摘要
观察人参-三七-川芎提取物对过氧化氢(H_(2)O_(2))诱导的内皮细胞衰老SIRT1-自噬通路的影响。该研究以血管内皮细胞作为研究对象,过氧化氢诱导内皮细胞衰老作为模型,实验分为空白对照组(control),模型组(model)、模型+DMSO组(DMSO),白藜芦醇组(RESV)及人参-三七-川芎提取物低(GNS-L)、中(GNS-M)、高(GNS-H)剂量组,药物干预时间为24 h。应用倒置显微镜观察细胞生长状态,透射电子显微镜观察自噬小体的形成,免疫荧光染色观察细胞微管相关蛋白1轻链3β(LC3)的变化,自噬双标腺病毒(mRFP-GFP-LC3)双荧光体系融合蛋白示踪自噬流,单丹磺酰尸胺(dansylcadaverine,MDC)染色检测自噬囊泡,蛋白质免疫印迹(Western blot)检测蛋白沉默信息调节因子2相关酶1(sirtuin 1,SIRT1)、泛素结合蛋白p62及LC3Ⅱ的表达。H_(2)O_(2)造模后,细胞生长速度放慢,细胞贴壁能力下降,SA-β-gal染色蓝染细胞增多,胞质内出现一定数量的双层膜结构的自噬小体及次级溶酶体,自噬流水平略增。给予药物干预后,与模型组相比,各剂量组细胞形态改善,贴壁能力尚可,细胞膜相对完整光滑,SA-β-gal染色蓝染细胞减少,胞质中出现较多自噬小体、自噬空泡及次级溶酶体,自噬溶酶体数量增多,自噬流增加,LC3及MDC染色的荧光强度明显提高,SIRT1及LC3Ⅱ蛋白表达增加,同时p62蛋白表达下调,表明用药后可以提高细胞自噬水平。中药人参-三七-川芎提取物可以延缓血管内皮细胞的衰老,且药物干预后,自噬流及相关蛋白SIRT1、LC3Ⅱ及p62变化明显,自噬水平明显提升,且EX527削弱了中药延缓血管衰老的作用,中药人参-三七-川芎提取物可能通过SIRT1自噬通路延缓血管内皮细胞的衰老。
This study aims to explore the effect of extract of Ginseng Radix et Rhizoma,Notoginseng Radix et Rhizoma,and Chuanxiong Rhizoma(hereinafter referred to as GNS)on the SIRT1-autophagy pathway of endothelial cell senescence induced by hydrogen peroxide(H_(2)O_(2)).To be specific,vascular endothelial cells were classified into the blank control group(control),model group(model),model+DMSO group(DMSO),resveratrol group(RESV),and GNS low-dose(GNS-L),medium-dose(GNS-M),and high-dose(GNS-H)groups.They were treated with H_(2)O_(2) for senescence induction except the control.After intervention of cells in each group with corresponding drugs for 24 h,cell growth status was observed under an inverted microscope,and the formation of autophagosome under the transmission electron microscope.In addition,the changes of microtubule-associated protein 1 light chain 3β(LC3 B)were detected by immunofluorescence staining.The autophagy flux was tracked with the autophagy double-labeled adenovirus(mRFP-GFP-LC3)fusion protein.Dansylcadaverine(MDC)staining was employed to determine the autophagic vesicles,and Western blot the expression of sirtuin 1(SIRT1),ubiquitin-binding protein p62,and LC3Ⅱ.After H_(2)O_(2) induction,cells demonstrated slow growth,decreased adhesion ability,raised number of SA-β-gal-stained blue ones,a certain number of autophagosomes with bilayer membrane and secondary lysosomes in the cytoplasm,and slight rise of autophagy flux level.Compared with the model group,GNS groups showed improved morphology,moderate adhesion ability,complete and smooth membrane,decreased SA-β-gal-stained blue cells,many autophagosomes,autophagic vesicles,and secondary lysosomes in the cytoplasm,increased autophagolysosomes,autophagy flux level,and fluorescence intensity of LC3 B and MDC,up-regulated expression of SIRT1 and LC3Ⅱ,and down-regulated expression of p62,suggesting the improvement of autophagy level.GNS can delay the senescence of vascular endothelial cells.After the intervention,the autophagy flux and related proteins SIRT1,LC3Ⅱand p62 changed significantly,and the autophagy level increased significantly.However,EX527 weakened the effect of Chinese medicine in delaying vascular senescence.GNS may delay the senescence of vascular endothelial cells through the SIRT1 autophagy pathway.
作者
修成奎
付莹坤
王强
王雪
胡艳红
吴烨
杨静
雷燕
XIU Cheng-kui;FU Ying-kun;WANG Qiang;WANG Xue;HU Yan-hong;WU Ye;YANG Jing;LEI Yan(Beijing Key Laboratory of Research of Chinese Medicine on Preventional and Treatment for Major Diseases,Experimental Research Center,China Academy of Chinese Medical Sciences,Beijing 100700,China;Guang′anmen Hospital,China Academy of Chinese Medical Sciences,Beijing 100056,China;Wangjing Hospital,China Academy of Chinese Medical Sciences,Beijing 100102,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2021年第23期6216-6223,共8页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81503448,82074260)
中国中医科学院自主选题研究项目(ZZ2018014)。
关键词
衰老
血管老化
内皮细胞衰老
人参-三七-川芎提取物
senescence
vascular senescence
senescence of endothelial cells
extract of Ginseng Radix et Rhizoma,Notoginseng Radix et Rhizoma,and Chuanxiong Rhizoma
