基于线粒体形态特征与UPLC-Triple-TOF-MS整合分析的人参/三七心肌保护物质辨析研究  

Identification of cardioprotective substances in Panax ginseng/P.notoginseng based on mitochondrial morphological characteristics and UPLC-Triple-TOF-MS

作　　者：史雪敬 陈彦羽 蔡思怡 邵青[2] 赵筱萍[1,3] SHI Xue-jing;CHEN Yan-yu;CAI Si-yi;SHAO Qing;ZHAO Xiao-ping(School of Basic Medical Sciences,Zhejiang Chinese Medical University,Hangzhou 310053,China;College of Pharmaceutical Sciences,Zhejiang University,Hangzhou 310058,China;Academy of Chinese Medical Science,Zhejiang Chinese Medical University,Hangzhou 310053,China)

机构地区：[1]浙江中医药大学基础医学院,浙江杭州310053 [2]浙江大学药学院,浙江杭州310058 [3]浙江中医药大学中医药科学院,浙江杭州310053

出　　处：《中国中药杂志》2024年第10期2766-2775,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82073999)。

摘　　要：人参为补气强身之要药,三七具有散血定痛之功,常用于治疗心脉瘀阻所致的心肌缺血性疾病。线粒体功能障碍常伴随线粒体形态的异常改变,其可塑性和动态性在心血管疾病中至关重要。该研究将原代乳鼠心肌细胞缺氧培养4 h再复氧2 h,构建心肌细胞缺氧复氧损伤模型。采用荧光探针MitoTracker Deep Red标记心肌细胞线粒体,Hoechst 33342标记细胞核,使用高内涵荧光成像系统采集心肌细胞荧光图像,运用ImagePro Plus软件对图像自动批量处理并提取线粒体形态特征的量化参数,选取代表性参数用于评价线粒体形态功能改变进而筛选人参、三七有效组分;进一步整合UPLC-Triple-TOF-MS技术,筛选人参、三七有效成分。结果显示组分RS-2、RS-4、SQ-1、SQ-4可以同时显著提高线粒体3个形态参数值,增加线粒体长度、分支程度和面积,有效改善缺氧复氧损伤引起的心肌细胞线粒体形态改变。对有效组分的主要成分筛选发现20(R)-人参皂苷Rg_(3)、人参皂苷Re、七叶胆苷ⅩⅦ对心肌细胞线粒体保护作用最佳,进一步研究表明20(R)-人参皂苷Rg_(3)可能通过上调视神经萎缩蛋白1(optic atrophy 1,OPA1)和线粒体融合蛋白2(mitofusin 2,MFN2)表达,人参皂苷Re和七叶胆苷ⅩⅦ通过上调OPA1蛋白表达,促进线粒体膜融合,减轻线粒体损伤,从而发挥心肌细胞的保护作用。该研究为从人参、三七中发现创新药物提供了实验依据,也为中药心肌保护有效组分与有效成分的规模化筛选提供了新方法。Panax ginseng is reputed to be capable of replenishing healthy Qi and bolstering physical strength,and P.notoginseng can resolve blood stasis and alleviate pain.P.ginseng and P.notoginseng are frequently employed to treat ischemic heart diseases caused by blockages in the heart vessels.Mitochondrial dysfunction often coexists with abnormal mitochondrial morphology,and mitochondrial plasticity and dynamics play key roles in cardiovascular diseases.In this study,primary neonatal rat cardiomyocytes were exposed to 4 hours of hypoxia(H) followed by 2 hours of reoxygenation(R).MitoTracker Deep Red and Hoechst 33342 were used to label mitochondria and nuclei,respectively.Fluorescence images were then acquired using ImageXpress Micro Confocal.Automated image processing and parameter extraction/calculation were carried out using ImagePro Plus.Subsequently,representative parameters were selected as indicators to assess alterations in mitochondrial morphology and function.The active compounds of P.ginseng and P.notoginseng were screened out and identified based on the UPLC-Triple-TOF-MS results and mitochondrial morphometric parameters.The findings demonstrated that RS-2,RS-4,SQ-1,and SQ-4 significantly increased the values of three key morphometric parameters,including mitochondrial length,branching,and area,which might contribute to rescuing morphological features of myocardial cells damaged by H/R injury.Among the active components of the two medicinal herbs,20(R)-ginsenoside Rg_3,ginsenoside Re,and gypenoside ⅩⅦ exhibited the strongest protective effects on mitochondria in cardiomyocytes.Specifically,20(R)-ginsenoside Rg_(3) might upregulate expression of optic atrophy 1(OPA1) and mitofusin 2(MFN2),and ginsenoside Re and gypenoside ⅩⅦ might selectively upregulate OPA1 expression.Collectively,they promoted mitochondrial membrane fusion and mitigated mitochondrial damage,thereby exerting protective effects on cardiomyocytes.This study provides experimental support for the discovery of novel therapeutic agents for

关 键 词：人参 三七 线粒体形态 缺氧复氧 活性成分 心肌保护 荧光成像 

分 类 号：R285.5[医药卫生—中药学]