基于Nrf2/HO-1/NQO1信号通路探讨益肾通络方改善糖尿病肾脏病小鼠氧化应激损伤的机制  

Yishen Tongluo Prescription Ameliorates Oxidative Stress Injury in Mouse Model of Diabetic Kidney Disease via Nrf2/HO-1/NQO1 Signaling Pathway

作　　者：张轶斐 白雪慧 曹梓静 张泽钰 唐靖怡 席俊羽 张术姣 张帅星 谢亦冉 吴宇琪 刘忠杰[1,2] 刘伟敬 ZHANG Yifei;BAI Xuehui;CAO Zijing;ZHANG Zeyu;TANG Jingyi;XI Junyu;ZHANG Shujiao;ZHANG Shuaixing;XIE Yiran;WU Yuqi;LIU Zhongjie;LIU Weijing(Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100029,China;Key Laboratory of Chinese Internal Medicine of Ministry of Education,Beijing University of Chinese Medicine,Beijing 100029,China;Beijing Puren Hospital,Beijing 100062,China)

机构地区：[1]北京中医药大学东直门医院,北京100029 [2]北京中医药大学中医内科学教育部重点实验室,北京100029 [3]北京普仁医院,北京100062

出　　处：《中国实验方剂学杂志》2025年第5期41-51,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(82374382,82074361,82274293);中央高校基本科研业务费专项资金资助项目揭榜挂帅项目(2023-JYB-JBZD-037);国家中医药管理局高水平医院临床研究和成果转化能力提升试点项目揭榜挂帅项目(DZMGXZYY-23002);中华中医药学会青年求实项目(ZSL-003-02)。

摘　　要：目的:基于核因子E_(2)相关因子2/血红素加氧酶-1/NAD(P)H醌还原酶1(Nrf2/HO-1/NQO1)信号通路,探讨益肾通络方对糖尿病肾脏病(DKD)小鼠氧化应激损伤的干预作用及机制。方法:采用SPF级雄性C57BL/6小鼠进行研究,分为正常组10只,造模组50只,采用腹腔注射链脲佐菌素(STZ)建立DKD模型。造模组随机分为模型组、司美格鲁肽组(40μg·kg^(-1))、益肾通络方高、中、低剂量组(18.2、9.1、4.55 g·kg^(-1)),每组10只,连续给药12周。留取标本后测定小鼠血糖、24 h尿蛋白;计算肾脏指数(KI);检测肌酐(SCr)、尿素氮(BUN)、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST);苏木素-伊红(HE)、过碘酸-希夫(PAS)、过碘酸-六胺银(PASM)、马松(Masson)染色观察各组肾组织病理改变;酶联免疫吸附测定法(ELISA)试剂盒检测β_(2)-微球蛋白(β_(2)-MG)、中性粒细胞明胶酶相关脂质运载蛋白(NGAL)、肾损伤分子-1(KIM-1)、肝脂肪酸结合蛋白(L-FABP)、一氧化氮合酶(NOS)、谷胱甘肽(GSH)、总抗氧化能力(T-AOC)和8-羟基脱氧鸟苷(8-OHdG)表达水平;免疫组化染色观察Kelch样环氧氯丙烷相关蛋白1(Keap1)、丙二醛(MDA)蛋白表达;实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测Nrf2/HO-1/NQO1 mRNA和蛋白表达水平。结果:与正常组比较,DKD模型组小鼠血糖、24 h尿蛋白水平、KI、SCr、BUN、ALT升高,肾小球肥大、肾小管扩张、基底膜增厚、系膜增生、胶原沉积,β_(2)-MG、NGAL、KIM-1、L-FABP水平增高,NOS、8-OHdG水平升高,GSH、T-AOC水平降低,MDA、Keap1表达增多,Nrf2、HO-1、NQO1、GCLC均表达降低(P<0.05);与模型组比较,司美格鲁肽及益肾通络方组中、高剂量组血糖、24 h尿蛋白水平、KI、SCr、BUN、ALT降低,肾组织病理损伤有不同程度的改善,β_(2)-MG、NGAL、KIM-1、L-FABP表达降低,NOS、8-OHdG水平降低,GSH、T-AOC水平升高,MDA、Keap1表达减少,Nrf2、HO-1、NQO1Objective:To investigate the effect and mechanism of Yishen Tongluo prescription in protecting mice from oxidative stress injury in diabetic kidney disease(DKD)via the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/NAD(P)H quinone oxidoreductase 1(NQO1)signaling pathway.Methods:Specific pathogen-free(SPF)male C57BL/6 mice were assigned into a control group(n=10)and a modeling group(n=50).The DKD model was established by intraperitoneal injection of streptozotocin.The mice in the modeling group were randomized into a model group,a semaglutide(40μg·kg^(-1))group,and high-,medium-,and low-dose(18.2,9.1,4.55 g·kg^(-1),respectively)Yishen Tongluo prescription groups,with 10 mice in each group.The treatment lasted for 12 weeks.Blood glucose and 24-h urine protein levels were measured,and the kidney index(KI)was calculated.Serum levels of creatinine(SCr),blood urea nitrogen(BUN),alanine aminotransferase(ALT),and aspartate aminotransferase(AST)were assessed.The pathological changes in the renal tissue were evaluated by hematoxylin-eosin,periodic acid-Schiff,periodic acid-silver methenamine,and Masson’s trichrome staining.Enzyme-linked immunosorbent assay kits were used to measure the levels ofβ2-microglobulin(β2-MG),neutrophil gelatinase-associated lipocalin(NGAL),kidney injury molecule-1(KIM-1),liver fatty acid-binding protein(L-FABP),nitric oxide synthase(NOS),glutathione(GSH),total antioxidant capacity(T-AOC),and 8-hydroxy-2'-deoxyguanosine(8-OHdG).Immunohistochemical staining was performed to examine the expression of Kelch-like ECH-associated protein 1(Keap1)and malondialdehyde(MDA).Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot were employed to determine the mRNA and protein levels,respectively,of factors in the Nrf2/HO-1/NQO1 signaling pathway.Results:Compared with the control group,the DKD model group showed rises in blood glucose,24-h urine protein,KI,SCr,BUN,and ALT levels,along with glomerular hypertrophy,renal tubular dilation,thic

关 键 词：糖尿病肾脏病 氧化应激 基于核因子E_(2)相关因子2(Nrf2)/血红素加氧酶-1(HO-1)/NAD(P)H醌还原酶1(NQO1)信号通路 益肾通络方 

分 类 号：R285[医药卫生—中药学] R289[医药卫生—中医学] R259