目的 研究地龙来源短肽（Leu-Leu-Ala-Pro-Pro，LLAPP）对溃疡性结肠炎（UC）的治疗作用，结合网络药理学及实验研究初步阐明其作用机制。方法 通过葡聚糖硫酸钠盐（DSS）自由饮水建立小鼠UC模型，观察LLAPP（12、6、3 mg·kg_-1）对UC小鼠的体质量、结肠长度、疾病活动指数（DAI）评分、存活率、脾脏指数的影响，通过苏木素-伊红（HE）染色观察结肠组织病理变化，酶联免疫吸附试验（ELISA）检测血清中白细胞介素-6（IL-6）、白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）的水平。通过网络药理学预测LLAPP治疗UC的潜在靶点。采用脂多糖（LPS）刺激RAW 264.7巨噬细胞建立细胞炎症模型，加入LLAPP（100、200、400 μmol·L^-1）处理24 h，运用实时荧光定量PCR（qRT-PCR）检测TNF-α、IL-6 mRNA的表达水平，Western blotting法检测UC小鼠结肠组织和RAW 264.7细胞中核因子κB（NF-κB） P65和p-P65的蛋白表达情况。结果 与模型组比较，LLAPP治疗可以显著改善UC小鼠体质量降低、结肠缩短、DAI评分下降、脾脏肿大（P＜0.01），降低死亡率； HE染色结果显示LLAPP治疗减轻了结肠黏膜的炎症细胞浸润、隐窝结构破坏； ELISA结果显示LLAPP降低了小鼠血清中IL-1β、IL-6、TNF-α的水平（P＜0.01）。网络药理学研究提示LLAPP治疗作用可能与调控NF-κB信号通路有关。细胞实验中，与模型组比较，LLAPP浓度相关性地抑制细胞中TNF-α、IL-6的mRNA表达（P＜0.01），LLAPP显著下调结肠组织和细胞中p-P65的蛋白表达（P＜0.01）。结论 LLAPP能有效缓解UC症状，其治疗作用可能与抑制NF-κB信号通路活化，减少IL-1β、IL-6、TNF-α等促炎因子的释放有关。

Objective To investigate the therapeutic effect of earthworm-derived small molecular peptide (LLAPP) on ulcerative colitis (UC) and preliminarily elucidate its mechanism of action using network pharmacology. Methods A mouse model of UC was established by administering dextran sulfate sodium (DSS) in drinking water. The effects of LLAPP on colon length, disease activity index (DAI) score, survival rate, and spleen index were evaluated. Pathological changes in colon tissue were observed via hematoxylin and eosin (HE) staining. Serum levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) were measured using enzyme-linked immunosorbent assay (ELISA). Potential targets of LLAPP in UC treatment were identified through network pharmacology. A cellular inflammation model was established by stimulating RAW 264.7 macrophages with lipopolysaccharide (LPS), and different doses of LLAPP were administered. The mRNA expression of TNF-α and IL-6 was detected using real-time quantitative PCR (qRT-PCR), and the protein expression of P65, and phosphorylated P65 (p-P65) in colon tissues and RAW 264.7 cells was measured via Western blotting. Results LLAPP treatment ameliorated colon shortening (P < 0.01), reduced DAI scores, improved survival rates, and alleviated spleen enlargement (P < 0.01) in UC mice. HE staining showed that LLAPP reduced inflammatory cell infiltration and crypt structural damage in the colonic mucosa. ELISA results indicated that LLAPP decreased serum levels of IL-1β, IL-6, and TNF-α (P < 0.01). Network pharmacology suggested that the therapeutic effect of LLAPP might be associated with the regulation of the nuclear factor kappa B (NF-κB) signaling pathway. LLAPP concentration-dependently inhibited mRNA expression of TNF-α and IL-6 in cells (P < 0.01). Western blotting analysis revealed that LLAPP downregulated p-P65 protein expression in colon tissues and cells (P < 0.01). Conclusion Earthworm-derived small molecular peptide (LLAPP) effectively alleviates symptoms of ulcerative colitis, potentially by inhibiting the activation of the NF-κB signaling pathway and reducing the release of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α.

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国家自然科学基金资助项目（82225047、82170274）；国家重点研发计划资助项目（2022YFC3501703）；浙江省自然科学基金资助项目（LZ24H280003）