The incidence of ulcerative colitis (UC) is on the rise globally and the development of drugs targeting UC is urgent. Finding the target of action of natural products is important for drug discovery, elucidation of drug action mechanism, and disease mechanism. San-Ye-Qing (SYQ), is an ancient herbal medicine, but whether the powder of its rhizome has pharmacological effects against UC and its mechanism of action are not clear. To evaluate the therapeutic effectiveness of rhizome powder of SYQ in treating UC, and conduct an isolation and characterization of the chemical constituents of the powder. Further, screen the most potent compounds among them and determine the potential mechanism for treating UC. In vivo, the therapeutic effect of SYQ's rhizome powder on UC was assessed by mice's body weight, DAI score, colon length, tissue MPO activity, serum inflammatory markers, etc. Additionally, HPLC was used to isolate and identify the specific chemical components of SYQ's rhizome powder. Then, the most effective compounds and their therapeutic targets were analysed and screened in SYQ rhizome powder using network pharmacology, combined with CCK-8 assay, NO release assay and molecular docking assay, in conjunction with CETSA, DARTS, SPR and enzyme activity assay. Finally, the biological effects of the key compound on the targets were validated using Western blot and ELISA. In vivo, SYQ rhizome powder effectively restored mice's body weight, lowered DAI and pathological score, downregulated the expression of inflammatory biomarkers, and restored colon length, as well as the colonic epithelial and mucus barriers. Afterward, 9 compounds were isolated and identified from the powder of the rhizomes of SYQ by HPLC. Nicotiflorin is the primary compound in SYQ with the highest concentration. According to both CCK-8 and NO release tests, Nicotiflorin is also the most efficacious compound. Combined with network pharmacological prediction, molecular docking analysis, CETSA, DARTS, SPR and enzyme activity assay, Nicotiflorin may ultimately suppress inflammation by targeting p65 and inhibiting the NF-κB pathway, thereby attenuating the activation of NLRP3 inflammasome. To verify this conclusion, Western blot and ELISA experiments were conducted. Our results suggest that the extract from SYQ rhizomes has therapeutic properties for UC. Its active ingredient Nicotiflorin exerted potent anti-UC effects by binding to p65 and inhibiting the activation of NF-κB and NLRP3 inflammasomes.

中文翻译：

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三叶青中烟花苷抗溃疡性结肠炎的作用机制


全球溃疡性结肠炎（UC）的发病率呈上升趋势，开发针对UC的药物刻不容缓。寻找天然产物的作用靶点对于药物发现、阐明药物作用机制和疾病机制具有重要意义。三叶清（SYQ）是一种古老的草药，但其根茎粉末是否具有治疗UC的药理作用及其作用机制尚不清楚。评价SYQ根茎粉治疗UC的疗效，并对粉的化学成分进行分离和表征。此外，筛选其中最有效的化合物并确定治疗 UC 的潜在机制。体内实验通过小鼠体重、DAI评分、结肠长度、组织MPO活性、血清炎症标志物等评价SYQ根茎粉对UC的治疗效果，并采用HPLC对SYQ根茎粉的具体化学成分进行分离鉴定。根茎粉。然后，利用网络药理学，结合CCK-8测定、NO释放测定和分子对接测定，并结合CETSA、DARTS、SPR和酶活性测定，对SYQ根茎粉中最有效的化合物及其治疗靶点进行分析和筛选。最后，使用蛋白质印迹和 ELISA 验证了关键化合物对靶标的生物学效应。在体内，SYQ根茎粉有效恢复小鼠体重，降低DAI和病理评分，下调炎症生物标志物的表达，恢复结肠长度以及结肠上皮和粘液屏障。采用HPLC从SYQ根茎粉末中分离鉴定出9个化合物。 烟花苷是SYQ中浓度最高的主要化合物。根据 CCK-8 和 NO 释放测试，烟花苷也是最有效的化合物。结合网络药理学预测、分子对接分析、CETSA、DARTS、SPR和酶活性测定，烟花苷最终可能通过靶向p65并抑制NF-κB通路，从而减弱NLRP3炎症小体的激活来抑制炎症。为了验证这一结论，进行了Western blot和ELISA实验。我们的结果表明 SYQ 根茎提取物具有治疗 UC 的功效。其活性成分 Nicotiflorin 通过与 p65 结合并抑制 NF-κB 和 NLRP3 炎症小体的激活而发挥有效的抗 UC 作用。