降钙素基因相关肽通过PKC-mitoKATP通路抑制缺氧/复氧诱导的caspase-3和caspase-9酶活性升高

Calcitonin gene-related peptide inhibits anoxia/reoxygenation-induced increases in the enzymatic activity of caspase-3 and caspase-9 through the protein kinase C-mitochondrial ATP sensitive potassium channel pathway

目的研究降钙素基因相关肽(calcitonin gene-related peptide,CGRP)对缺氧/复氧(anoxia/reoxygenation,A/R)诱导大鼠心肌细胞凋亡的影响及其相关的信号转导通路。方法选用60只健康1~3 d龄SD大鼠,建立心肌细胞体外原代培养模型,采用随机数字表法将心肌细胞分成10组,并进行分组实验(每组6孔):①对照组;②A/R组;③CGRP组(CGRP+A/R组);④特异性CGRP受体阻滞剂组(CGRP8-37+CGRP+A/R组);⑤线粒体ATP敏感性钾通道(ATP sensitive potassium channel,KATP)阻滞剂组(5-HD+CGRP+A/R组);⑥线粒体KATP激动剂组(DZ+A/R组);⑦蛋白激酶A阻滞剂组(H-89+CGRP+A/R组);⑧蛋白激酶C阻滞剂组(chelerythine+CGRP+A/R组);⑨非特异性KATP阻滞剂组(glibenclamide+CGRP+A/R组);⑩细胞膜KATP阻滞剂组(HMR-1098+CGRP+A/R组)。采用分光光度法分别检测各组心肌细胞A/R损伤后caspase-3、caspase-9酶活性。 结果与对照组比较,A/R组心肌细胞中caspase-3、caspase-9的酶活性均明显升高(P<0.05)。与A/R组比较,CGRP+A/R组心肌细胞中caspase-3、caspase-9的酶活性均明显降低(P<0.05),使用10-6 mol/L CGRP8-37阻滞剂该作用可被逆转(P<0.05)。此外,与A/R组比较,DZ+A/R组心肌细胞中caspase-3、caspase-9的酶活性均明显降低(P<0.05)。与CGRP+A/R组比较,glibenclamide+CGRP+A/R组和5-HD+CGRP+A/R组心肌细胞中caspase-3、caspase-9的酶活性均明显升高(P<0.05),而HMR-1098+CGRP+A/R组心肌细胞中caspase-3、caspase-9的酶活性并未明显升高(P>0.05)。与CGRP+A/R组比较,chelerythine+CGRP+A/R组心肌细胞中caspase-3、caspase-9的酶活性出现明显升高(P<0.05),H-89+CGRP+A/R组心肌细胞中caspase-3、caspase-9的酶活性有升高的趋势,但差异无统计学意义(P>0.05)。 结论CGRP主要通过激活蛋白激酶C-线粒体敏感性钾通道(protein kinase C-mitochondrial KATP,PKC-mitoKATP),降低凋亡蛋白酶caspase-3、caspase-9的表达,来减轻心肌细胞A/R损伤,发挥抗心肌细胞凋亡作用。

ObjectiveTo investigate the effect of calcitonin gene-related peptide(CGRP)on the apoptosis of rat cardiomyocytes induced by anoxia/reoxygenation(A/R)and its related signaling pathways.MethodsA total of 60 male healthy SD rats aging 1 d to 3 d were selected to establish a primary model of cardiomyocytes in vitro.The animals were randomly divided ten groups before A/R experiments(n=6):①a control group,②an A/R group,③a CGRP+A/R group,④a CGRP8-37+CGRP+A/R group,⑤an 5-HD+CGRP+A/R group,⑥a DZ+A/R group,⑦an H-89+CGRP+A/R group,⑧a chelerythine+CGRP+A/R group,⑨a glibenclamide+CGRP+A/R group and⑩an HMR-1098+CGRP+A/R group.The activities of caspase-3 and caspase-9 were measured by spectrophotometry.ResultsCompared with the control group,the A/R group showed significantly increased activities of caspase-3 and caspase-9(P<0.05).Compared with the A/R group,the CGRP+A/R group demonstrated significantly decreased activities of caspase-3 and caspase-9(P<0.05),which were reversed by the treatment of 10-6mol/L CGRP8-37(P<0.05).In addition,the activities of caspase-3 and caspase-9 in the DZ+A/R group was remarkably lower than those in the A/R group(P<0.05).Compared with the CGRP+A/R group,the glibenclamide+CGRP+A/R and 5-HD+CGRP+A/R groups demonstrated significantly increased activities of caspase-3 and caspase-9(P<0.05),while not significant increases were found in the activities of caspase-3 and caspase-9 in the HMR-1098+CGRP+A/R group(P>0.05).Compared with the CGRP+A/R group,the chelerythine+CGRP+A/R group produced significantly increased activities of caspase-3 and caspase-9(P<0.05),while the activities of caspase-3 and caspase-9 in the H-89+CGRP+A/R group were slightly increased without statistical differences(P>0.05).ConclusionsCGRP relieves cardiomyocyte A/R injury and plays an important role in cardiomyocyte apoptosis by activating protein kinase C-mitochondrial ATP sensitive potassium channel and decreasing the expression of caspase-3 and caspase-9.