瘦素激活PI3K/AKT信号促进小鼠心肌细胞衰老的初步研究

Activation of PI3K/AKT signaling pathway by leptin promotes MCM senescence of mouse cardiomyocytes

目的:探讨瘦素在小鼠心肌细胞(mouse cardiomyocyte,MCM)衰老中的作用及调控机制。方法:qPCR检查瘦素刺激后MCM中衰老相关指标p16、p21和衰老相关分泌表型(senescence-associated secretory phenotype,SASP)的mRNA表达量;Western blot检测p16、p21、γ-H2AX、PI3K、AKT、p-PI3K和p-AKT的蛋白表达量;β-半乳糖苷酶染色检测MCM衰老。PI3K抑制剂(LY294002)预处理2 h后再给予瘦素刺激,qPCR和Western blot检测p16和p21的m RNA和蛋白表达水平;qPCR检查SASP的mRNA表达水平;β-半乳糖苷酶染色检测MCM衰老。结果:瘦素刺激MCM后,p16和p21的m RNA及蛋白表达增加,同时γ-H2AX的蛋白水平也显著上升,SASP[白介素(interleukin,IL)-1β、肿瘤坏死因子(tumor necrosis factor,TNF)-α、IL-6和单核细胞趋化蛋白(monocyte chemoattractant protein,MCP)-1)]的mRNA水平上调,PI3K/AKT信号通路的蛋白磷酸化水平升高,β-半乳糖苷酶染色显示MCM发生衰老。PI3K抑制剂预处理2 h后,p16和p21的m RNA和蛋白水平明显降低,同时γ-H2AX的蛋白水平也显著下调;SASP mRNA水平降低;β-半乳糖苷酶染色显示MCM衰老缓解。结论:瘦素通过活化PI3K/AKT信号通路分泌SASP(IL-1β、TNF-α、IL-6和MCP-1)调控MCM衰老的发展进程。

Objective:To explore the role and regulation mechanism of leptin in senescence of mouse cardiomyocytes(MCM).Methods:The mRNA expression levels of senescence related indicators p16,p21,and senescence-associated secretory phenotype(SASP)in leptin stimulated MCM were examined by qPCR;the protein expressions of p16,p21,γ-H2AX,PI3K,AKT,p-PI3K,and p-AKT were detected by Western blot;the senescence of MCM was detected byβ-galactosidase staining.PI3K inhibitor(LY294002)was pretreated for 2 h and then stimulated with leptin,the mRNA and protein levels of p16 and p21 were detected by qPCR and Western blot;the mRNA levels of SASP were examined by qPCR;MCM senescence was detected byβ-galactosidase staining.Results:In MCM stimulated by leptin,the mRNA and protein levels of p16 and p21,as well as the protein level ofγ-H2AX increased,the mRNA levels of SASP[(interleukin,IL)-1β,tumor necrosis facto(r TNF)-α、IL-6,monocyte chemoattractant protein(MCP)-1]were up-regulated,the phosphorylation levels of proteins in PI3K/AKT signaling pathway increased;andβ-galactosidase staining showed the senescence of MCM.When pretreated with PI3K inhibitor for 2 h,the mRNA and protein levels of p16 and p21,as well as the protein level ofγ-H2AX were down-regulated,and the expressions of SASP mRNA were down-regulated,the senescence of MCM was alleviated.Conclusion:Leptin regulates the progression of MCM senescence by activating PI3K/AKT signaling pathway and promoting SASP(IL-1β,TNF-α,IL-6 and MCP-1)secretion.