基于TLR4/MyD88/NF-κB信号通路探讨参苓白术散在治疗腹泻型肠易激综合征中的作用机制

The Mechanism of Shenling Baizhu San in the Treatment of Diarrhea-Predominant Irritable Bowel Syndrome Based on the TLR4/MyD88/NF-κB Signaling Pathway

摘要: 背景：腹泻型肠易激综合征(IBS-D)是一种以慢性腹痛和腹泻为特征的功能性胃肠道疾病，炎症是其主要致病因素。参苓白术散(SLBZS)作为一种传统中药方剂，在缓解IBS-D症状方面展现出潜力，但其作用机制仍不完全明了。本研究通过TLR4/MyD88/NF-κB信号通路探讨SLBZS治疗IBS-D的分子机制，在大鼠模型中进行研究。方法：采用束缚应激结合寒凉通便和不规则饮食的方法建立IBS-D大鼠模型，随后使用低、中、高剂量的SLBZS进行治疗。评估一般行为、粪便含水量、布里斯托尔便形量表评分、小肠蠕动速率。通过苏木精–伊红(HE)染色评估结肠炎症。采用ELISA测定血清中CD4+、CD8+、IL-1β和IL-6水平。通过RT-qPCR和Western blot分别分析结肠组织中TLR4、MyD88和NF-κB的mRNA和蛋白表达。结果：与正常对照组相比，IBS-D大鼠表现为毛发凌乱、嗜睡和水样便，粪便含水量显著增加(P < 0.0001)，布里斯托尔评分升高(P < 0.0001)，小肠蠕动速率加快(P < 0.0001)。结肠组织表现为明显的炎性细胞浸润，血清中CD4+、CD8+ (P < 0.0001)、IL-1β和IL-6水平升高(P < 0.001或P < 0.0001)，TLR4、MyD88和NF-κB的mRNA和蛋白表达上调(P < 0.0001)。中剂量和高剂量SLBZS显著改善了大鼠的毛发状态、精神状态和便质，减少了粪便含水量和布里斯托尔评分(P < 0.01或P < 0.0001)，减慢了小肠蠕动速率(P < 0.01)，并缓解了结肠炎症。这些剂量还降低了血清中CD4、CD8 (P < 0.01至P < 0.0001)、IL-1β和IL-6水平(P < 0.05至P < 0.0001)，并下调了TLR4、MyD88和NF-κB的mRNA (P < 0.05至P < 0.0001)和蛋白表达(P < 0.0001或P < 0.001)。低剂量SLBZS未显示出显著效果(P > 0.05)。结论：SLBZS，特别是中高剂量，通过抑制TLR4/MyD88/NF-κB信号通路、减少肠道炎症和调节免疫反应，改善了IBS-D大鼠的症状。这些结果阐明了SLBZS的分子机制，并支持其作为IBS-D治疗药物的潜力。

Abstract: Background: Diarrhea-predominant irritable bowel syndrome (IBS-D) is a functional gastrointestinal disorder characterized by chronic abdominal pain and diarrhea, with inflammation as a key pathogenic factor. Shenling Baizhu San (SLBZS), a traditional Chinese herbal formula, shows promise in alleviating IBS-D symptoms, but its mechanisms remain incompletely understood. This study investigates the molecular mechanisms of SLBZS in treating IBS-D via the TLR4/MyD88/NF-κB signaling pathway in a rat model. Methods: An IBS-D rat model was induced using restraint stress combined with cold-pungent purgation and irregular diet, followed by treatment with low, medium, and high doses of SLBZS. General behavior, fecal water content, Bristol stool form scale scores, and small intestinal transit rate were assessed. Colonic inflammation was evaluated using hematoxylin-eosin (HE) staining. Serum levels of CD4+, CD8+, IL-1β, and IL-6 were measured by ELISA. mRNA and protein expression of TLR4, MyD88, and NF-κB in colon tissue were analyzed using RT-qPCR and Western blot, respectively. Results: Compared to normal controls, IBS-D rats exhibited disheveled fur, lethargy, and watery stools, with significantly increased fecal water content (P < 0.0001), elevated Bristol scores (P < 0.0001), and accelerated small intestinal transit rate (P < 0.0001). Colonic tissue showed marked inflammatory cell infiltration, with elevated serum CD4+, CD8+ (P < 0.0001), IL-1β, and IL-6 levels (P < 0.001 or P < 0.0001), and upregulated TLR4, MyD88, and NF-κB mRNA and protein expression (P < 0.0001). Medium and high-dose SLBZS significantly improved fur condition, mental status, and stool consistency, reduced fecal water content and Bristol scores (P < 0.01 or P < 0.0001), slowed intestinal transit rate (P < 0.01), and alleviated colonic inflammation. These doses also decreased serum CD4, CD8 (P < 0.01 to P < 0.0001), IL-1β, and IL-6 levels (P < 0.05 to P < 0.0001), and downregulated TLR4, MyD88, and NF-κB mRNA (P < 0.05 to P < 0.0001) and protein expression (P < 0.0001 or P < 0.001). Low-dose SLBZS showed no significant effects (P > 0.05). Conclusion: SLBZS, particularly at medium and high doses, ameliorates IBS-D symptoms in rats by suppressing the TLR4/MyD88/NF-κB signaling pathway, reducing intestinal inflammation, and modulating immune responses. These findings elucidate the molecular mechanisms of SLBZS and support its potential as a therapeutic agent for IBS-D.