Mechanism of Modified Zhenwutang in Delaying Progression of Chronic Renal Failure Based on Network Pharmacology， Molecular Docking， and Experimental Verification

ZHANG Yuanyuan; JIN Peipei; LI Wanzhe; GUO Dengzhou

doi:10.13422/j.cnki.syfjx.20230719

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Mechanism of Modified Zhenwutang in Delaying Progression of Chronic Renal Failure Based on Network Pharmacology， Molecular Docking， and Experimental Verification

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- Mechanism of Modified Zhenwutang in Delaying Progression of Chronic Renal Failure Based on Network Pharmacology， Molecular Docking， and Experimental Verification
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 16, Pages: 123-133(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.20230719
  CLC： R284;R285;R289;R287;R22;R2-031;R33;R24
- Published：20 August 2023，
  
  Published Online：15 June 2023，
  
  Received：11 April 2023，
- 稿件说明：
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张圆圆,靳培培,李宛哲等.基于网络药理学、分子对接和实验验证探讨加味真武汤延缓慢性肾功能衰竭进展的作用机制[J].中国实验方剂学杂志,2023,29(16):123-133.

ZHANG Yuanyuan,JIN Peipei,LI Wanzhe,et al.Mechanism of Modified Zhenwutang in Delaying Progression of Chronic Renal Failure Based on Network Pharmacology， Molecular Docking， and Experimental Verification[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(16):123-133.
张圆圆,靳培培,李宛哲等.基于网络药理学、分子对接和实验验证探讨加味真武汤延缓慢性肾功能衰竭进展的作用机制[J].中国实验方剂学杂志,2023,29(16):123-133. DOI： 10.13422/j.cnki.syfjx.20230719.

ZHANG Yuanyuan,JIN Peipei,LI Wanzhe,et al.Mechanism of Modified Zhenwutang in Delaying Progression of Chronic Renal Failure Based on Network Pharmacology， Molecular Docking， and Experimental Verification[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(16):123-133. DOI： 10.13422/j.cnki.syfjx.20230719.

摘要

目的

基于网络药理学初步预测加味真武汤治疗慢性肾功能衰竭（CRF）的活性成分、作用靶点及信号通路，通过分子对接和动物实验验证探讨其延缓疾病进展的可能作用机制。

方法

通过本草组鉴（HERB）数据库获取加味真武汤的有效成分及靶点；通过基因数据库（GeneCards）获取CRF疾病靶点；通过Venny 2.1软件获得交集靶点基因，结合STRING数据库绘制蛋白质-蛋白质相互作用网络，并通过Cytoscape 3.9.1软件筛选加味真武汤治疗CRF的核心作用靶点；使用Metascape数据库对交集基因进行基因本体（GO）分析和京都基因与基因组百科全书（KEGG）富集分析。选取关键作用靶点与药物活性成分通过AutoDockTools 1.5.6软件进行分子对接验证；采用腺嘌呤灌胃12周的方法建立实验性CRF大鼠模型，给予加味真武汤和盐酸贝那普利干预4周。给药结束后处死大鼠，通过免疫组织化学（IHC）、免疫荧光（IF）、实时荧光定量聚合酶链式反应（Real-time PCR）、蛋白免疫印迹（Western blot）法检测大鼠肾组织中脯氨酸羟化酶1（PHD1）、脯氨酸羟化酶2（PHD2）、低氧诱导因子-1

（HIF-1

），

-平滑肌肌动蛋白（

-SMA）表达水平进行动物实验验证。

结果

通过HERB数据库获得加味真武汤药物靶点基因426个；通过GeneCards获取CRF相关靶点基因2 698个；药物疾病交集基因154个；核心靶点8个，其中白蛋白（ALB）、蛋白激酶B1（Akt1）、肿瘤坏死因子（TNF）、白细胞介素-6（IL-6）、胰岛素（INS）、血管内皮生长因子A（VEGFA）、肿瘤蛋白p53（TP53）、白细胞介素-1

