南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (7): 1051-1062.doi: 10.12122/j.issn.1673-4254.2023.07.01

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六神丸治疗小鼠结肠炎相关性结直肠癌的作用机制：基于网络药理学和体内验证方法

张雪芳，陈延华，李宗恒，尚 靖，袁泽婷，邓皖利，骆 莺，韩 娜，殷佩浩，殷 军   

  1. 沈阳药科大学中药学院，辽宁 本溪 117004；上海中医药大学附属普陀医院普外科，上海 200062；上海中医药大学上海市中医药研究院中西医结合肿瘤介入研究所，上海 200062；上海市长宁区妇幼保健院检验科，上海 200000
  • 出版日期:2023-07-20 发布日期:2023-07-20

Analysis of therapeutic mechanism of Liushen Wan against colitis-associated colorectal cancer based on network pharmacology and validation in mice

ZHANG Xuefang, CHEN Yanhua, LI Zongheng, SHANG Jing, YUAN Zeting, DENG Wanli, LUO Ying, HAN Na, YIN Peihao, YIN Jun   

  1. School of Traditional Chinese Medicine, Shengyang Pharmaceutical University, Benxi 117004, China; Department of General Surgery, Putuo Hospital, Shanghai University of Traditional Chinese Medicine; Interventional Cancer Institute of Chinese Integrative Medicine, Shanghai Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China; Clinical Laboratory, Shanghai Changning Maternity and Infant Health Hospital, Shanghai 200000, China
  • Online:2023-07-20 Published:2023-07-20

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摘要： 目的 通过网络药理学和体内实验探讨六神丸治疗结肠炎相关性结直肠癌（CAC）的潜在作用机制。方法 运用TCMSP、BATMAN-TCM、CNKI、PubMed、Genecards、OMIM和TTD数据库获取六神丸和CAC相关靶点，利用Venny在线作图网站得到六神丸-CAC共同靶点。然后，通过Cytoscape 3.8.2构建PPI网络筛选六神丸治疗CAC的核心靶点，并利用DAVID数据库进行GO和KEGG富集分析。体内实验验证分析，将C57BL/6J小鼠随机分为3组：对照组（Ctrl）、模型组（AOM/DSS）和六神丸组（LSW），采用AOM/DSS构建CAC小鼠模型。通过小鼠体质量变化、疾病活动指数评分、结肠长度、肿瘤大小和肿瘤数量等评估六神丸对CAC的治疗作用；HE染色、RT-qPCR方法检测六神丸对CAC小鼠炎症介质的作用；免疫组织化学染色、TUNEL染色评估六神丸对AOM/DSS诱导结肠肿瘤细胞增殖和凋亡的影响；免疫组织化学、Western blot方法检测六神丸对CAC小鼠TLR4蛋白表达的影响。结果 通过网络药理学方法获得69个六神丸-CAC共同靶点，构建PPI网络进行核心靶点筛选，得到33个核心靶点。KEGG通路富集分析显示，六神丸可能通过Toll样受体信号通路作用于CAC。体内研究结果表明，与模型组相比，六神丸治疗能够显著抑制小鼠结肠炎相关肿瘤的发生，减少肿瘤数量和负荷（P<0.05），明显改善小鼠结肠组织病理学变化，下调促炎细胞因子mRNA水平，抑制结肠肿瘤细胞增殖（P<0.01）并促进其凋亡（P<0.001）；同时，六神丸还显著降低了结肠组织中TLR4蛋白表达（P<0.05）。结论 六神丸可能通过调控Toll样受体信号TLR4的表达，减轻小鼠肠道炎症，抑制结肠肿瘤细胞增殖并促进其凋亡来发挥抗CAC作用。

关键词: 六神丸；网络药理学；结肠炎相关性结直肠癌；Toll样受体信号通路；Toll样受体4

Abstract: Objective To explore the therapeutic mechanism of Liushen Wan (LSW) against colitis-associated colorectal cancer (CAC) by network pharmacology. Methods TCMSP, BATMAN-TCM, CNKI, PubMed, Genecards, OMIM, and TTD databases were used to obtain the related targets of LSW and CAC. The common targets of LSW and CAC were obtained using Venny online website. The PPI network was constructed using Cytoscape 3.8.2 to screen the core targets of LSW in the treatment of CAC. GO and KEGG enrichment analysis were conducted using DAVID database. The therapeutic effect of LSW on CAC was evaluated in a C57BL/6J mouse model of AOM/DSS-induced CAC by observing the changes in body weight, disease activity index, colon length, and size and number of the tumor. HE staining and RT-qPCR were used to analyze the effect of LSW on inflammatory mediators. Immunohistochemistry and TUNEL staining were used to evaluate the effect of LSW on the proliferation and apoptosis of AOM/DSS-treated colon tumor cells. Immunohistochemistry and Western blotting were used to detect the effects of LSW on the expression of TLR4 proteins in CAC mice. Results Network pharmacology analysis identified 69 common targets of LSW and CAC, and 33 hub targets were screened in the PPI network. KEGG pathway enrichment analysis suggested that the effect of LSW on CAC was mediated by the Toll-like receptor signaling pathway. In the mouse model of AOM/DSS-induced CAC, LSW significantly inhibited colitis-associated tumorigenesis, reduced tumor number and tumor load (P<0.05), obviously improved histopathological changes in the colon, downregulated the mRNA levels of pro-inflammatory cytokines, and inhibited the proliferation (P<0.01) and promoted apoptosis of colon tumor cells (P<0.001). LSW also significantly decreased TLR4 protein expression in the colon tissue (P<0.05). Conclusion LSW can inhibit CAC in mice possibly by regulating the expression of TLR4 to reduce intestinal inflammation, inhibit colon tumor cell proliferation and promote their apoptosis.

Key words: Liushen Wan; network pharmacology; colitis-associated colorectal cancer; Toll-like receptor signaling pathway; TLR4