大鼠心肌细胞氧糖剥夺-复氧复糖时p38MAPK信号通路与钙超载的关系

Relationship between p38MAPK signaling pathway and calcium over-loading during oxygen-glucose deprivation and restoration in cardiomyocytes of rats

目的评价大鼠心肌细胞氧糖剥夺-复氧复糖时p38丝裂原活化蛋白激酶(p38MAPK)信号通路与钙超载的关系。方法取出生1~3d的SD大鼠,分离培养心肌细胞,采用随机数字表法分为3组(n=27):对照组(C组)、氧糖剥夺-复氧复糖组(OGD/R组)、p38MAPK抑制剂SB203580组(SB组)。采用缺氧小室对细胞进行氧糖剥夺6h复氧复糖2h。SB组氧糖剥夺前采用10μmol/LSB203580孵育1h。复氧复糖2h时,倒置显微镜下观察心肌细胞形态,采用CCK-8法检测细胞活力,采用2,4-二硝基苯肼显色法测定上清液LDH释放率,采用流式细胞术测定细胞钙离子浓度,采用Westernblot法测定磷酸化p38MAPK(p-p38MAPK)和p38MAPK表达水平,计算p-p38MAPK/p38MAPK比值。结果与C组比较,OGD/R组和SB组LDH释放率、细胞内钙离子浓度和p-p38MAPK/p38MAPK比值升高,细胞活力降低(P<0.05);与OGD/R组比较,SB组LDH释放率、细胞钙离子浓度和p-p38MAPK/p38MAPK比值降低,细胞活力升高(P<0.05),细胞形态接近正常,数量增多。结论p38MAPK信号通路激活后可介导钙超载,参与大鼠心肌细胞氧糖剥夺-复氧复糖损伤的病理生理机制。

Objective To evaluate the relationship between p38mitogen-activated protein kinase (p38MAPK) signaling pathway and calcium over-loading during oxygen-glucose deprivation and restoration (OGD/R) in cardiomyocytes of rats. Methods Cardiomyocytes obtained from Sprague-Dawley rats, aged 1-3 days, were cultured and divided into 3 groups (n=27 each) using a random number table method: control group (group C), group OGD/R and p38MAPK inhibitor SB203580 group (group SB). The cells were subjected to OGD for 6 h followed by restoration of O2-glucose supply for 2 h. Cells were incubated for 1 h with 10 μmol/L SB203580 in group SB.At 2 h of restoration of O2-glucose supply, cell morphology was observed under an inverted microscope, cell viability was measured by the CCK-8 method, the release of lactate dehydrogenase (LDH) in the supernatant was determined by 2, 4-dinitrobenzene chromogenic method, intracellular calcium concentration was determined by flow cytometry, and the expression of phosphorylated p38MAPK (p-p38MAPK) and p38MAPK was detected using Western blot.The LDH release rate and p-p38MAPK/p38MAPK ratio were calculated. Results Compared with group C, the LDH release rate, intracellular calcium concentration and p-p38MAPK/p38MAPK were significantly increased, and the cell viability was markedly decreased in group OGD/R and group SB (P<0.05). Compared with OGD/R group, the LDH release rate, intracellular calcium concentration and p-p38MAPK/p38MAPK ratio were significantly decreased, the cell viability was increased (P<0.05), the cell morphology was nearly normal, and the number of cells was increased in group SB. Conclusionp 38MAPK signaling pathway can mediate calcium overload after being activated and is involved in the pathophysiological mechanism of OGD/R in cardiomyocytes of rats.