蛇伤胶囊对竹叶青蛇伤血管内皮细胞TM／PC系统的影响

摘要

Objective To investigate the mechanism of Sheshang capsule treating the coagulation disorder induced by Trimeresurus stejnegeri snakebites . Methods The New Zealand white rabbits were chosen .They were dis‐tributed into the Chinese Medicine group and the blank group randomly .Each group had 10 rabbits .696 mg・kg -1・d-1 Sheshang capsule solution and normal saline were given by gavage twice a day for five days respectively .2 hours after the last gavage ,the rabbits'blood was collected through cardiac puncture .Then ,the drug serum and blank serum were made by the normal method .Cytotoxicity assay was made by Thiazolyl Blue Tetrazolium Bromide assay(MTT) and the concentration of snake venom in our study was determined as 5 μg/ml .The human umbilical vein endothelial cells(HUVEC) were distributed into the blank group (KB) ,the model group (MX) and the low , middle and high dose drug serum group (DY ,ZY ,GY) after conventional culture and passage and each group had 10 samples .KB was cultured with 10% blank rabbit serum .MX was cultured with 5μg/ml of snake venom in addi‐tion .DY ,ZY and GY were cultured with the same composition as MX in the first 6 hours and then they were cul‐tured with 5% ,10% and 15% drug serum respectively in addition .After 72 hours culture ,the medium was collect‐ed to measure the level of thrombomodulin(TM) ,thrombin‐thrombomodulin(T‐TM) ,activated protein C(APC) , protein S(PS) and endothelial protein C receptor(EPCR) by enzyme linked immunosorbent assay(ELISA) . Re‐sults The content of TM ,T‐TM ,APC ,EPCR and PS in MX increased significantly compared with KB(all P<0.05 ,P<0.01 in T‐TM ,EPCR and PS) .The content of TM ,T‐TM ,APC ,EPCR and PS in DY ,ZY and GY all de‐creased compared with MX and ZY decreased significantly(all P<0.05 ,P<0.01 in T‐TM and EPCR) . Conclu‐sion Trimeresurus stejnegeri venom could damage vascular endothelial cells and increase the risk of bleeding .The Sheshang capsule could improve our coagulation function through its effect on TM/PC system of vascular endothelialcells .This is a part of mechanism of Sheshang capsule treating the coagulation disorder induced by T rimeresurus stej negeri snakebites .%目的：研究蛇伤胶囊治疗竹叶青蛇伤凝血障碍的机制。方法选取新西兰大白兔20只，随机分为中药组和空白组，每组10只，分别以696 mg・ kg －1・d －1蛇伤胶囊药液和生理盐水灌胃，每天2次，连续5天，末次灌胃2 h后心脏采血，常规方法制备含药血清和空白血清。采用噻唑蓝法对竹叶青蛇毒进行细胞毒性试验，确定蛇毒干预浓度为5μg／ml。人脐静脉血管内皮细胞常规培养、传代后，随机分为空白组（KB组），模型组（MX组），低、中、高剂量含药血清组（DY 组、ZY 组、GY 组）5组，每组10个样本。KB组加入10％正常兔血清培养，MX组在KB组的基础上加入5μg／ml竹叶青蛇毒液培养，DY组、ZY组、GY组在M X基础上培养6 h后分别加入5％、10％、15％的含中药兔血清继续培养。各组分别培养72 h后收集细胞培养液，以酶联免疫吸附法检测 TM 、T‐TM 、APC、EPCR、PS水平。结果 MX组的 TM 、T‐TM 、APC、EPCR、PS水平相对 KB组均显著升高（均P＜0.05，其中 T‐TM 、EPCR和PS的 P值＜0.01）；DY 组、ZY组、GY组的 TM 、T‐TM 、APC、EPCR、PS水平相对MX组均出现不同程度的降低，其中ZY组降低效果显著（均 P＜0.05，其中 T‐TM 和 EPCR的 P值＜0.01）。结论竹叶青蛇毒可造成血管内皮细胞损伤，增加机体出血风险；蛇伤胶囊可通过作用血管内皮细胞 T M／PC系统改善机体的凝血功能，是治疗竹叶青蛇伤凝血障碍的部分机制。