目的 基于肠道菌群和代谢组学探讨荆防合剂抗血栓的作用机制。方法 48只大鼠随机分为对照组、模型组及荆防合剂低、中、高剂量组和阿司匹林组，每组8只。采用ip角叉菜胶及低温处理诱导大鼠血栓，造模前连续给药7 d，造模后48 h麻醉处死，取大鼠尾巴进行苏木素-伊红（hematoxylin-eosin，HE）和Masson染色，结肠进行AB-PAS染色；取血浆检测凝血四项；ELISA检测血浆中血栓烷B₂（thromboxane B₂，TXB₂）、6-酮-前列环素F1α（6-keto-PGF1α）、抗凝血酶III（antithrombin III，ATIII）、纤维蛋白原降解产物（fibrinogen degradation products，FDP）和血清中肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、γ干扰素（interferon-γ，IFN-γ）、白细胞介素-1β（interleukin-1β，IL-1β）、IL-6及肠道分泌型免疫球蛋白A（secretory immunoglobulin A，SIgA）含量；检测肠道黏膜屏障指标内毒素（endotoxin，LPS）、二胺氧化酶（diamine oxidase，DAO）、D-乳酸（D-lactic acid，D-LA）含量；qRT-PCR和Western blotting检测肠道紧密连接蛋白1（zonula occludens proteins-1，ZO-1）和咬合蛋白（Occludin）基因和蛋白表达；基于16S rDNA高通量测序技术和非靶向代谢组学联合测定大鼠肠道菌群和内源性代谢物变化。结果 荆防合剂可显著降低血栓大鼠的相对黑尾长度（P＜0.05、0.01），减少尾巴炎性浸润现象及胶原含量（P＜0.05、0.01），回调凝血活酶时间（activated partial thromboplastin time，APTT）、凝血酶时间（thrombin time，TT）、纤维蛋白原（fibrinogen，FIB），凝血因子TXB2、6-keto-PGF1α、ATⅢ、FDP（P＜0.05、0.01），炎性因子TNF-α、IFN-γ、IL-1β、IL-6（P＜0.05、0.01）及肠道SIgA水平（P＜0.05），降低肠黏膜损伤因子LPS、DAO、D-LA水平（P＜0.05、0.01），升高肠道紧密连接蛋白表达水平（P＜0.05、0.01）。16S rDNA和血清非靶向代谢组学结果显示，荆防合剂可显著改善血栓大鼠体内的肠道菌群紊乱，其代谢相关通路可能与不饱和脂肪酸、亚油酸代谢等有关。结论 荆防合剂对角叉菜胶引起的大鼠血栓有较好的治疗作用，能够减轻大鼠尾巴病理改变，缓解凝血因子、炎性因子失调，改善肠道屏障损伤及肠道菌群紊乱，影响不饱和脂肪酸等相关代谢通路。

Objective To explore the antithrombotic mechanism of Jingfang Mixture (荆防合剂) based on intestinal flora and metabolomics. Methods A total of 48 rats were divided into control group, model group, Jingfang Mixture low-, medium-, high-dose groups and aspirin group, with eight rats in each group. Thrombus was caused by intraperitoneal injection of carrageenan combined with low-temperature induction. The drug was administered 7 d before modeling, anesthetized and sacrificed 48 h after modeling. The tail of rats was taken for hematoxylin-eosin (HE) and Masson staining, and the colon was stained for AB-PAS staining; Pasma was taken for four coagulation items; ELISA was used to detect the contents of thromboxane B₂ (TXB₂), 6-keto-PGF1α, antithrombin Ⅲ (ATⅢ), fibrinogen degradation products (FDP) in plasma and tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1β (IL-1β), IL-6 and intestinal secretory immunoglobulin A (SIgA) in serum; The contents of endotoxin (LPS), diamine oxidase (DAO) and D-lactic acid (D-LA) in intestinal mucosal barrier indicators were measured; qRT-PCR and Western blotting were used to detect the gene and protein expressions of intestinal zonula occludens proteins-1 (ZO-1) and Occludin; Changes in rat intestinal flora and endogenous metabolites were measured based on 16S rDNA high-throughput sequencing technology and non-targeted metabolomics. Results Jingfang Mixture could significantly reduce the relative black tail length of thrombotic rats (P < 0.05, 0.01), reduce inflammatory infiltration and collagen content in tail (P < 0.05, 0.01), regulate the levels of activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (FIB), coagulation factors TXB2, 6-keto-PGF1α, ATIII, FDP (P < 0.05, 0.01), inflammatory factors TNF-α, IFN-γ, IL-1β, IL-6 (P < 0.05, 0.01) and intestinal SIgA (P < 0.05), reduce the levels of intestinal mucosa damage factors LPS, DAO, D-LA (P < 0.05, 0.01), and increase the expression levels of intestinal tight junction protein gene and protein (P < 0.05, 0.01). 16S rDNA and serum non-targeted metabolomic results showed that Jingfang Mixture could significantly improve intestinal flora disorders in rats with thrombosis, and its metabolic pathways may be related to the metabolism of unsaturated fatty acids and linoleic acid. Conclusion Jingfang Mixture has a good therapeutic effect on thromboticrats caused by carrageenan, as well as reducing pathological changes in rat tails, alleviating the imbalance of coagulation factors and inflammatory factors, improving intestinal barrier damage and intestinal flora disorders, and affecting related metabolic pathways such as unsaturated fatty acids.

R285.5

山东省自然基金创新发展联合基金项目（ZR2022LZY021）