低氧预处理对氧糖剥夺人脑微血管内皮细胞线粒体呼吸功能的保护作用

Protective effects of hypoxia pretreatment on mitochondrial respiration function in oxygen-glucose deprivation human brain microvascular endothelial cell

目的通过观察低氧预处理对氧糖剥夺人脑微血管内皮细胞(human brain microvascular endothelial cell,HBMVEC)低氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)和线粒体琥珀酸脱氢酶复合体亚基2(succinate dehydrogenase complex iron sulfur subunit B,SDHB)表达水平以及线粒体ATP合成的影响,评价低氧预处理对氧糖剥夺HBMVEC的保护作用.方法离体培养HBMVEC,采用随机数字表法将HBMVEC分为5组(每组6孔):正常对照组(A组)、氧糖剥夺组(B组)、氧糖剥夺+低氧预处理组(C组)、氧糖剥夺+低氧预处理+二甲基草酰甘氨酸(dimethyloxalylglycine,DMOG)组(D组)和氧糖剥夺+低氧预处理+2-ME组(E组).给予低氧预处理及HIF-1α抑制剂和稳定剂后,对HBMVEC进行氧糖剥夺,使用Annexin V检测细胞凋亡和坏死;用ATP检测试剂盒测定细胞内ATP含量;采用Western blot法测定HIF-1α、SDHB蛋白表达.结果与A组比较,B组、C组、D组和E组HIF-1α表达水平明显升高,ATP含量明显减少,细胞凋亡率明显升高(P<0.05),B组、C组和E组SDHB表达水平明显降低(P<0.05);与B组比较,C组和D组HIF-1α和SDHB表达水平明显升高,ATP含量明显增加,细胞凋亡率明显降低(P<0.05).结论低氧预处理可通过调节HIF-1α表达改善氧糖剥夺所致的HBMVEC线粒体呼吸链复合体Ⅱ的损伤,发挥对氧糖剥夺HBMVEC模型的保护作用.

Objective The objective of this research is to investigate the protective effects of hypoxia pretreatment on oxygen-glucose deprivation human brain microvascular endothelial cell(HBMVEC)via detecting mitochondrial function and hypoxia-inducible factor-1α(HIF-1α)and succinate dehydrogenase complex iron sulfur subunit B(SDHB)expression in oxygen-glucose deprivation HBMVEC.Methods Cultured HBMVEC were randomly divided into 5 groups:sham group(group A),oxygen-glucose deprivation group(group B),oxygen-glucose deprivation+hypoxia pretreatment group(group C),oxygen-glucose deprivation+hypoxia pretreatment+dimethyloxalylglycine(DMOG)(group D)and oxygen-glucose deprivation+hypoxia pretreatment+2-ME(group E).Then,cell apoptosis was detected by Annexin V kit.ATP content was analyzed by ATP assay kit.The level of HIF-1α and SDHB expression were determined by Western blot.Results Compared with group A,the expression of HIF-1αwas up-regulated,ATP content was significantly decreased and cell apoptosis was increased in group B,group C,group D and group E(P<0.05),the expression of SDHB was significantly down-regulated in group B,group C and group E(P<0.05).Compared with group B,the expression of HIF-1α and SDHB was up-regulated,ATP content was significantly increased and cell apoptosis was decreased in group C and group D(P<0.05).Conclusions Hypoxia pretreatment can protect mitochondrial respiration function in oxygen-glucose deprivation HBMVEC via promoting HIF-1α and SDHB expression.