丹参内生细菌 TD16 发酵产物对小鼠尾静脉血栓形成的抑制作用

Inhibitory effects of the fermentation products of Salvia miltiorrhiza endophytic bacteria TD16 on tail vein thrombosis formation in mice

摘要

[目的]本文旨在探究丹参内生细菌 TD16 的发酵提取物(TDFE)对角叉菜胶诱导小鼠尾静脉血栓形成的抑制作用及其潜在机制。[方法]将 KM 小鼠随机分为正常组(Control 组)、模型组(Model 组)、阿司匹林阳性对照组(Aspirin 组，10mg·kg^-1)和 TDFE 低剂量组(TDFE-L 组，50mg·kg^-1)、中剂量组(TDFE-M 组，100mg·kg^-1)、高剂量组(TDFE-H 组，200mg·kg^-1)，灌胃给药 7d 后构建小鼠尾静脉血栓模型。通过测定黑尾率、血栓长度、凝血功能指标(凝血酶原时间 PT、活化部分凝血酶原时间 APTT、凝血酶时间 TT 和纤维蛋白原 FIB)、血清肝功能指标(天门冬氨酸氨基转移酶 AST、丙氨酸氨基转移酶 ALT、白蛋白 ALB、总胆红素 TBIL)、炎症因子(白细胞介素 IL-6、IL-1β 和肿瘤坏死因子 TNF-α)、血清纤溶指标(组织型纤溶酶原激活剂 t-PA、血栓素 B₂ TXB₂ 和纤溶酶原激活抑制剂 PAI-1)以及肝脏和鼠尾组织的病理学分析等从多维度评价 TDFE 的体内抗血栓作用，并结合 UPLC-MS 和网络药理学初步解析活性成分及作用靶点。[结果]与 Model 组相比，TDFE-L 组血栓显著减少(P<0.05)，TDFE-M 组和 TDFE-H 组血栓极显著减少(P<0.01)，Aspirin 组几乎无明显血栓;各给药组小鼠的肝脏损伤均显著减轻，其中 TDFE-L 组和 Aspirin 组效果相当。凝血功能方面，各给药组小鼠的 PT、APTT 和 TT 均显著延长(P<0.05)，FIB 含量降低。血清炎症因子 IL-6、IL-1β 和 TNF-α 含量也显著降低(P<0.05)。纤溶指标方面，t-PA 浓度增加，而 TXB₂ 和 PAI-1 浓度显著下降(P<0.05)。同时，血清中 AST、ALT 活性等肝代谢相关指标也显著降低(P<0.05)。UPLC-MS 鉴定出 α-亚麻酸(11.61%)、异香兰素(6.31%)等 19 种主要成分。网络药理学显示其可通过 IL-6、过氧化物酶体增殖物激活受体 γ (PPARG)、细胞间黏附分子 1(ICAM-1)等靶点调控脂质代谢和动脉粥样硬化通路发挥抗血栓作用。[结论]丹参内生细菌 TD16 发酵产物能有效抑制角叉菜胶诱导的小鼠尾静脉血栓形成，在血栓病治疗领域具有潜在的应用价值。

Abstract

[Objectives]The present study aimed to explore the inhibitory effect and underlying mechanism of the fermented extract from endophytic bacterium TD16 (TDFE) the root of Salvia miltiorrhiza on carrageenan-induced tail vein thrombosis in mice. [Methods]KM mice were randomly divided into normal group (Control group), model group (Model group), aspirin-positive control group (Aspirin group, 10mg·kg^-1), and TDFE low-dose group (TDFE-L, 50mg·kg^-1), medium-dose group (TDFE-M, 100mg·kg^-1) and high-dose group (TDFE-H, 200mg·kg^-1). After continuous gavage administration for 7 days, a mouse tail vein thrombosis model was established. The in vivo antithrombotic effect of TDFE was evaluated through comprehensive analysis of multiple dimensions, including black tail rate, thrombus length, coagulation indices (prothrombin time, PT; activated partial thromboplastin time, APTT; thrombin time, TT; fibrinogen, FIB), serum liver function indices (aspartate aminotransferase, AST; alanine aminotransferase, ALT; albumin, ALB; total bilirubin, TBIL), inflammatory factors (interleukins IL-6, IL-1β and tumor necrosis factor TNF-α), serum fibrinolytic indexes (tissue-type fibrinogen activator, t-PA; thromboxane B₂, TXB₂; fibrinogen activator inhibitor, PAI-1), as well as pathological analysis of liver and rat tail tissues. The UPLC-MS and network pharmacology were combined to analyze the chemical components and potential targets of TDFE. [Results]Compared with the Model group, the thrombus in the TDFE-L group was significantly reduced (P<0.05). The TDFE-M and TDFE-H groups exhibited very limited thrombosis (P<0.01), and the Aspirin group showed almost no visible thrombosis. The liver damage in mice from each administration group was significantly mitigated, with the most pronounced effect observed in the TDFE-L and the Aspirin group. Regarding coagulation function, PT, APTT and TT were significantly prolonged in mice from each administration group (P<0.05), accompanied by a decrease in FIB content. Furthermore, a significant decrease in the serum concentrations of inflammatory cytokines IL-6, IL-1β, and TNF-α was observed (P<0.05). Regarding fibrinolysis index, the concentration of t-PA increased, while the concentrations of TXB₂ and PAI-1 significantly decreased (P<0.05). Concurrently, serum AST, ALT activities and other liver metabolism-related indices demonstrated a significant decrease (P<0.05). A comprehensive comparison of the TDFE-H group and the Aspirin group revealed that the former exhibited a superior overall effect. UPLC-MS identified 19 major components, including α-linolenic acid (11.61%) and isovanillin (6.31%). Network pharmacology analysis suggested that TDFE might exert antithrombotic effects by regulating lipid metabolism and atherosclerosis pathways through targets such as IL-6, peroxisome proliferator-activated receptor γ (PPARG), and intercellular adhesion molecule-1 (ICAM-1). [Conclusions]The fermentation product of S. miltiorrhiza endophytic bacteria TD16 could effectively inhibit carrageenan-induced tail vein thrombosis in mice, indicating its potential application value in the treatment of thrombosis.

关键词

丹参 / 内生细菌 / 发酵产物 / 尾静脉血栓 / 角叉菜胶 / 抗血栓

Key words

Salvia miltiorrhiza / endophytic bacteria / fermentation product / tail vein thrombosis / carrageenan / antithrombosis

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基金资助

江苏省农业科技自主创新项目(CX(22)3174)

国家中医药管理局高水平中医药重点学科建设项目(ZYYZDXK-2023293)

企业合作项目(20231020)

南京农业大学大学生创新创业项目(202414XX012)