目的 基于腸道菌群和代謝組學(xué)探討荊防合劑抗血栓的作用機(jī)制。方法 48只大鼠隨機(jī)分為對照組、模型組及荊防合劑低、中、高劑量組和阿司匹林組，每組8只。采用ip角叉菜膠及低溫處理誘導(dǎo)大鼠血栓，造模前連續(xù)給藥7 d，造模后48 h麻醉處死，取大鼠尾巴進(jìn)行蘇木素-伊紅（hematoxylin-eosin，HE）和Masson染色，結(jié)腸進(jìn)行AB-PAS染色；取血漿檢測凝血四項(xiàng)；ELISA檢測血漿中血栓烷B₂（thromboxane B₂，TXB₂）、6-酮-前列環(huán)素F1α（6-keto-PGF1α）、抗凝血酶III（antithrombin III，ATIII）、纖維蛋白原降解產(chǎn)物（fibrinogen degradation products，F(xiàn)DP）和血清中腫瘤壞死因子-α（tumor necrosis factor-α，TNF-α）、γ干擾素（interferon-γ，IFN-γ）、白細(xì)胞介素-1β（interleukin-1β，IL-1β）、IL-6及腸道分泌型免疫球蛋白A（secretory immunoglobulin A，SIgA）含量；檢測腸道黏膜屏障指標(biāo)內(nèi)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檢測腸道緊密連接蛋白1（zonula occludens proteins-1，ZO-1）和咬合蛋白（Occludin）基因和蛋白表達(dá)；基于16S rDNA高通量測序技術(shù)和非靶向代謝組學(xué)聯(lián)合測定大鼠腸道菌群和內(nèi)源性代謝物變化。結(jié)果 荊防合劑可顯著降低血栓大鼠的相對黑尾長度（P＜0.05、0.01），減少尾巴炎性浸潤現(xiàn)象及膠原含量（P＜0.05、0.01），回調(diào)凝血活酶時(shí)間（activated partial thromboplastin time，APTT）、凝血酶時(shí)間（thrombin time，TT）、纖維蛋白原（fibrinogen，F(xiàn)IB），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）及腸道SIgA水平（P＜0.05），降低腸黏膜損傷因子LPS、DAO、D-LA水平（P＜0.05、0.01），升高腸道緊密連接蛋白表達(dá)水平（P＜0.05、0.01）。16S rDNA和血清非靶向代謝組學(xué)結(jié)果顯示，荊防合劑可顯著改善血栓大鼠體內(nèi)的腸道菌群紊亂，其代謝相關(guān)通路可能與不飽和脂肪酸、亞油酸代謝等有關(guān)。結(jié)論 荊防合劑對角叉菜膠引起的大鼠血栓有較好的治療作用，能夠減輕大鼠尾巴病理改變，緩解凝血因子、炎性因子失調(diào)，改善腸道屏障損傷及腸道菌群紊亂，影響不飽和脂肪酸等相關(guān)代謝通路。

Objective To explore the antithrombotic mechanism of Jingfang Mixture (荊防合劑) 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

山東省自然基金創(chuàng)新發(fā)展聯(lián)合基金項(xiàng)目（ZR2022LZY021）