基于Keap1/Nrf2/Ho-1信号转导通路探讨糖肾宁防治糖尿病肾病的作用机制  

Discussion on the mechanism of Tangshenning in the prevention and treatment of diabetic kidney disease based on Keap1/Nrf2/Ho-1 signaling pathway

作　　者：刘运华[1] 张新雪[1] 高坤[1] 王思童 张新江 王舒月 周楷栋 靳鸽 周鑫[1] 蔡炎沫 赵宗江[1] LIU Yun-hua;ZHANG Xin-xue;GAO Kun;WANG Si-tong;ZHANG Xin-jiang;WANG Shu-yue;ZHOU Kai-dong;JIN Ge;ZHOU Xin;CAI Yan-mo;ZHAO Zong-jiang(Beijing University of Chinese Medicine,Beijing 100029,China)

机构地区：[1]北京中医药大学,北京100029

出　　处：《中华中医药杂志》2023年第5期2409-2417,共9页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家重点研发计划项目子课题(No.2018YFC1704304)。

摘　　要：目的:通过网络药理学和体内外实验探究糖肾宁对糖尿病肾病(DKD)大鼠肾脏的保护作用和对糖基化终末产物(AGEs)诱导的HK-2细胞损伤的影响,以及对Kelch样环氧氯丙烷相关蛋白-1(Keap1)/核因子E_(2)相关因子2(Nrf2)/血红素加氯酶1(Ho-1)信号转导通路的调节作用。方法:通过TCMSP筛选糖肾宁药物靶点,通过GenGards、OMIM、drugbank、TTD、Disgenet数据库筛选DKD作用靶点,取公共靶点进行PPI网络分析并进行GO和KEGG预测。将40只SD大鼠,随机分为正常组10只,造模组30只;造模组给予高糖高脂饲料喂养8周后,一次性腹腔注射链脲佐菌素(35 mg/kg)构建DKD模型;将成模动物随机分为模型组,厄贝沙坦(25 mg·kg^(-1)·d^(-1))组,糖肾宁组(1.05 g·kg^(-1)·d^(-1)),每组8只;正常组与模型组灌胃等体积的去离子水;观察大鼠一般状况,称体质量、测24 h尿蛋白定量;干预16周后取材,收集血清检测血尿素(Urea)、血肌酐(Scr),血脂(TG、TC、LDL)、氧化应激(ROS、NO、SOD)指标;采用HE染色、Mallory染色和透射电镜观察肾组织病理形态;Western Blot和RT-PCR检测大鼠肾组织中Keap1、Nrf2、Ho-1、Gpx4蛋白及mRNA表达。体外培养HK-2细胞,并分为空白组、AGEs组(200μg/mL)、无药血清组、糖肾宁组,各组细胞培养48 h后,使用CCK-8法检测细胞活性,荧光探针法观察活性氧表达情况。结果:网络药理分析预测糖肾宁可能通过影响氧化还原相关信号通路,调控AGEs诱导的氧化应激反应,防治DKD肾损伤。体内实验中,与模型组比较,糖肾宁组大鼠一般状态改善,24 h尿蛋白定量显著减少(P<0.01),血清Scr含量降低(P<0.05),Urea有降低趋势,TC、TG、LDL显著降低(P<0.01,P<0.05),ROS水平显著降低(P<0.05),病理染色显示肾间质纤维化明显减轻,电镜观察发现足突融合、粘连情况减轻,糖肾宁组肾组织Keap1蛋白和mRNA表达降低(P<0.01),Nrf2、Ho-1、Gpx4蛋白及mRNA表达显著升高(P<0.01,P<0.05)。体外实验中,Objective:To explore the protective effect of Tangshenning on the kidneys of DKD rats,the effect on AGEsinduced HK-2 cell damage,and the regulatory effect on Keap1/Nrf2/Ho-1 signaling pathway through network pharmacology and in vitro and in vivo experiments.Methods:Tangshenning drug targets were screened by TCMSP,DKD targets were screened by GenGards,OMIM,drugbank,TTD,and Disgenet databases,and the two were intersected for PPI network analysis and target prediction by GO and KEGG.Forty SD rats were randomly divided into 10 normal groups and 30 modules were made;After the module was fed with high-sugar and high-fat feed for 8 weeks,a one-time intraperitoneal injection of Streptozotocin(35 mg/kg)was used to construct a DKD model.The model animals were randomly divided into model group,irbesartan(25 mg·kg^(-1)·d^(-1))group,and Tangshenning group(1.05 g·kg^(-1)·d^(-1))according to body weight,8 rats in each group.Normal group and model group animals gavage equal volume of deionized water.The general condition of rats was observed,weighed and quantified in urine for 24 h;After the intervention of 16 weeks,serum was collected to detect Urea,Scr,blood lipids(TG,TC,LDL),and oxidative stress indicators(ROS,NO,SOD);HE staining,Mallory staining and transmission electron microscopy were used to observe the pathological morphology of renal tissue.Western Blot and RT-PCR detected Keap1,Nrf2,Ho-1,Gpx4 protein and mRNA expression in rat kidney tissue.HK-2 cells were cultured in vitro and divided into:normal group,AGEs group(200μg/mL),drug-free serum group,Tangshenning serum group,and after 48 h of cell culture,the cell activity of each group was detected by CCK-8 method,and the expression of reactive oxygen species in each group was observed by fluorescent probe method.Results:Network pharmacological analysis predicted that Tangshenning may regulate and regulate AGEs-induced oxidative stress by affecting redox-related signaling pathways to prevent and treat DKD kidney injury.In vivo experiments,compared with the model group

关 键 词：糖尿病肾病 糖肾宁 肾痿 氧化应激 HK-2 Kelch样环氧氯丙烷相关蛋白1/核因子E_(2)相关因子2/血红素加氯酶1 

分 类 号：R285.5[医药卫生—中药学]