Molecular Mechanism of Tangbikang Granules Against Diabetic Peripheral Neuropathy： Based on Network Pharmacology and Experimental Verification

ZHANG Yaqi; QIN Lingling; BAI Huizhong; ZHANG Chengfei; ZHANG Qiue; ZUO Xinwei; JIANG Shengyuan; ZHAO Yi; LIU Tonghua; MU Xiaohong

doi:10.13422/j.cnki.syfjx.20230722

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Molecular Mechanism of Tangbikang Granules Against Diabetic Peripheral Neuropathy： Based on Network Pharmacology and Experimental Verification

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- Molecular Mechanism of Tangbikang Granules Against Diabetic Peripheral Neuropathy： Based on Network Pharmacology and Experimental Verification
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 9, Pages: 81-90(2023)
- 作者机构：
  
  1.北京中医药大学东直门医院，北京 100700
  2.北京中医药大学科技处，北京 100029
  3.教育部高等学校学科创新引智基地，北京 100029
  4.北京中医药大学生命科学学院，北京 100029
  5.北京中医药大学中医学院，北京 100029
  6.北京中医药大学中医养生学北京市重点实验室，北京 100029
  7.科技部中医药防治糖尿病国际联合研究中心，北京 100029
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20230722
  CLC： R2-0;R22;R242;R2-031;R285.5
- Published：05 May 2023，
  
  Published Online：01 February 2023，
  
  Received：21 September 2022，
- 稿件说明：
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张亚奇,秦灵灵,白惠中等.基于网络药理学和实验验证探讨糖痹康颗粒治疗糖尿病周围神经病变的分子机制[J].中国实验方剂学杂志,2023,29(09):81-90.

ZHANG Yaqi,QIN Lingling,BAI Huizhong,et al.Molecular Mechanism of Tangbikang Granules Against Diabetic Peripheral Neuropathy： Based on Network Pharmacology and Experimental Verification[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(09):81-90.
张亚奇,秦灵灵,白惠中等.基于网络药理学和实验验证探讨糖痹康颗粒治疗糖尿病周围神经病变的分子机制[J].中国实验方剂学杂志,2023,29(09):81-90. DOI： 10.13422/j.cnki.syfjx.20230722.

ZHANG Yaqi,QIN Lingling,BAI Huizhong,et al.Molecular Mechanism of Tangbikang Granules Against Diabetic Peripheral Neuropathy： Based on Network Pharmacology and Experimental Verification[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(09):81-90. DOI： 10.13422/j.cnki.syfjx.20230722.

摘要

目的

基于网络药理学和体内实验验证探讨糖痹康颗粒治疗糖尿病周围神经病变的相关分子机制。

方法

通过中药系统药理学数据库与分析平台（TCMSP）平台查找糖痹康颗粒组方中各味中药所含的活性成分及其对应的靶点基因，TCMSP中未收录的中药则通过文献查询有效成分后通过SwissADME类药性分析后上传SwissTargetPrediction数据库预测相关靶点基因。通过GeneCard数据库收集糖尿病周围神经病变（DPN）的相关疾病靶点基因。将药物基因与疾病基因取交集获得糖痹康颗粒治疗DPN的核心靶点基因。将核心靶点基因上传Metascape平台进行基因本体（GO）、京都基因与基因组百科全书（KEGG）富集分析。高糖高脂饲料诱导加小剂量链脲佐菌素（STZ）腹腔注射建立糖尿病大鼠模型，成模后进行糖痹康颗粒高、中、低剂量组（2.5、1.25、0.625 g·kg

-1

）12周连续药物干预。通过功能学检测感觉神经传导速度（SNCV）电生理测定评估神经传导速度改变，坐骨神经苏木素-伊红（HE）染色光镜下观察组织形态评估神经损伤程度，实时荧光定量聚合酶链式反应（Real-time PCR）检测大鼠坐骨神经组织腺苷一磷酸活化蛋白激酶（AMPK）通路相关靶向分子的基因表达，蛋白免疫印迹法（Western blot）检测大鼠坐骨神经组织中AMPK、磷酸化腺苷一磷酸活化蛋白激酶（p-AMPK）的蛋白表达水平。

结果

筛选出槲皮素、山柰酚、

β-

谷甾醇、水蛭蝶啶A、豆甾醇、黄芩素等主要活性成分，主要作用于白细胞介素-6（IL-6）、肿瘤坏死因子（TNF）、蛋白激酶B（Akt）、JUN、HSP90AA1等关键靶点和AMPK、核转录因子-

B（NF-

B）、Janus 激酶/信号转导子与转录激活因子（JAK/STAT）等信号通路。分子对接结果表明

-谷甾醇、豆甾醇等药物活性成分与IL-6、TNF、JUN、HSP90AA1等作用靶点分子有较好的结合性。动物实验结果显示，与正常组比较，模型组大鼠坐骨神经SNCV显著降低（

