低氧诱导因子-1α对SH-SY5Y细胞兴奋性毒性损伤的调节作用

Regulatory Role of HIF-1α in the SH-SY5Y Cells Excitotoxic Injury

探讨低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)在喹啉酸诱导人神经母细胞瘤SH-SY5Y细胞损伤中的作用。将体外培养的人神经母细胞瘤SH-SY5Y细胞分为对照组、喹啉酸低、中、高剂量组及HIF-1α抑制剂二甲氧基雌二醇(2-methoxyestradiol,2ME2)预处理喹啉酸高剂量组,采用噻唑蓝还原法和乳酸脱氢酶漏出率检测法测定细胞损伤程度,Hoechst 33342单荧光染色法观察细胞凋亡,免疫荧光染色法检测HIF-1α在细胞内的表达,免疫印迹法检测细胞HIF-1α、蛋白激酶B(protein kinase B,Akt)、磷酸化Akt(p-Akt)、Bcl-2和Bax的表达。结果显示,喹啉酸可剂量、时间依赖性地诱导SH-SY5Y细胞损伤,导致细胞凋亡。同时,喹啉酸可使SH-SY5Y细胞HIF-1α表达上调并发生核转位、p-Akt表达增加及Bax/Bcl-2表达比例增加,而2ME2可抑制喹啉酸诱导的SH-SY5Y细胞损伤及降低HIF-1α、p-Akt和Bax/Bcl-2的表达。由此说明,HIF-1α/Akt通路介导了喹啉酸诱导SHSY5Y的细胞凋亡。HIF-1α抑制剂(2ME2)能够减轻喹啉酸致SH-SY5Y细胞损伤程度,减少细胞凋亡。

To investigate the role of HIF-1α in SH-SY5Y cell injury induced by quinolinic acid, SH-SY5Y cells were divided into control group, quinolinic acid low, medium and high dosage groups and HIF-1α inhibitor （2ME2） pretreated group; the cell viability was determined by MTT reduction assay and LDH assay; cell apop- tosis was determined by Hoechst 33342 staining; the intracellular distribution of HIF-ls was examined by HIF-1α immunostaining and the expressions of HIF-ls, Akt, p-Akt, Bcl-2 and Bax were determined by Western blot. The results showed that quinolinic acid induced SH-SY5Y cell damage in a dosage- and time-dependent manner, leading to apoptosis. In addition, quinolinic acid enhanced HIF-ls expression and nuclear translocation. The p-Akt expression and Bax/Bcl-2 ratio were increased by quinolinc acid in SH-SY5Y cells, and 2ME2 inhibited quinolinic- acid induced SH-SY5Y cells damage and reduced the expression of HIF-ls, p-Akt and Bax/Bcl-2. These results indicated that HIF-1α/Akt pathway mediates quinolinic acid induced cell apoptosis in SH-SY5Y cells, and HIF-ls inhibitor （2ME2） can lower the degree of SH-SY5Y cells damage induced by quinolinic acid, reducing apoptosis.