睾酮通过减少氧化应激干预血管内皮细胞衰老

摘要

目的：观察睾酮对过氧化氢（ H 2O 2）诱导衰老的人脐静脉内皮细胞（ HUVECs）的影响。方法用SA－βgal细胞化学检测法观察不同浓度睾酮及雄激素受体拮抗剂、雌激素受体拮抗剂对H 2O 2诱导的HUVECs衰老的干预作用，用2，7二氯氢化荧光素二酯（ H2 DCF－DA）染色检测细胞内活性氧簇（ ROS ）水平，用免疫印迹分析（ Western 印迹）法检测细胞中去磷酸化 Rb 蛋白水平。结果睾酮在3．0×10－9、3．0×10－8、3．0×10－7 mol／L等3种浓度下均具有延缓 H2 O2诱导的 HUVECs的 SA－βgal阳性率明显增加的趋势；与3．0×10－8 mol／L睾酮干预组相比，予10－6 mol／L ICI182，780预处理，HUVECs的SA－βgal阳性率明显增加，ROS水平增加，去磷酸化Rb蛋白水平明显增加。结论睾酮对H 2O 2诱导的 HUVECs衰老的影响与其剂量有关；生理浓度睾酮具有延缓H 2 O2诱导的HUVECs衰老的趋势，其作用可能是通过转化为雌激素，作用于雌激素受体，减少氧化应激和调节去磷酸化Rb蛋白产生。%Objective To investigate the effects of testosterone on the senescence of human umbilical vein endothelial cells ( HU-VECs) induced by hydrogen peroxide ( H2 O2) and initially investigate the mechanism of act of testosterone at physiological concentration on it.Methods Effects of testosterone at different concentrations with or without its antagonist Flutamide or estrogen receptors antagonist ICI1827,80 on the senescence of HUVECs induced by H 2O 2 were studied by means of SA-βgalactosidase staining ,the level of reactive oxy-gen species(ROS) were also detected by 2,7-dichlorodihydrofluorescein diacetate staining and the expression of hypophosphorylation of pRb by Western blot.Results H2O2(60 μmol/L,72 h)increased positive rate of SA-βgalactosidase staining of HUVECs,as well as increased ROS level and hypophosphorylation of pRb expression .Testosterone at low concentrations (3×10-9 mol/L,3×10-8 mol/L and 3×10-7 mol/L) delaying increased positive rate of SA-βgalactosidase staining of HUVECs induced by H 2 O2 ,most obviously at concentration of 3×10-8mol/L (but P>0.05 versus H2O2stimulated group),whereas higher concentration(3×10 -6mol/L) had opposite effect(70.20±3.40 for SA-βgalactosidase staining method,P<0.001 versus H2O2 stimulated group).The estrogen receptors antagonist ICI182,170 rather than androgen antagonist Flutamide at concentration of 10-6 mol/L inhibited the delaying trend induced by testosterone at concentration of 3×10-8 mol/L,as well as increased ROS level and hypophosphorylation of Rb expression in HUVECs (67.00±2.82 for SA-βgalactosidase staining method, 2.89±0.10 for H2DCF-DA staining method,10.24±0.20 for Western blot,P<0.01 versus testosterone at concentration of 3×10-8 mol/L group) .Conclusions Testosterone modulates the senescence of HUVECs induced by H 2 O2 in a dose-related manner .And at physiological concentrations,testosterone has the trend of delaying it ,mainly by transforming to estrogen acting on estrogen receptors decreasing oxidative stress and modulating hypophosphorylation of Rb expression consequently .