热量限制缓解血管紧张素Ⅱ诱导的心肌细胞肥大作用

Caloric restriction alleviates angiotensin-Ⅱ-induced cardiomyocyte hypertrophy

目的探讨热量限制(caloric restriction,CR)对血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)诱导的大鼠心肌细胞肥大的影响及可能机制。方法将H9c2细胞分为6组:正常对照组、AngⅡ组(1μmol/L)、热量限制组(CR)、CR+AngⅡ组、NF-κB抑制剂组(CR+AngⅡ+Ss)、NF-κB激动剂组(CR+AngⅡ+Bet)。将各组细胞进行相应的药物处理后,用CCK-8法检测细胞活力;鬼笔环肽染色检测心肌细胞表面积;细胞培养液中乳酸脱氢酶(LDH)、髓过氧化物酶(MPO)及氧化应激指标(ROS、SOD、MDA)的检测采用相应试剂盒;RT-qPCR法检测心肌细胞中肥大相关基因(ANP、BNP、β-MHC)与焦亡相关基因(NLRP3、ASC、GSDMD、caspase1、IL-18与IL-1β)的mRNA表达;Western blot法检测心肌细胞中NLRP3、ASC、GSDMD、caspase1以及NF-κB的蛋白表达水平。结果(1)AngⅡ可诱导H9c2心肌细胞表面积及肥大标志基因(ANP、BNP、β-MHC)的mRNA表达量较对照组显著增加,而这种改变在CR干预下被减弱。(2)CR能逆转AngⅡ诱导的肥大心肌细胞中ROS、MDA、LDH、MPO含量的增加及SOD含量的减少。(3)与AngⅡ组相比,CR处理使AngⅡ诱导的肥大心肌细胞中焦亡相关基因NLRP3、ASC、GSDMD、caspase1及NF-κB的mRNA表达量与蛋白表达量明显降低。(4)NF-κB抑制剂水杨酸钠(Ss)使NLRP3、GSDMD的mRNA表达水平较CR+AngⅡ组进一步降低,而NF-κB激动剂白桦脂酸(BA)则逆转了CR的这种保护作用。结论CR可以明显缓解AngⅡ诱导的大鼠心肌细胞肥大,CR的心肌细胞保护作用可能与调控NF-κB途径介导的细胞焦亡有关。

Objective To investigate the effect of calorie restriction(CR)cardiomyocyte hypertrophy induced by angiotensinⅡ(AngⅡ)and the possible mechanisms involved.Methods H9c2 cells were divided into six groups:Control,AngⅡ(1μmol/L),CR,CR+AngⅡ,CR+AngⅡ+Ss and CR+AngⅡ+Bet groups.The cell viability of each group was detected using the CCK-8 method after corresponding drug treatments.The surface area of cardiomyocytes was detected by TRITC-phalloidin staining.The concentrations of lactic dehydrogenase(LDH),myeloperoxidase(MPO),Reactive oxygen species(ROS),Superoxide dismutase(SOD),and Malondialdehyde(MDA)in cell culture supernatants were detected with kits.The mRNA expression of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),myosin heavy chain-β(β-MHC),NOD-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein(ASC),gasdermin-D(GSDMD),caspase1,interleukin-18(IL-18),and interleukin-1β(IL-1β)was detected by RT-qPCR,and the protein levels of NLRP3,ASC,GSDMD,caspase1 and Nuclear factor-κB(NF-κB)in cardiomyocytes were detected by Western blot.Results The surface area of H9c2 cardiomyocytes and the mRNA expression levels of hypertrophy markers(ANP,BNP,β-MHC)were significantly increased in AngⅡ-induced H9c2 cardiomyocytes compared with the controls,and these changes were attenuated by CR intervention.CR reversed the AngⅡ-induced increase in LDH and MPO in myocardium mast cells.The mRNA expression levels of NLRP3,ASC,GSDMD,caspase1,IL-18,and IL-1βand the protein levels of NLRP3,ASC,GSDMD,caspase1,and NF-κB were significantly decreased in AngⅡ-induced hypertrophied cardiomyocytes under CR intervention compared with AngⅡonly.An NF-κB inhibitor further reduced the mRNA expression of NLRP3 and GSDMD compared with expression in the CR+AngⅡgroup,while an NF-κB agonist blocked the effect of CR on the mRNA expression of coke-death related genes.Conclusions CR reduced the cardiomyocyte hyperplasia induced by AngⅡin rats,and it may protect cardiomyocytes by inhibiting pyroptosis through the regulation of NF-κB.