黄芪甲苷与人参皂苷Rg1配伍抗PC12细胞氧糖剥夺/复糖复氧后自噬性损伤相互作用的研究  

Interaction of Astragaloside Ⅳ combined with Ginsenoside Rg1 against autophagy injury of PC12 cells induced by oxygen glucose deprivation/reoxygenation

作　　者：丁煌[1] 李静娴[1] 唐标[1] 刘晓丹[1] 李玲[1] 唐映红[1] 邓常清[1] 黄小平[1] 

机构地区：[1]湖南中医药大学分子病理实验室,中西医结合心脑疾病防治湖南省重点实验室,细胞生物学与分子技术湖南省高校重点实验室,湖南长沙410208

出　　处：《中国药理学通报》2017年第2期235-243,共9页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No81573875,81503385);湖南省高校创新平台开放基金资助项目(No14K068);湖南省科技厅资助项目(No2014SK3001);湖南省研究生科研创新项目(NoCX2016B372);湖南省教育厅资助项目(No14C0844);湖南省中医药管理局重点项目(No201301,201508);中医内科重大疾病防治及成果转化省部共建教育部重点实验室开放课题(NoZYNK201405);“中西医结合防治心脑血管疾病的相关基础研究”湖南省高校科技创新团队;“中医药防治心脑血管疾病基础研究”湖南省自然科学创新群体基金

摘　　要：目的探讨黄芪甲苷和人参皂苷Rg1配伍对PC12细胞氧糖剥夺后再复糖复氧(oxygen glucose deprivation/reoxygenation,OGD/R)诱导的细胞自噬性损伤的影响及其相互作用。方法以PC12细胞建立OGD/R自噬性损伤模型,分别设立黄芪甲苷和人参皂苷Rg1不同剂量,药物干预细胞后,以激光共聚焦显微镜检测自噬体评价药物对细胞自噬性损伤的作用,并计算药物的半数抑制浓度(median inhibitory concentration,IC_(50))。以黄芪甲苷和人参皂苷Rg1的IC_(50)为1个剂量单位,按Isobologram法分别设立黄芪甲苷与人参皂苷Rg1不同比例(1∶2、1∶1、2∶1)的配伍,研究药物对细胞自噬的影响,计算各比例配伍的IC_(50),采用Isobologram及95%可信区间和相互作用指数γ分析黄芪甲苷与人参皂苷Rg1配伍抑制自噬的相互作用性质。在此基础上,以黄芪甲苷与人参皂苷Rg1的IC_(50)剂量进行1∶1配伍,以细胞存活率、乳酸脱氢酶(lactate dehydrogenase,LDH)漏出率、自噬体数量及p62蛋白表达评价药物配伍对细胞损伤和细胞自噬的影响。结果黄芪甲苷与人参皂苷Rg1抑制自噬的IC_(50)及其95%可信区间分别为(27.22±0.614)mg·L^(-1)[25.96,29.03]、(13.68±1.334)mg·L^(-1)[10.27,16.95]。黄芪甲苷与人参皂苷Rg1在1∶1配伍时呈现协同增效作用,而黄芪甲苷与人参皂苷Rg1在1∶2和2∶1配伍时,呈拮抗作用。以黄芪甲苷和人参皂苷Rg1的IC_(50)剂量进行1∶1配伍验证,结果显示单用黄芪甲苷和人参皂苷Rg1以及配伍均能增强细胞存活率,减轻LDH漏出,减少自噬体数量和LC3-Ⅱ蛋白数量,增加p62蛋白表达,且配伍组效应强于黄芪甲苷与人参皂苷Rg1单用组。析因设计实验分析表明,以黄芪甲苷和人参皂苷Rg1的IC_(50)剂量进行1∶1配伍时,对细胞存活、LDH漏出及自噬体形成均存在交互作用。结论在缺糖缺氧2h再复糖复氧24 h,细胞出现过度自噬和细胞损伤,黄芪甲苷和人参皂苷Rg1以IC_(50)剂量进�Aim To probe the effect and the interac-tion of Astragaloside Ⅳ and Ginsenoside Rg1 on auto-phagy injury of PC1 2 cells induced by oxygen glucose deprivation /reoxygenation（OGD /R）.Methods Auto-phagy injury model of PC1 2 cells induced by OGD /R was established,Astragaloside Ⅳ and Ginsenoside Rg1 were divided into different dosages to interfere PC1 2 cells,the autophagosome was detected to assess the effect of drugs on autophagy injury with confocal micro-sopy,and median inhibitory concentration （IC50 ）of Astragaloside Ⅳ and Ginsenoside Rg1 was calculated. Taking IC50 of Astragaloside Ⅳ and Ginsenoside Rg1 as 1 unit,the combinations of different ratios of Astragalo-side Ⅳ and Ginsenoside Rg1 （1 ∶2,1 ∶1 ,2 ∶1 ） were set up according to Isobologram method, the effect of the drugs against cells autophagy injury was determined and IC50 of different ratio combinations was acquired,then the nature of interaction was analysed that Astragaloside Ⅳ combined with Ginsenoside Rg1 inhibited autophagy through Isobologram analysis, 95% confidence and interaction index γ.On this ba-sis,using the combination of IC50 that Astragaloside Ⅳcombined with Ginsenoside Rg1 exerting synergy effect to assess the effect on cell injury and autophagy via cell survival rate,lactate dehydrogenase （LDH） leakage rate,the number of autophagosome and the expression of p62 protein.Results IC50 and 95% confidence that Astragaloside Ⅳ and Ginsenoside Rg1 inhibited autophagy was （27.22 ±0.61 4）mg ·L^-1 [25.96, 29.03],（1 3.68 ±1 .334）mg·L^-1 [1 0.27,1 6.95], respectively.Astragaloside Ⅳ combined with Ginsen-oside Rg1 obtained a synergitic effect under 1 ∶1 ratio while presented the antagonism under 1 ∶2 or 2 ∶1 . The verification results showed that Astragaloside Ⅳ, Ginsenoside Rg1 and the combination could enhance cell survival rate,relieve LDH leakage,reduce the numbers of autophagosome and LC3-Ⅱ,and increase the expression of p62;the effects of the combination were better than those of d

关 键 词：黄芪甲苷 人参皂苷RG1 配伍 PC12细胞 细胞自噬 Isobologram分析 相互作用 

分 类 号：R284.1[医药卫生—中药学] R289.1[医药卫生—中医学]