摘要
目的:筛选诃子制草乌特征性成分中的主要毒性作用成分和减毒作用成分。方法:采用均匀设计法安排实验,3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法考察诃子制草乌中4种特征性成分的不同配比对H9c2心肌细胞存活率的影响;应用Data Processing System(DPS)数据处理软件,对不同配比的细胞存活率进行逐步回归并绘制等高线图,分析4种特征性成分对H9c2心肌细胞毒性的影响及交互作用关系;以细胞存活率、乳酸脱氢酶(LDH)漏出率、细胞核平均荧光强度、线粒体膜电位为指标进一步对分析结果进行实验验证。结果:4种指标性成分中,存在于草乌中的新乌头碱、苯甲酰新乌头原碱均可明显降低H9c2心肌细胞存活率,以新乌头碱对毒性的贡献更大;诃子中的没食子酸、鞣花酸均可提高模型组(新乌头碱+苯甲酰新乌头原碱)细胞存活率,且没食子酸的作用强于鞣花酸。从交互作用的程度来看,具有显著降低H9c2心肌细胞存活率的交互项为新乌头碱和苯甲酰新乌头原碱;而交互项新乌头碱和鞣花酸、苯甲酰新乌头原碱和没食子酸可明显提高H9c2心肌细胞存活率。实验证实,新乌头碱和苯甲酰新乌头原碱共同作用,与对照组相比,细胞存活率明显降低(P<0.01),LDH漏出率和细胞核平均荧光强度增加(P<0.01),线粒体膜电位降低(P<0.01);在新乌头碱和苯甲酰新乌头原碱混合物中单独加入没食子酸或鞣花酸均可提高细胞存活率(P<0.01),升高线粒体膜电位(P<0.05或P<0.01),降低LDH的漏出和细胞核平均荧光强度(P<0.01);同时加入没食子酸和鞣花酸,其毒性作用进一步减弱。结论:诃子制草乌中的新乌头碱和苯甲酰新乌头原碱均具有一定的心肌毒性,以新乌头碱对毒性的贡献更大,两者具有协同作用;没食子酸和鞣花酸均可降低由两种乌头碱类成分产生的毒性作用,以没食子酸的减毒作用贡献更大,两者具有协同作用。
Objective:To screen the components promoting or reducing myocardial toxicity of H9 c2 cardiomyocytes from the characteristic components in Aconitii Kusnezoffii Radix processed with Chebulae Fructus.Methods:The experiment was arranged by uniform design,and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)method was used to detect the effects of different component combinations on the survival rate of H9 c2 cardiomyocytes.Taking the cell survival rate as the index,Data Processing System(DPS)software was used to establish a regression equation and draw a contour map to analyze the influence of four characteristic components on the toxicity of H9 c2 cardiomyocytes and the interactions among the components.Taking the cell survival rate,lactate dehydrogenase(LDH)leakage rate,average fluorescence intensity of nucleus and mitochondrial membrane potential as the indicators,experiments were performed to further verify the results of DPS analysis.Results:Among the four characteristic components,mesaconitine and benzoylmesaconitine in Aconitii Kusnezoffii Radix could significantly reduce the survival rate of H9 c2 cardiomyocytes,and mesaconitine contributed more to the toxicity.Both gallic acid and ellagic acid,which were from Chebulae Fructus,could improve the cell survival rate of the model group(mesacontine+benzoylmesacontine),and the effect of gallic acid was stronger than that of ellagic acid.The interaction terms that could significantly reduce the survival rate of H9 c2 cardiomyocytes were mesaconitine and benzoylmesaconitine.The interaction of mesaconitine with ellagic acid,and benzoylmesaconitine with gallic acid could significantly improve the survival rate of H9 c2 cardiomyocytes.It was further confirmed by experiments that the combination of mesaconitine and benzoylmesaconitine could significantly reduce the cell survival rate of H9 c2 cardiomyocytes(P<0.01),increase the leakage rate of LDH and average fluorescence intensity of nucleus(P<0.01),and reduce mitochondrial membrane potential(P<0.01).Adding gallic acid or ellagic acid alone to the mixture of mesaconitine and benzoylmesaconitine could reduce the toxicity to H9 c2 cardiomyocytes,which was manifested as increasing the cell survival rate(P<0.01)and mitochondrial membrane potential(P<0.05 or P<0.01),reducing the leakage of LDH and average fluorescence intensity of nucleus(P<0.01).When both gallic acid and ellagic acid were added,the toxic effects were further attenuated.Conclusion:Among the characteristic components in Aconitii Kusnezoffii Radix processed with Chebulae Fructus,mesaconitine and benzoylmesaconitine are both cytotoxic to H9 c2,while mesaconitine has a greater contribution to toxicity,and the combination has a synergistic effect to the cytotoxicity.Either gallic acid or ellagic acid can reduce the toxicity induced by the two aconitine components,and the attenuation effect of gallic acid is stronger.When the two are used in combination,the attenuation effect is further enhanced.
作者
韩舒
包丽媛
韩喜桃
唐雅楠
刘子琴
王宏月
张凤婷
米硕
李飞
杜红
Han Shu;Bao Liyuan;Han Xitao;Tang Ya’nan;Liu Ziqin;Wang Hongyue;Zhang Fengting;Mi Shuo;Li Fei;Du Hong(Beijing University of Chinese Medicine,Beijing 100029,China)
出处
《中国药师》
CAS
2022年第1期27-33,共7页
China Pharmacist
基金
国家自然科学基金项目(编号:81774004)
国家重点研发计划项目(编号:2018YFC1706303)。
关键词
诃子制草乌
均匀设计
特征性成分
H9C2心肌细胞
Aconitii Kusnezoffii Radix processed with Chebulae Fructus
Uniform design
Characteristic components
H9c2 cardiomyocytes
