N-乙酰半胱氨酸通过抑制活性氧-p38通路减轻缺氧复氧诱导的乳鼠心肌细胞凋亡

目的 探讨N-乙酰半胱氨酸(N-acetylcysteine,NAC)抑制缺氧复氧(hypoxia-reoxygenation,H/R)诱导的乳鼠心肌细胞凋亡的机制.方法 心肌细胞培养48 h后随机分为对照组、缺氧复氧组(H/R组)、缺氧复氧+NAC组(100 p.mol/L)(H/R+NAC组).H/R组心肌细胞先缺氧6 h,随后复氧72 h,H/R+NAC组在H/R组细胞培养液中加NAC(100 μmol/L).采用锥虫蓝检测心肌细胞活性.流式细胞仪与Annexin V测定细胞早期凋亡.TUNEL检测细胞晚期凋亡.活性氧绿色荧光显色试剂检测活性氧(reactive oxygen species,ROS)浓度.RT-PCR检测bcl2、bax基因mRNA水平.Western blot检测bel2、bax、p38与pp38基因蛋白水平.结果 H/R组有活性的细胞数量为74.9%,显著低于对照组(93.5%,P<0.01),H/R+NAC组有活性的细胞数为89.9%,显著高于H/R组(P<0.01).H/R组早期凋亡的心肌细胞数为25.2%,显著高于对照组(6.5%,P<0.01),H/R+NAC组早期凋亡的细胞数为11.1%,显著低于H/R组(P<0.01).H/R组晚期凋亡的心肌细胞数为33.5%,显著高于对照组(3.5%,P<0.01),H/R+NAC组晚期凋亡的细胞数为13.5%,显著低于H/R组(P<0.01).H/R组心肌细胞ROS产生显著高于对照组,H/R+NAC组心肌细胞ROS产生显著低于H/R组.H/R组pp38/p38条带密度比值(13.40)也显著高于对照组(3.89).H/R+NAC组pp38/p38条带密度比值(1.95)显著低于H/R组(13.4),P<0.01.H/R组bcl2 mRNA与蛋白水平显著低于对照组,bax mRNA与蛋白水平显著高于对照组.H/R+NAC组bcl2 mRNA与蛋白水平显著高于H/R组.H/R+NAC组bcl2/bax mRNA水平比值(1.79)显著高于H/R组(1.22),P<0.05,但仍低于对照组(1.85).H/R+NAC组bcl2/bax条带密度比值(0.71)显著高于H/R组(0.50),P<0.05,但仍低于对照组(2.53).结论 NAC通过抑制ROS-p38通路减轻缺氧复氧诱导的乳鼠心肌细胞凋亡,这一作用具有潜在的临床应用价值.

The anti-apoptotic effects of N-acetylcysteine in neonatal rat cardiomyocytes underwent hypoxia-reoxygenation injury

Objective To evaluate the effects of N-acetylcysteine (NAC) on hypoxia-reoxygenation (H/R) injury induced apoptosis in neonatal rat cardiomyocytes. Methods Neonatal rat cardiomyocytes were cultured for 48 h and then randomized into control group, H/R group and H/R + NAC group. Cardiomyocytes underwent hypoxia for 6 h, reoxygenation for 72 h in the absence (H/R group) or presence ( H/R + NAC group) of NAC (100 μmol/L). Cell viability was assayed with trypan blue staining. Early stage of apoptosis was detected by flow cytometry with Annexin Ⅴ, late stage of apoptosis was assessed by TUNEL staining. ROS in culture medium was assayed by Image-iT? LIVE green reactive oxygen species detection kit bcl2 and bax mRNA levels were determined by real-time quantitative PCR (RT-PCR). Bcl2, bax, p38 and pp38 protein levels were measured by Western blot. Results The percentage of viable cardiomyocytes (93. 5% , 74. 9% , 89. 9% ) was significantly reduced while percentage of early stage of apoptotic cardiomyocytes (6. 5% , 25. 2% and 11. 1% ) and late stage of apoptotic cardiomyocytes (3. 5% ,33. 5% and 13. 5% ) were significantly increased in H/R group compared to control group and these changes could be largely reversed by NAC (all P < 0. 01). Significantly increased ROS generation in H/R group could also be attenuated by NAC (P < 0. 01). The band density ratio of pp38 and p38 was significantly upregulated in H/R group (13.4 vs. 3. 89), the mRNA and protein expressions of bcl2 were significantly lower and bax expressions were significantly higher in H/R group than those in control group and these changes could also be attenuated by NAC. Conclusion NAC significantly reduced apoptosis through inhibiting the phosphorylation of p38 signal pathway.