维生素C对乌头碱中毒导致乳鼠心肌细胞凋亡的影响及机制分析
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摘要
目的观察维生素C对乌头碱导致心肌细胞损伤的防护作用并探讨可能的机制。方法提取并培养乳鼠原代心肌细胞,采用蛋白质印迹法验证浓度梯度的乌头碱对心肌细胞凋亡的影响;加入浓度梯度的维生素C,用同样的实验方法观察其对乌头碱中毒导致心肌细胞凋亡的改善作用。利用蛋白质印迹法筛选出在整个中毒解救过程中p53蛋白表达的变化;探讨维生素C联合应用p53抗体对乌头碱导致心肌细胞凋亡的影响。结果乌头碱能够浓度梯度性地促进Bad蛋白表达并下调Bcl-2蛋白表达(P<0.01),说明其能诱导心肌细胞凋亡。维生素C可以减弱乌头碱中毒心肌细胞中Bad蛋白的表达而增强Bcl-2的表达(P <0.05),说明其能减弱乌头碱中毒引起的细胞凋亡。浓度梯度的维生素C与乌头碱合用时,p53蛋白的表达随着维生素C浓度的增加而降低(P <0. 01)。加入外源性p53抗体,可以减弱乌头碱中毒引起的细胞凋亡,增强维生素C的解救作用(P<0.01)。结论维生素C可以减轻乌头碱的毒性作用,这可能与维生素C抑制了p53信号通路有关。
Objective To investigate the effect of vitamin C on cardiomyocyte apoptosis caused by aconitine and to analyze the potential mechanism. Methods Primary cardiomyocytes of suckling mice were extracted and cultured. Western blotting was used to detect the effects of aconitine and vitamin C on cell growth and the effect of vitamin C combined with p53 on cell apoptosis induced by aconitine. Results Aconitine could increase Bad expression and reduce Bcl-2 expression( P < 0. 01),suggesting it induced the apoptosis of cardiomyocytes.Vitamin C could reduce Bad expression and increase Bcl-2 expression in cardiomyocytes cultured with aconitine(P <0.05); it could alleviate the toxicity of aconitine. Under gradient concentrations of vitamin C plus aconitine,the expression level of p53 in cells decreased with the concentration of vitamin C(P <0. 01). P53 anti-body could reduce the toxicity of aconitine and enhance the effect of vitamin C(P < 0. 01). Conclusion Vitamin C can alleviate the toxicity of aconitine on cardiomyocytes, which may be mediated by the p53 pathway.
Objective To investigate the effect of vitamin C on cardiomyocyte apoptosis caused by aconitine and to analyze the potential mechanism. Methods Primary cardiomyocytes of suckling mice were extracted and cultured. Western blotting was used to detect the effects of aconitine and vitamin C on cell growth and the effect of vitamin C combined with p53 on cell apoptosis induced by aconitine. Results Aconitine could increase Bad expression and reduce Bcl-2 expression( P < 0. 01),suggesting it induced the apoptosis of cardiomyocytes.Vitamin C could reduce Bad expression and increase Bcl-2 expression in cardiomyocytes cultured with aconitine(P <0.05); it could alleviate the toxicity of aconitine. Under gradient concentrations of vitamin C plus aconitine,the expression level of p53 in cells decreased with the concentration of vitamin C(P <0. 01). P53 anti-body could reduce the toxicity of aconitine and enhance the effect of vitamin C(P < 0. 01). Conclusion Vitamin C can alleviate the toxicity of aconitine on cardiomyocytes, which may be mediated by the p53 pathway.
引文
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[2] Li XM, Liu J, Pan FF, et al. Quercetin and aconitine synergistically induces the human cervical carcinoma HeLa cell apoptosis via endoplasmic reticulum(ER)stress pathway[J]. PloS one, 2018,13(1):e0191062. DOI:10.1371/journal. pone. 0191062.
