蠲脉Ⅰ号化痰消食拆方促进蛋白激酶B表达抑制内皮细胞自噬性死亡  

Study of Phlegm-resolving and Digestion-promoting Separated Recipe from Juanmai No.1 Prescription for Promoting Protein Kinase B Expression and Inhibiting Autophagy-induced Death of Human Umbilical Vein Endothelial Cells

作　　者：白晔[1] 李菲[1] 黄凤媛 邓柏杨[2] 李熙灿[3] 陈东风[1] 黎晖[1] 邓汝东[1] 周健洪[1] 

机构地区：[1]广州中医药大学解剖学教研室,广东广州510006 [2]广西中医药大学附属瑞康医院,广西南宁530011 [3]广州中医药大学中药学院,广东广州510405

出　　处：《广州中医药大学学报》2014年第1期79-84,94,共7页Journal of Guangzhou University of Traditional Chinese Medicine

基　　金：广西壮族自治区自然科学基金重点项目(编号:2010GXNSFD013051)

摘　　要：【目的】探讨蠲脉Ⅰ号化痰消食拆方(简称化痰消食方)对自噬损伤内皮细胞保护作用的物质基础,以及对蛋白激酶B(PKB)和自噬相关的微管相关蛋白LC3、泛素结合蛋白P62表达的影响。【方法】采用递增极性溶剂法分别分离提取化痰消食方(成分为半夏、茯苓、枳实、白术、山楂、神曲)的石油醚、乙酸乙酯、乙醇以及水部位。将4个部位的提取物分别作用于过氧化氢诱导的自噬性损伤内皮细胞,应用四甲基偶氮唑盐(MTT)法观察内皮细胞的活性,采用GC-MS(gas chromatography-mass spectrometry)分析有效部位的化学成分,并采用Western blot法观察有效部位对PKB表达及自噬的影响。【结果】石油醚部位为化痰消食方保护内皮细胞自噬性损伤的活性部位;GS-MS检测发现石油醚部位主要成分为多种脂肪酸;Western blot分析结果显示:石油醚部位能上调PKB表达、下调LC3表达,抑制LC3-Ⅰ向LC3-Ⅱ转化,并使细胞内P62表达水平升高。【结论】石油醚部位多种脂肪酸为化痰消食方有效部位,能促进细胞内PKB表达,抑制细胞内的自噬水平,从而拮抗内皮细胞的自噬性死亡。Objective To investigate the mechanism of phlegm-resolving and digestion-promoting separated recipe （PDSR） from Juanmai No.1 Prescription for protecting human umbilical vein endothelial cells （HUVECs） from autophagic deaths, and to observe its effect on the expression of protein kinase B （PKB）, microtubule-associated protein light chain 3 （LC3） and Ubiquitin-binding protein P62. Methods Different constituents were extracted from PDSR （composed of Rhizoma Pinelliae, Poria, Fructus Aurantii Immaturus, Rhizoma Atracty｜odis Macrocephalae, Fructus Crataegi and Massa Medicata Fermentata） by increasing polar solvent method with petroleum ether, acetic ether, ethanol and water, respectively. And then the four extracts were used for the incubation of of HUVECs with autophagic damage induced by hydrogen peroxide. The activity of the cultured HUVECs was observed by methyl thiazolyl tetrazolium （MTT） assay. The chemical constituents from effective fractions of PDSR were analyzed by gas chromatography- mass spectrometry （GC-MS）, and their effects on the expression of LC3, P62 and PKB protein were observed by Western blotting method. Results The constituents extracted from PDSR with petroleum ether were the active fraction for protecting HUVECs from autophagic deaths. The results of GS-MS showed that the petroleum ether fraction mainly consisted of fatty

关 键 词：蠲脉I号化痰消食拆方 内皮细胞 自噬 细胞培养 

分 类 号：R285.5[医药卫生—中药学]