N-乙酰半胱氨酸对糖氧剥夺诱导星形胶质细胞损伤的抑制作用及机制研究

Study of Protect Effect and Its Medhanism of N-acetyl-L-cysteine Protecting Astrocyte Injury Induced by Glucose and Oxygen Deprivation

目的探讨N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)对糖氧剥夺(oxygen and glucose deprivation,OGD)诱导的星形胶质细胞损伤过程中AKT信号通路的影响及其意义。方法以原代培养的星形胶质细胞建立OGD模型,并分为4组:对照组,OGD组,NAC组和NAC+AKT特异性抑制剂MK-2206组(NAC+MK-2206组);细胞培养24 h后,采用噻唑蓝(MTT)法检测细胞存活率、流式细胞仪技术分析细胞凋亡比例,试剂盒检测细胞内SOD活性和丙二醛(MDA)含量,蛋白免疫印迹法检测细胞内AKT、磷酸化AKT(p-AKT)、mTORC1、磷酸化mTORC1(p-mTORC1)、胞浆型磷脂酶A2(c LPA2)、caspase3及Bcl-2的表达水平。结果与OGD组相比,NAC组的细胞存活率,p-AKT、p-mTORC1及Bcl-2表达和SOD活性增加,细胞凋亡比例,cPLA2、caspase3表达,MDA含量降低。与NAC组相比,NAC+MK-2206组的细胞存活率,p-AKT、p-mTORC1及Bcl-2表达和SOD活性降低,细胞凋亡比例、cPLA2、MDA含量、caspase3表达增加。结论 NAC缓解OGD诱导AKT信号通路抑制作用,降低cPLA2诱导细胞凋亡作用。

OBJECTIVE To investigate the role of AKT signaling pathway in protecting cell injury by N-acetyl-L-cysteine（NAC） induced by oxygen and glucose deprivation（OGD） in astrocytes. METHODS The astrocytes were used to establish an OGD model. The astrocytes were divided into four groups including control group, OGD group, NAC group and NAC＋MK-2206 group. MTT was used to detect cell survival rate. Apoptosis was analyzed by flow cytometry. SOD activity detection kit（WST-8） and lipid peroxidation superoxide dismutase assay kit was used to analyze the SOD activity and malondialdehyde（MDA） content respectively. Western blot was applied to test expression level of AKT and phosphorylated AKT（p-AKT）, mTORC1, phosphorylated mTORC1（p-mTORC1）, cytosolic phospholipase A2（cPLA2）, caspase3 and Bcl-2. RESULTS Compared with OGD group, the cell survival rate, the expression of p-AKT, p-mTORC1 and Bcl-2, the activity of SOD increased in NAC group, but the the proportion of apoptosis, expression of cPLA2 and caspase3, MDA content decreased. Compared with NAC group, the cell survival rate, the expression of p-AKT, p-mTORC1 and Bcl-2, the activity of SOD decreased in NAC＋MK-2206 group, but the the proportion of apoptosis, expression of cPLA2 and caspase3, MDA content increased. CONCLUSION NAC alleviate the inhibition of AKT signaling pathway and reduce the expression of cPLA2 and apoptosis by OGD.