EGCG降低2型糖尿病大鼠肝细胞凋亡与内质网应激蛋白PERK及GRP78表达的实验研究  

Experimental Study on the Effect of Epigallocatechin Gallate on Hepatocyte Apoptosis and the Expression of Endoplasmic Reticulum Stress Protein PERK and GRP78 in Type 2 Diabetic Rats

作　　者：罗梓人 贾旭 刘红[3] 高瑛[3] 买文丽[3] 刘行海[3] 刘华[3] 郑倩[3] LUO Zi-ren;XU Jia;LIU Hong;GAO Ying;MAI Wen-li;LIU Xing-hai;LIU Hua;ZHENG Qian(School of Pharmacy,North Sichuan Medical College,Nanchong 637000,China;Department of Pharmacy,North Sichuan Medical College,Nanchong 637000,China;Department of Pharmacology,North Sichuan Medical College,Nanchong 637000,China)

机构地区：[1]川北医学院药学院,四川南充637000 [2]川北医学院药剂科,四川南充637000 [3]川北医学院生理学教研室,四川南充637000

出　　处：《茶叶通讯》2020年第4期665-674,共10页Journal of Tea Communication

基　　金：四川省教育厅重点项目(17ZA0168、17ZA0167);南充市科技厅课题(18SXH20224、18SXHZ0293)。

摘　　要：为探讨表没食子儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)对2型糖尿病(type 2 diabetes mellitus,T2DM)大鼠肝脏细胞凋亡的作用及其机制与内质网应激蛋白PERK和GRP78表达的关系,采用注射小剂量链脲佐菌素联合喂养高脂饮食建立T2DM大鼠模型,成模后随机分为模型组(MOR组)、EGCG1低剂量组和EGCG2高剂量组,同时设置正常对照组(NOR组)。EGCG1组与EGCG2组干预12周,检测各组大鼠空腹血糖(fasting blood glucose,FBG)、空腹胰岛素水平(fasting insulin,FINS)和计算胰岛素敏感指数(insulin sensitivity index,ISI),观察EGCG对大鼠糖代谢的影响;HE染色和透射电镜分别观察肝细胞形态和超微结构变化,原位末端转移酶标记技术(TUNEL)方法检测各组大鼠肝细胞凋亡改变,免疫组化和实时荧光定量PCR分别检测肝脏PERK和GRP78蛋白的表达和mRNA水平。结果显示,比较MOR组,EGCG1组大鼠FBG显著降低(P<0.01),EGCG2组大鼠FBG与FINS均显著降低(P<0.01),ISI在高浓度EGCG组明显降低(P<0.01)。HE染色可见模型组肝脏结构存在明显病理损伤,EGCG1和EGCG2组均可观察到肝细胞结构的改善。透射电镜下模型组细胞核染色质聚集,呈现细胞凋亡的改变,不同浓度EGCG干预后肝细胞凋亡细胞减少。TUNEL法测定NOR组有较少细胞凋亡,MOR组AI明显高于NOR组(P<0.01),EGCG1、EGCG2组与MOR组比较AI降低,差异有统计学意义(P<0.05,P<0.01)。免疫组化显示MOR组PERK和GRP78蛋白表达显著高于NOR组(P<0.01),EGCG1与EGCG2组PERK和GRP78蛋白表达减少,低于模型组(P<0.05,P<0.01)。RT-PCR结果显示,MOR组大鼠肝脏内质网应激蛋白PERK和GRP78的mRNA水平明显高于NOR组(P<0.01),与MOR组相比,不同浓度EGCG干预后肝脏细胞中PERK和GRP78的mRNA水平降低,EGCG1组P<0.05,EGCG2组P<0.01。表明EGCG可通过降低内质网应激蛋白PERK和GRP78的表达,抑制肝脏细胞内质网应激水平,从而减轻2型糖尿病大鼠肝细胞凋亡的发生。To explore the effect of epigallocatechin gallate(EGCG)on apoptosis of liver cells in type 2 diabetes mellitus(T2DM)rats and the relationship between its mechanism and the expression of endoplasmic reticulum stress proteins perk and GRP78,T2DM rat model was established by injecting low-dose streptozotocin combined with a high-fat diet.The rats were randomly divided into model group(MOR group),EGCG1 low-dose group and EGCG2 high-dose group,and normal control group(NOR group)was set up.EGCG1 group and EGCG2 group were treated for 12 weeks,the fasting blood glucose(FBG)and fasting insulin(FINS)levels of rats of each group were measured,and insulin sensitivity index(ISI)was calculated,the effects of EGCG on glucose metabolism in rats were observed.HE staining and transmission electron microscopy were used to observe the morphological and ultrastructural changes of liver cells,In situ terminal transferase labeling(TUNEL)method was used to detect the changes of hepatocytes apoptosis.Immunohistochemistry and real-time fluorescent quantitative PCR were used to determine liver endoplasmic reticulum stress protein expression and mRNA level of PERK and GRP78.The results showed that compared with the MOR group,FBG of EGCG1 group was significantly reduced(P<0.01),FBG and FINS of the EGCG2 group were significantly reduced(P<0.01).ISI in high concentration EGCG group was decreased significantly(P<0.01).HE staining showed that the liver structure of model group had obvious pathological damage,and the improvement of hepatocyte structure could be observed in EGCE1 and EGCG2 groups.Under the transmission electron microscope,the chromatin in nucleus of model group was aggregated,and the apoptosis of hepatocytes decreased after different concentrations of EGCG.TUNEL method.The AI in the MOR group was significantly higher than that in the NOR group(P<0.01).Compared with the MOR group,the AI of EGCG1 and EGCG2 group was lower than that in MOR group,and the difference was statistically significant(P<0.05,P<0.01).Immunohistochemistry sho

关 键 词：表没食子儿茶素没食子酸酯 2型糖尿病 内质网应激 肝脏细胞 凋亡 

分 类 号：S571.1[农业科学—茶叶生产加工]