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朱媛媛, 潘龙, 韩曼, 宋延平.基于TLR4/MyD88/NF-κB通路与肠道菌群探究仲叶止泻合剂对脾虚泄泻小鼠的作用机制[J].湖南中医药大学学报英文版,2025,45(11):2044-2053.[Click to copy
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| 基于TLR4/MyD88/NF-κB通路与肠道菌群探究仲叶止泻合剂对脾虚泄泻小鼠的作用机制 |
| 朱媛媛,潘龙,韩曼,宋延平 |
| (陕西中医药大学, 陕西 咸阳 712046;陕西怡柏源生物医药科技有限公司, 陕西 西安 710061;陕西省中医药研究院, 陕西 西安 710003) |
| 摘要: |
| 目的 基于Toll样受体4/髓样分化初级反应蛋白88/核因子-κB(TLR4/MyD88/NF-κB)通路与肠道菌群探讨仲叶止泻合剂对脾虚泄泻小鼠的作用及机制。方法 灌胃大黄水煎液以制备脾虚泄泻模型；随机将60只雄性KM小鼠分为6组，包括空白组，模型组，阳性药组及仲叶止泻合剂高、中、低剂量组，每组10只；造模成功后，各给药组灌胃对应药物治疗7 d。对比治疗前后小鼠体质量、腹泻情况、脾脏指数；HE染色观察各组小鼠结肠组织形态学变化；16S rDNA高通量测序检测各组小鼠肠道菌群结构变化；ELISA检测各组小鼠血清中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量；Western blot检测各组小鼠结肠组织中TLR4、MyD88蛋白表达水平；免疫荧光检测各组小鼠结肠组织中核因子-κB(NF-κB) p-p65蛋白入核情况。结果 与空白组比较，模型组小鼠体质量、脾脏指数均降低（P<0.05）；粪便含水率、腹泻指数、血清中IL-6及TNF-α含量、结肠组织中TLR4及MyD88蛋白表达水平、结肠组织核内NF-κB p-p65相对荧光强度均升高（P<0.05）。与模型组比较，阳性药组及仲叶止泻合剂不同剂量组小鼠体质量、脾脏指数均升高（P<0.05）；粪便含水率、腹泻指数、血清中IL-6及TNF-α含量、结肠组织中TLR4及MyD88蛋白表达水平、结肠组织核内NF-κB p-p65相对荧光强度均降低（P<0.05）。与阳性药组及仲叶止泻合剂高、中剂量组比较，仲叶止泻合剂低剂量组小鼠粪便含水率、腹泻指数、血清中IL-6及TNF-α含量、结肠组织中TLR4及MyD88蛋白表达水平、结肠组织核内NF-κB p-p65相对荧光强度均降低（P<0.05）。16S r DNA结果显示，仲叶止泻合剂可调控小鼠肠道菌群结构，恢复α和β多样性，提高有益菌相对丰度，降低有害菌相对丰度。结论 仲叶止泻合剂能缓解脾虚泄泻小鼠的腹泻症状，改善结肠组织病理损伤，这一作用可能是通过下调TLR4/MyD88/NF-κB信号通路蛋白表达，并改善肠道菌群实现的。 |
| 关键词: 腹泻 仲叶止泻合剂 脾虚泄泻 绿原酸 TLR4/MyD88/NF-κB通路 肠道菌群 |
| DOI：10.3969/j.issn.1674-070X.2025.11.004 |
| Received:July 18, 2025 |
| 基金项目:陕西省中医药管理局项目(ZYJXG-Y23009)。 |
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| Exploring the mechanism of action of Zhongye Zhixie Mixture on mice with spleen-deficiency diarrhea based on the TLR4/MyD88/NF-κB pathway and gut microbiota |
| ZHU Yuanyuan, PAN Long, HAN Man, SONG Yanping |
| (Shaanxi University of Chinese Medicine, Xianyang, Shaanxi 712046, China;Shaanxi Yibaiyuan Biomedical Technology Co., Ltd., Xi'an, Shaanxi 710061, China;Shaanxi Academy of Traditional Chinese Medicine, Xi'an 710003, China) |
| Abstract: |
| Objective To investigate the effects and mechanism of Zhongye Zhixie Mixture（ZYZXM） on mice with spleen-deficiency diarrhea based on the Toll-like receptor 4/myeloid differentiation primary response protein 88/nuclear factor-kappa B（TLR4/MyD88/NF-κB） pathway and gut microbiota. Methods Gavage administration of rhubarb decoction was performed to establish a spleen-deficiency diarrhea model. Sixty male KM mice were randomly divided into six groups, including a blank group, a model group, a positive drug group, and high-, medium-, and low-dose groups of ZYZXM, with 10 mice in each group. After successful model establishment, each drug administration group received corresponding medication via gavage for 7 days. The body weight,diarrhea condition, and spleen index of the mice were compared before and after treatment. Hematoxylin-eosin（HE） staining was used to observe the morphological changes in the colon tissues of mice in each group. 16S rDNA high-throughput sequencing was employed to check alterations in the gut microbiota structure of mice in each group. Enzyme-linked immunosorbent assay（ELISA）was utilized to measure the levels of interleukin-6（IL-6） and tumor necrosis factor-α（TNF-α） in the serum of mice in each group.Western blot analysis was performed to examine the protein expression levels of TLR4 and MyD88 in the colon tissues of mice in each group. Immunofluorescence was used to check the nuclear translocation of nuclear factor-kappa B（NF-κB） p-p65 protein in the colon tissues of mice in each group. Results Compared with the blank group, the mice in the model group exhibited decreases in body weight and spleen index（P＜0.05）, while increases were observed in fecal water content, diarrhea index, serum levels of IL-6 and TNF-α, protein expression levels of TLR4 and MyD88 in colon tissues, and relative fluorescence intensity of nuclear NF-κB p-p65 in colon tissues（P＜0.05）. In comparison with the model group, the mice in the positive drug group and different-dose groups of ZYZXM showed increases in body weight and spleen index（P＜0.05）, along with decreases in fecal water content, diarrhea index,serum levels of IL-6 and TNF-α, protein expression levels of TLR4 and MyD88 in colon tissues, and relative fluorescence intensity of nuclear NF-κB p-p65 in colon tissues（P＜0.05）. When compared with the positive drug group and the high-and medium-dose groups of ZYZXM, the low-dose group of ZYZXM demonstrated decreases in fecal water content, diarrhea index, serum levels of IL-6 and TNF-α, protein expression levels of TLR4 and MyD88 in colon tissues, and relative fluorescence intensity of nuclear NF-κB p-p65 in colon tissues（P＜0.05）. The results of 16S rDNA sequencing indicated that ZYZXM could regulate the gut microbiota structure in mice, restore α and β diversity, increase the relative abundance of beneficial bacteria, and decrease the relative abundance of harmful bacteria. Conclusion ZYZXM can alleviate diarrhea symptoms in mice with spleen-deficiency diarrhea and improve pathological damage in colon tissues. This effect may be achieved by down-regulating the expression of proteins in the TLR4/MyD88/NF-κB signaling pathway and improving the gut microbiota. |
| Key words: diarrhea Zhongye Zhixie mixture spleen-deficiency diarrhea chlorogenic acid TLR4/MyD88/NF-κB gut microbiota |
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