摘要
目的观察泻火解毒法对竹叶青蛇毒导致血管内皮炎症损伤相关趋化因子及黏附分子的影响。方法①动物实验：取健康新西兰大白兔50只，按计算机产生的随机数字分为正常对照组、模型组，以及蛇伤胶囊低、中、高剂量组，每组10只。采用经兔右后腿皮下注射0．75mL／kg竹叶青蛇毒液的方法复制竹叶青蛇伤动物模型；对照组给予等量生理盐水。制模后6h，蛇伤胶囊低、中、高剂量组分别给予蛇伤胶囊溶液174、348、522mg·kg^-1·d^-1（用生理盐水稀释为17．4、34．8和52．2g／L的药液，灌胃量均为10mL·kg^-1·d^-1）；模型组和正常对照组灌胃等量生理盐水；均每日灌胃1次，连续灌胃1周。于末次灌胃后24h经耳缘静脉采血，离心取血清备用。同时取兔腹主动脉全段，保存于液氮中备用。②细胞实验：以改进的Eagle培养液（MEM）培养人脐静脉内皮细胞（HUVEC）24h，按计算机产生的随机数字分为空白对照组、模型组和蛇伤胶囊药物血清低、中、高剂量组，每组10孔。于细胞培养液中加入5mg／L竹叶青蛇毒液复制竹叶青蛇毒中毒细胞模型。培养6h后，模型组和空白对照组给予10％正常兔血清，药物组给予5％、10％和15％含药血清。培养72h后，收集细胞提取总RNA。用实时荧光定量聚合酶链反应（qPCR）检测兔腹主动脉及HUVEC白细胞介素-8（IL-8）、单核细胞趋化因子-1（MCP-1）、细胞间黏附分子-1（ICAM-1）和血管内皮细胞黏附分子-1（VCAM-1）的mRNA表达水平；用酶联免疫吸附试验（ELISA）检测兔血清E-选择素（CD62E）含量。结果模型组腹主动脉和细胞IL-8、MCP-1、ICAM-1和VCAM-1的mRNA表达水平及CD62E含量均较对照组显著升高[以正常对照组和空白对照组IL-8、MCP-1、ICAM-1和VCAM-1的mRNA表达水平均为1，动物模型组上述指标的mRNA表达水平（2^-ΔΔCt）分别为3．96±0．39、3．07±0．27、3．71±0．26、3．94±0．26，细胞模型组上述指标的mRNA表达水平（2^-ΔΔCt）分别为3．53±0．70、2．24±0．48、3．13±0．44、2．80±0．13，均P〈0．01]，模型组血清CD62E含量亦较正常对照组显著升高（μg／L：1．31±0．22比0．82±0．13，P〈0．01），蛇伤胶囊低、中、高剂量组腹主动脉和细胞IL-8、MCP-1、ICAM-1和VCAM-1的mRNA表达水平均较模型组明显降低[腹主动脉：IL-8mRNA（2^-ΔΔCt）为1．13±0．19、1．26±0．16、1．27±0．17比3．96±0．39，MCP-1mRNA（2^-ΔΔCt）为1．79±0．24、2．22±0．38、1．76±0．19比3．07±0．27，ICAM-1mRNA（2^-ΔΔCt）为2．05±0．11、1．68±0．09、2．37±0．48比3．71±0．26，VCAM-1mRNA（2^-ΔΔCt）为1．59±0．08、1．40±0．11、1．84±0．11比3．94±0．26；细胞：IL-8mRNA（2^-ΔΔCt）为2．33±0．59、2．82±0．82、2．51±0．77比3．53±0．70，MCP-1mRNA（2^-ΔΔCt）为1．59±0．35、1．48±0．36、1．54±0．29比2．24±0．48，ICAM-1mRNA（2^-ΔΔCt）为1．46±0．38、1．77±0．65、1．73±0．50比3．13±0．44，VCAM-1mRNA（2^-ΔΔCt）为2．49±0．24、2．18±0．19、2．45±0．24比2．80±0．13，均P〈0．05]，蛇伤胶囊中、高剂量组血清CD62E含量均较模型组明显降低（μg，L：1．01±0．14、1．04±0．13比1．31±0．22，均P〈0．01），但3个剂量组比较差异均无统计学意义（均P〉0．05）。结论泻火解毒法能通过抑制竹叶青蛇伤引起的炎症反应治疗竹叶青蛇伤血管内皮损伤。
Objective To investigate the influence of the purging fire and removing toxin method on chemokines and adhesion factors related to vascular endothelialitis injury induced by toxin of trimeresurus stejnegeri bite. Methods （1） Animal experiment： 50 healthy New Zealand white rabbits were chosen. According to random numbers generated by statistical software, they were divided into normal control group, model group, low, middle and high dose Sheshang capsule groups, 10 in each group. Trimeresurus stejnegeri bite model was replicated by injecting 0.75 mL/kg snake venom into subcutaneous tissues of rabbits＇ right hind legs. And the same volume of normal saline was injected into the rabbit in the normal control group. After the model was established for 6 hours, the rabbits in low, middle and high dose Sheshang capsule groups received 174, 348 and 522 mg·kg^-1· d^-1 of Sheshang capsule solution respectively （the content of capsules was dissolved in normal saline to make liquid with 17.4, 34.8 and 52.2 g/L Sheshang solution respectively, so the volume of garage of each group was 10 mL·kg^-1· d^-1）; in the model and normal control groups, the same amount of normal saline was given by gavage, once daily for consecutive one week. 24 hours after the last gavage,the blood of the rabbits was collected through an auricular border vein and the serum was separated by centrifuge ready for use. Meanwhile, the whole abdominal aorta segment of the rabbit was harvested and kept them in liquid nitrogen ready for use. （2） Cell experiment： human umbilical vascular endothelial cell （HUVEC） was cultured with MEM for 24 hours. The solution was replaced and according to the random number generated by statistical software, the cells were divided into blank control group, model group and low, middle, high dose Sheshang capsule medicinal serum groups, 10 wells in each