抑制泛素特异性蛋白酶7改善血管紧张素Ⅱ诱导的心肌细胞肥大

Inhibition of Ubiquitin-Specific Protease 7 Improves AngiotensinⅡ-Induced Cardiomyocyte Hypertrophy

目的探究泛素特异性蛋白酶7(USP7)抑制剂FT671在血管紧张素Ⅱ(AngⅡ)诱导的H9c2细胞肥大中的作用和潜在机制。方法将H9c2细胞随机分为四组:对照组、FT671组、AngⅡ组、FT671+AngⅡ组。利用α-actinin细胞免疫荧光染色检测心肌细胞表面积;Western blot检测心房钠尿肽(ANP)、脑钠肽(BNP)、B细胞淋巴瘤-2(Bcl-2)、B细胞淋巴瘤-2相关X蛋白(Bax)、白细胞介素-6(IL-6)、单核细胞趋化蛋白-1(MCP-1)、肿瘤坏死因子-α(TNF-α)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)以及NADPH氧化酶4(Nox4)的蛋白表达水平;RT-PCR检测ANP、BNP、β-肌球蛋白重链(β-MHC)、IL-6、MCP-1、TNF-α的mRNA表达;TUNEL染色检测细胞凋亡情况;细胞计数试剂8(CCK8)实验检测各组细胞活力;活性氧(ROS)染色检测细胞内氧化损伤水平。结果与对照组相比,AngⅡ组USP7蛋白表达明显增加,而使用FT671后,USP7表达明显被抑制。与AngⅡ组相比,FT671+AngⅡ组心肌细胞面积减小;ANP、BNP、Bax的蛋白表达减少,ANP、BNP、β-MHC、Bax、IL-6、MCP-1以及TNF-α的mRNA表达减少;Bcl-2的蛋白和mRNA表达均增加。与AngⅡ组相比,FT671+AngⅡ组TUNEL阳性细胞明显减少,心肌细胞活力增强,ROS生成减少,Nox4、NLRP3蛋白表达减少,差异具有统计学意义(P<0.05)。结论FT671通过抑制Nox4/ROS/NLRP3炎症通路减轻AngⅡ诱导的心肌细胞中的氧化应激和炎症反应,从而减轻心肌细胞肥大。

Objective To investigate the effect and mechanism of ubiquitin-specific protease 7(USP7)inhibitor FT671 in cardiomyocyte hypertrophy induced by angiotensinⅡ(AngⅡ).Methods H9c2 cardiomyocytes were randomly divided into four groups:control group,FT671 group,AngⅡgroup,FT671+AngⅡgroup.The surface area of cardiomyocytes was detected by immunofluorescence staining ofα-actinin.The protein expression of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),B-cell lymphoma-2(Bcl-2),B-cell lymphoma-2-associated X protein(Bax),interleukin-6(IL-6),monocyte chemoattractant protein-1(MCP-1),tumor necrosis factor-α(TNF-α),NOD-like receptor thermal protein domain associated protein 3(NLRP3)and nicotinamide adenine dinucleotide phosphate oxidase 4(Nox4)were detected by Western blot.The mRNA levels of ANP,BNP,β-myosin heavy chain(β-MHC),IL-6,MCP-1,TNF-αwere detected by real-time PCR.TUNEL staining was used to evaluate cell apoptosis.Cell counting kit-8(CCK8)was used to detect cell viability.The fluorescent probe 2’,7’-dichlorofluorescein diacetate(DCFH-DA)was used to measure intracellular reactive oxygen species(ROS)formation.Results There was a significant increase in USP7 protein expression in the AngⅡgroup,which was evidently inhibited by the USP7 inhibitor FT671.Compared with AngⅡgroup,cardiomyocyte surface area was significantly reduced in FT671+AngⅡgroup;the protein expression of ANP,BNP and Bax,as well as mRNA level of ANP,BNP,β-MHC,Bax,IL-6,MCP-1,TNF-α,were decreased in FT671+AngⅡgroup.In addition,the protein expression and mRNA level of Bcl-2 were increased in FT671+AngⅡgroup.Additionally,compared with AngⅡgroup,TUNEL positive cells was significantly reduced,cell viability was improved and ROS generation was inhibited in FT671+AngⅡgroup.Further studies showed the protein expression of NLRP3 and Nox4 was decreased after FT671 treatment in AngⅡinduced cardiomyocyte hypertrophy.Conclusion The USP7 inhibitor FT671 attenuated oxidative stress and inflammatory response in AngⅡ-induced cardiomyocyte by inhibiting the Nox4/ROS/NLRP3 inflammatory pathway,thereby reducing cardiomyocyte hypertrophy.