摘要
目的 探讨棕榈油酸(POA)对心肌缺氧/复氧损伤后心肌细胞焦亡的影响.方法 实验设置对照组(Control)、缺氧/复氧组(HR)、棕榈油酸处理组(HR+POA)、溶剂无水乙醇(ET)对照组(HR+ET);采用CCK-8法检测心肌细胞活力,Hoechst33342/PI双染和乳酸脱氢酶(LDH)试验检测心肌细胞焦亡水平.ELISA法检测细胞培养上清液中炎症因子白细胞介素(IL)-1β、IL-18释放量,qRT-PCR法和Western blot分别检测细胞焦亡相关分子NLRP3、ASC、Caspase-1、GS-DMD、1L-1β及IL-18的mRNA和蛋白相对表达水平.结果 CCK-8结果显示,与HR组相比,加入不同浓度POA干预,POA浓度在25～100 μmol/L时能增加缺氧/复氧心肌细胞活力;构建心肌细胞缺氧/复氧模型,与对照组相比,HR组中 NLRP3、ASC、Cleaved-caspase 1、GSDMD-N、IL-Iβ 及 IL-18基因的蛋白和mRNA表达增加(P＜0.05),Hoechst33342/PI心肌细胞染色阳性百分比增加(P＜0.05),且LDH、IL-Iβ及IL-18 释放增加(P＜0.05);100 μmol/L POA 干预后,与 HR+ET 组相比,HR+POA 组中 NLRP3、ASC、Cleaved-caspase 1、GSDMD-N、IL-Iβ及IL-18的蛋白和mRNA表达降低(P＜0.05),Hoechst33342/PI心肌细胞染色阳性百分比降低,且LDH、IL-Iβ及IL-18水平降低(P＜0.05).结论 棕榈油酸可能通过抑制缺氧/复氧后心肌细胞焦亡和炎症反应,改善心肌细胞缺氧/复氧损伤.
Abstract
Objective To investigate the effect of palmitoleic acid(POA)on pyroptosis of cardiomyocytes after hy-poxia/reoxygenation-induced injury in the human myocardium.Methods The experiment comprised a control group(Control,normal culture),a hypoxia/reoxygenation group(HR),a palmitoleic acid-treated group(HR+POA),and an anhydrous ethanol control group(HR+ET).Cardiomyocytes viability was assessed using CCK-8 assay,and the level of pyroptosis of cardiomyocytes was measured through the double staining with Hoechst33342/PI and LDH assay.ELISA was employed to detect the release of inflammatory factors IL-1 β and IL-18 in the cell culture supernatant.qRT-PCR and Western blot were utilized to determine the relative expression levels of mRNA and protein of pyroptosis-related genes,namely NLRP3,ASC,Caspase-1,GSDMD,IL-1 β and IL-18,respective-ly.Results CCK-8 assay showed that the survival of hypoxic/reoxygenated cardiomyocytes increased with the ad-dition of POA at concentrations ranging from 25 to 100 μmol/L,as compared to the HR group;a hypoxia/reoxy-genation model of cardiomyocyte was established.The expression of protein and mRNA increased in NLRP3,ASC,Cleaved caspase-1,GSDMD-N,IL-Iβ and IL-18 vs the control group(P＜0.05),the positive percentage of Ho-echst33342/PI staining in cardiomyocytes increased significantly(P＜0.05),the release of LDH,IL-Iβ,and IL-18 increased(P＜0.05).After intervention with 100 µmol/L POA,the protein and mRNA expression levels of NLRP3,ASC,Cleaved caspase-1,GSDMD-N,IL-Iβ,and IL-18 were significantly reduced in the HR+POA group vs HR+ET group(P＜0.05).The positive percentage of Hoechst33342/PI staining in cardiomyocytes de-creased significantly,and the levels of LDH,IL-Iβ and IL-18 significantly decreased(P＜0.05).Conclusion Palmitoleic acid may alleviate hypoxia/reoxygenation-induced injury of cardiomyocytes by inhibiting pyroptosis and inflammatory response after hypoxia/reoxygenation in human myocardium.
基金项目
国家自然科学基金(81960047)
国家自然科学基金(82160086)
贵州省科学技术基金(黔科合平台人才GCC2022040-1)
国家科技期刊平台
NSTL
万方数据