表没食子儿茶素没食子酸酯对程序性坏死的抑制作用  

Inhibitory effect of epigallocatechin gallate on necroptosis

作　　者：温静 胡爽 范晓迪[1] 李澎[1] WEN Jing;HU Shuang;FAN Xiaodi;LI Peng(Institute of Basic Medical Sciences,Xiyuan Hospital of China Academy of Chinese Medical Sciences,Beijing Key Laborato-ry of Pharmacology of Chinese Materia Medica,Beijing 100091,China;Graduate School of China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]中国中医科学院西苑医院基础医学研究所,北京市中药药理重点实验室,北京100091 [2]中国中医科学院研究生院,北京100700

出　　处：《中国病理生理杂志》2023年第5期811-818,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81274196;No.81973594)。

摘　　要：目的:观察表没食子酸儿茶素没食子酸酯(EGCG)的抗程序性坏死作用并探讨其机制。方法:培养L929小鼠成纤维肉瘤细胞和3T3小鼠成纤维细胞,以不同浓度的EGCG作用24 h,MTT法观察EGCG毒性。复制TNFα诱导的L929细胞程序性坏死、Z-VAD诱导的L929细胞程序性坏死和TNFα复合Z-VAD诱导的3T3细胞程序性坏死三种经典模型,以EGCG干预,MTT法测定细胞活力,计算EGCG对程序性坏死的抑制率,相差显微镜观察细胞形态。在TNFα诱导的L929细胞模型中,Western blot法检测受体相互作用蛋白1(RIP1)和RIP3的表达,荧光显微镜观察线粒体形态,流式细胞术检测线粒体和细胞内超氧阴离子含量。结果:EGCG在100 mg/L以下对L929和3T3细胞没有毒性;在12.5~50 mg/L对程序性坏死导致的细胞活力下降具有显著抑制作用,并且改善细胞形态,同时可以抑制RIP1和RIP3的表达增加、线粒体的过度分裂及线粒体和细胞内超氧阴离子浓度的升高,这些作用均呈浓度依赖性。结论:EGCG具有显著的抗程序性坏死作用,这一作用与其对程序性坏死通路的阻抑有关。AIM:To observe the anti-necroptosis effect of(−)-epigallocatechin gallate(EGCG)and further study its mechanism.METHODS:Mouse fibrosarcoma L929 cells and mouse 3T3 fibroblasts were cultured,and the toxici⁃ties of EGCG to these cells were gauged by MTT assay.Three classical necroptosis models,including L929 cell necropto⁃sis induced by TNFα,L929 cell necroptosis induced by Z-VAD,and 3T3 cell necroptosis induced by TNFαplus Z-VAD,were duplicated,and the effects of EGCG on cell death were evaluated by MTT assay and phase-contrast microscopy.The therapeutic mechanism of EGCG was further studied in the L929 cell model induced by TNFα.The expression levels of re⁃ceptor-interacting protein 1(RIP1)and RIP3 were analyzed by Western blot,the morphological changes of mitochondria were observed by fluorescence microscopy,and the levels of mitochondrial and cellular superoxide anions were measured by flow cytometry.RESULTS:Treatment with EGCG showed no toxicities to L929 and 3T3 cells at the concentration up to 100 mg/L.It inhibited the decrease in cell viability,and ameliorated the cell morphology in the three classical necropto⁃sis models.In L929 cell necroptosis model induced by TNFα,EGCG prevented the increases in RIP1 and RIP3 expres⁃sion levels,the over-fragmentation of mitochondria,and the elevation of superoxide anions in the mitochondria and cells.These ameliorative effects of EGCG all presented concentration-dependently in the range from 12.5 to 50 mg/L.CON⁃CLUSION:EGCG has a strong inhibitory effect on necroptosis,which may be associated with its blocking activity on the necroptosis pathway.

关 键 词：表没食子酸儿茶素没食子酸酯 程序性坏死 受体相互作用蛋白 氧自由基 

分 类 号：R285.5[医药卫生—中药学] R329.28[医药卫生—中医学]