目的 探讨补阳还五汤（Buyang Huanwu Decoction，BYHWT）对大鼠脑缺血再灌注损伤（cerebral ischemia-reperfusion injury，CIRI）后肠道黏膜屏障损伤的保护作用及机制。方法 采用网络药理学筛选BYHWT治疗CIRI后肠道黏膜损伤的靶点，并进行富集分析。采用改良的Zea-Longa线栓法制备大鼠CIRI模型，随机分为模型组和BYHWD低、中、高剂量（1.29、2.57、5.14 g/kg）组，每组6只，另取假手术组大鼠6只。连续给药5 d后，测定神经功能缺损评分和脑梗死面积；采用苏木素-伊红（hematoxylin-eosin，HE）、TUNEL染色及电镜观察结肠组织病理、细胞凋亡及超微结构变化；免疫组化和Western blotting检测结肠组织闭合蛋白（Occludin）、紧密连接蛋白-1（zonula occludens-1，ZO-1）蛋白表达；非靶向代谢组学分析肠道内容物，联合网络药理学构建“代谢物-反应-酶-基因”网络，并利用分子对接、ELISA及Western blotting验证关键靶点。结果 网络药理学获得BYHWT治疗CIRI后肠道黏膜损伤的22个核心靶点，涉及脂质代谢、丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）等通路。与假手术组比较，模型组大鼠神经功能损伤加重（P＜0.001），脑梗死面积增加（P＜0.05），结肠黏膜结构破坏，Occludin与ZO-1表达降低（P＜0.01），细胞凋亡增加（P＜0.05）；与模型组比较，BYHWT显著改善以上指标（P＜0.05、0.01）。代谢组学发现9个差异代谢物回调，主要富集于类固醇激素、胆汁酸和花生四烯酸代谢途径。代谢组学与网络药理学整合分析提示BYHWT通过调控花生四烯酸/二高-γ-亚麻酸-前列腺素代谢通路发挥作用。分子对接显示前列腺素E₁（prostaglandin E₁，PGE₁）与前列腺素内过氧化物合酶2（prostaglandin-endoperoxide synthase 2，PTGS2）结合良好，BYHWT干预后大鼠结肠组织中PGE₁水平显著升高（P＜0.05），PTGS2蛋白表达水平显著降低（P＜0.05）。结论 BYHWT可能通过调节花生四烯酸/二高-γ-亚麻酸-PTGS2-前列腺素代谢网络，改善CIRI后肠道黏膜屏障损伤。

Objective To investigate the protective effect and mechanism of Buyang Huanwu Decoction (补阳还五汤, BYHWT) on intestinal mucosal barrier injury following cerebral ischemia-reperfusion injury (CIRI) in rats. Methods Network pharmacology was employed to screen potential targets of BYHWT for treating intestinal mucosal injury post-CIRI, followed by enrichment analysis. The improved Zea Longa suture method was used to prepare a rat CIRI model, which was randomly divided into model group and BYHWD low-, medium-, and high-dose (1.29, 2.57, 5.14 g/kg) groups, with six rats in each group, and six rats in sham group. After continuous administration for 5 d, the neurological deficit score and cerebral infarction area were measured. Hematoxylin-eosin (HE), TUNEL staining and electron microscopy were used to observe the pathological, apoptotic and ultrastructural changes of colon tissue. Immunohistochemistry and Western blotting were used to detect the protein expressions of Occludin and zonula occludin-1 (ZO-1) in colon tissue. Non-targeted metabolomics was performed on intestinal contents, and the results were integrated with network pharmacology to construct a “metabolite-reaction-enzyme-gene” network. Key pathways were validated using molecular docking, ELISA and Western blotting. Results Network pharmacology identified 22 core targets of BYHWT for treating post-CIRI intestinal mucosal injury, involving pathways such as lipid metabolism and mitogen activated protein kinase (MAPK). Compared with sham group, model group showed aggravated neurological damage (P < 0.001), increased cerebral infarction area (P < 0.05), disrupted colon mucosal structure, decreased expressions of Occludin and ZO-1 (P < 0.01), and increased cell apoptosis (P < 0.05). Compared with model group, BYHWT significantly improved the above indicators (P < 0.05, 0.01). Metabolomics discovered nine differential metabolite downregulation pathways, mainly enriched in steroid hormones, bile acids and arachidonic acid metabolism pathways. The integrated analysis of metabolomics and network pharmacology suggested that BYHWT exerted its effect by regulating arachidonic acid/dihydro-γ-linolenic acid prostaglandin metabolic pathway. Molecular docking showed that prostaglandin E₁ (PGE₁) bind well to prostaglandin endoperoxide synthase 2 (PTGS2). After BYHWT intervention, the level of PGE₁ in colon tissue was significantly increased (P < 0.05), while the expression of PTGS2 protein was significantly decreased (P < 0.05). Conclusion BYHWT may alleviate intestinal mucosal barrier injury following CIRI by modulating arachidonic acid/dihomo-γ-linolenic acid-PTGS2-prostaglandin metabolic network.

R285.5

辽宁省科技计划联合计划项目（2024-MSLH-247）