利拉鲁肽通过调控自噬和Na^(+),K^(+)-ATPase活性抑制高糖诱导的心肌细胞肥大

Liraglutide inhibits high glucose-induced cardiomyocyte hypertrophy via modulating autophagy and Na^(+),K^(+)-ATPase activity

目的探讨利拉鲁肽(liraglutide,LRG)抑制高糖(HG)诱导的心肌细胞肥大的可能机制。方法体外培养H9c2细胞,分为对照(CON)组、HG组、低、中、高剂量LRG(LRG-L、LRG-M、LRG-H)组、LRG-H+自噬抑制剂3-甲基腺嘌呤(3-MA)组。鬼笔环肽染色观察细胞表面积;试剂盒测定细胞膜Na^(+),K^(+)-ATPase(NKA)活性;Real time-PCR和Western blot测定NKAα1、NKAα2 mRNA和蛋白表达;单丹磺胺戊二胺(MDC)荧光染色观察自噬囊泡数量;Western blot测定肥大标志基因(ANP、β-MHC)、自噬标志基因(Beclin-1、LC3、p62)蛋白表达。随后将H9c2细胞分为CON组、HG组、LRG-H组、LRG-H+Sirt1抑制剂EX 527组、LRG-H+AMPK抑制剂Compound C(CC)组,再次检测上述指标;Western blot测定Sirt1/AMPK/mTOR通路相关蛋白表达。结果LRG可抑制心肌细胞肥大,下调ANP、β-MHC蛋白表达;LRG可促进NKA活性恢复,上调NKAα2 mRNA和蛋白表达;LRG可增加自噬囊泡数量,上调Beclin-1、LC3-Ⅱ/LC3-Ⅰ蛋白表达,下调p62蛋白表达;LRG可上调Sirt1、p-AMPK/AMPK蛋白表达,下调p-mTOR/mTOR蛋白表达;使用自噬抑制剂3-MA、Sirt1抑制剂EX 527、AMPK抑制剂CC则部分逆转了LRG的作用。结论LRG可抑制高糖所致心肌细胞肥大,其机制与调控Sirt1/AMPK/mTOR通路激活自噬及促进NKA活性恢复有关。

Aim To investigate the mechanism through which liraglutide(LRG)inhibited high glucose(HG)-induced cardiomyocyte hypertrophy.Methods Cultured H9c2 were divided into control(CON)group,HG group,low-,middle-and high-dose LRG(LRG-L,LRG-M and LRG-H)groups,LRG-H+autophagy inhibitor trimethyladenine(3-MA)group.The relative cell surface change was assessed phalloidin staining.Membrane bound Na^(+),K^(+)-ATPase(NKA)activity was detected by the kit.The mRNA and protein expression of NKAα1 and NKAα2 was determined by Real time-PCR and Western blot,respectively.The number of autophagic vesicles was observed by monodansylcadaverin(MDC)staining.The protein expression of hypertrophic marker genes(ANP andβ-MHC)and autophagy marker genes(Beclin-1,LC3 and p62)was detected by Western blot.Subsequently,H9c2 cells were divided into CON group,HG group,LRG-H group,LRG-H+Sirt1 inhibitor EX 527 group,LRG-H+AMPK inhibitor Compound C(CC)group,and the above indicators were re-tested.The expression of Sirt1/AMPK/mTOR signaling related proteins was detected by Western blot.Results LRG inhibited cardiomyocyte hypertropy,and down-regulated the protein expression of ANP andβ-MHC.LRG increased the membrane bound NKA activity,and up-regulated the mRNA and protein expression of NKAα2.LRG increased the number of autophagic vesicles,up-regulated the protein expression of Beclin-1 and LC3-Ⅱ/LC3-Ⅰ,and down-regulated the protein expression of p62.LRG up-regulated the protein expression of Sirt1 and p-AMPK/AMPK,and down-regulated the protein expression of p-mTOR/mTOR.However,these effects were largely abolished by the autophagy inhibitor 3-MA,Sirt1 inhibitor EX 527 and AMPK inhibitor CC.Conclusions LRG attenuates HG-induced cardiac hypertrophy via regulating Sirt1/AMPK/mTOR pathway,activating autophagy and restoring NKA activity.