[3]张翅,潘德林,朱雅宁,等.附子中6种生物碱质谱裂解机制及醇解产物研究[J].中国中医急症,2016,25(12):2278-2282.DOI:10.3969/j. issn. 1004-745X. 2016.12.021.Zhang C, Pan DL, Zhu YN, et al. Study on the fragmentation behavior and alcoholysates of aconitum alkaloids in aconitum carmichaeli debx[J]. Journal of Emergency in Traditional Chinese Medicine, 2016, 25(12):2278-2282. DOI:10. 3969/j. issn.1004-745X.2016.12.021.
[4]杨武斌,王平.乌头碱药理作用及毒性研究进展[J].时珍国医国药,2014,25(2):427-429. DOI:10. 3969/j. issn. 1008-0805.2014.02.077.Yang WB, Wang P. Pharmacology effect and toxicity of aconitine[J].Lishizhen Medicine and Materia Medica Research, 2014,25(2):427-429. DOI:10.3969/j.issn. 1008-0805.2014.02.077.
[5]费巧玲,张雪,董艳红,等.乌头碱配伍人参皂苷Rg_1对心肌细胞毒效浓度研究[J].时珍国医国药,2015,26(11):2610-2613.Fei QL, Zhang X, Dong YH, et al. Cytotoxic effect study of aconitine combined with ginsenoside Rg_1 on cardiomyocytes[J]. Lishizhen Medicine and Materia Medica Research, 2015,26(11):2610-2613.
[6]卿楠,颉永乐.急性乌头碱中毒导致致命性心律失常1例[J].中国中医急症,2013,22(7):1270. DOI:10.3969/j. issn. 1004-745X.2013.07.105.Qing N, Xie YL. A case of fatal arrhythmia caused by acute aconitine poisoning[J]. Journal of Emergency in Traditional Chinese Medicine, 2013,22(7):1270. DOI:10. 3969/j. issn. 1004-745X.2013.07.105.
[7]田野,潘德田,刘会勇.乌头碱中毒致心律失常30例[J].临床内科杂志,2013,30(2):133-134. DOI:10.3969/j. issn.1001-9057.2013.02.023.Tian Y, Pan DT, Liu HY. 30 cases of arrhythmia caused by aconitine poisoning[J]. Journal of Clinical Internal Medicine,2013,30(2):133-134. DOI:10. 3969/j. issn. 1001-9057. 2013.02.023.
[8]赵永堂.新乌头碱、乌头碱与次乌头碱在附子不同炮制法中含量变化的实验研究[J].中国医药导报,2012,9(14):27-28.DOI:10.3969/j. issn. 1673-7210.2012.14.011.Zhao YT. Experimental study of the content of new aconitine,aconitine and hypoaconitine in different processing method of aconite[J]. China Medical Herald, 2012,9(14):27-28. DOI:10.3969/j. issn. 1673-7210.2012.14. 011.
[9]温善姗,万海同,张宇燕,等.乌头碱对心衰细胞强心作用的量-时-效关系的实验研究[J].中国中医急症,2012,21(4):562-564. DOI:10.3969/j. issn. 1004-745X.2012.04.026.Wen SS, Wan HT, Zhang YY, et al. Experimental study of aconitine on dose-time-effect relationship in cardiac effect of heart failure cells[J]. Journal of Emergency in Traditional Chinese Medicine, 2012,21(4):562-564. DOI:10. 3969/j. issn. 1004-745X.2012.04.026.
[10]宋昆,李长罗,何芳.双黄连粉针剂联合胺碘酮治疗乌头碱中毒致严重室性心律失常的临床研究[J].中国医药导报,2012,9(22):97-98. DOI:10.3969/j. issn. 1673-7210.2012.22.042.Song K, Li CL, He F. Clinical study of Shanghuanglian Powder Injection combined with Amiodarone on serious ventricular arrhythmia induced by aconitine poisoning[J]. China Medical Herald, 2012,9(22);97-98. DOI:10.3969/j. issn. 1673-7210.2012.22.042.
