阿托伐他汀保护高糖环境下心肌微血管内皮细胞损伤的机制研究

摘要

目的 观察阿托伐他汀对高糖环境下心肌微血管内皮细胞损伤的作用,并探讨其机制.方法 培养SD大鼠心肌微血管内皮细胞,将细胞分成对照组、高血糖组、单纯阿托伐他汀处理组、阿托伐他汀+高糖组,使用超化物测定试剂盒检测自由基(RO S)水平,以末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)检测细胞凋亡.利用短RNA干扰(siRNA)技术,下调内皮细胞蛋白激酶B1(Akt1)和β1-整合素(β1-Integrin)的表达,采用Western blot检测小GTP结合蛋白解离刺激物(SmgGDS)表达水平.结果 (1)高糖组ROS水平高于对照组(t=4.154,P<0.01),阿托伐他汀组和阿托伐他汀+高糖组ROS水平均低于高糖组(t=4.233和2.893,均P<0.05).(2)高葡萄糖组细胞凋亡比例高于对照组(t=4.058,P<0.01),阿托伐他汀组和阿托伐他汀+高糖组细胞凋亡比例均低于高葡萄糖组(t=4.157和2.601,均P<0.05).(3)转染Akt1-siRNA后,与模拟组相比,高糖组和高糖+阿托伐他汀组的Akt1的表达水平均降低(t=4.058和4.167,均P<0.01);转染β1-integrin-siRNA后,与模拟组相比,高糖组和高糖+阿托伐他汀组的β1-integrin的表达水平均降低(t=4.073和4.215,均P<0.01).(4)阿托伐他汀低剂量组(1μmol/L)和阿托伐他汀高剂量组(10μmol/L)心肌微血管内皮细胞的SmgGDS相对表达水平均高于对照组(t=2.671和2.832,均P<0.05),高剂量组的SmgGDS相对表达水平高于低剂量组(t=2.612,P<0.05);转染Akt1-siRNA后,高糖组和高糖+阿托伐他汀组的SmgGDS表达水平均降低,高糖+阿托伐他汀+模拟组的SmgGDS表达水平高于高糖+模拟组(t=4.051,P<0.01);转染β1-integrin-siRNA后,高糖组和高糖+阿托伐他汀组的SmgGDS表达水平均低于高糖+阿托伐他汀+模拟组,高糖+阿托伐他汀+模拟组的SmgGDS表达水平高于高糖+模拟组(t=4.068,P<0.01);高糖组和高糖+阿托伐他汀组Akt磷酸化随时间变化的表达水平显示10 min后,高糖+阿托伐他汀组Akt磷酸化表达水平高于5 min后(t=2.608,P<0.05),15 min后Akt磷酸化表达水平高于10 min后(t=3.127,P<0.05);β1-integrin-siRNA转染高糖组,与高糖+模拟组比较,p-Akt/t-Akt降低(t=3.371,P<0.05).结论 阿托伐他汀可以保护高糖环境下心肌微血管内皮细胞免受氧化应激和凋亡,这种保护作用部分可能是通过他汀依赖的β1-Integrin/Akt1途径,上调SmgGDS实现的.%Objective To investigate the protective mechanisms of Atorvastatin against high glucose environment-induced injuries of myocardial microvascular endothelial cells. Methods Myocardial microvascular endothelial cells(MMECs)in SD rat were cultured and divided into groups of control group ,hyperglycemia group ,atorvastatin group ,and atorvastatin + high glucose group. The level of reactive oxygen species (ROS)was assayed using Superoxide Assay Kit. Apoptosis of cells was detected by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL) . The expression levels of Akt1 and β1-Integrin were assayed by short-interfering RNA (siRNA ) technique ,and the levels of small GTP-binding protein dissociation stimulator (SmgGDS) expression were measured using Western blot. Results (1)The level of ROS was higher in the high glucose group than in the control group(t=4.154 ,P <0.01) ,and lower in both Atorvastatin group and the Atorvastatin + high glucose group than in the high glucose group (t= 4.233 and 2.893 ,both P <0.05). (2)The proportion of apoptotic cells was higher in the high glucose group than in the control group(t= 4.058 ,P < 0.01) ,and lower in both Atorvastatin group and the Atorvastatin + high glucose group than in the high glucose group(t=4.157 and 2.601 ,both P<0.05).(3)The expression level of Akt1 was lower in the high glucose group and the high glucose + Atorvastatin group than in the mock control group after transfection of Akt1-siRNA(t=4.058 and 4.167 ,both P<0.01).The expression level of β1-integrin was lower in the high glucose group and the high glucose + atorvastatin group than in the mock control group after transfection of β1-integrin-siRNA (t=4.073 and 4.215 , both P<0.01). (4)Western blot analysis showed the following results. First ,the relative expression levels of SmgGDS in both the low dose(1 μmol/L)and high dose(10 μmol/L)of atorvastatin group were higher than in the control group (t= 2.671 and 2.832 ,both P < 0.05).Second ,the relative expression level of SmgGDS in the high dose group were higher than in the low dose group (t=2.612 , P< 0.05 ). Third ,after transfection of Akt1-siRNA ,the expression level of SmgGDS in the high glucose + Atorvastatin group and the high glucose group was decreased ;and the level was higher in the high glucose + atorvastatin + mock group than in the high glucose + mock group(t=4.051 ,P<0.01).Fourth ,after transfection of β1-integrin-siRNA ,the expression level of SmgGDS was lower in high glucose + Atorvastatin group and the high glucose group than in the high glucose +Atorvastatin + mock group ;the level was higher in the high glucose + Atorvastatin + mock group than in the high glucose + mock group(t= 4.068 ,P < 0.01).Fifth ,the expression level of Akt phosphorylation in the high glucose group and the high glucose + Atorvastatin group was higher at 10 minutes than at five minutes(t=2.608 ,P<0.05) ,and higher at 15 minutes than at 10 minutes(t=3.127 ,P <0.05). After transfection of β1-integrin-siRNA ,the expression level of p-Akt /t-Akt was lower in the high glucose group than in the high glucose + mock group(t= 3.371 ,P < 0.05). Conclusions Atorvastatin treatment protects myocardial microvascular endothelial cells possibly by up-regulating SmgGDS through β1-integrin/Akt1 against high glucose environment-induced oxidative stress and apoptosis injuries.