国际麻醉学与复苏杂志   2019, Issue (2): 0-0
    
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
程雪赟, 原大江1()
1.山西医科大学
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
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摘要：

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； ② A/R组； ③ CGRP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。
关键词： 降钙素基因相关肽；　缺氧/复氧损伤；　ATP敏感性钾通道；　半胱氨酸天冬氨酸蛋白
Abstract:

Ｏｂｊｅｃｔｉｖｅ　To 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.　Methods　A 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): ① a control group, ② an A/R group, ③ a CGRP+A/R group, ④ a CGRP8-37+CGRP+A/R group, ⑤ an 5-HD+CGRP+A/R group, ⑥ a DZ+A/R group, ⑦ an H-89+CGRP+A/R group, ⑧ a chelerythine+CGRP+A/R group, ⑨ a glibenclamide+CGRP+A/R group and ⑩ an HMR-1098+CGRP+A/R group. The activities of caspase-3 and caspase-9 were measured by spectrophotometry.　Results　Compared 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).　Conclusions　CGRP 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.
Key words: Calcitonin gene-related peptide;　Anoxia-reoxygenation injury;　ATP sensitive potassium channel;　Caspases;　Cardiomyocytes