山楂酸通过抑制细胞焦亡以减轻心肌缺血再灌注损伤的机制研究

Maslinic acid alleviates myocardial ischemia/reperfusion injury by inhibiting pyroptosis

目的探讨山楂酸对心肌缺血再灌注(IR)损伤中细胞焦亡和炎性反应的作用机制。方法H9C2心肌细胞随机分为对照组、对照+山楂酸组、缺氧复氧(HR)组、HR+山楂酸组,通过缺氧4h、复氧12h构建HR损伤模型。将48只雄性SD大鼠随机分为假手术组、IR组、IR+山楂酸组、IR+山楂酸+Tri组,每组12只。通过结扎大鼠心脏左前降支30min、再灌注2h构建IR损伤模型。检测心肌细胞上清液和血清乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)、白细胞介素(IL)-1β、IL-18水平,使用药物-分子对接预测山楂酸与NOD样受体热蛋白结构域相关蛋白3(NLRP3)的结合位点和结合力,Westernblot检测NF-κB抑制蛋白α(IκBα)、NF-κBP65、NLRP3、凋亡相关斑点样蛋白(ASC)、消皮素D-N端片段(GSDMD-N)表达情况。结果与对照组比较,HR组细胞活力明显降低,磷酸化IκBα、磷酸化NF-κBP65蛋白表达、LDH、IL-1β和IL-18水平明显升高(P<0.05);与HR组比较,HR+山楂酸组细胞活力明显升高,磷酸化IκBα、磷酸化NF-κBP65蛋白表达、LDH、IL-1β和IL-18水平明显降低(P<0.05)。与假手术组比较,IR组磷酸化IκBα、磷酸化NF-κBP65、NLRP3、ASC、GSDMD-N蛋白表达、血清CK-MB、LDH、IL-1β和IL-18水平明显升高(P<0.05);与IR组比较,IR+山楂酸组磷酸化IκBα、磷酸化NF-κBP65、NLRP3、ASC、GSDMD-N蛋白表达、血清CK-MB、LDH、IL-1β和IL-18水平明显降低(P<0.05);与IR+山楂酸组比较,IR+山楂酸+Tri组磷酸化IκBα、磷酸化NF-κBP65、NLRP3、ASC、GSDMD-N蛋白表达明显升高,血清CK-MB、LDH、IL-1β和IL-18水平明显升高[(1681.00±136.20)U/Lvs(1251.00±213.60)U/L,(1776.00±185.80)U/Lvs(1330.00±172.50)U/L,4.32±0.45vs2.95±0.26,3.89±0.20vs2.47±0.29,P<0.05]。结论山楂酸可靶向干预NLRP3活性,从而抑制炎性反应和细胞焦亡,最终有效减轻心肌IR损伤。

Objective To investigate the mechanism of maslinic acid on pyroptosis and inflammato-ry response in myocardial ischemia/reperfusion (IR)injury.Methods H9C2 cardiomyocytes were randomly divided into control group,control±maslinic acid group,hypoxia reoxygenation(HR)group,and HR+maslinic acid group.Cellular model of HR injury was constructed by hypoxia for 4 h and then reoxygenation for 12 h.Forty-eight maee SD rats were randomly divided into Sham group,IRgroup,IR-maslinic acid group,IR+maslinic acid+Tri group(n=12).Rat model of myocardial 1R injury was established by ligating the left anterior descending branch for 30 min followed by reperfusion for 2 h.The viability of cardiomyocytes was detected,the levels of LDH,CK-MB,IL-1β and IL-18 in the supernatant of cardiomyocytes and rat serum samples were detected in each group.Drug-molecular docking was performed to predict the binding site and binding force of maslinic acid and NOD-likc receptor thermal protein domain-associated protein 3(NLRP3).Western blotting was used to detect KBa,NFkB P65,NLRP3,apoptosis-associated speck-like protein(ASC),and gasdermid D-N terminal(GSDMD-N)in each group of cardiomyo-cytes and myocardial tissues.Results Compared with the Control group,significantly reduced cell viability,enhanced protein levels of p-IκBa,p-NF-κB P65 and higher releases of IDH,IL-1β and IL-18 were observed in the HR group(P<0.05).Maslinic acid treatment reversed HR-induced changes in above indicators(P<0.05).Compared with the Sham group,the protein levels of p-κBa,p-NF-κB P65,NLRP3,ASC,GSDMD-N and the releases of serum CK-MB,LDH,IL-1β and IL-1β were significantly increased in the IR group(P<0.05).Maslinic acid treatment also reversed above indicators induced by 1R injury(P<0.05).The protein levels of p-IκBa,p-NF-κB P65,NLRP3,ASC and GSDMD-N were significantly increased,and the releases of serum CK-MB,LDH,IL-1β and IL-18 were also elevated in the IR±maslinic acid±Tri group than the IR+maslinic acid group(1681.00±136.20 U/L vs 1251.00±213.60 U/L,1776.00±185.80 U/L vs 1330.00±172.50 U/L,4.32±0.45 vs 2.95±0.26,3.89±0.20 vs 2.47±0.29,P<0.05).Conclusion Maslinic acid can show target intervention in NLRP3 activity,thereby inhibiting inflammatory re-sponse and cell pyroptosis,and ultimately attenuate myocardial IR injury effectively.