Exploring the Mechanism of Action of <italic style="font-style: italic">Qizhu Kang'ai Formula </italic>（芪术抗癌方） for the Treatment of Colorectal Cancer based on Network Pharmacology and Experimental Validation

SUN Ruolan; LIANG Yan; ZHAO Fan; ZHU Shijiao; WAN Linlu; WANG Xu; YIN Qihang; YIN Gang; TANG Decai

doi:10.13288/j.11-2166/r.2024.04.012

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Exploring the Mechanism of Action of Qizhu Kang'ai Formula （芪术抗癌方） for the Treatment of Colorectal Cancer based on Network Pharmacology and Experimental Validation

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- Exploring the Mechanism of Action of Qizhu Kang'ai Formula （芪术抗癌方） for the Treatment of Colorectal Cancer based on Network Pharmacology and Experimental Validation
- Journal of Traditional Chinese Medicine Vol. 65, Issue 4, Pages: 404-413(2024)
- 作者机构：
  
  1.南京中医药大学中医学院，江苏省南京市栖霞区仙林大道138号，210023
  2.南京中医药大学药学院
- 作者简介：
- 基金信息：
- DOI：10.13288/j.11-2166/r.2024.04.012
  CLC：
- Received：23 March 2023，
  
  Revised：2023-12-09，
  
  Published：17 February 2024
- 稿件说明：
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孙若岚,梁研,赵凡等.基于网络药理学和实验验证探讨芪术抗癌方治疗结直肠癌的作用机制[J].中医杂志,2024,65(04):404-413.

SUN Ruolan,LIANG Yan,ZHAO Fan,et al.Exploring the Mechanism of Action of Qizhu Kang'ai Formula （芪术抗癌方） for the Treatment of Colorectal Cancer based on Network Pharmacology and Experimental Validation[J].Journal of Traditional Chinese Medicine,2024,65(04):404-413.
孙若岚,梁研,赵凡等.基于网络药理学和实验验证探讨芪术抗癌方治疗结直肠癌的作用机制[J].中医杂志,2024,65(04):404-413. DOI： 10.13288/j.11-2166/r.2024.04.012.

SUN Ruolan,LIANG Yan,ZHAO Fan,et al.Exploring the Mechanism of Action of Qizhu Kang'ai Formula （芪术抗癌方） for the Treatment of Colorectal Cancer based on Network Pharmacology and Experimental Validation[J].Journal of Traditional Chinese Medicine,2024,65(04):404-413. DOI： 10.13288/j.11-2166/r.2024.04.012.

摘要

目的

探讨芪术抗癌方治疗结直肠癌的潜在分子机制。

方法

应用网络药理学对芪术抗癌方中活性成分和治疗结直肠癌的作用靶点进行分析，对芪术抗癌方治疗结直肠癌的关键靶点进行基因功能注释（GO）和京都基因组百科全书（KEGG）通路富集分析。运用分子对接预测核心活性成分与关键靶点的结合活性。通过建立结肠癌原位移植瘤模型小鼠验证网络药理学研究得出的芪术抗癌方干预结直肠癌的关键靶点。48只小鼠随机分为假手术组，模型组，5-氟尿嘧啶（5-Fu）组，芪术抗癌方低、中、高剂量组，每组8只。除假手术组外，其余各组进行结肠癌原位移植瘤造模。造模后隔天开始5-Fu组给予5-Fu 30 mg/kg，每3天1次，腹腔注射；芪术抗癌方低、中、高剂量组分别给予芪术抗癌方2.925、5.85、11.7 g/（kg·d）灌胃，假手术组和模型组每天以0.1 ml/10 g生理盐水灌胃，共21 天。比较各组原位瘤质量及肿瘤肝转移灶数目，HE染色观察结肠肿瘤组织的病理变化，Western blot法检测结肠肿瘤组织中非受体型蛋白酪氨酸磷酸酶1（PTPN1）、黏着斑蛋白（vinculin）、整合素亚单位αν、整合素亚单位β3、E-钙黏蛋白（E-cadherin）的蛋白表达。

结果

网络药理学筛选得到芪术抗癌方干预结直肠癌的核心活性成分前六位为槲皮素、山柰酚、芹菜素、木犀草素、黄芩苷元、熊果酸，其作用靶点有212个，排列前3位的环加氧酶1（PTGS1）、环加氧酶2（PTGS2）、非受体型蛋白酪氨酸磷酸酶1（PTPN1），可能是芪术抗癌方治疗结直肠癌的关键靶点。这些关键靶点主要在磷脂酰肌醇3-激酶/蛋白激酶B（PI3K-Akt）信号通路、黏着斑和黏附连接中显著富集。分子对接结果显示，除PTGS1与槲皮素、山柰酚、芹菜素的结合活性较好外（结合能≥

7.0 kcal/mol），PTGS1与木犀草素、黄芩苷元、熊果酸，PTGS2、PTPN1与6个核心活性成分均有强烈的结合活性（结合能＜

7.0 kcal/mol）。实验验证结果显示，与假手术组比较，模型组小鼠结肠肿瘤组织PTPN1、vinculin、整合素亚单位αν、整合素亚单位β3蛋白表达显著升高，E-cadherin表达显著降低（

