胰高血糖素样肽-1对AGEs诱导H9C2心肌细胞自噬的影响  

The protective effect of glucogon like peptide-1 on H9C2 cardimyocytes against AGEs-induced injury by inhibiting excessive autophagy

作　　者：张军[1,2] 谷翔[1,2] 黄问银[2] 张普华[2] 于欢[3] 殷嫦嫦[3] 王丽丽[3] 毛卫未[2] 

机构地区：[1]南昌大学研究生院医学部,江西南昌330006 [2]九江学院附属医院心内科,江西九江332000 [3]九江学院转化医学实验室,江西九江332000

出　　处：《中国药理学通报》2017年第3期348-353,共6页Chinese Pharmacological Bulletin

基　　金：国家自然科学(地区)基金项目(No 81660152);江西省教育厅科技项目(No GJJ09350)

摘　　要：目的观察胰高血糖素样肽-1(GLP-1)对晚期糖基化终产物(AGEs)诱导H9C2心肌细胞自噬(Autophagy)的影响,初步探讨GLP-1心肌细胞保护作用与自噬活性关系。方法将传代培养H9C2心肌细胞随机分4组:(1)Control组:加入0.9%生理盐水;(2)AGEs组:加入100 mg·L^(-1)AGEs;(3)AGEs+GLP-1组:同时加入100 mg·L^(-1)AGEs和10 nmol·L^(-1)GLP-1;(4)AGEs+GLP-1+Rapamycin组:同时加入100 mg·L^(-1)AGEs、10 nmol·L^(-1)GLP-1及5μmol·L^(-1)Rapamycin(自噬诱导剂)。各组在经上述预处理24 h后,分别用CCK-8法检测细胞存活率Hoechst 33258试剂盒检测细胞的凋亡率,DCFH-DA荧光探针检测细胞内活性氧(ROS)水平,流式细胞术检测细胞自噬溶酶体形成,Western blot法检测自噬相关蛋白(LC3Ⅱ,Beclin-1)表达。结果 (1)与Control组相比,AGEs组细胞生存率明显减少,细胞内ROS水平明显增高,细胞自噬溶酶体水平及自噬相关蛋白(LC3Ⅱ、Beclin-1)表达均明显增加;(2)与AGEs组相比,AGEs+GLP-1组细胞生存率明显增高,细胞内ROS水平、细胞自噬溶酶体水平及自噬相关蛋白(LC3Ⅱ、Beclin-1)表达均明显下降;(3)与AGEs组相比,AGEs+GLP-1+rapamycin组中细胞内ROS水平降低,而细胞生存率、细胞自噬溶酶体水平及自噬相关蛋白(LC3Ⅱ、Beclin-1)表达未见差异。结论 (1)AGEs可导致H9C2心肌细胞内ROS升高,诱导细胞损伤并激活细胞自噬;(2)GLP-1对AGEs诱导的H9C2心肌细胞损伤具有保护作用,其保护机制可能与抑制细胞自噬活性有关。Aim Toinvestigatetheeffectofglucogon like peptide-1 on H9 C2 cardiomyocytes against AGEs-induced injury and potential protective mechanisms of autophagy.Methods CulturedH9C2cardiomyocytes were respectively incubated with 0. 9% NaCl,100 mg ·L-1 AGEs,100 mg · L-1 AGEs ＋10 nmol · L-1 GLP-1 ,100 mg·L-1 AGEs＋10 nmol·L-1 GLP-1 ＋5μmol·L-1 rapamycinfor 24 h.Intracellular ROS pro-duction was measured by DCFH-DA fluorescent probe. Cell viability was quantified by CCK-8 assay.Nucleus morphology was observed under fluorescence micro-scope after being incubated with Honchest 33258.The formations of autolysosomes were detected by flow cy-tometry using the PH-sensitive fluorescent dye acridine orange（OA）.Western blot was applied to assess theexpression of autophagy-associated protein including LC3Ⅱ,Beclin-1.Results AGEsimpairedcellviabil-ity,increased intracellular ROS production,enhanced formation of autolysosomes and up-regulated expression of LC3Ⅱand Beclin-1;however,the effect were re-versed remarkably by GLP-1 .Furthermore,effect of GLP-1 was inhibited by rapamycin（autophagy agonist） in cell viability,amount of autolysosomes,and expres-sion of autophagy-associated protein but not in intracel-lularROSproduction.Conclusion GLP-1mayprotect H9 C2 cardiomyocytes against AGEs-induced injury partly by inhibiting excessive autophagy.

关 键 词：胰高血糖素样肽素-1 糖基化终产物 H9C2心肌细胞 自噬 活性氧 细胞保护 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R329.25[医药卫生—基础医学]