[11] Sun GB, Sun H, Meng XB, et al. Aconitine-induced Ca2+overload causes arrhythmia and triggers apoptosis through p38MAPK signaling pathway in rats[J]. Toxicol Appl Pharmacol,2014,279(1):8-22. DOI:10.1016/j. taap. 2014.05.005.
[12]徐盈,陈国兵,吴海燕,等.乌头碱中毒致严重心律失常151例分析[J].临床荟萃,2012,27(1):67-68.Xu Y, Chen GB, Wu HY, et al. Analysis of 151 cases of severe arrhythmia caused by aconitine poisoning[J]. Clinical Focus,2012,27(1):67-68.
[13]乔尚,尚佳,董巧玲,等.激动M3-mAchR对乌头碱诱发大鼠心律失常的保护机制的研究[J].临床和实验医学杂志,2017,16(24):2408-2411. DOI:10. 3969/j. issn. 1671-4695.2017.24.005.Qiao S, Shang J, Dong QL, et al. The protective mechanism via activation of M3 receptor in aconitine-induced arrhythmias in rats[J].Journal of Clinical and Experimental Medicine, 2017,16(24):2408-2411. DOI:10.3969/j. issn. 1671-4695.2017.24.005.
[14]陈国烽,王亚军.维生素C在新陈代谢中的生理功能[J].中国食物与营养,2014,20(1):71-74. DOI:10. 3969/j. issn. 1006-9577.2014.01.017.Chen GF, Wang YJ. Physiological function of vitamin C in metabolism[J]. Food and Nutrition in China, 2014,20(1):71-74. DOI:10.3969/j. issn. 1006-9577.2014.01.017.
[15] Shi B, Su Y, Chang S, et al. Vitamin C protects piglet liver against zearalenone-induced oxidative stress by modulating expression of nuclear receptors PXR and CAR and their target genes[J].Food Funct, 2017,8(10):3675-3687.
[16]朱云鸿,徐瑞容,黄红铭,等.维生素C对硼替佐米诱导多发性骨髓瘤细胞株KM-3凋亡作用的影响[J].南通医学院学报,2009,29(1):14-17. DOI:10.3969/j. issn. 1674-7887.2009.01.005.Zhu YH, Xu RR, Huang HM, et al. Effect of vitamin C on bortezomib-induced apoptotic death in multiple myeloma cell line KM-3[J]. Journal of Nantong University(Medical Sciences),2009,29(1):14-17. DOI:10.3969/j. issn. 1674-7887.2009.01.005.
[17]曾尟枚,向光大.维生素C对2型糖尿病患者葡萄糖负荷后血管内皮依赖性功能的保护作用[J].医药导报,2008,27(12):1465-1468. DOI:10.3870/j. issn. 1004-0781.2008.12. 015.Zeng XM, Xiang GD. Effect of antioxidant vitamin C on vascular endothelial function during acute hyerglycemia in patients with type 2diabetes[J]. Herald of Medicine,2008,27(12):1465-1468. DOI:10.3870/j. issn. 1004-0781.2008.12.015.
[18]温宝伶,余雷,方艳,等.维生素C治疗百草枯中毒大鼠的量效关系[J].中南大学学报:医学版,2016,41(12):1323-1327.DOI:10.11817/j. issn. 1672-7347.2016.12.012.Wen BL, Yu L, Fang Y, et al. Dose-effect relationship between vitamin C and paraquat poisoning rats[J]. Journal of Central South University(Medical Science), 2016,41(12):1323-1327. DOI:10.11817/j. issn. 1672-7347.2016.12.012.
[19]刘梦思,刘云,刘海文,等.维生素C、E对铅中毒大鼠心脏活动的影响[J].昆明医科大学学报,2013,34(1):56-58.DOI:10.3969/j. issn. 1003-4706.2013.01.015.Liu MS, Liu Y, Liu HW, et al. The effect of vitamin C and vitamin E on cardiac function in lead poisoning rats[J]. Journal of Kunming Medical University, 2013,34(1):56-58. DOI:10.3969/j. issn. 1003-4706.2013.01.015.
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