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

ZHANG Yuanyuan; JIN Peipei; BIAN Dong; GUO Dengzhou

doi:10.13422/j.cnki.syfjx.20230615

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

更新时间：2023-07-18

- Mechanism of Modified Zhenwutang in Delaying Renal Interstitial Fibrosis in Chronic Renal Failure Based on Ang Ⅱ/AT1R/NOX4 Signaling Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 16, Pages: 89-99(2023)
- 作者机构：
  
  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
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20230615
  CLC： R284;R285;R289;R287;R22;R2-031;R33;R24
- Received：06 April 2023，
  
  Published Online：05 May 2023，
  
  Published：20 August 2023
- 稿件说明：
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张圆圆,靳培培,边东等.基于AngⅡ/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[J].中国实验方剂学杂志,2023,29(16):89-99.

ZHANG Yuanyuan,JIN Peipei,BIAN Dong,et al.Mechanism of Modified Zhenwutang in Delaying Renal Interstitial Fibrosis in Chronic Renal Failure Based on Ang Ⅱ/AT1R/NOX4 Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(16):89-99.
张圆圆,靳培培,边东等.基于AngⅡ/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[J].中国实验方剂学杂志,2023,29(16):89-99. DOI： 10.13422/j.cnki.syfjx.20230615.

ZHANG Yuanyuan,JIN Peipei,BIAN Dong,et al.Mechanism of Modified Zhenwutang in Delaying Renal Interstitial Fibrosis in Chronic Renal Failure Based on Ang Ⅱ/AT1R/NOX4 Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(16):89-99. DOI： 10.13422/j.cnki.syfjx.20230615.

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（AngⅡ）、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-

（TGF-

）、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-

、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-

mRNA表达水平；蛋白质免疫印迹法（Western blot）检测AT1R、NOX4、TGF-

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（

0.01）；模型组实验大鼠Cr、BUN含量水平显著升高（

0.01），TP、ALB含量水平显著降低（

0.01）；模型组实验大鼠血清AngⅡ含量显著升高（

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、肾小囊壁层囊壁外、肾小球基底膜和肾小管基底膜胶原纤维明显增多；模型组实验大鼠AT1R、NOX4在肾小球、肾小管表达明显增强，TGF-

在肾小管表达明显增强，COL1A1、COL3A1在肾间质表达明显增强；模型组实验大鼠AT1R、TGF-

mRNA表达显著增强（

0.01），NOX4 mRNA表达显著减弱（

0.01）；AT1R、NOX4、TGF-

蛋白表达显著增强（

0.01）。②与模型组比较，加味真武汤干预后，24 h-UTP显著降低（

0.01）；Cr、BUN含量水平显著降低（

0.01），TP、ALB含量水平显著升高（

0.01）；AngⅡ含量显著下降（

0.01）；肾脏病理损害减轻；AT1R、NOX4、TGF-

、COL1A1、COL3A1在肾小球、肾小管、肾间质达减弱；AT1R、TGF-

mRNA表达显著下降（

0.01），NOX4 mRNA表达显著升高（

0.01）；AT1R、NOX4、TGF-

蛋白表达显著减弱（

0.01）；中药组表现出明显的量效趋势。

结论

加味真武汤可能通过降低CRF大鼠血清及肾组织中AngⅡ、AT1R、NOX4、TGF-

表达，延缓肾间质纤维化进展，从而减轻肾脏病理损害，减少蛋白尿，保护肾功能，达到延缓CRF进展的目的，且中药组具有量效趋势。

Abstract

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 angiotensin Ⅱ （Ang Ⅱ）， angiotensin Ⅱ type 1 receptor （AT1R）， nicotinamide adenine dinucleotide phosphate （NADPH） oxidase 4 （NOX4）， transforming growth factor-

（TGF-

）， type I collagen （COL1A1）， and type Ⅲ collagen （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 （

=10） and an experimental group （

=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， 13

week， and 17

week of the experiment， 24 hour urinary protein quantification （24 h-UTP） was measured. At the end of the 17th 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 Ang Ⅱ. 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-

， 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-

. Western blot was conducted to measure the protein expression levels of AT1R， NOX4， and TGF-

Result

① Compared with the normal group， the model group showed a significant increase in 24 h-UTP （

0.01）. The levels of Cr and BUN in the model group were significantly higher （

0.01）， while the levels of TP and ALB were significantly lower （

0.01）. The serum Ang Ⅱ level in the model group was significantly elevated （

0.01）. The model group exhibited widening of the renal glomerular mesangial space， necrotic glomeruli， increased interstitial width with extensive inflammatory cell infiltration， brownish precipitates blocking the renal tubular lumens， irregular renal tubules， and significant deposition of collagen fibers in the renal interstitium. Additionally， the collagen fibers around the renal vessels， outside the parietal layer of the renal sacs， glomerular basement membrane， and tubular basement membrane increased significantly. The expression of AT1R and NOX4 in the glomeruli and renal tubules of the model group was significantly enhanced， and TGF-

expression also significantly increased in the renal tubules. The expression of COL1A1 and COL3A1 in the renal interstitium significantly increased. The mRNA expression of AT1R and TGF-

in the model group significantly increased （

0.01）， while NOX4 mRNA expression significantly decreased （

0.01）. The protein expression of AT1R， NOX4， and TGF-

was significantly enhanced （

0.01）. ② Compared with the model group， modified Zhenwutang significantly reduced 24h-UTP （

0.01）， decreased levels of Cr and BUN （

0.01）， increased levels of TP and ALB （

0.01）， reduced serum Ang Ⅱ level （

0.01）， alleviated renal pathological damage， reduced expression of AT1R， NOX4， TGF-

， COL1A1， and COL3A1 in the glomeruli， renal tubules， and renal interstitium， reduced mRNA expression of AT1R and TGF-

（

0.01）， increased NOX4 mRNA expression （

0.01）， and weakened protein expression of AT1R， NOX4， and TGF-

（

0.01）. The modified Zhenwutang groups showed a significant dose-effect trend.

Conclusion

Modified Zhenwutang may delay renal interstitial fibrosis in CRF rats by reducing the expression of Ang Ⅱ， AT1R， NOX4， and TGF-

in the serum and renal tissues， thereby alleviating renal pathological damage， reducing proteinuria， protecting renal function， and delaying the progression of CRF. The modified Zhenwutang group exhibited a dose-effect trend.

关键词

Keywords

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