降糖速率通过ERK1/2通路影响心肌细胞凋亡

Glucose-lowering rate influences cardiomyocyte apoptosis via ERK1/2 pathway

目的 通过降低心肌细胞培养基中葡萄糖浓度,观察不同降糖速率下,细胞外信号调节激酶（ERK1/2）通路抑制前后心肌细胞凋亡程度、肿瘤坏死因子α（TNF-α）表达的变化,探索降糖速率对心肌细胞损伤程度及其炎性分泌功能的影响及机制.方法 原代培养并鉴定Wistar乳鼠心肌细胞,在葡萄糖浓度为25 mmol/L培养基中培养72 h后,降低培养基浓度.根据培养基中葡萄糖浓度将细胞随机分为5组,即：A组为对照组,B、C、D、E组为实验组,A组维持25 mmol/L葡萄糖浓度不变,B组葡萄糖浓度调整为20mmol/L（降糖速率为5 mmol/L）,C组葡萄糖浓度调整为15 mmol/L（降糖速率为10 mmol/L）,D组葡萄糖浓度调整为10 mmol/L（降糖速率为15 mmol/L）,E组葡萄糖浓度调整为5 mmol/L（降糖速率为20mmol/L）,分别于调整葡萄糖浓度后3、24、48 h采用细胞计数试剂盒（CCK8）检测细胞存活率;Annexin V-PI染色后,流式细胞仪和激光共聚焦显微镜检测细胞凋亡情况;ELISA方法检测TNF-α水平;Western印迹法测定ERK1/2蛋白及其磷酸化水平.将U0126加入5组中,重复上述方法检测心肌细胞凋亡程度、TNF-α表达的变化.结果 不同降糖速率组,同一时间点,以A组为对照组,B组心肌细胞存活率增高,而C、D、E组逐渐降低（P＜0.05）,B、C、D组TNF-α浓度逐渐升高,E组降低（P＜0.01）.培养24 h时以A组为对照组,B组凋亡率显著降低[（23.9±1.27）％,P＜0.05],p-ERK1/2表达增加（P＜0.05）,C、D、E组凋亡率增高[C组（39.9±2.12）％,D组（45.7±3.62）％,E组（56.6±3.80）％,P＜O.05],C组ERK1/2磷酸化水平最低,D、E组ERK1/2磷酸化水平逐渐增加（P＜0.05）.加入U0126后,各实验组较之前心肌细胞的存活率均提高（P＜0.01）,TNF-α浓度均降低（P＜0.05）;与对照组比较,仅E组在培养24 h及48 h时,较A组心肌细胞存活率降低,凋亡率[（38.9±1.53）％]增高（P＜0.05）,TNF-α浓度增高（P＜0.05）,其余各组不同培养时间点差异均无统计学意义（P＞0.05）.结论 不同降糖速率下心肌细胞的凋亡、炎症因子TNF-α的表达与ERK1/2通路有关;降糖过程中,ERK1/2通路参与了降糖速率过快对心肌细胞的促凋亡过程.降糖过程中,TNF-α的分泌不仅与渗透压变化有关,亦依靠ERK1/2通路的作用.

Objective To explore the effects of extracellular regulated protein kinase 1/2 （ERK1/2） signal pathway on cardiomyocyte apoptosis and tumor necrosis factor-α （TNF-α） expression at different glucose-lowing rates,and the influence of glucose-lowing rate on cardiomyocyte injury and inflammatory secretion function,as well as its mechanism.Methods Cardiomyocytes of Wistar neonate rat were maintained in medium supplemented with 25 mmol/L glucose for 72 h.Then the medium was changed to different concentrations of glucose and all cells were divided into five groups.Group A was control group whose medium supplemented with 25 mmol/L glucose.Medium of group B,C,D,E was supplemented with 20,15,10,5 mmol/L glucose （glucose-lowing rate was 5,10,15,20 mmol/L） respectively.Survival rate of cardiomyocyte was measured by CCK8 kit.Cardiomyocyte apoptosis was measured by flow cytometry instrument and laser confocal microscope after Annexin V-PI.TNF-α was measured by ELISA.ERK1/2 protein and phosphorylation were measured by Western blot.Cardiomyocyte apoptosis and TNF-α levels were measured again after U0126 was added.Results At the same time point,along with the glucose-lowing rate increased,survival rate of cardiomyocyte in group A was increased and those in group C,D,E were decreased （P＜ 0.05）.TNF-α concentration was increased in group B,C,D and decreased in group E.After 24 h,apoptosis rate decreased in group B and increased in group C,D,E （P＜0.05）.ERK1/2 phosphorylation level increased in group B,D,and E（P＜0.05）.The ERK1/2 phosphorylation level in group B was the lowest.After U0126 was added,survival rates of cardiomyocyte in all groups were increased （P＜0.01） while TNF-α concentrations were decreased （P＜0.05）.In every group,survival rate of eardiomyocyte after 48 h was lower than that after 3 h and 24 h,while TNF-α concentration was higher （P＜0.05）.Conclusion Influence of glucose-lowering rate for cardiomyocyte apoptosis and TNF-o is caused by ERK1/2 pathway.In the glucose-lowering course,ERK1/2 pathway promotes cardiomyocytes apoptosis and TNF-α secretion is related with not only osmotic pressure,but also ERK1/2 signal pathway activation as well.