降钙素基因相关肽通过调控PI3K/Akt/mTOR信号通路对缺氧/复氧心肌细胞自噬的影响  

Effect of calcitonin gene-related peptide on autophagy in hypoxic/reoxygenated cardiomyocytes through regulation of PI3K/Akt/mTOR signaling pathway

作　　者：董立博 原大江[2] Dong Libo;Yuan Dajiang(Department of Anesthesiology,Shanxi Medical University,Taiyuan 030000,Shanxi,China;Department of Critical Care Medicine,Second Hospital of Shanxi Medical University,Taiyuan 030000,Shanxi,China)

机构地区：[1]山西医科大学麻醉学院,太原030000 [2]山西医科大学第二医院重症医学科,太原030000

出　　处：《中华危重病急救医学》2025年第1期53-58,共6页Chinese Critical Care Medicine

基　　金：山西省自然科学基金(20210302123269)。

摘　　要：目的探讨降钙素基因相关肽(CGRP)对缺氧/复氧(H/R)心肌细胞自噬的影响及其与磷脂酰肌醇3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路的关系。方法常规体外培养大鼠心肌细胞株H9c2,当细胞生长至80%融合度时传代,用于实验。①CGRP剂量筛选实验:将细胞分为空白对照组、H/R组及不同剂量CGRP预处理组。将H9c2细胞置于密闭缺氧箱缺氧处理2 h后,置于常规培养箱中复氧处理12 h,制备H/R模型;CGRP预处理组于制模前分别给予0.01、0.1、0.5、1、5、10μmol/L CGRP预处理;空白对照组不给予任何处理。采用细胞计数试剂盒-8(CCK-8)检测细胞存活率,筛选出最适宜的药物剂量。②干预实验:另取H9c2细胞,分为空白对照组、H/R组、CGRP+H/R组和CGRP+PI3K靶向抑制剂ly294002(LY)+H/R组。H/R组制备细胞H/R模型;CGRP+H/R组和CGRP+LY+H/R组分别于制模前给予1μmol/L CGRP单独或联合10μmol/L LY处理12 h;空白对照组常规培养,不给予任何处理。采用CCK-8检测细胞存活率;采用比色法检测细胞乳酸脱氢酶(LDH)释放水平;采用蛋白质免疫印迹试验(Western blotting)检测细胞中自噬相关蛋白〔自噬效应蛋白Beclin-1、微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、自噬蛋白p62〕及PI3K/Akt/mTOR信号通路蛋白〔磷酸化Akt(p-Akt)、磷酸化mTOR(p-mTOR)〕表达。结果①CGRP剂量筛选实验结果:与空白对照组比较,H/R组细胞存活率显著下降;而给予0.1、0.5、1、5μmol/L CGRP预处理后,细胞存活率显著升高,以1μmol/L CGRP的干预效果最佳,与H/R组比较差异有统计学意义〔(74.23±6.18)%比(23.43±4.09)%,P<0.01〕,故作为后续实验的干预剂量。②干预实验结果:与空白对照组比较,H/R组细胞存活率显著降低,LDH释放水平显著升高,Beclin-1、LC3-Ⅱ的蛋白表达显著上调,p62、p-Akt、p-mTOR的蛋白表达显著下调,说明心肌细胞在H/R处理后发生死亡,并伴随自噬水平升高,且该过程与PI3Objective To investigate the effects of calcitonin gene-related peptide(CGRP)on autophagy in hypoxic/reoxygenated(H/R)cardiomyocytes and its relationship with the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.MethodsThe rat cardiomyocyte cell line H9c2 was routinely cultured in vitro and passaged for experiments when the cells grew to 80%fusion.①CGRP dosage screening experiment:the cells were divided into blank control group,H/R group and different dosages of CGRP pretreatment groups.H9c2 cells were placed in a closed hypoxia chamber for 2 hours and then reoxygenated in a conventional incubator for 12 hours to prepare the H/R model.The CGRP pretreatment groups were pretreated with 0.01,0.1,0.5,1,5,and 10μmol/L CGRP before the modeling process.The blank control group was not given any treatment.Cell counting kit-8(CCK-8)was used to detect the cell survival rate,and the most suitable drug dosage was screened out.②Intervention experiment:H9c2 cells were divided into blank control group,H/R group,CGRP+H/R group,and CGRP+PI3K target inhibitor ly294002(LY)+H/R group.H/R group was prepared as cellular H/R model.CGRP(1μmol/L)alone or in combination with LY(10μmol/L)was administered to CGRP+H/R group and CGRP+LY+H/R group,respectively,prior to the preparation of cellular H/R model.The blank control group was cultured routinely without treatment.The cell survival rate was detected by CCK-8.The level of lactate dehydrogenase(LDH)release was detected by colorimetric assay.The expressions of autophagy-related proteins[autophagy effector protein Beclin-1,microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ),autophagy protein p62]and PI3K/Akt/mTOR signaling pathway proteins[phosphorylated Akt(p-Akt),phosphorylated mTOR(p-mTOR)]were detected by Western blotting.Results①Results of CGRP dosage screening experiment:compared with the blank control group,the cell survival rate of the H/R group decreased significantly;and after giving 0.1,0.5,1,5μmol/L CGRP

关 键 词：降钙素基因相关肽 缺氧/复氧损伤 自噬 PI3K/Akt/mTOR信号通路 

分 类 号：R614[医药卫生—麻醉学]