处理后的HT22细胞,凋亡阳性细胞明显减少(P<0.01);免疫荧光结果表明GSPE预处理能抑制HT22细胞中Bax、cleaved caspase-3、LC3Ⅱ和Beclin1的表达并促进Bcl-2的表达。结论:OGD对HT22细胞损伤明显,GSPE预处理可以减轻OGD对HT22细胞损伤并显著提高细胞活性,其作用机制可能与其抑制细胞凋亡和细胞自噬有关。

The effects of grape seed proanthocyanidin extract on oxygen-glucose deprivation injury in HT22 cells

Objective:The effects of grape seed proanthocyanidin extract(GSPE)in HT22 cells were studied by exploring the expression of apoptotic/autophagy-related proteins after oxygen-glucose deprivation(OGD).Methods:HT22 cells were divided into control group,OGD group and OGD+GSPE group.The cell viability of HT22 was detected by CCK8 kit after different OGD time intervention(4,6,8 h)and we determined the OGD time that will be used in the subsequent experiments;Then,at the same OGD time,the viability of HT22 cells was detected by CCK8 kit which were treated with different concentrations of GSPE(20,40,100,200μmol/L)and the best concentration of GSPE was determined;HT22 cells were immunostained with Anti-Tubulin antibody to visualize the morphology;AnnexinⅤ-FITC/PI staining and TUNEL staining were performed to detect cell apoptosis;Immunofluorescence staining was used to detect the expression of Bax,Bcl-2,cleaved caspase-3,LC3II and Beclin1;The relative activity of caspase-3 was detected by caspase-3 assay kit.Results:After observation of cell morphology,we found the control group had large cell body and complete cell morphology,a lot of cells in the OGD group were shriveled into clusters and had small cell body.However,compared with the OGD group,the cell morphology in the OGD+GSPE group was significantly improved;Observation of apoptosis showed that apoptotic positive cells were significantly decreased in OGD+GSPE group(P<0.01).Immunofluorescence staining showed that GSPE pretreatment can decrease the expression of Bax,cleaved caspase-3,LC3Ⅱ,Beclin1 and increase the expression of Bcl-2.Conclusion:OGD can damage HT22 cells and GSPE pretreatment can significantly protect HT22 cells from the invasion of OGD and markedly improve cell viability.Furthermore,its mechanism may be related to the inhibition of apoptosis and autophagy.