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 active β-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 active β-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 active β-catenin、 p-GSK3β表达降低（P < 0.05）。与对照组比较，［Leu31，Pro34］-NPY 显著增加心肌细胞 active β-catenin、p-GSK3β表达（P < 0.05），促进细胞核β-catenin 积累。与［Leu31，Pro34］-NPY 组比较，BIBO3304 抑制细胞核β-catenin 表达，ICG001 显著缓解 ［Leu31，Pro34］-NPY诱导的心肌细胞肥大和细胞活力下降（P < 0.01）。结论：NPY通过Y1受体转导激活β-catenin信号通路介导心肌细胞损伤和心肌纤维化。
Abstract:
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）and β-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 and β-MHC in the cardiomyocytes were detected by qPCR，and the cell viability of H9C2 was measured by CCK-8. The expressions of active β-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. Nuclear β- catenin accumulation was estimated by immunofluorescence staining. ICG001，a specific β-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+ISO group，and the expression of cardiac hypertrophy related gene were decreased（P < 0.01）. Compared with the control group，［Leu31，Pro34］-NPY increased ANP and β-MHC mRNA levels， and decreased cell viability in H9C2 cardiomyocytes（P < 0.01）. Compared with the control group，the protein expressions of active β- catenin and p-GSK3β/t-GSK3β were increased in the heart of mice from ISO group（P < 0.05）. Compared with the ISO group，the expressions of active β-catenin and p-GSK3β were decreased in BIBO3304+ISO group（P < 0.05）. Compared with the control group， ［Leu31，Pro34］-NPY increased expressions of active β-catenin and p-GSK3β，as well as facilitated nuclear β-catenin accumulation in the cardiomyocytes（P < 0.05）. Compared with［Leu31，Pro34］-NPY group，BIBO3304 decreased β-catenin expression in the nucleus， and ICG001 effectively alleviated cardiomyocyte hypertrophy and cell viability reduction（P < 0.01）. Conclusion：NPY/Y1 receptor mediates cardiomyocyte injury and fibrosis through β-catenin pathway.
