龙蛭汤含药血清联合3-MA、白藜芦醇对氧化应激环境下血管内皮细胞自噬及管腔形成的影响  

Effect of Longzhi Decoction-Containing Serum Combined with 3-MA and Resveratrol on Autophagy and Tubular Formation of Vascular Endothelial Cells under Oxidative Stress

作　　者：黄显雯 刘启华[1] 陈永斌[1] 罗捷 李南方 邹玲 罗燕萍 HUANG Xianwen;LIU Qihua;CHEN Yonghin;LU Jie;LI Nanfang;ZOU Ling;LUO Yanping(The First Affiliated Hospital of Guangxi Medical University,Nanning 530021,Guangxi,China;Guangxi Medical University,Nanning 530021,Guangxi,China)

机构地区：[1]广西医科大学第一附属医院中医科,广西南宁530021 [2]广西医科大学,广西南宁530021

出　　处：《中华中医药学刊》2020年第10期59-63,I0023-I0025,共8页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金(81560790);广西自然科学基金面上项目(2014GxNSFAA118277)。

摘　　要：目的观察龙蛭汤含药血清联合3-甲基腺嘌呤(3-Methyladenine,3-MA)、白藜芦醇(Resveratrol,Res)对过氧化氢(H2O2)诱导的氧化应激环境下血管内皮细胞(HUVECs)自噬及管腔形成的影响。方法SPF级SD大鼠分别予生理盐水、龙蛭汤水煎液灌胃7 d,取腹主动脉血制备含药血清,分装备用。通过建立H2O2诱导的HUVECs氧化应激自噬模型,分别加入正常对照组(Control组)、生理盐水含药血清组(空白血清组)、3-MA(3-MA组)、3-MA+10%龙蛭汤含药血清(3-MA+LZD组)、Res(Res组)、Res+10%龙蛭汤含药血清(Res+LZD组);CCK8、划痕实验、基质胶管型形成实验分别检测HUVECs增殖、迁移及管型形成能力,细胞免疫荧光检测自噬表达强度,Western Blot比较各组细胞Beclin-1、LC3Ⅱ/Ⅰ、VEGF的蛋白表达水平。结果与空白血清组相比,Res组与Res+LZD组的细胞增殖数、迁移距离及管型形成数均明显增多,LC3荧光表达增强,Beclin-1、LC3Ⅱ/Ⅰ、VEGF蛋白水平升高,3-MA组的自噬、VEGF蛋白水平均降低;与3-MA组、Res组相比,加入龙蛭汤含药血清后的细胞增殖、迁移、管型形成数增多,3-MA+LZD组自噬荧光表达增强,Beclin-1、LC3Ⅱ/Ⅰ、VEGF蛋白水平升高;Res+LZD组下调Beclin-1、LC3Ⅱ/Ⅰ蛋白水平,与自噬荧光表达趋势一致,同时VEGF蛋白表达水平上调。结论龙蛭汤含药血清通过调节Beclin-1、LC3Ⅱ/Ⅰ自噬蛋白水平调控自噬通路,提高血管内皮细胞活力,促进内皮细胞迁移及管型形成,可能是血管内皮细胞氧化应激损伤后的血管新生的重要作用机制。Objective To observe the effects of Longzhi Decoction(LZD)-containing serumcombined with 3-methyladenine(3-MA)and resveratrol(Res)on autophagy expression and tubular formation of vascular endothelial cells(HUVECs)induced by hydrogen peroxide(H2O2).Methods SD rats of SPF grade were treated with normal saline and Longzhi Decoction for 7 days.The aortic blood was taken out to prepare serum containing LZD and used for equipment.By establishing H2O2-induced oxidative stress autophagy model of HUVECs,normal culture serum(normal control group),saline-containing serum group(blank serum group),3-MA(3-MA group),3-MA+10% LZD(3-MA+LZD group),Resgroup,Res+10% LZD(Res+LZD group).CCK8,scratch test and Matrigel tube formation test were used to detect the proliferation,migration and tubular formation ability of HUVECs.Cell immunofluorescence was used to detect the expression of autophagy.Western Blot was used to compare the protein expression levels of Beclin-1,LC3 Ⅱ/Ⅰ and VEGF in each group.Results Compared with those of the blank serum group,the number of cell proliferation,migration distance and tubular formation increased significantly in Res group and Res+LZD group.The fluorescence expression of LC3 increased,the levels of Beclin-1,LC3 Ⅱ/Ⅰ and VEGF protein increased,and the levels of autophagy and vascular endothelial growth protein decreased in 3-MA group.Compared with those of the 3-MA group and Res group,the number of cell proliferation,migration and tubular formation increased after adding the serum of LZD.In 3-MA+LZD group,the expression of autophagic fluorescence increased,the levels of Beclin-1,LC3 Ⅱ/Ⅰ and VEGF protein increased.In Res+LZD group,the levels of Beclin-1 and LC3 Ⅱ/Ⅰ protein were down-regulated,which was consistent with the trend of autophagic fluorescence expression,while the expression of VEGF protein was up-regulated.Conclusion LZD can enhance the viability of vascular endothelial cells and promote the migration and tubular formation of endothelial cells by regulating the expression lev

关 键 词：龙蛭汤含药血清 3-MA RESVERATROL 自噬 血管管型形成 

分 类 号：R285.5[医药卫生—中药学]