0.01）；有髓神经纤维排列紊乱、稀松，轴索断裂，甚至髓鞘脱失；坐骨神经AMPK

、AMPK

、过氧化物酶体增殖物激活受体

共激活因子-1

（PGC-1

）、去乙酰化酶3（SirT3）、线粒体转录因子A（TFAM）mRNA表达及坐骨神经蛋白表达水平的p-AMPK/AMPK明显降低（

0.05，

0.01）。与模型组比较，糖痹康颗粒治疗后各组SNCV显著升高（

0.01）；神经形态接近正常组，排列较为紧密；神经AMPK

、AMPK

、PGC-1

、SirT3、TFAM mRNA表达明显升高（

0.05，

0.01）；糖痹康颗粒高、中剂量组p-AMPK/AMPK显著升高（

0.01），糖痹康颗粒低剂量组与模型组蛋白表达差异无统计学意义。

结论

糖痹康颗粒治疗DPN具有多通路-多靶点的特性，其机制可能与糖痹康颗粒激活并调控AMPK/PGC-1

/SirT3信号通路发挥对DPN的治疗作用相关，为后续糖痹康颗粒干预DPN更深入的研究提供实验依据。

Abstract

Objective

To explore the mechanism of Tangbikang granules （TBK） against diabetic peripheral neuropathy （DPN） based on network pharmacology and

in-vivo

experiment.

Method

The active components in medicinals of TBK and their target genes were searched from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）. The active components of the medicinals which are not included in TCMSP were searched from previous research. After the analysis of drug-likeness by SwissADME， the target genes of them were predicted with SwissTargetPrediction. DPN-related target genes were retrieved from GeneCards. The common targets of the disease and the prescription were the hub genes of TBK against DPN， which were uploaded to Metascape for Gene Ontology （GO） term enrichment and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis. High-sugar and high-fat diet and low-dose streptozotocin （STZ，

） were employed to induce diabetes in rats， and then the model rats were respectively treated with low-dose （0.625 g·kg

-1

）， medium-dose （1.25 g·kg

-1

）， and high-dose （2.5 g·kg

-1

） TBK for 12 weeks. Sensory nerve conduction velocity （SNCV） was evaluated. After hematoxylin and eosin （HE） staining， the sciatic nerve was observed under light microscope to examine the nerve damage. Real-time PCR was performed to detect the gene expression of adenosine monophosphate-activated protein kinase （AMPK） pathway-related targets in rat sciatic nerve， and Western blot to measure the protein expression of AMPK and phosphorylated （p）-AMPK in rat sciatic nerve.

Result

The main active components of TBK， such as quercetin， kaempferol，

-sitosterol， leech pteridine A， stigmasterol， and baicalein were screened out， mainly acting on interleukin-6 （IL-6）， tumor necrosis factor （TNF）， protein kinase B （Akt）， JUN， and HSP90AA1 and signaling pathways such as AMPK， nuclear factor-

B （NF

-κ

B）， and Janus kinase/signal transducer and activator of transcription （JAK/STAT）. Molecular docking results showed that

-sitosterol and stigmasterol had high binding affinity with IL-6， TNF， JUN， and HSP90AA1. As for the animal experiment， compared with the normal group， model group had low SNCV of sciatic nerve （

0.01）， disordered and loose myelinated nerve fibers with axonotmesis and demyelinization， low mRNA expression of AMPK

， AMPK

， peroxisome proliferator-activated receptor

coactivator-1

（PGC-1

）， Sirtuin 3 （SirT3）， mitochondrial transcription factor A （TFAM）， and low p-AMPK/AMPK ratio in sciatic nerve （

0.05，

0.01）. Compared with the model group， TBK of the three doses raised the SNCV （

0.01）， restored nerve morphology and nerve compactness， and increased the mRNA expression of AMPK

， AMPK

， PGC-1

， SirT3， and TFAM （

0.05，

0.01）. The ratio of p-AMPK/AMPK in the high-dose and medium-dose TBK groups was higher than that in the model group （

0.01）， while the protein expression in the low-dose TBK group was insignificantly different from that in the model group.

Conclusion

TBK exerts therapeutic effect on DPN through multiple pathways and targets. The mechanism is that it activates and regulates AMPK/PGC-1

/SirT3 signaling， which lays a basis for further study of TBK in the treatment of DPN.

关键词

糖痹康颗粒网络药理学糖尿病周围神经病变动物实验分子机制系统药理学数据库与分析平台（TCMSP）SwissTargetPrediction数据库

Keywords

Tangbikang granulesnetwork pharmacologydiabetic peripheral neuropathyanimal experimentsmolecular mechanismTraditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）SwissTargetPrediction

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