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目的探讨中药单体前胡丙素(Pra-C)对缺血/再灌注(I/R)心肌细胞保护作用的机制。方法将培养的乳鼠心肌细胞随机分为I/R组、Pra-C预处理组和对照组。用全自动生化仪测定各组乳酸脱氢酶(LDH)的漏出量(n=5)。半定量RT-PCR检测钠钙交换蛋白(NCX)mRNA的转录(n=3),其活性以液闪仪测定Na+依赖的45Ca2+摄取率表示(n=5)。细胞膜钙ATP酶(PMCA)的活性以定磷比色法测定(n=5)。结果I/R组LDH的漏出量显著增加(P<0.01),Pra-C预处理组与I/R组比较LDH的漏出量显著降低(P<0.01)。I/R组NCX Na+依赖的45Ca2+摄取率比对照组显著增高(P<0.01),Pra-C预处理组较I/R时45Ca2+摄取率降低(P<0.01)。I/R组NCXmRNA的水平比对照组显著高(P<0.01);Pra-C预处理组较I/R组NCX mRNA的水平显著低(P<0.01)。各组间PMCA的活性无显著性差异。结论Pra-C可显著减轻I/R损伤;其对I/R心肌的保护与下调细胞膜NCX mRNA的转录和其蛋白的活性有关。
Objective To investigate the mechanism of protective effect of Pra-C (Pra-C) on ischemia/reperfusion (I/R) cardiomyocytes. Methods The cultured neonatal rat cardiomyocytes were randomly divided into I/R group, Pra-C pretreatment group and control group. The amount of lactate dehydrogenase (LDH) leakage in each group was determined using an automatic biochemical analyzer (n=5). Semi-quantitative RT-PCR was used to detect the transcription of sodium-calcium exchange protein (NCX) mRNA (n=3). The activity was measured by liquid scintillation assay and Na+-dependent 45Ca2+ uptake rate (n=5). The activity of cell membrane calcium ATPase (PMCA) was determined by a constant phosphorus method (n=5). Results The leakage of LDH in I/R group was significantly increased (P<0.01), and the leakage of LDH was significantly decreased in Pra-C pretreatment group and I/R group (P<0.01). The rate of 45Ca2+ uptake by NCX Na+ in the I/R group was significantly higher than that in the control group (P<0.01). The 45Ca2+ uptake rate in the Pra-C pretreatment group was lower than that in the I/R group (P<0.01). The level of NCX mRNA was significantly higher in the I/R group than in the control group (P<0.01). The level of NCX mRNA in the Pra-C pretreatment group was significantly lower than that in the I/R group (P<0.01). There was no significant difference in the activity of PMCA between the groups. Conclusion Pra-C can significantly reduce I/R injury; its protection of I/R myocardium is related to the down-regulation of NCX mRNA transcription and its protein activity.