＜0.05）；与模型组相比，芪术抗癌方高、中剂量组原位瘤质量降低，肝转移结节数减少（

＜0.05），芪术抗癌方各剂量组及5-Fu组PTNP1、vinculin、整合素亚单位αν、整合素亚单位β3、E-cadherin表达水平呈现不同程度的回调，芪术抗癌方各剂量组各指标改变呈一定的剂量依赖（

＜0.05）。HE染色观察到模型组肿瘤细胞核多呈分裂象，芪术抗癌方各剂量组存在不同程度的肿瘤细胞核碎裂，中、高剂量组更多。

结论

芪术抗癌方能够抑制结直肠癌原位瘤生长和肝转移，其作用机制可能是通过PTPN1调节了肿瘤细胞-细胞间和肿瘤细胞-细胞外基质间的黏附。

Abstract

Objective

To explore the potential molecular mechanism of

Qizhu Kang'ai Formula

（芪术抗癌方， QZKAF） for the treatment of colorectal cancer （CRC）.

Methods

Network pharmacology was used to analyze the active ingredients and targets of QZKAF for CRC， and analyze the key targets of QZKAF for the treatment of CRC by gene function annotation （GO） and Kyoto Encyclopedia of Genomes （KEGG） pathway enrichment analysis. Molecular docking was applied to predict the binding activity of the core active ingredients to the key targets. A orthotopic transplantation tumor mice model of CRC was established to validate the key targets of QZKAF for CRC obtained from network pharmacology analysis. Forty-eight mice were randomly divided into the sham operation group， the model group， the 5-fluorouracil （5-Fu） group， and the QZKAF low-， medium-， and high-dose groups， with 8 mice in each group. Except for the sham operation group， the remaining groups underwent colon cancer orthotopic transplantation tumor modeling. The 5-Fu group was given 30 mg/kg of 5-Fu by intraperitoneal injection once every 3 days on the alternate day after modeling， while the QZKAF low-， medium-， and high-dose groups were given 2.925， 5.85， and 11.7 g/（kg·d） of QZKAF by gastric gavage， respectively， and the sham-operation group and the model group were gavaged with 0.1 ml/10 g of normal saline every day， all for 21 days. The in situ tumors mass and the number of liver metastases were compared between the groups. The pathological changes of colon tumor tissues were observed by HE staining， and the protein expression of protein tyrosine phosphatase nonreceptor type 1 （PTPN1）， vinculin， integrin subunit αν， integrin subunit β3， and E-cadherin were detected in colon tumor tissues by Western blot.

Results

Network pharmacology screening yielded that the top six core active ingredients of QZKAF intervening in CRC were quercetin， kaempferol， apigenin， luteolin， baicalein and ursolic acid. There were 212 targets of action， and the ranked top three were prostaglandin endoperoxide synthase 1 （PTGS1）， prostaglandin endoperoxide synthase 2 （PTGS2）， and PTPN1， which may be the key targets of QZKAF in the treatment of CRC. These key targets were significantly enriched mainly in phosphatidylinositol 3-kinase/protein kinase B （PI3K-Akt） signaling pathway， focal adhesion and adhesion junction. Molecular docking results： except for PTGS1 with better binding activity to quercetin， kaempferol， and apigenin （binding energy ≥

7.0 kcal/mol）， PTGS1 showed strong binding activity to lignans， baicalein， ursolic acid， as well as PTGS2 and PTPN1 to the six core active ingredients （binding energy ＜

7.0 kcal/mol）. Experimental validation results： the protein expression of PTPN1， vinculin， integrin subunit αν， integrin subunit β3 in the colon tumor tissues of mice in the model group significantly increased， and the expression of E-cadherin significantly decreased compared to those in the sham operation group （

＜0.05）. Compared to those in the model group， the mass of the in situ tumors was reduced， and the number of hepatic metastasis nodules decreased in the high- and medium-dose QZKAF groups （

＜0.05）； the expression levels of PTNP1， vinculin， integrin subunit αν， integrin subunit β3 and E-cadherin in all QZKAF groups and 5-Fu group showed different degrees of retracement， and the changes of the indicators in all QZKAF groups showed a certain degree of dose-dependence （

＜0.05）. HE staining showed that the nuclei of tumor cells in the model group were mostly schizophrenic， and there were different degrees of nuclear fragmentation of tumor cells in all QZKAF groups with more in the medium- and high-dose groups.

Conclusion

QZKAF could inhibit the growth of in situ tumors and liver metastasis of CRC. Its mechanism might be related to the regulation of tumor cell-cell and tumor cell-extracellular matrix adhesion by PTPN1.

关键词

Keywords

references

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Exploring the Mechanism of Action of Qizhu Kang'ai Formula （芪术抗癌方） for the Treatment of Colorectal Cancer based on Network Pharmacology and Experimental Validation

DOI：10.13288/j.11-2166/r.2024.04.012

摘要

Abstract

关键词

Keywords

references