目的 研究参附黄方对脓毒症肠损伤的治疗作用及其潜在机制。方法 采用超高效液相色谱-串联质谱（ultra-performance liquid chromatography-tandem mass spectrometry，UPLC-MS/MS）法检测参附黄方的主要入血成分。通过网络药理学分析参附黄方的物质基础和潜在作用机制。为验证网络药理学预测结果的可靠性，采用分子对接技术分析参附黄方核心成分与目标靶点的相互作用。通过盲肠结扎穿孔（cecal ligation and puncture，CLP）手术构建小鼠脓毒症模型，给予参附黄方干预后，采用苏木素-伊红（hematoxylin-eosin，HE）染色评估参附黄方对脓毒症小鼠肠损伤的治疗效果；采用ELISA检测血清中白细胞介素-1β（interleukin-1β，IL-1β）和IL-6的水平；采用免疫荧光法检测肠道屏障蛋白紧密连接蛋白-1（zonula occludens-1，ZO-1）和闭合蛋白（Occludin）的表达；采用TUNEL染色和免疫组化法评估细胞凋亡及消皮素D（gasdermin D，GSDMD）蛋白表达；采用qRT-PCR检测回肠组织IL-1β、半胱氨酸天冬氨酸蛋白酶-1（cystein-asparate protease-1，Caspase-1）、NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor thermal protein domain associated protein 3，NLRP3）、GSDMD 的mRNA表达；采用Western blotting检测回肠组织NLRP3、Caspase-11、GSDMD-NT、凋亡相关斑点样蛋白（apoptosis-associated speck，ASC）、Caspase-1 p20、IL-1β的蛋白表达。结果 UPLC-MS/MS检测到参附黄方18种主要的入血成分，主要有人参皂苷Rf、(＋)-儿茶素-5-O-葡萄糖苷、人参皂苷Rg₁、大黄酸和表儿茶素。网络药理学结果显示，IL-1β和Caspase-1为关键靶点；KEGG富集分析显示靶点显著富集于细胞焦亡、NLRP3炎症小体等通路。分子对接结果证实核心活性成分与关键靶点蛋白（IL-1β、Caspase-1、NLRP3、GSDMD）具有良好的结合活性。动物实验结果显示，与模型组比较，参附黄方治疗显著减轻脓毒症小鼠的肠损伤，显著降低小鼠血清中促炎因子水平（P＜0.05、0.001），增加肠道屏障蛋白的表达，显著减少回肠组织细胞凋亡（P＜0.001），显著下调回肠组织中焦亡关键蛋白表达（P＜0.05、0.01、0.001）。结论 参附黄方可通过抑制GSDMD介导的细胞焦亡信号通路改善脓毒症小鼠肠损伤。

Objective To study the therapeutic effect and potential mechanism of Shenfuhuang Formula (参附黄方) on sepsis-induced intestinal injury. Methods Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to detect the main blood components of Shenfuhuang Formula. The material basis and potential mechanism of Shenfuhuang Formula were analyzed through network pharmacology. To verify the reliability of network pharmacology prediction results, molecular docking technology was used to analyze the interaction between the core components of Shenfuhuang Formula and the target. A mouse sepsis model was constructed by cecal ligation and puncture (CLP) surgery, and after intervention with Shenfuhuang Formula, the therapeutic effect of Shenfuhuang Formula on intestinal injury in septic mice was evaluated using hematoxylin-eosin (HE) staining; ELISA was used to detect the levels of interleukin-1β (IL-1β) and IL-6 in serum; Immunofluorescence method was used to detect the expressions of intestinal barrier proteins zonula occludin-1 (ZO-1) and occludin; Cell apoptosis and gasdermin D (GSDMD) protein expression were evaluated using TUNEL staining and immunohistochemistry; qRT-PCR was used to detect the mRNA expressions of IL-1β, cysteine aspartate protease-1 (Caspase-1), NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and GSDMD in ileum; Western blotting was used to detect the protein expressions of NLRP3, Caspase-11, GSDMD-NT, apoptosis-associated speck (ASC), Caspase-1 p20 and IL-1 β in ileum. Results A total of 18 main blood components in Shenfuhuang Formula were detected by UPLC-MS/MS, including ginsenoside Rf, (+)-catechin-5-O-glucoside, ginsenoside Rg₁, rhein and epicatechin. The results of network pharmacology showed that IL-1β and Caspase-1 were key targets; KEGG enrichment analysis showed that the target was significantly enriched in pathways such as cell apoptosis and NLRP3 inflammasome. The molecular docking results confirmed that the core active ingredient had good binding activity with key target proteins (IL-1β, Caspase-1, NLRP3, GSDMD). The animal experiment results showed that compared with model group, Shenfuhuang Formula treatment significantly reduced intestinal injury in sepsis mice, significantly reduced the levels of pro-inflammatory factors in serum of mice (P < 0.05, 0.001), increased the expressions of intestinal barrier proteins, significantly reduced cell apoptosis in ileum (P < 0.001), and significantly down-regulated the expressions of key proteins of pyroptosis in ileum (P < 0.05, 0.01, 0.001). Conclusion Shenfuhuang Formula could improve intestinal injury in sepsis mice by inhibiting GSDMD-mediated pyroptosis signaling pathway.

R285.5

国家自然科学基金面上项目（82174157）；国家自然科学基金面上项目（82474315）；北京市自然科学基金资助项目（7242219）；北京市医院管理中心“青苗”计划专项经费资助（QML20231002）