泻火解毒法对竹叶青蛇伤血管内皮细胞NF-kB信号通路相关因子的影响

Influence of Purging Fire and Removing Toxin Therapy on Related Factors of Vascular Endothelial Cells NF-kB Signaling Pathway in Trimeresurus Stejnegeri Bites

目的：观察泻火解毒法对竹叶青蛇伤血管内皮细胞核转录因子-kB（NF-kB）信号通路相关因子的影响。方法：根据前期研究复制竹叶青蛇伤动物及细胞模型。均设置空白组（KB）、模型组（MX）和低、中、高剂量蛇伤胶囊药液组（DY、ZY、GY）5组,每组10样本。在动物实验中,KB经兔右后腿皮下注射0.75 mL/kg生理盐水,6 h后灌胃10 mL/kg生理盐水。MX、DY、ZY和GY经兔右后腿皮下注射0.75 mL/kg竹叶青蛇毒液,6 h后分别灌胃10 mL/kg生理盐水及低、中、高剂量药液。各组每日灌胃1次,连续灌胃1周后采血并分离出血清待测。细胞实验中,KB加入10%正常兔血清培养,MX在KB的基础上加入5μg/mL竹叶青蛇毒液培养,DY、ZY和GY在MX基础上培养6 h后分别加入5%、10%和15%的含中药兔血清继续培养。各组培养72 h后,收集人脐静脉血管内皮细胞（HUVEC）及培养液检测。酶联免疫吸附法检测兔血清中NF-kB、IkBs激酶（IKK）、NF-kB诱导激酶（NIK）的含量。蛋白免疫印迹法测定各组细胞IKK、NIK、NF-kB p65的蛋白表达,激光共聚焦显微镜检测NF-kB p65在各组细胞的表达。结果：动物实验中,MX组IKK、NIK和NF-kB p65含量较KB组均出现了显著升高（P〈0.01）;ZY组和GY组的IKK、NIK和NF-kB p65含量较MX组均出现显著降低（P〈0.05或P〈0.01）。细胞实验中,MX组HUVEC中IKK、NIK和NF-kB p65的蛋白表达较空白组显著升高（P〈0.05或P〈0.01）,DY组和ZY组HUVEC中IKK、NIK和NF-kB p65蛋白表达较MX组显著减少（P〈0.05或P〈0.01）;激光共聚焦显微镜下MX组HUVEC中NF-kB p65的表达显著高于KB组（P〈0.01）,而DY组和ZY组HUVEC中NF-kB p65的表达较MX组显著下降（P〈0.01）。结论：NF-kB信号通路是竹叶青蛇伤血管内皮细胞炎症损伤的关键,泻火解毒法可通过调控该信号通路来治疗竹叶青蛇伤血管内皮细胞炎症损伤。

Objective： To investigate the influence of purging fire and removing toxinon therapy on the related factors of vascular endothelial cells nuclear transcription factor kappa B（ NF-kB） signaling pathway in Trimeresurus stejnegeri Bites. Methods：The animal and cell models were established according to the former study. The blank group（ KB）,the model group（ MX） and the low,middle and high dose Sheshang capsule liquid groups（ DY,ZY,GY） were set up.There were 10 samples in each group. In vivo,KB was injected 0. 75 mL/kg normal saline into rabbits’ right hind legs,and received 10 mL/kg normal saline by gavage after 6 hours. MX,DY,ZY and GY were injected 0. 75 mL/kg Trimeresurus Stejnegeri venom into rabbits’ right hind legs. After 6 hours,MX received 10 mL/kg normal saline by gavage,and Sheshang Capsule groups with different doses received 10 mL/kg low,middle and high dose Sheshang Capsule liquid by gavage respectively. The gavage was given once a day for a week. 24 hours after the last gavage,the blood of the rabbits was collected through auricular vein and the serum was separated. In vitro,KB was cultured with 10% blank rabbit serum. MX was cultured with 5 μg/mL of snake venom in addition. DY,ZY and GY were cultured with the same composition as MX in the first 6 hours and then they were cultured with 5%,10% and 15% drug serum respectively in addition. After 72 hours culture,the human vascular endothelial cells（ HUVEC） and medium were collected to measure. The level of IkBs kinase（ IKK）,nuclear transcription factor kappa B（ NF-kB） and NF-kB induced kinase（ NIK） in rabbit serum were measured by enzyme linked immunosorbent assay（ ELISA）. Protein expressions of IKK,NIK and NF-kBp65 in vascular endothelial cells of each group wre detected by Western blot. T he expression of NF-kB p65 in vascular endothelial cells was detected by confocal laser scanning microscope（ LSCM）. Results： In animal experiment,the levels of IKK,NIK and NF-kB p65 in MX group increased significantly compared with those of KB group（ P 〈 0. 01）. In ZY group and GY group,the levels of IKK,NIK and NF-kB p65 all decreased compared with those of MX group（ P 〈 0. 05 or P 〈 0. 01）. In vitro cell experiment,the protein expressions of IKK,NIK and NF-kB p65 in vascular endothelial cells in MX group increased significantly compared with those of KB group（ P 〈 0. 05 or P 〈 0. 01）. In DY group and ZY group,the protein expressions of IKK,NIK and NF-kB p65 all decreased compared with those of MX group（ P 〈 0. 05 or P 〈 0. 01）. The expression of NF-kB p65 in HUVEC in MX group was significantly higher than that in KB group under LSCM（ P 〈 0. 01）. In DY group and ZY group,the expressions of NF-kB p65 in HUVEC were decreased compared with those of the MX group（ P 〈 0. 01）. Conclusion： NF-kB signaling pathway is the key to inflammatory injury induced by Trimeresurus stejnegeri bites in vascular endothelial cell. The therapy of purging fire and removing toxin could treat inflammatory injury of the vascular endothelial cell induced by Trimeresurus stejnegeri bites through the regulation of NF-kB signaling pathway.