普罗布考对非对称性二甲基精氨酸诱导的人脑微血管内皮细胞凋亡的作用

Effects of probucol on apoptosis of human brain microvascular endothelial cells induced by asymmetric dimethylarginine

目的:探讨普罗布考对非对称性二甲基精氨酸(ADMA)诱导的人脑微血管内皮细胞(HBMEC)凋亡的保护作用。方法:取HBMEC分为不同的处理组。①治疗组:加入30μmol/L ADMA及普罗布考,其中普罗布考又分为10^-8、10^-7、10^-6、10^-5 mol/L 4个剂量。②ADMA组:仅加入30μmol/L ADMA。③普罗布考组:仅加入10^-5 mol/L普罗布考。④对照组:不加药物。分别采用MTT法检测细胞活性,比色法测定LDH释放量,ELISA法测定BDNF。另取HBMEC分为对照组、ADMA组、ADMA+普罗布考组、普罗布考组,ADMA剂量30μmol/L,普罗布考剂量10^-5 mol/L。分别采用比色法测定Caspase-3蛋白活性,ELISA法测定MDA,DCFH-DA法测定ROS,RT-PCR法测定bax、bcl-2、eNOS mRNA表达水平。结果:与对照组相比,ADMA组LDH释放量升高,细胞活性和BDNF降低(P<0.05);与ADMA组相比,各ADMA+普罗布考组LDH释放量降低,细胞活性和BDNF升高(P<0.05)。析因分析可知ADMA主效应使MDA、ROS释放量、bax mRNA表达、Caspase-3蛋白活性升高,bcl-2、eNOS mRNA表达降低,且ADMA与普罗布考存在交互作用(P<0.001)。结论:普罗布考能有效对抗ADMA诱导的HBMEC损伤。

Aim:To investigate the effects of probucol on apoptosis of human brain microvascular endothelial cells(HBMEC)induced by asymmetric dimethylarginine(ADMA).Methods:In experimentⅠ,HBMEC were divided into different groups,treatment group[30μmol/L ADMA and probucol(10^-8,10^-7,10^-6,10-5 mol/L)were given],ADMA group(only 30μmol/L ADMA was given),probucol group(only 10-5 mol/L probucol was given),and control group(without drugs).Cell viability of HBMEC was measured by MTT method.The release of LDH was measured by colorimetry.BDNF was measured by ELISA.In experimentⅡ,HBMEC were divided into four groups,control group,ADMA group,ADMA+probucol group,and probucol group.The dose of ADMA and probucol was 30μmol/L and 10-5 mol/L.The activity of Caspase-3 was detected by colorimetry.MDA was measured by ELISA method.ROS was measured by DCFH-DA method.The expressions of bax,bcl-2,eNOS mRNA were evaluated by RT-PCR.Results:Compared with control group,the release of LDH in ADMA group was higher,but cell activity and the concentration of BDNF in ADMA group were lower(P<0.05).Compared with ADMA group,the release of LDH in ADMA+probucol groups were lower,but cell activity and the concentration of BNDF were higher(P<0.05).According to factorial analysis,MDA,the release of ROS,the expression of bax mRNA and Caspase-3 protein activity were increased,and the expressions of bcl-2 and eNOS mRNA were decreased by ADMA(P<0.001).There was interaction between ADMA and probucol(P<0.001).Conclusion:Probucol could protect HBMEC from the injury induced by ADMA.