基于Keap1-Nrf2/ARE信号传导通路探讨黄芪多糖改善干燥综合征模型大鼠心功能的机制  

Astragalus Polysaccharides Improved the Cardiac Function in Sjgren′s Syndrome Model Rats Based on Keap 1-Nrf2/ARE Signaling Pathway:a Mechanism Exploration

作　　者：王芳[1] 刘健[2] 叶英法 张晓军[1,3] 万磊[2] 郑力 

机构地区：[1]安徽中医药大学研究生部,合肥230031 [2]安徽中医药大学第一附属医院风湿免疫科,合肥230031 [3]湖北中医药大学研究生处,武汉430065 [4]Department of Radiation Biology,City of Hope National Medical Center and Beckman Research Institute

出　　处：《中国中西医结合杂志》2014年第5期566-574,共9页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家中医药重点学科中医痹病学建设项目(No.国中医药发[2009]30号);国家中医药管理局科研课题专项基金(No.04-05LP27);安徽省科技厅科研计划项目(No.11010402170);安徽中医内科应用基础与开发研究省级实验室项目(No.科条[2008]150号);安徽中医学院科技创新团队项目(No.2010TD005)

摘　　要：目的基于Keap1-Nrf2/ARE信号传导通路探讨黄芪多糖改善干燥综合征(Sjgren′s syndrome,SS)模型大鼠心功能变化的机制。方法将48只Wistar雄性大鼠按随机数字表法分为4组:空白对照组(空白组)、模型对照组(模型组)、黄芪多糖组(中药组)和羟氯喹组(西药组),每组12只,分别向每只大鼠(空白组除外)两后足跖部注射与弗氏完全佐剂充分乳化后的颌下腺蛋白混合抗原0.1mL,诱导SS模型。致炎后第19天开始干预,空白组及模型组均给予等量生理盐水(1mL/100g),其余组分别给予黄芪多糖(1mg/100g)、羟氯喹(0.031 25g/kg),各组每天干预1次,连续干预30天。给药结束后观察大鼠的体质量变化、饮水量变化、颌下腺指数、脾指数、腺体组织学变化;采用有创血流动力学监测SS模型大鼠心功能的变化;采用ELISA法检测血清中活性氧(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、总抗氧化能力(total antioxidant capacity,TAC)、TNF-α、IL-35;HE染色观察心肌组织的病理学改变;免疫组化染色观察ROS、活性氮自由基(reactive nitrogen species,RNS)、谷胱甘肽(glutathione,GSH)、硫氧还蛋白(thioredoxin,TRX)表达;实时荧光定量PCR(real time fluorescence quantitative PCR,RTFQ-PCR)检测Keap1、Nrf2、ARE mRNA蛋白的表达;Western blot方法测定大鼠心肌组织γ-谷氨酰半胱氨酸合成酶(γ-glutamic acid and a half long glycine synthetase,γ-GCS)、血红素氧合酶1(heme oxygenase-1,HO-1)蛋白表达水平。结果与空白组比较,模型组大鼠饮水量、颌下腺指数、脾指数、HR、心脏指数(HI)、左室收缩期压(LVSP)、左室舒张期压(LVEDP)、MDA、ROS、TNF-α、ROS蛋白表达、RNS蛋白表达、Keap1mRNA、MafmRNA、Nfr2mRNA、HO-1蛋白表达、γ-GCS蛋白表达升高(P<0.01),体质量、左室内压上升下降最大速率(±dp/dtmax)、SOD、TAC、IL-35、GSH、TRX蛋白表达降低(P<0.01)。与模型组比较Objective To explore the mechanism of Astragalus polysaccharides （APS） for improving the cardiac function of Sjogren＇s syndrome （SS） model rats based on Keapl-Nrf2/ARE signaling pathway. Methods Totally 48 male Wistar rats were randomly divided into four groups by random digit table, i.e., the blank control group,the model control group,the APS group, and the hydroxychloroquine group,12 in each group. Except those in the blank control group, 0.1 mL mixed antigen protein of sufficiently emulsified Freund＇s complete adjuvant and submandibular gland protein was injected from two feet plantar to induce SS model. The intervention was started from 19th day after inflammation induction. Equal volume of normal saline was given to rats in the blank control group （1 mL/100 g）, APS was administered to those in the APS group （1 mg/100 g）, and hydroxychloroquine （0.03 125 g/kg） was administered to those in the hydroxychloroquine group. All rats were intervened once per day for 30 consecutive days. Changes of rats＇ body mass and drinking water quantity, submandibular gland index, spleen index, histological changes of glands were observed. Changes of the heart function were monitored using invasive hemodynamics. Serum reactive oxygen species （ROS）, malondialdehyde （MDA）, superoxide dismutase （SOD）, total antioxidant capacity （TAC）, tumor necrosis factor alpha （TNF-α）, and interleukin- 35 （IL-35）were detected using ELISA method. The pathological changes were observed using HE staining. The protein expression of ROS, reactive nitrogen species （RNS）, glutathione （GSH）, and thioredoxin （TRX） were observed by immunohistochemical staining. The mRNA expression of Keapl, Nrf2, and ARE was detected using real time fluorescent quantitative PCR. The protein expression levels of γ-glutamic acid and a half long glycine synthetase （γ-GCS） and heme oxygenase 1 （HO-1） in the myocardial tissue were determined by Western blot method. Results Compared with the blank control

关 键 词：干燥综合征 心功能 氧化应激 黄芪多糖 Keapl—Nrf2 ARE信号通路 

分 类 号：R580[医药卫生—内分泌]