桃红四物汤加减方通过抑制中性粒细胞胞外诱捕网形成对大鼠骨折后深静脉血栓形成的影响

Eff ect of modifi ed Taohong Siwu decoction on deep vein thrombosis formation afterfracture in rats by inhibiting the formation of neutrophil extracellular traps

目的 探究桃红四物汤加减方通过抑制中性粒细胞胞外诱捕网(NETs)形成对大鼠骨折后深静脉血栓形成的实验研究。方法 将24只大鼠随机分为空白对照组、模型对照组、阳性药物组、中药组,每组6只。制备机械损伤性大鼠股骨骨折模型,对照组和模型对照组给于生理盐水灌胃,阳性药物组给予利伐沙班片,中药组按照方剂1 mL·100 kg^(-1)大鼠体质量灌胃。造模后7 d眼球取血,检测各组抗凝指标水平;ELISA法检测各组血清肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、瓜氨酸化组蛋白H3(Cit-H3)、髓过氧化物酶(MPO)和D-二聚体(DD)水平;检测血清超氧化物歧化酶(SOD)活力水平和丙二醛(MDA)水平;观察左股静脉组织标本HE染色形态。结果 与空白对照组比较,模型对照组血清APTT、PT和TT水平均降低,DD水平升高(P<0.01);与模型对照组比较,阳性药物组与中药组的血清APTT、PT和TT水平均升高,DD水平降低(P<0.05或0.01);与空白对照组比较,模型对照组血清中SOD活力水平降低,MDA含量升高(P<0.01);与模型对照组比较,阳性药物组与中药组血清中SOD活力水平升高,MDA含量降低(P<0.05或0.01);与空白对照组比较,模型对照组血清中IL-6和TNF-α含量升高(P<0.01);与模型对照组比较,阳性药物组与中药组IL-6和TNF-α含量降低,且中药组低于阳性药组(P<0.05或0.01);与空白对照组比较,模型对照组血清中MPO水平升高,Cit-H3水平降低(P<0.01);与模型对照组比较,阳性药物组与中药组的血清MPO、Cit-H3水平均降低,且中药组低于阳性药物组(P<0.01);空白对照组静脉形态结构正常,无炎症细胞浸润;DVT组可见血栓形成,静脉内膜且有损伤,血管内皮细胞出现剥脱现象,血管壁炎性细胞浸润;阳性药物组仍可见血栓,但小于模型对照组;中药组血栓小于模型对照组。结论 桃红四物汤加减方对大鼠深静脉血栓的形成具有防治作用,其机制可能是通过抑制中性粒细胞胞外诱捕网的形成抑制炎症发生,从而干预血栓的形成。

Objective To explore the eff ect of modifi ed Taohong Siwu decoction on deep venous thrombosis after fracture in rats by inhibiting the formation of neutrophil extracellular traps(NETs).Methods A total of 24 SD rats were randomly divided into the blank control group,the model control group,the positive drug group and the Chinese medicine group,with 6 rats in each group.Rat model of femoral fracture with mechanical injury was prepared.The blank control group and the model control group received normal saline by gavage, the positive drug group received rivaroxaban tablets, and the Chinese medicine group administered1 mL·100 kg^(-1) of rat body weight by gavage according to the formula. Blood sampls were taken from the eyeball at 7 days aftermodeling, and the levels of anticoagulant indicators in each group were detected. The levels of tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6), citrullinated histone H3 (Cit-H3), myeloperoxidase (MPO) and D-dimer (DD) were detected by Enzyme-LinkedImmunosorbent Assay (ELISA). The serum levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured.The HE staining morphology of the left femoral vein tissue specimen was observed. Results Compared with the blank controlgroup, serum levels of activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) in the modelcontrol group decreased, and level of D-dimer (DD) increased (P <0.01). Compared with the model control group, serum levels ofAPTT, PT and TT in the positive drug group and the Chinese medicine group increased, and the level of DD decreased (P <0.05 orP <0.01). Compared with the blank control group, the serum SOD activity level in the model control group decreased, and MDAcontent increased (P <0.01). Compared with the model control group, the serum SOD activity level in the positive drug group andthe Chinese medicine group increased, and MDA content decreased (P <0.05 or P <0.01). Compared with the blank control group, theserum levels of IL-6 and TNF-α in the model control group increased (P <0.01). Compared with the model control group, the levelsof IL-6 and TNF-α in the positive drug group and the Chinese medicine group decreased, and the Chinese medicine group was lowerthan the positive drug group (P <0.05 or P <0.01). Compared with the blank control group, the serum level of MPO in the modelcontrol group increased, and the level of Cit-H3 decreased (P <0.01). Compared with the model control group, in the positive druggroup and the Chinese medicine group, the serum levels of MPO and Cit-H3 decreased, and the Chinese medicine group was lowerthan the positive drug group (P <0.01). In the blank control group, the morphology and structure of the veins were normal, and therewas no infl ammatory cell infi ltration. In the model control group, thrombosis could be seen, with damage to the vascular intima, thephenomenon of endothelial cell detachment in blood vessels, and infl ammatory cell infi ltration in the vascular wall. In the positivedrug group, thrombus could still be seen, but it was smaller than that in the model control group. The thrombus in the Chinesemedicine group was smaller than that in the model control group. Conclusion Modifi ed Taohong Siwu decoction has the preventiveand therapeutic eff ect on the formation of deep venous thrombosis in rats, and its mechanism may be to inhibit infl ammation byinhibiting the formation of NETs, thereby intervening in the formation of thrombosis.