神经肽Y/Y1受体激活β-catenin信号通路介导心肌细胞损伤
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江苏医药职业学院

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]江苏省高等学校自然科学研究面上项目(20KJB310029);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)


Neuropeptide Y/Y 1 receptor induced cardiomyocyte injury through β-catenin signaling pathway
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Jiangsu Vocational College of Medicine,Yancheng,224005

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    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小鼠皮下异丙肾上腺素(Isoprenaline, ISO)构建心肌损伤模型,采用腹腔注射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/day ISO)、BIBO3304+ISO组(0.1 mg/kg/day BIBO3304+20 mg/kg/day ISO)、BIBO3304组(0.1 mg/kg/day BIBO3304),每组10只,连续给药14天。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ANP、β-MHC mRNA表达。采用Y1受体特异性激活剂[Leu31,Pro34]-NPY刺激H9C2细胞,检测心肌肥大基因ANP、β-MHC mRNA表达;CCK8检测心肌细胞活力。Western blot检测各组小鼠心肌组织和H9C2细胞active β-catenin、p-GSK3β、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:与对照组相比,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组对比,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)。与对照组相比,[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)。与对照组相比,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组相比,BIBO3304+ISO组心肌组织active β-catenin、p-GSK3β表达降低(p<0.05)。与对照组相比,[Leu31,Pro34]-NPY显著增加心肌细胞active β-catenin、p-GSK3β表达(p<0.05)。与对照组相比,[Leu31, Pro34]-NPY促进细胞核β-catenin积累;与[Leu31, Pro34]-NPY组相比,BIBO3304抑制细胞核β-catenin表达。与[Leu31, Pro34]-NPY组相比,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: In this study, we aimed to explore the effect and mechanism of NPY/Y1 receptor (YIR) 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 Y1R antagonist BIBO3304 by intraperitoneal injection. Forty male C57BL/6J mice were randomly divided into 4 groups: control group (Saline), ISO group (20 mg/kg/day ISO), BIBO3304+ISO group (0.1 mg/kg/day BIBO3304+20 mg/kg/day ISO), and BIBO3304 group (0.1 mg/kg/day BIBO3304). All the drug was administered continuously for 14 days. H9C2 cells were cultured and directly treated with the specific Y1R agonist [Leu31, Pro34]-NPY in vitro. Real-time PCR was used to investigate the expression of NPY, ANP, β-MHC mRNA in the heart of mice and/or in H9C2 cells. Western blot analysis was used to detect the protein expression of NPY, active β-catenin, p-GSK3β and total-GSK3β in the heart and/or H9C2 cells. HE and Masson staining were used to observe the changes of myocardial fiber structure and the degree of myocardial fibrosis. The viability of H9C2 cardiomyocytes was detected by CCK8. Nuclear β-catenin accumulation was detected by immunofluorescence staining. ICG001, a specific β-catenin inhibitor was used to treat H9C2, and then the cardiomyocyte hypertrophy and cell viability induced by [Leu31, Pro34]-NPY were further examined. Results: Compared with the Saline group, cardiac NPY mRNA and protein expressions increased significantly in ISO group (p<0.05), in which it also exhibited myocardial fiber arrangement disorder, cardiomyocyte necrosis, high degree of myocardial fibrosis and increased gene expressions of cardiac hypertrophy. Compared with the ISO group, the myocardial damage and fibrosis were effectively alleviated in BIBO3304+ISO group, and the expressions of cardiac hypertrophy gene decreased significantly (p<0.01). With [Leu31, Pro34]-NPY direct treatment, the level of ANP and β-MHC mRNA expression increased in H9C2, while the cardiomyocytes viability decreased (p<0.01). It showed that the protein expression levels of active β-catenin and p-GSK3β/t-GSK3β increased in the heart of mice from ISO group (p<0.05) and [Leu31, Pro34]-NPY induced H9C2 cells (p<0.05). The administration of BIBO3304 reversed the protein changes (p<0.05). [Leu31, Pro34]-NPY facilitated β-catenin accumulation in the nucleus, while BIBO3304 decreased β-catenin expression in the nucleus. Compared with the [Leu31, Pro34]-NPY group, ICG001 could effectively alleviated cardiomyocyte hypertrophy and cell viability reduction (p<0.01). Conclusion: These findings suggested NPY/Y1 receptor mediated cardiac injury and fibrosis through β-catenin pathway.

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  • 收稿日期:2023-07-19
  • 最后修改日期:2023-11-16
  • 录用日期:2024-02-27
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