碘化N-正丁基氟哌啶醇通过蛋白激酶C途径保护缺氧复氧心肌细胞

The protective effect of N-n-butyl haloperidol iodide F_2 on hypoxia-reoxygenation injury of cardiomyocyte via PKC-α

目的研究碘化N-正丁基氟哌啶醇(F2)对缺氧复氧(H/R)所致心肌细胞损伤的作用及蛋白激酶C(PKC)活性的影响。方法用1～3d的Sprague-Dawley(SD)新生乳鼠进行心肌细胞原代培养,取生长4～5d的心肌细胞制作H/R模型。采用非放射性同位素法测定PKC活性、蛋白免疫印迹法检测PKC蛋白转位;双抗体夹心光化学测定法和酶联免疫吸附试验(ELISA)方法分别检测培养心肌细胞上清液中肌钙蛋白(cTnI)浓度和肿瘤坏死因子-α(TNF-α)分泌观察心肌细胞损伤状况。结果心肌细胞缺氧复氧时,PKC总活性明显升高,PKCα和βⅡ均发生转位。F2和cPKC特异性抑制剂G6976可减少cTnI泄漏和TNF-α的分泌,减轻心肌细胞H/R所致的损伤;PKC激动剂thymeleatoxin(TXA)拮抗F2的心肌保护作用。结论 F2对培养心肌细胞H/R损伤具有保护作用,可能与其抑制钙依赖PKCα转位有关。

Objective To observe the change of Protein Kinase C （PKC） activity, translocation oi PKC iso- forms-a, ]3 ]~ in primary cultured cardiomyocytes induced by Hypoxia/ reoxygenation （H/R）, which is helpful to elucidate the molecular mechanism of N-n-butyl haloperidoi iodide F2 in a cardiomyocyte H/R model. Methods Neonatal rat cardiomyocytes were primarily cultured and H/R model was preparaed, PKC activity was measured by non-radioactive detection of PKC, and the translocation pattern of PKCa and 13 [[ isoforms was assessed by frac tionated Western-blot analysis. Measurements included cardiac troponin I （eTnI） release were measured by two site sandwich immunoassay and levels of tumor necrosis factor-a （TNF （z）were measured by sandwich enzyme linked immunoabsorbent assay （ELISA） method to assess the degree of injury of cultured cardiomyocytes. Results In primary cultured cardiomyocytes exposed to H/R, PKC activity significantly increased after 2 h of hypoxia and 30 rain of reoxygenation, PKCc~ and 131I showed significant translocation from the soluble to the particulate fraction after H/R. Treatment with the conventional PKC inhibitor G66976 and F2 reduced the levels of cTnI and TNF-a,which is blocked by the PKCa activator thymeleatoxin. Conclusion Myocardial protection of F2 depended on PKC~ inhibition.