group. Trimeresurus stejnegeri toxin cell model was reproduced by addition of 5 mg/L snake venom into the cell culture medium. After 6-hour culture, the cells of model group and blank control group received 10% normal rabbit serum, and the cells of low, middle and high dose Sheshang medicinal serum capsule groups received serum containing 5%, 10% and 15% drug, respectively. After culture for 72 hours, the cells were collected and the total RNA was extracted. The real-time fluorescent quantitative polymerase chain reaction （qPCR） was used to detect the levels of mRNA of interleukin-8 （IL-8）, monocyte chemoattractant protein-1 （MCP-1）, intercellular adhesion molecule-1 （ICAM-1） and vascular endothelial cell adhesion molecule-1 （VCAM-1） in the vascular endothelial cells of rabbit aorta abdominalis and human umbilical vein, and the content of serum E-select element （CD62E） was measured by enzyme linked immunosorbent assay （ELISA）. Results In model group, the expression levels of mRNA in IL-8, MCP-1, ICAM-1, VCAM-1 and the content of CD62E were all increased significantly in the endothelial cells of rabbit aorta abdominalis and HUVEC compared with those in control group [when the mRNA expression levels of IL-8, MCP-1, ICAM-1 and VCAM-1 in normal and blank control group were all being 1, the mRNA expression levels （2^-ΔΔCt） of the above mentioned inflammatory factors and adhesion molecule in animal model group were 3.96± 0.39, 3.07 ±0.27, 3.71 ± 0.26, 3.94 ± 0.26, and the mRNA expression levels （2^-ΔΔCt） of the above mentioned inflammatory factors and adhesion molecule in HUVEC model group were 3.53±0.70, 2.24±0.48, 3.13±0.44, 2.80±0.13, respectively, all P 〈 0.01]. The content of CD62E in serum was increased signifieantly in model group eompared with that in normal control group （μg/L： 1.31± 0.22 vs. 0.82 ± 0.13, P 〈 0.01）, the mRNA expression levels of IL-8, MCP-1, ICAM-1 and VCAM-1 were decreased significantly in low, middle, high dose Sheshang capsule groups eompared with those in model group in endothelial cells of aorta abdominalis of rabbits and HUVEC [abdominal aorta： IL-8 mRNA （2^-ΔΔCt） were 1.13 ± 0.19, 1.26 ± 0.16, 1.27 ± 0.17 vs. 3.96 ± 0.39, MCP-1 mRNA （2^-ΔΔCt） were 1.79 ± 0.24, 2.22 ± 0.38, 1.76±0.19 vs. 3.07±0.27, ICAM-1 mRNA （2^-ΔΔCt） were 2.05±0.11, 1.68±0.09, 2.37±0.48 vs. 3.71±0.26, VCAM-1 mRNA （2-aact） were 1.59±0.08, 1.40±0.11, 1.84±0.11 vs. 3.94±0.26; HUVEC： IL-8 mRNA （2^-ΔΔCt） were 2.33±0.59, 2.82±0.82, 2.51±0.77 vs. 3.53±0.70, MCP-1 mRNA （2^-ΔΔCt） were 1.59±0.35, 1.48±0.36, 1.54±0.29 vs. 2.24±0.48, ICAM-1 mRNA （2^-ΔΔCt） were 1.46±0.38, 1.77±0.65, 1.73±0.50 vs. 3.13±0.44, VCAM-1 mRNA （2^-ΔΔCt） were 2.49±0.24, 2.18±0.19, 2.45±0.24 vs. 2.80±0.13, all P 〈 0.05]. The contents of CD62E were decreased signifieantly in middle, high dose Sheshang capsule groups compared with the content in model group （pg/L： 1.01±0.14, 1.04±0.13 vs. 1.31±0.22, all P 〈 0.01）, but there were no statistical significant differences among the three drug group （all P 〉 0.05）. Conclusion The therapy of purging fire and removing toxin can treat vascular endothelial iniury by inhibiting the inflammatory response induced by Trimeresurus stejnegeri bites.
出处
《中国中西医结合急救杂志》
CAS
CSCD
北大核心
2017年第2期192-195,共4页
Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care
基金
国家自然科学基金（81302978）
福建省自然科学基金（2012J01379）
福建中医药大学校管课题重点学科专项（X2014033-学科,X2014034-学科,X2014035-学科）
关键词
泻火解毒法
蛇伤胶囊
竹叶青蛇
炎症反应
血管内皮损伤
Therapy of purging fire and removing toxin
Sheshang capsule
Trimeresurus stejnegeri
Inflammatory response
Vascular endothelial injury
