吉林大学学报(医学版) ›› 2023, Vol. 49 ›› Issue (3): 675-681.doi: 10.13481/j.1671-587X.20230316

• 基础研究 • 上一篇    下一篇

二甲双胍对H9C2心肌细胞肥大的改善作用及其机制

黄德盛1,赵亚楠1,陈云1,孙艺瑶1,覃伟林1,刘谋杰2,谢菊华1()   

  1. 1.沈阳医学院组织胚胎学教研室,辽宁 沈阳 110034
    2.中国医科大学附属第一医院心内科,辽宁 沈阳 110034
  • 收稿日期:2022-08-01 出版日期:2023-05-28 发布日期:2023-06-20
  • 通讯作者: 谢菊华 E-mail:xjh814@126.com
  • 作者简介:黄德盛(1996-),男,辽宁省大连市人,在读硕士研究生,主要从事心血管疾病基础和临床方面的研究。
  • 基金资助:
    辽宁省科技厅科学技术计划项目(2018225013);辽宁省沈阳市“高层次创新人才计划”项目(RC190477);沈阳医学院科学研究基金项目(20181005);沈阳医学院2021年校级大学生科研项目(20219051)

Improvement effect of metformin on hypertrophy of H9C2 cardiomyocytes and its mechanism

Desheng HUANG1,Yanan ZHAO1,Yun CHEN1,Yiyao SUN1,Weilin QIN1,Moujie LIU2,Juhua XIE1()   

  1. 1.Department of Tissue and Embryology, Shenyang Medical College, Shenyang 110034, China
    2.Department of Cardiology, First Affiliated Hospital, China Medical University, Shenyang 110034, China
  • Received:2022-08-01 Online:2023-05-28 Published:2023-06-20
  • Contact: Juhua XIE E-mail:xjh814@126.com

摘要:

目的 探讨二甲双胍对异丙肾上腺素(ISO)诱导的H9C2心肌细胞肥大的作用,并阐明其可能的分子机制。 方法 培养H9C2细胞,采用50 μmol·L-1 ISO干预H9C2细胞建立心肌细胞肥大模型作为ISO组;不予处理的细胞作为对照组。Western blotting法检测2组H9C2心肌细胞中脑钠肽(BNP)蛋白表达水平。将H9C2心肌细胞分为对照组、ISO组、ISO+二甲双胍组和二甲双胍组。采用CCK-8法检测各组H9C2细胞活力,选取二甲双胍的最佳药物干预浓度和时间,采用结晶紫染色观察各组H9C2心肌细胞形态表现和横截面积,采用实时荧光定量PCR(RT-qPCR)法检测各组细胞中心房钠尿肽(ANP)mRNA表达水平,线粒体荧光探针观察各组细胞中线粒体形态表现,Western blotting法检测各组细胞中线粒体融合标志性蛋白神经萎缩蛋白1(OPA1)和线粒体融合蛋白(MFN)2蛋白表达水平。 结果 与对照组比较,ISO 组H9C2心肌细胞横截面积增加(P<0.05),细胞中BNP蛋白表达水平升高(P<0.05),提示心肌细胞肥大模型造模成功。CCK-8法检测,2 mmol·L-1 二甲双胍作用8 h时细胞活力恢复效果最佳。RT-qPCR法检测,与对照组比较,ISO组细胞中ANP mRNA表达水平升高(P<0.05);与ISO组比较,二甲双胍+ISO组和二甲双胍组细胞中ANP mRNA表达水平降低(P<0.05)。线粒体荧光探针观察,对照组细胞中线粒体呈绿色荧光,多为短棒状,少数线粒体丝线状相连;与对照组比较,ISO组H9C2细胞中点状线粒体明显增加,ISO+二甲双胍组和二甲双胍组细胞中点状线粒体明显减少。Western blotting法检测,与对照组比较,ISO组细胞中OPA1和MFN2蛋白表达水平降低(P<0.05);与ISO组比较,ISO+二甲双胍组和二甲双胍组细胞中OPA1和MFN2蛋白表达水平升高(P<0.05)。 结论 二甲双胍可改善ISO诱导的H9C2心肌细胞肥大,其机制可能与其上调OPA1和MFN2蛋白表达及促进线粒体融合有关。

关键词: 心肌细胞肥大, 二甲双胍, 线粒体融合, 异丙肾上腺素, 心房钠尿肽

Abstract:

Objective To discuss the effect of metformin on the hypertrophy of the H9C2 cardiomyocytes induced by isoproterenol (ISO), and to clarify its possible molecular mechanism. Methods The H9C2 cells were cultured; 50 μmol·L-1 ISO was used to establish the cardiomyocyte hypertrophy model,and they were used as ISO group;the cells without any treatment were used as control group. The expression levels of brain natriuretic peptide (BNP) in the H9C2 cardiomyocytes in two groups were detected by Western blotting method.The cells were divided into control group, ISO group, ISO+metformin group, and metformin group.The viabilities of the H9C2 cardiomyocytes in two groups were detected by CCK-8 method; the optimal drug intervention concentration and time of metformin were selected;crystal violet staining was used to observe the morphology and cross-sectional area of the H9C2 cardiomyocytes;real-time fluorescence qualitative PCR(RT-qPCR) method was used to detect the expression levels of atrial natriuretic peptide (ANP) in the cells in various groups;the morphology of mitochondriums in the H9C2 cardiomyocytes was observed by fluorescence probes; Western blotting method was used to detect the expression levels of optic atrophy 1 (OPA1) and mitofusin(MFN) proteins in the cells in various groups. Results Compared with control group,the cross-sectional area of the H9C2 cardiomyocytes in ISO group was increased significantly (P<0.05), and the expression level of BNP protein was increased significantly (P<0.05), suggesting that the cardiomyocyte hypertrophy model was successfully modeled. The RT-qPCR results showed that compared with control group, the expression level of ANP mRNA in the cells in ISO group was increased(P<0.05); compared with ISO group, the expression levels of ANP mRNA in the cells in metformin+ISO and metformin groups were decreased(P<0.05). The mitochondrium fluorescence probe observing results showed that the punctate mitochondrum in control group showed green fluorescence, mostly short rod-shaped, and a few mitochondrum were filamentously connected;compared with control group, the number of punctate mitochondrum in the H9C2 cells in ISO group was significantly increased,and the numbers of punctate mitochondrium in ISO+meformin and meformin groups were significantly decreased.The Western blotting results showed that compared with control group,the expression levels of OPA1 and MFN2 proteins in the cells in ISO group were decreased (P<0.05);compared with ISO group, the expression levels of OPA1 and MFN2 proteins in the cells in ISO+metformin and metformin groups were increased(P<0.05). Conclusion Metformin can alleviate the ISO-induced hypertrophy of the H9C2 cardiomyocytes,and its mechanism may be related to improving the mitochondrium fusion by up-regulating the expression levels of OPA1 and MFN2 proteins.

Key words: Cardiomyocyte hypertrophy, Metformin, Mitochondrial fusion, Isoproterenol, Atrial natriuretic peptide

中图分类号: 

  • R329.28