基于TLR4/MyD88/MAPK信号通路探讨脉络舒通丸抗血栓性浅静脉炎的作用机制研究

Mechanism of Mailuo Shutong Pill on anti-superficial thrombophlebitis based on TLR4/MyD88/MAPK signaling pathway

关键词:  脉络舒通丸  血栓性浅静脉炎  TLR4/MyD88/MAPK信号通路  作用机制

Key words:  Mailuo Shutong Pill  superficial thrombophlebitis  TLR4/MyD88/MAPK signaling pathway  mechanism

摘要:

[目的] 探讨脉络舒通丸抗血栓性浅静脉炎（STP）的作用机制。[方法] 将36只日本大耳白兔随机分为正常组、模型组、脉络舒通丸低、中、高剂量组、地奥司明组（阳性药对照），每组6只。除正常组外，其余各组均采用耳缘静脉注射20%甘露醇建立STP兔模型。造模同时各给药组连续灌胃给药14 d。苏木精-伊红（HE）染色法检测各组兔耳组织病理变化；酶联免疫吸附（ELISA）法检测各组血清中白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的水平；测定凝血4项；蛋白免疫印迹（Western Blot）法检测耳组织Toll样受体4（TLR4）、髓样分化因子88（MyD88）、细胞外信号调节激酶（ERK1/2）、p38丝裂原活化蛋白激酶（p38MAPK）和c-氨基酸末端激酶（JNK）蛋白表达及相关磷酸化水平。[结果] 与正常组比较，模型组病理损伤评分显著增加（P<0.01）；血清中IL-1β、TNF-α和IL-6的表达水平明显升高（P<0.05）；活化部分凝血酶时间（APTT）、凝血酶原时间（PT）、凝血酶时间（TT）缩短，纤维蛋白原（FIB）含量升高（P<0.05）；TLR4、MyD88、丝裂原活化蛋白激酶（MAPK，包括p38MAPK、ERK1/2和JNK）蛋白及相关磷酸化水平均显著上调（P<0.01）。与模型组比较，脉络舒通丸低、中、高剂量组、地奥司明组兔耳组织病理损伤显著改善，病理损伤评分明显降低（P<0.05或P<0.01）；血清中IL-1β、TNF-α和IL-6的表达水平显著降低（P<0.05或P<0.01）；APTT、PT、TT明显延长，FIB含量明显降低（P<0.05或P<0.01）；TLR4、MyD88、MAPK（p38MAPK、ERK1/2和JNK）蛋白及相关磷酸化水平均显著下调（P<0.05或P<0.01）。[结论] 脉络舒通丸可能通过抑制TLR4/MyD88/MAPK信号通路，减轻炎症反应，改善凝血功能，从而发挥抗血栓性浅静脉炎作用。

Abstract:

[Objective] To investigate the mechanism of Mailuo Shutong Pill on superficial thrombophlebitis. [Methods] A total of 36 Japanese white rabbits were randomly divided into normal group, model group, low, medium, high dose of Mailuo Shutong Pill group, and positive drug(Diosmin) group, with 6 rabbits in each group. Except the normal group, the rabbit model of superficial thrombophlebitis was established by injecting 20% mannitol into bilateral auricular vein of other groups. At the same time, the treated groups were respectively administrated through gavage once a day for 14 consecutive days. Pathological changes of rabbit ear tissue were detected by hematoxylin and eosin(HE) staining. The levels of interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) in serum were detected by enzyme-linked immunosorbent assay(ELISA). The semi-automatic hemagglutination analyzer was used to measure indicators of coagulation function. Expressions levels of toll-like receptor 4(TLR4) and myeloid differentiation factor 88(MyD88) were detected by western blotting. The protein expression and phosphorylation level of extracellular signal-regulated kinase(ERK1/2), p38 mitogen-activated protein kinase(p38MAPK) and c-amino acid terminal kinase(JNK) were detected by western blotting. [Results] The model group demonstrated increase in pathological injury score(P<0.01), significant decline in serum content of inflammatory factors IL-1β, TNF-α and IL-6(P<0.05);shortened activated partial thromboplastin time, prothrombin time(PT), and thrombin time(TT), while increased fibrinogen(FIB) content(P<0.05);increased the protein expression and the phosphorylation levels of TLR4, MyD88, MAPK(p38MAPK, ERK1/2 and JNK, P<0.01) compared with the normal group. While Mailuo Shutong Pill significantly reduced the score of pathological injury and improved the pathological injury of ear tissue(P<0.05 or P<0.01);significantly decreased the expression levels of IL-1β, TNF-α and IL-6 in serum(P<0.05 or P<0.01);prolonged APTT, PT and TT and decreased FIB content(P<0.05 or P<0.01);down-regulated the protein expression and phosphorylation level of TLR4, MyD88, MAPK(p38MAPK, ERK1/2 and JNK, P<0.05 or P<0.01) compared with the model group. [Conclusion] Mailuo Shutong Pill was able to inhibit the inflammatory response and improve blood coagulation function in a model rabbit with superficial thrombophlebitis, and the mechanism may be related to the inhibition of TLR4/MyD88/MAPK signaling pathway activation.