tirzepatide对AngⅡ诱导的心肌细胞肥大的抑制作用及机制研究

Inhibitory Effect and Mechanism of Tirzepatide on Angiotensin Ⅱ-Induced Cardiomyocyte Hypertrophy

目的探讨tirzepatide(TZP)对血管紧张素Ⅱ(AngⅡ)诱导新生大鼠心肌细胞肥大的影响及机制研究。方法分离新生大鼠心肌细胞并进行体外培养,采用AngⅡ(1μmol/L)刺激心肌细胞24 h建立心肌细胞肥大模型,使用TZP(100 nmol/L)和AngⅡ(1μmol/L)共同孵育新生大鼠心肌细胞24 h。实验随机分为4组:正常对照组、TZP组、AngⅡ组和AngⅡ+TZP组。采用鬼笔环肽染色评估单个心肌细胞肥大程度;TUNEL检测心肌细胞凋亡情况;CCK-8法检测心肌细胞活力;2,7-二氯二氢荧光素二乙酸酯荧光探针检测活性氧水平;逆转录聚合酶链反应检测新生大鼠心肌细胞中心房利尿钠肽、脑钠肽、β-肌球蛋白重链、B细胞淋巴瘤-2相关X蛋白(Bax)、B细胞淋巴瘤-2(Bcl-2)、NADPH氧化酶(Nox)2、Nox4和NADPH氧化酶活化蛋白1(NOXA2)、超氧化物歧化酶2(SOD2)、NADPH醌氧化还原酶1(NQO1)和谷胱甘肽过氧化物酶(GSH-Px)的mRNA表达水平;Western blot检测新生大鼠心肌细胞中脑钠肽、Bax、Bcl-2、SOD2、NQO1和GSH-Px蛋白质的表达水平。此外,逆转录聚合酶链反应和Western blot检测各组心肌细胞中的Beclin-1和p62的mRNA和蛋白质表达水平。结果TZP可显著降低AngⅡ诱导的心肌细胞肥大标志物和凋亡标志物的蛋白质及mRNA表达水平的升高,下调促氧化因子Nox2、Nox4和NOXA2的mRNA表达水平,促进抗氧化因子SOD2、NQO1和GSH-Px的mRNA表达水平。以上作用与正常对照组比较差异均有统计学意义(P<0.05)。同时,Western blot和自噬小体检测证实了TZP主要通过抑制细胞内自噬途径来拮抗AngⅡ诱导的心肌细胞肥大。结论TZP可缓解AngⅡ诱导的心肌细胞肥大,并通过抑制自噬途径改善AngⅡ诱导的心肌细胞肥大作用。

Objective To investigate the effect and mechanism of tirzepatide(TZP)in angiotensinⅡ(AngⅡ)-induced neonatal rat cardiomyocyte hypertrophy.Methods Neonatal rat cardiomyocytes were isolated and cultured in vitro,AngⅡ(1μmol/L)was used to stimulate cardiomyocytes for 24 h to establish a cardiomyocyte hypertrophy model,and neonatal rat cardiomyocytes were co-incubated using TZP(100 nmol/L)and AngⅡ(1μmol/L)for 24 h.The experiment will be randomly divided into four groups:control group,TZP group,AngⅡgroup,and AngⅡ+TZP group.Phalloidin staining was used to assess the degree of hypertrophy of individual cardiomyocytes;TUNEL staining was used to detect cardiomyocyte apoptosis;CCK-8 was used to detect cardiomyocyte activity;DCFH-DA fluorescent probe was used to detect the level of reactive oxygen species(ROS);and reverse transcription-polymerase chain reaction was used to detect the mRNA levels of atrial natriuretic peptide,brain natriuretic peptide,andβ-myosin heavy chain(β-MHC),B-cell lymphoma-2 associated X protein(Bax),B-cell lymphoma-2(Bcl-2),reduced nicotinamide adenine dinucleotide phosphate oxidase(Nox)2,Nox4,reduced nicotinamide adenine dinucleotide phosphate oxidase activator 2(NOXA2),superoxide dismutase 2(SOD2),NADPH quinone oxidoreductase 1(NQO1)and glutathione peroxidase(GSH-Px).The expression levels of brain natriuretic peptide,Bax,Bcl-2,SOD2,NQO1 and GSH-Px proteins were detected by Western blot in neonatal rat cardiomyocyte.In addition,reverse transcription-polymerase chain reaction and Western blot were performed to detect the mRNA and protein expression levels of Beclin-1 and p62 in cardiomyocytes of each group.Results TZP can significantly reduce the increase in protein and mRNA expression levels of cardiomyocyte hypertrophy markers and apoptosis markers induced by AngⅡ,down-regulate the mRNA expression levels of pro-oxidant factors Nox2,Nox4 and NOXA2,and promote the antioxidant factors SOD2,NQO1 and GSH-Px mRNA expression levels.Compared with the control group,the above effects were statistically significant(P<0.05).Meanwhile,Western blot and autophagosome assay confirmed that TZP antagonized AngⅡ-induced cardiomyocyte hypertrophy mainly by inhibiting intracellular autophagy pathway.Conclusion TZP can alleviate AngⅡ-induced cardiomyocyte hypertrophy and improve the effect of AngⅡ-induced cardiomyocyte hypertrophy by inhibiting autophagy.