HIF-1α在七氟醚预处理减轻大鼠皮质神经元凋亡中的作用：与Bid、Bim和Puma的关系

Role of HIF-1α in reduction of apoptosis in cortical neurons of rats by sevoflurane preconditioning： the relationship with Bid, Bim and Puma

目的 评价缺氧诱导因子-1α（HIF-1α）在七氟醚预处理减轻大鼠皮质神经元凋亡中的作用.方法 新生SD大鼠,培养原代皮质神经元,以1×106个/ml的密度接种于6孔板（2 ml/孔）,采用随机数字表法,分为4组（n=15）：正常对照组（C组）、缺氧复氧组（A/R组）、七氟醚预处理组（SP组）和HIF-1α抑制剂2-甲氧基雌二醇组（H组）.采用氧糖缺失90 min,复氧复糖24 h的方法制备皮质神经元缺氧复氧损伤模型;SP组通人2.0％七氟醚2h,随后采用PBS洗涤3次,每次5 min,结束后立即制备缺氧复氧损伤模型;H组加入5 μmol/L2-甲氧基雌二醇孵育72 h时进行七氟醚预处理.采用膜联蛋白V/碘化丙啶双染流式细胞术检测神经元凋亡情况,采用免疫蛋白印迹法测定神经元Bid、Bim、Puma和活化的caspase-3的表达水平.结果 与C组比较,A/R组神经元凋亡率升高,Bid、Bim、Puma和活化的caspase-3表达上调（P＜0.05）;与A/R组比较,SP组和H组神经元凋亡率降低,SP组Bid、Bim、Puma和活化的caspase-3表达下调（P＜0.05）;与SP组比较,H组神经元凋亡率升高,Bid、Bim、Puma和活化的caspase-3表达上调（P＜0.05）.结论 HIF-1α介导了七氟醚预处理减轻大鼠神经元凋亡的过程,其机制与下调Bid、Bim和Puma表达有关.

Objective To evaluate the role of hypoxia inducible factor-1α （HIF-1α） in reduction of apoptosis in cortical neurons of rats by sevoflurane preconditioning.Methods Primary cortical neurons obtained from neonatal Sprague-Dawley rats were seeded in 6-well plates （2 ml/well）,and randomly divided into 4 groups （n =15 each） using a random number table：control group （C group）,anoxiareoxygenation （A/R） group,sevoflurane preconditioning group （SP group） and HIF-1α inhibitor 2-methoxyestradiol group （H group）.The neurons were subjected to O2-glucose deprivation for 90 min followed by restoration of O2-glucose supply for 24 h.In group SP,the neurons were exposed to 2.0％ sevoflurane for 2 h followed by 5 min washout for 3 times,and then sevoflurane preconditioning was performed immediately.In group H,sevoflurane preconditioning was performed at 72 h of incubation with 5 μmol/L 2-methoxyestradiol.The apoptosis in neurons was assessed using Annexin V-FITC/PI assay,and apoptosis rate was calculated.The expression of Bid,Bim,Puma and activated caspase-3 in neurons was detected by Western blot.Results Compared with group C,apoptosis rate was significantly increased,and the expression of Bid,Bim,Puma and activated caspase-3 was up-regulated in group A/R.Compared with group A/R,apoptosis rate was significantly decreased,and the expression of Bid,Bim,Puma and activated caspase-3 was down-regulated in group SP.Compared with group SP,apoptosis rate was significantly increased,and the expression of Bid,Bim,Puma and activated caspase-3 was up-regulated in group H.Conclusion HIF-1α mediates reduction of apoptosis in rat neurons by sevoflurane preconditioning,and down-regulated expression of Bid,Bim,and Puma is involved in the mechanism.