百蕊草通过调节肠道菌群和调控EGFR/PI3K/Akt信号通路改善小鼠抗生素相关性腹泻

doi:10.12122/j.issn.1673-4254.2025.02.09

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (2): 285-295.doi: 10.12122/j.issn.1673-4254.2025.02.09

• • 上一篇

百蕊草通过调节肠道菌群和调控EGFR/PI3K/Akt信号通路改善小鼠抗生素相关性腹泻

徐皓男¹(), 张放³, 黄钰莹², 姚其盛⁴, 管悦琴⁴, 陈浩¹^,²^,⁵()

^1.安徽科技学院，动物科学学院，安徽凤阳 233100
^2.安徽科技学院，生命与健康科学学院，安徽凤阳 233100
^3.安徽科技学院，食品工程学院，安徽凤阳 233100
^4.安徽九华华源药业有限公司，安徽滁州 239000
^5.安徽中医药大学药学院，安徽合肥 230012

收稿日期:2024-11-25 出版日期:2025-02-20 发布日期:2025-03-03
通讯作者: 陈浩 E-mail:2770863077@qq.com;chenhao@ahstu.edu.cn
作者简介:徐皓男，在读硕士研究生，E-mail: 2770863077@qq.com
基金资助:
国家自然科学基金青年项目(31802244);安徽省中医药传承创新研究项目(2024CCCX256);安徽省博士后科研资助项目(2024C853);安徽省自然科学基金(2020085QH395);安徽省重点研究与开发计划项目(202004a07020031);安徽省高校自然科学研究重大项目(KJ2020ZD011)

Thesium chinense Turcz. alleviates antibiotic-associated diarrhea in mice by modulating gut microbiota structure and regulating the EGFR/PI3K/Akt signaling pathway

Haonan¹ XU¹(), Fang³ ZHANG³, Yuying² HUANG², Qisheng⁴ YAO⁴, Yueqin⁴ GUAN⁴, Hao CHEN¹^,²^,⁵()

^1.College of Animal Science, Anhui Science and Technology University, Fengyang 233100, China
^2.College of Life and Health Sciences, Anhui Science and Technology University, Fengyang 233100, China
^3.College of Food Engineering, Anhui Science and Technology University, Fengyang 233100, China
^4.Anhui Jiuhua Huayuan Pharmaceutical Co. , Ltd. , Chuzhou 239000, China
^5.School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China

Received:2024-11-25 Online:2025-02-20 Published:2025-03-03
Contact: Hao CHEN E-mail:2770863077@qq.com;chenhao@ahstu.edu.cn
Supported by:
Natural Science Foundation for the Youth of China(31802244)

摘要/Abstract

摘要：

目的基于网络药理学与16S rRNA高通量测序探究百蕊草（TCT）治疗抗生素相关性腹泻（AAD）的作用机制。方法通过网络药理学收集百蕊草与AAD的共有靶点与基因，构建蛋白质相互作用网络，使用KEGG通路富集分析筛选关键信号通路，使用AutoDock Vina进行分子对接验证，并使用GROMACS进行分子动力学模拟以验证活性成分与核心靶点的静态及动态结合变化。动物实验部分采用灌胃盐酸林可霉素建立AAD小鼠模型，40只KM小鼠随机分为空白对照组、自然恢复组（NR）、TCT低剂量组（TCT-L）、TCT高剂量组（TCT-H），雌雄各半（n=10）。实验期间每天分别灌胃给予1%羧甲基纤维素钠和1.5 g/kg和3 g/kg的百蕊草凝胶溶液，观察并记录小鼠体质量变化和腹泻情况。使用HE染色观察各组小鼠结肠病理变化，使用ELISA法检测炎症因子IL-6和TNF-α含量，使用16S rRNA测序技术检测小鼠肠道菌群的变化，采用Western blotting检测各组小鼠EGFR、p-EGFR、PI3K、p-PI3K、Akt、p-Akt蛋白的表达。结果网络药理学筛选得到66个百蕊草活性成分，并预测得到998个相关靶点，与1304个AAD靶点基因取交集得到68个潜在靶点，其中核心靶点有EGFR、STAT3、PIK3CA等。KEGG富集分析提示百蕊草主要通过PI3K/Akt信号通路发挥作用。分子对接结果显示核心靶点EGFR与coniferin结合自由能最低，分子动力学模拟结果显示EGFR与coniferin在10 ns时保持稳定的构象，Gibbs自由能结果显示，当蛋白质回旋半径值为3.11~3.15且均方根偏差值为0.35~0.5时，EGFR与coniferin复合物处于相对稳定的构象状态。动物实验显示，百蕊草可改善小鼠结肠组织形态，减少炎症因子分布，降低结肠组织中TNF-α和IL-6水平（P<0.001）；提高肠道菌群多样性（P<0.05），调节关键菌群如联合乳杆菌属、拟杆菌属等的丰度；降低结肠组织p-EGFR、p-PI3K、p-Akt蛋白水平（P<0.01）。结论百蕊草能通过调节肠道菌群结构，调控EGFR/PI3K/Akt信号通路，降低TNF-α和IL-6炎症因子水平，达到治疗AAD的作用。

Abstract:

