基于Ang Ⅱ/AT1R/NOX4信号通路探讨加味真武汤延缓慢性肾功能衰竭肾间质纤维化机制  

Mechanism of Modified Zhenwutang in Delaying Renal Interstitial Fibrosis in Chronic Renal Failure Based on AngⅡ/AT1R/NOX4 Signaling Pathway

作　　者：张圆圆 靳培培 边东 郭登洲[2] ZHANG Yuanyuan;JIN Peipei;BIAN Dong;GUO Dengzhou(Graduate School,Hebei University of Chinese Medicine,Shijiazhuang 050200,China;The First Affiliated Hospital of Hebei University of Chinese Medicine/Hebei Provincial Hospital of Traditional Chinese Medicine,Shijiazhuang 050011,China)

机构地区：[1]河北中医药大学研究生学院,石家庄050091 [2]河北中医药大学第一附属医院/河北省中医院,石家庄050011

出　　处：《中国实验方剂学杂志》2023年第16期89-99,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：河北省中医药管理局科研计划项目(2023021);河北省自然科学基金项目(H2022423367);河北中医学院博士研究生创新项目(XCXZZBS2023009)。

摘　　要：目的:通过观察加味真武汤对腺嘌呤诱导慢性肾功能衰竭(CRF)大鼠血清及肾组织中血管紧张素Ⅱ(Ang Ⅱ)、血管紧张素Ⅱ1型受体(AT1R)、还原型辅酶Ⅱ(NADPH)氧化酶4(NOX4)、转化生长因子-β_(1)(TGF-β_(1))、Ⅰ型胶原蛋白(COL1A1)、Ⅲ型胶原蛋白(COL3A1)表达的影响,探讨加味真武汤延缓CRF肾间质纤维化的可能作用机制。方法:将50只SPF级雄性SD大鼠按照随机数字表法分为正常组10只、造模组40只,将大鼠适应性饲养1周后采用腺嘌呤150 mg·kg^(-1)·d^(-1)灌胃的方法建立实验性CRF大鼠模型。造模完成后随机选取正常组和造模组大鼠各3只取材,检测造模是否成功。造模成功后,将造模组大鼠按照随机数字表法分成模型组、中药低剂量组、中药中剂量组、中药高剂量组、盐酸贝那普利组各6只,进行药物灌胃,每日1次,治疗4周。于实验第1周末、第13周末、第17周末检测24 h尿蛋白定量(24 h-UTP),第17周各组大鼠麻醉后取材,腹主动脉取血后离心取上清检测血清总蛋白(TP)、白蛋白(ALB)、肌酐(Cr)、尿素氮(BUN);酶联免疫吸附测定法(ELISA)检测血清AngⅡ表达水平;苏木素-伊红(HE)、马松(Masson)染色法观察肾组织病理改变;免疫组织化学(IHC)法观察AT1R、NOX4、TGF-β_(1)、COL1A1、COL3A1表达情况;实时荧光定量聚合酶链式反应法(Real-time PCR)观察AT1R、NOX4、TGF-β mRNA表达水平;蛋白质免疫印迹法(Western blot)检测AT1R、NOX4、TGF-β_(1)表达水平。结果:(1)与正常组比较,模型组实验大鼠24 h-UTP显著增高(P<0.01);模型组实验大鼠Cr、BUN含量水平显著升高(P<0.01),TP、ALB含量水平显著降低(P<0.01);模型组实验大鼠血清AngⅡ含量显著升高(P<0.01);模型组实验大鼠肾小球球囊间隙明显增宽,可见坏死肾小球,肾间质明显增宽伴有大量炎细胞浸润,大量肾小管管腔有褐色沉淀物阻塞,无规整肾小管,肾间质有大量胶原纤维沉积,肾脏血管周围、Objective:To explore the underlying mechanism of modified Zhenwutang in delaying renal interstitial fibrosis in chronic renal failure(CRF)by observing the effects of modified Zhenwutang on the expression of angiotensinⅡ(AngⅡ),angiotensinⅡtype 1 receptor(AT1R),nicotinamide adenine dinucleotide phosphate(NADPH)oxidase 4(NOX4),transforming growth factor-β_(1)(TGF-β_(1)),type I collagen(COL1A1),and typeⅢcollagen(COL3A1)in the serum and renal tissues of adenine-induced CRF rats.Method:Fifty male SPF-grade SD rats were randomly divided into a normal group(n=10)and an experimental group(n=40)using a random number table.After one week of adaptive feeding,the experimental CRF model was established in rats by administering adenine at 150 mg·kg^(-1)·d^(-1) orally.Three rats from each group were randomly selected to evaluate the model induction.After successful modeling,rats in the experimental group were randomly divided into a model group,low-,medium,and high-dose modified Zhenwutang groups,and a benazepril hydrochloride group,with six rats in each group.The rats were orally administered the corresponding drugs once daily for four weeks.At the end of the first week,13th week,and 17th week of the experiment,24 hour urinary protein quantification(24 h-UTP)was measured.At the end of the 17^(th) week,the rats were euthanized,and blood samples were collected from the abdominal aorta for the measurement of total protein(TP),albumin(ALB),creatinine(Cr),and blood urea nitrogen(BUN)in the serum.Enzyme-linked immunosorbent assay(ELISA)was used to measure the expression levels of serum AngⅡ.Hematoxylin-eosin(HE)staining and Masson's trichrome staining were performed to observe the pathological changes in renal tissues.Immunohistochemistry(IHC)was performed to observe the expression of AT1R,NOX4,TGF-β_(1),COL1A1,and COL3A1.Real-time fluorescence-based quantitative polymerase chain reaction(Real-time PCR)was used to observe the mRNA expression levels of AT1R,NOX4,and TGF-β_(1).Western blot was conducted to measure the p

关 键 词：慢性肾功能衰竭 加味真武汤 肾间质纤维化 血管紧张素Ⅱ 血管紧张素Ⅱ1型受体 还原型辅酶Ⅱ(NADPH)氧化酶4(NOX4) 

分 类 号：R284[医药卫生—中药学] R285[医药卫生—中医学] R289R287R22R2-031R33R24