神经肽Y/Y1受体激活β⁃catenin信号通路介导心肌细胞损伤  

Neuropeptide Y/Y1 receptor activiation mediates cardiomyocyte injury throughβ⁃catenin signaling pathway

作　　者：胡君 戴丽 陈霞 任志欣 钱炳俊 HU Jun;DAI Li;CHEN Xia;REN Zhixin;QIAN Bingjun(School of Public Health and Management,Jiangsu Vocational College of Medicine,Yancheng 224005,China)

机构地区：[1]江苏医药职业学院公共卫生与管理学院,江苏盐城224005

出　　处：《南京医科大学学报（自然科学版）》2024年第3期313-321,共9页Journal of Nanjing Medical University(Natural Sciences)

基　　金：江苏省高等学校自然科学研究项目(20KJB310029,21KJB310012);盐城市科技计划项目(YCBK2023030);江苏医药职业学院高层次人才科研启动项目(20200026)。

摘　　要：目的:研究神经肽Y(neuropeptide Y,NPY)/Y1受体信号转导在心肌细胞损伤中的作用及机制。方法:C57BL/6J小鼠皮下注射异丙肾上腺素(isoprenaline,ISO)构建心肌损伤模型,腹腔注射Y1受体特异性拮抗剂BIBO3304干预。小鼠随机分为对照组(生理盐水)、ISO组[20 mg/(kg·d)ISO]、BIBO3304+ISO组[0.1 mg/(kg·d)BIBO3304+20 mg/(kg·d)ISO]、BIBO3304组[0.1 mg/(kg·d)BIBO3304],每组10只,连续给药14 d。实时定量PCR和Western blot检测小鼠心肌组织中NPY表达。HE染色和Masson染色观察各组小鼠心肌纤维结构变化和纤维化程度;定量PCR检测小鼠心肌肥大基因心房钠尿肽(atrial natriuretic peptide,ANP)、β⁃肌球蛋白重链(β⁃myosin heavy chain,β⁃MHC)mRNA表达。采用Y1受体特异性激活剂[Leu31,Pro34]⁃NPY刺激H9C2细胞,检测ANP、β⁃MHC mRNA表达;CCK⁃8检测心肌细胞活力。Western blot检测各组小鼠心肌组织和H9C2细胞中activeβ⁃catenin、磷酸化糖原合成酶激酶⁃3β(phospho⁃glycogen synthesis kinase 3β,p⁃GSK3β)、总糖原合成酶激酶⁃3β(total glyco⁃gen synthesis kinase 3β,t⁃GSK3β)蛋白表达。免疫荧光染色检测心肌细胞β⁃catenin入核情况。利用β⁃catenin特异性抑制剂ICG001处理细胞,检测[Leu31,Pro34]⁃NPY诱导的心肌细胞肥大和细胞活力变化。结果:与对照组比较,ISO组小鼠心肌组织NPY mRNA和蛋白表达均显著增加(P<0.05),心肌纤维排列紊乱,心肌纤维化程度高,心肌肥大基因表达增加。与ISO组比较,BIBO3304+ISO组小鼠心肌损伤和纤维化得到有效缓解,心肌肥大基因表达下降(P<0.01)。与对照组比较,[Leu31,Pro34]⁃NPY增加H9C2细胞ANP、β⁃MHC mRNA表达,降低心肌细胞活力(P<0.01)。与对照组比较,ISO组小鼠心肌组织activeβ⁃catenin、p⁃GSK3β表达明显上调,p⁃GSK3β/t⁃GSK3β增加;与ISO组比较,BIBO3304+ISO组心肌组织activeβ⁃catenin、p⁃GSK3β表达降低(P<0.05)。与对照组比较,[Leu31,Pro34]⁃NPY显著增加心Objective:To explore the effect and mechanism of neuropeptide Y(NPY)/Y1 receptor signaling in myocardial injury.Methods:The C57BL/6J mice model of cardiac injury was established by subcutaneous injection with isoproterenol(ISO),which was subsequently treated with the specific NPY/Y1 receptor antagonist BIBO3304 by intraperitoneal injection.C57BL/6J mice were randomly divided into 4 groups:control group(Saline),ISO group[20 mg/(kg·d)ISO],BIBO3304+ISO group[0.1 mg/(kg·d)BIBO3304+20 mg/(kg·d)ISO],and BIBO3304 group[0.1 mg/(kg·d)BIBO3304].All the drug was administered continuously for 14 days.The expression of NPY mRNA and protein in the heart of mice were detected by qPCR and Western blot,respectively.The change of myocardial fiber structure and myocardial fibrosis were observed by HE and Masson staining.Levels of cardiac hypertrophy related gene,atrial natriuretic peptide(ANP)andβ⁃myosin heavy chain(β⁃MHC)mRNA in the heart of mice were also estimated by qPCR.[Leu31,Pro34]⁃NPY,a specific Y1 receptor agonist,was used to directly treat H9C2 cells in vitro.The mRNA levels of ANP andβ⁃MHC in the cardiomyocytes were detected by qPCR,and the cell viability of H9C2 was measured by CCK⁃8.The expressions of activeβ⁃catenin,phospho⁃glycogen synthesis kinase 3β(p⁃GSK3β)and total glycogen synthesis kinase 3β(t⁃GSK3β)in the heart and H9C2 cells were analyzed by Western blot.Nuclearβ⁃catenin accumulation was estimated by immunofluorescence staining.ICG001,a specificβ⁃catenin inhibitor,was used to treat H9C2,and the cardiomyocyte hypertrophy and cell viability induced by[Leu31,Pro34]⁃NPY were further examined.Results:Compared with the control group,cardiac NPY mRNA and protein expressions were increased significantly in ISO group(P<0.05),in which it exhibited myocardial fiber arrangement disorder,high degree of myocardial fibrosis and increased expression of cardiac hypertrophy related gene.Compared with the ISO group,the myocardial damage and fibrosis were effectively alleviated in BIBO3304+I

关 键 词：NPY/Y1受体 β⁃catenin信号通路 心肌损伤 心肌纤维化 

分 类 号：R541.78[医药卫生—心血管疾病]