Objective To investigate the therapeutic mechanism of Thesium chinense Turcz. (TCT) for antibiotic-associated diarrhea (AAD). Methods Network pharmacology, KEGG pathway enrichment analysis and molecular docking were used to identify the shared targets and genes of TCT and AAD, the key signaling pathways and the binding between the active components in TCT and the core protein targets. In a Kunming mouse model of AAD established by intragastric administration of lincomycin hydrochloride, the effects of daily gavage of 1% carboxymethyl cellulose sodium or TCT gel solutions at 1.5 g/kg and 3 g/kg (n=10) on body weight and diarrhea were observed. HE staining, ELISA, 16S rRNA sequencing, and Western blotting were used to examine pathologies, expression levels of IL-6 and TNF-α, changes in gut microbiota, and protein expressions of EGFR, p-EGFR, PI3K, p-PI3K, Akt, and p-Akt in the colon tissues of the mice. Results We identified a total of 66 active components of TCT and 68 core targets including EGFR, STAT3 and PIK3CA. KEGG pathway enrichment analysis suggested that the therapeutic effects of TCT was mediated primarily through the PI3K/Akt signaling pathway. Molecular docking showed that EGFR had the highest binding affinity with coniferin, and the EGFR-coniferin complex maintained a stable conformation at 10 ns, whose stability was also confirmed by Gibbs free energy analysis. In the mouse models of AAD, treatment with TCT significantly improved colonic tissue morphology, decreased colonic levels of TNF-α and IL-6, increased gut microbiota diversity, and modulated the relative abundances of the key genera including Lactobacillus and Bacteroides. TCT treatment also markedly reduced protein expressions of p-EGFR, p-PI3K and p-Akt in the colon tissues of the mice. Conclusion TCT can alleviate AAD in mice by modulating gut microbiota composition, regulating the EGFR/PI3K/Akt signaling pathway, and reducing TNF‑α and IL-6 expressions.

Key words: Thesium chinense Turcz., antibiotic-associated diarrhea, network pharmacology, molecular dynamics simulation, 16S rRNA gene sequencing, EGFR/PI3K/Akt signaling pathway

徐皓男, 张放, 黄钰莹, 姚其盛, 管悦琴, 陈浩. 百蕊草通过调节肠道菌群和调控EGFR/PI3K/Akt信号通路改善小鼠抗生素相关性腹泻[J]. 南方医科大学学报, 2025, 45(2): 285-295.

Haonan¹ XU, Fang³ ZHANG, Yuying² HUANG, Qisheng⁴ YAO, Yueqin⁴ GUAN, Hao CHEN. Thesium chinense Turcz. alleviates antibiotic-associated diarrhea in mice by modulating gut microbiota structure and regulating the EGFR/PI3K/Akt signaling pathway[J]. Journal of Southern Medical University, 2025, 45(2): 285-295.

图/表 8

图1 百蕊草治疗AAD的网络药理学分析

Fig.1 Network pharmacology analysis of Thesium chinense Turcz. (TCT) in treating AAD. A: PPI network. B: Core target map. C: "Disease-drug-compound-gene" network. D: KEGG pathway enrichment map. E: Heatmap of molecular docking results. F: Schematic diagram of EGFR binding with coniferin.

图2 EGFR与coniferin的分子对接及分子动力模拟分析

Fig.2 Molecular docking and molecular dynamics simulation analysis of EGFR with coniferin. A: RMSD analysis. B: Radius of gyration analysis of EGFR. C: Gibbs free energy landscape. D: MM/PBSA binding free energy plot.

图4 各组小鼠结肠组织

Fig.4 Pathological examination of the colonic tissue of the mice in each group (HE staining, original magnification:×200). Arrows indicated inflammatory cell infiltration.

图5 TCT对小鼠结肠TNF-α和IL-6水平的影响

Fig.5 ELISA for detecting TNF-α (A) and IL-6 (B) levels in mouse colon tissues in each group. ###P<0.001 vs control group, ***P<0.001 vs NR group (n=6).

图6 小鼠肠道菌群的alpha多样性影响

Fig.6 Changes of alpha diversity in the gut microbiota of the mice. A: ACE index. B: Chao index. C: Shannon index. D: Species rank abundance curve. *P<0.05, **P<0.01 (n=5).

图7 Beta多样性分析及菌群组成分析

Fig.7 Beta diversity analysis and microbiota composition analysis (n=5). A: PCoA plot. B: NMDS plot. C: Bar plot of genus-level community composition among samples. D: Bar plot of genus-level community composition among groups.

图8 肠道菌群LEfSe分析

Fig.8 LEfSe analysis of gut microbiota (n=5).

图9 各组小鼠结肠组织EGFR、PI3K、Akt及其磷酸化蛋白表达水平

Fig.9 Expression levels of EGFR, PI3K, Akt and their phosphorylated proteins in the colon tissues of the mice in each group. ###P<0.001 vs control group, **P<0.01, ***P<0.001 vs NR group (n=3).

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百蕊草通过调节肠道菌群和调控EGFR/PI3K/Akt信号通路改善小鼠抗生素相关性腹泻

Thesium chinense Turcz. alleviates antibiotic-associated diarrhea in mice by modulating gut microbiota structure and regulating the EGFR/PI3K/Akt signaling pathway

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