（IL-1

）等靶点可能与加味真武汤治疗CRF密切相关；KEGG富集分析预测加味真武汤治疗CRF的机制主要涉及脂质与动脉粥样硬化、HIF-1信号通路、细胞凋亡、核转录因子-

B（NF-

B）信号通路等通路。分子对接结果，加味真武汤的成分与核心作用靶点中的ALB、Akt1、TNF、IL-6、INS、VEGFA、TP53、IL-1

具有较为稳定的结合活性，其可能通过影响ALB、Akt1、TNF、IL-6、INS、VEGFA、TP53、IL-1

靶点蛋白进而调控炎症反应和细胞凋亡。动物模型验证结果，加味真武汤可降低CRF大鼠肾组织中HIF-1

、

-SMA表达水平，提高PHD1、PHD2表达水平，减轻肾组织细胞缺氧损伤，减少肌成纤维细胞生成，减缓CRF大鼠疾病进展。

结论

加味真武汤可能通过影响ALB、Akt1、TNF、IL-6、INS、VEGFA、TP53、IL-1

等靶点蛋白的表达及调控HIF-1等信号通路进而改善肾组织缺氧，抑制细胞转分化、细胞凋亡/焦亡、炎症反应，从而延缓CRF进展。

Abstract

Objective

To preliminarily predict the active ingredients， targets， and signaling pathways of modified Zhenwutang in the treatment of chronic renal failure （CRF） based on network pharmacology and explore its potential mechanism for delaying disease progression through molecular docking and animal experiments.

Method

The effective ingredients and targets of modified Zhenwutang were obtained from the HERB database. The targets related to CRF were obtained from the GeneCards. The intersection target genes were obtained using Venny 2.1 software and a protein-protein interaction （PPI） network was constructed using the STRING. The core targets for treating CRF with modified Zhenwutang were screened using Cytoscape 3.9.1 software. The intersection genes were analyzed using Metascape database for gene ontology （GO） analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis. Molecular docking validation was performed using AutoDockTools 1.5.6 software for the key targets and active ingredients. An experimental CRF model was established in rats by administering adenine via gavage for 12 weeks， followed by intervention with modified Zhenwutang and benazepril hydrochloride for four weeks. After treatment， the rats were euthanized， and immunohistochemistry （IHC）， immunofluorescence （IF）， real-time quantitative polymerase chain reaction （Real-time PCR）， and western blot were performed to detect the expression levels of prolyl hydroxylase domain-containing proteins 1 （PHD1）， prolyl hydroxylase domain-containing proteins 2 （PHD2）， hypoxia-inducible factor-1

（HIF-1

）， and

-smooth muscle actin （

-SMA） in the renal tissues of the rats.

Result

A total of 426 drug target genes of modified Zhenwutang were obtained from the HERB database. A total of 2 698 target genes related to CRF were obtained from the GeneCards database. There were 154 intersection genes between the drug and the disease. Eight core targets were identified， including albumin （ALB）， protein kinase B1 （Akt1）， tumor necrosis factor （TNF）， interleukin-6 （IL-6）， insulin （INS）， vascular endothelial growth factor A （VEGFA）， tumor protein p53 （TP53）， and interleukin-1

（IL-1

）， which might be closely related to the treatment of CRF with modified Zhenwutang. KEGG enrichment analysis predicted that the main mechanism of modified Zhenwutang in treating CRF involved lipid and atherosclerosis， HIF-1 signaling pathway， cell apoptosis， and nuclear factor kappa B （NF-

B） signaling pathway. Molecular docking results showed that the ingredients of modified Zhenwutang had stable binding activity with the core targets ALB， Akt1， TNF， IL-6， INS， VEGFA， TP53， and IL-1

， which may regulate inflammation and cell apoptosis by affecting the target proteins. The animal model validation results demonstrated that modified Zhenwutang could reduce the expression levels of HIF-1

and

-SMA in the renal tissues of CRF rats， increase the expression levels of PHD1 and PHD2， alleviate renal tissue hypoxia injury， reduce myofibroblast formation， and slow down the progression of CRF in rats.

Conclusion

Modified Zhenwutang may improve renal tissue hypoxia， inhibit cell transdifferentiation， cell apoptosis/necroptosis， and inflammation by affecting the expression of target proteins such as ALB， Akt1， TNF， IL-6， INS， VEGFA， TP53， and IL-1

， as well as regulating the HIF-1 signaling pathway， thus delaying the progression of CRF.

关键词

加味真武汤CRF网络药理学分子对接动物实验

Keywords

modified Zhenwutangchronic renal failurenetwork pharmacologymolecular dockinganimal experiments

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