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目的:研究碘化N-正丁基氟哌啶醇(F2)等钙拮抗剂通过肿瘤坏死因子(TNF)-α/NF-κB环路介导的非钙依赖机制对缺氧复氧(H/R)心肌细胞的保护作用。方法:在体外培养的H9C2细胞上,建立无钙H/R模型,实验分为对照组、无钙组、无钙H/R组、无钙H/R+DMSO组及无钙H/R+不同剂量钙拮抗剂组。ELISA检测培养上清液中TNF-α的释放量;实时荧光定量PCR检测培养心肌细胞中TNF-α的mRNA。Western blot验证p-IKB-α、IKB-α、p-p65、p65在H9C2中的表达。比色法检测培养上清液中大鼠乳酸脱氢酶(LDH)浓度。结果:无钙缺氧0.5 h复氧0.5 h可引起TNF-α升高(P<0.05),无钙1h TNF-α无变化。F2和维拉帕米(Ver)能剂量-依赖性抑制无钙H/R所致的TNF-α、LDH的生成以及p-IKB-α、p-p65表达。结论:F2和Ver通过对TNF-α/NF-KB环路的介导,在无钙条件下对心肌细胞H/R损伤起保护作用。
AIM: To investigate the effect of calcium antagonists such as N-butyl haloperidol iodide (F2) on hypoxia / reoxygenation (H / H) mediated by non-calcium-dependent mechanisms mediated by tumor necrosis factor (TNF) -α / NF- / R) cardiomyocyte protective effect. Methods: Calcium-free H / R model was established on H9C2 cells cultured in vitro. The experiment was divided into control group, calcium-free group, calcium-free H / R group, calcium-free H / Dosage groups of calcium antagonists. The release of TNF-α in the culture supernatant was detected by ELISA. The mRNA of TNF-α in cultured cardiomyocytes was detected by real-time fluorescence quantitative PCR. The expression of p-IKB-α, IKB-α, p-p65, p65 in H9C2 was confirmed by Western blot. Colorimetric assay of rat lactate dehydrogenase (LDH) concentration in culture supernatant. Results: After 0.5 h reoxygenation for 0.5 h without calcium and hypoxia, TNF-α was increased (P <0.05), and no changes were found in 1 h TNF-α. F2 and verapamil can dose-dependently inhibit the production of TNF-α, LDH and the expression of p-IKB-α, p-p65 induced by calcium-free H / R. Conclusion: F2 and Ver can protect H / R injury of cardiomyocytes under calcium-free condition mediated by TNF-α / NF-KB pathway.