大黄酸抑制过氧化氢诱导的人脐静脉内皮细胞凋亡及机制研究  

Rhein inhibits hydrogen peroxide-induced apoptosis of human umbilical vein endothelial cells and its mechanism

作　　者：邹进 万子琳 姚兰 徐梦瑶 王述蓉 ZOU Jin;WAN Zi-lin;YAO Lan;XU Meng-yao;WANG Shu-rong(Department of Pharmacy,Affiliated Hospital of Southwest Medical University,Lazhou 646000,China;College of Pharmacy,Southwest Medical University,Santai 621100,China;Department of Pharmacy,People′s Hospital of Santai County,Lazhou 646000,China)

机构地区：[1]西南医科大学附属医院药学部,四川泸州646000 [2]西南医科大学药学院,四川三台621100 [3]三台县人民医院药剂科,四川泸州646000

出　　处：《中国中药杂志》2021年第23期6204-6215,共12页China Journal of Chinese Materia Medica

基　　金：泸州市人民政府-西南医科大学联合项目(2020LZXNYDJ18)。

摘　　要：该研究探讨大黄酸(rhein,RH)对过氧化氢(hydrogen peroxide,H_(2)O_(2))诱导的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)凋亡和自噬的影响及其潜在的作用机制。用H_(2)O_(2)建立HUVECs的氧化损伤模型,分成不同的处理组,MTT法检测细胞生存率,Annexin V-FITC/PI双染法、Hoechst 33258荧光染色检测细胞凋亡;Ad-mCherry-GFP-LC3B腺病毒转染法检测自噬情况;Western blot法检测蛋白表达。结果表明RH预保护细胞,可提高H_(2)O_(2)损伤的HUVECs的细胞存活率,且呈剂量依赖性,减少凋亡相关蛋白Bax和cleaved caspase-3的表达,升高Bcl-2的的表达,降低Bax/Bcl-2的比值;可上调自噬标志性蛋白微管相关蛋白1轻链3(microtubule associated protein 1 light chain 3,LC3)-Ⅱ的表达,下调p62的表达。腺病毒转染结果显示,RH可使细胞绿色斑点和红色斑点增多,且黄色斑点也显著增多。而加入自噬抑制剂3-MA后,细胞自噬降低,凋亡增加。RH可使沉默信息调节因子2相关酶1(silent information regulator 2 related enzyme 1,SIRT1)蛋白表达增加;加入SIRT1抑制剂EX-527后,可减弱大黄酸的保护作用,细胞活力降低;加入自噬抑制剂3-MA对SIRT1蛋白的表达无影响,但加入EX-527后SIRT1、LC3-Ⅱ蛋白的表达降低,p62的表达升高。经RH处理后,磷酸腺苷活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)的磷酸化水平升高,哺乳动物雷帕霉素靶蛋白(mechanistic target of rapamycin,mTOR)的磷酸化水平降低,呈剂量依赖性;且这种作用能被AMPK抑制剂compound C减弱。RH可能通过SIRT1/AMPK/mTOR通路增强自噬,减少H_(2)O_(2)诱导的HUVECs凋亡。This study investigated the effect of rhein(RH)on the apoptosis and autophagy of human umbilical vein endothelial cells(HUVECs)induced by hydrogen peroxide(H_(2)O_(2))and its underlying mechanism.The oxidative damage model in HUVECs was established and the cells were divided into different treatment groups.Cell survival rate was detected by MTT assay,apoptosis by Annexin V-FITC/PI double staining and Hoechst 33258 fluorescence staining,autophagy by Ad-mCherry-GFP-LC3 B adenovirus transfection,and protein expression by Western blot.The results showed that RH could protect cells by increasing the cell survival rate in a dose-dependent manner,decreasing the expression of apoptosis-related proteins(Bax and cleaved caspase-3)and the ratio of Bax/Bcl-2,elevating the expression of Bcl-2,up-regulating the expression of microtubule-associated protein 1 light chain 3(LC3)-Ⅱ,and down-regulating the expression of p62.Adenovirus transfection results showed that RH could increase the green and red spots,as well as the yellow spots.However,after the addition of autophagy inhibitor 3-MA,autophagy was reduced and apoptosis was increased.RH could enhance the expression of silent information regulator 2 related enzyme 1(SIRT1).The addition of SIRT1 inhibitor EX-527 reduced the protective effect of RH and cell viability.The addition of 3-MA had no effect on the expression of SIRT1 protein,but the expression of SIRT1 and LC3-Ⅱproteins decreased and the expression of p62 increased after the addition of EX-527.After RH treatment,the phosphorylation of adenosine monophosphate-activated protein kinase(AMPK)increased,while that of the mechanistic target of rapamycin(mTOR)decreased in a dose-dependent manner.Moreover,this effect could be weakened by the AMPK inhibitor compound C.RH may enhance autophagy through SIRT1/AMPK/mTOR pathway to reduce H_(2)O_(2)-induced apoptosis of HUVECs.

关 键 词：大黄酸 HUVECS 凋亡 自噬 

分 类 号：R285[医药卫生—中药学]