目的 利用四维数据非依赖性采集（4D-DIA）定量蛋白质组学技术研究消痔丸治疗巴豆油诱导肛门肿胀类痔模型大鼠的作用机制。方法 建立巴豆油诱导的大鼠肛门肿胀类痔模型，采用4D-DIA非标定量蛋白质组学技术分析鉴定空白组、模型组、消痔丸1.62 g/kg组肛门直肠组织中的蛋白。以表达差异倍数≥1.2或差异倍数≤0.83且P＜0.05筛选差异表达蛋白，分析消痔丸显著干预的差异表达蛋白并对其进行基因本体（GO）功能富集分析和京都基因和基因组百科全书（KEGG）信号通路分析。通过Western blotting、RT-qPCR等实验手段验证消痔丸对AMP活化蛋白激酶（AMPK）/过氧化物酶体增殖物激活受体γ共激活因子1-α（PGC-1α）/核因子-κB（NF-κB）p65信号通路的调控作用。结果 筛选出模型组与对照组大鼠肛门直肠组织的显著差异表达蛋白共657个，消痔丸组与模型组显著差异表达蛋白共105个，消痔丸对其中38个差异表达蛋白有显著干预作用。生物信息学分析发现消痔丸可干预AMPK信号通路、自噬、甘油磷脂代谢、花生四烯酸代谢、三羧酸循环、铁死亡、肿瘤坏死因子（TNF）等通路过程。动物实验表明，与模型组比较，消痔丸各剂量组肛门直肠组织中PGC-1α、AMPK mRNA表达显著升高，NF-κB p65 mRNA表达显著降低（P＜0.05、0.01、0.001）。与模型组比较，消痔丸1.62、3.24 g/kg组肛门直肠组织中p-AMPK/AMPKα比值显著升高（P＜0.05、0.01）；消痔丸各剂量组PGC-1α表达显著升高，p-p65/p65值显著降低（P＜0.01、0.001）。结论 消痔丸通过调控AMPK/PGC-1α/NF-κB p65信号通路及花生四烯酸代谢，发挥抗炎及调控能量代谢的生物活性。

Objective To study the mechanism of Xiaozhi Pills in treatment of croton oil-induced anal swelling hemorrhoids model rats by 4D-DIA non-standard quantitative proteomics technology. Methods The rat model of anal swelling hemorrhoids induced by croton oil was established. The 4D-DIA non-standard quantitative proteomics technique was used to analyze and identify the proteins in the anorectal tissues of the control group, the model group, and the Xiaozhi Pills 1.62 g/kg group. The differentially expressed proteins (DEPs) were screened by expression fold change ≥ 1.2 or ≤ 0.83 and P ＜ 0.05. The DEPs significantly intervened by Xiaozhi Pills were analyzed, and GO function enrichment analysis and KEGG signal pathway analysis were carried out. Regulatory effect of Xiaozhi Pills on AMPK/PGC-1α/NF-κB p65 signaling pathway was verified by Western blotting, RT-qPCR. Results A total of 657 DEPs in anorectal tissues of rats in model group and control group were screened, and 105 DEPs in Xiaozhi Pills group and model group were screened. Xiaozhi Pills had a significant intervention effect on 38 DEPs. Bioinformatics analysis showed that Xiaozhi Pills could interfere with AMPK signaling pathway, autophagy, glycerophospholipid metabolism, arachidonic acid metabolism, tricarboxylic acid cycle, ferroptosis, TNF, and other pathways. Animal experiments showed that compared with the model group, the expression of PGC-1α and AMPK mRNA in the anorectal tissues of each dose group of Xiaozhi Pills significantly increased, while the expression of NF-κB p65 mRNA significantly decreased (P < 0.05, 0.01, 0.001). Compared with the model group, the ratios of p-AMPK/AMPKα in the anorectal tissues of the 1.62 and 3.24 g/kg groups of Xiaozhi Pills significantly increased (P < 0.05, 0.01), the expression of PGC-1α in each dose group of Xiaozhi Pills significantly increased, and the ratio of p-p65/p65 significantly decreased (P < 0.01, 0.001). Conclusion Xiaozhi Pills exerts anti-inflammatory and energy metabolism-regulating biological activities by regulating AMPK/PGC-1α/NF-κB p65 signaling pathway and arachidonic acid metabolism.

R285.5

国有资本经营预算支持省属企业科研项目（2024GZ031）