Objective To investigate the role of hypoxia-inducible factor-1α/ hexokinase Ⅱ (HIF-1α/HKⅡ) signaling pathway in the inhibition of hypoxia/reoxygenation (H/R)-induced apoptosis in the rat cardiomyocytes by hypoxic preconditioning (HPC).Methods Primarily cultured cardiomyocytes obtained from the neonatal rats were seeded in culture dishes at the density of 5× 105cells/ml.The cardiomyocytes were attached to the wall for 72 h and then randomly divided into 6 groups (n=15 each) using a random number table:control group (group C);group H/R;group HPC;HIF-1o inhibitor YC-1 group (group YC-1);group HPC + YC-1;dimethyl sulfoxide (DMSO) group.The ceils were exposed to D-Hank solution saturated with 95％ N2 and 5％ CO2 for 4 h,and then cultured in the normal culture atmosphere for 2 h.The cells in YC-1 and DMSO groups were incubated in the culture medium containing 10 μmol/l YC-1 and 0.1％ DMSO (100 μl) for 24 b,respectively.HPC was induced by 3 cycles of 10 min hypoxia followed by 30 min reoxygenation,and H/R injury model was then established in group HPC.In group HPC+YC-1,the cells were incubated for 5 min in the M199 culture medium supplemented with 10 μmol/L YC-1 and 10％ fetal bovine serum,and the other treatments were similar to those previously described in group HPC.After the end of treatments,the apoptosis in cardiomyocytes was detected by TUNEL,the expression of HIF-1α,HKⅡ and cytochrome c was detected by Western blot,and the mitochondrial membrane potential was quantitatively measured using fluorescence.Apoptotic rate was calculated.Results Compared with group C,the apoptotic rate was significantly increased,the mitochondrial membrane potential was significantly decreased,and the expression of HIF-1α,HKⅡ and cytochrome c was significantly up-regulated in group H/R (P＜0.05).Compared with group H/R,the apoptotic rate was significantly decreased,and the mitochondrial membrane potential was significantly increased,and the expression of HIF-1αt and HKⅡ was significantly up-regulated,and the expression of cytochrome c was significantly down-regulated in group HPC (P＜0.05).Compared with group HPC,the apoptotic rate was significantly increased,the mitochondrial membrane potential was significantly decreased,the expression of HIF-1α and HKⅡ was significantly down-regulated,and the expression of cytochrome c was significantly up-regulated in group HPC+YC-1 (P＜0.05).Conclusion The mechanism by which HPC inhibits H/R-induced apoptosis in rat cardiomyocytes is associated with activation of HIF-1α/HKⅡ signaling pathway.%目的 探讨缺氧诱导因子-1α/己糖激酶Ⅱ(HIF-1α/HKⅡ)信号通路在缺氧预处理抑制缺氧/复氧诱导大鼠心肌细胞凋亡中的作用.方法 原代培养乳鼠心肌细胞,接种于培养皿中,细胞浓度为5× 105个/ml,贴壁72 h时采用随机数字表分为6组(n=15):对照组(C组)、缺氧/复氧组(H/R组)、缺氧预处理组(HPC组)、HIF-1α抑制剂YC-1组(YC-1组)、缺氧预处理+YC-1组(HPC+YC-1组)和二甲基亚砜组(DMSO组).细胞置入经95％N2+5％CO2混合气体饱和的D-Hank液,在无氧环境中孵育4h后正常培养2h,以建立缺氧/复氧损伤模型.YC-1组和DMSO组分别用含10μmol/L YC-1的培养基、含0.1％二甲基亚砜(100 μ/)的培养基孵育24 h.HPC组在无氧环境中孵育10 min后复氧30 min,重复3次完成缺氧预处理,随后制备缺氧/复氧损伤模型.HPC+YC-1组采用含10 μmol/L YC-1+10％胎牛血清的M199培养基中孵育5 min,随后处理同HPC组.各组处理结束后,采用TUNEL法测定细胞凋亡率,采用Western blot法测定细胞HIF-1α、HKⅡ和细胞色素c的表达水平,采用免疫荧光法测定线粒体膜电位.结果 与C组比较,H/R组细胞凋亡率升高,线粒体膜电位降低,HIF-1α、HKⅡ和细胞色素c表达上调(P＜0.05).与H/R组比较,HPC组细胞凋亡率下降,线粒体膜电位升高,HIF-1α和HKⅡ表达上调,细胞色素c表达下调(P＜0.05).与HPC组比较,HPC+YC-1组细胞凋亡率升高,线粒体膜电位降低,HIF-1α和HKⅡ表达下调,细胞色素c表达上调(P＜0.05).结论 缺氧预处理抑制缺氧/复氧诱导大鼠心肌细胞凋亡的机制与激活HIF-1α/HKⅡ信号通路有关.
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