ABSTRACT
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 gavage 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-△ △Ct) 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-△ △Ct) 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 significantly in model group compared 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 compared with those in model group in endothelial cells of aorta abdominalis of rabbits and HUVEC [abdominal aorta:IL-8 mRNA (2-△ △Ct) were 1.13 ± 0.19,1.26 ± 0.16,1.27 ± 0.17 vs.3.96 ± 0.39,MCP-1 mRNA (2-△ △ Ct) were 1.79 ± 0.24,2.22 ± 0.38,1.76±0.19 vs.3.07±0.27,ICAM-1 mRNA (2 △△Ct) were 2.05±0.11,1.68±0.09,2.37±0.48 vs.3.71±0.26,VCAM-1 mRNA (2-△△Ct) were 1.59±0.08,1.40±0.11,1.84±0.11 vs.3.94±0.26;HUVEC:IL-8 mRNA (2-△△Ct) were 2.33±0.59,2.82±0.82,2.51±0.77 vs.3.53±0.70,MCP-1 mRNA (2-△△Ct) were 1.59±0.35,1.48±0.36,1.54±0.29 vs.2.24±0.48,ICAM-1 mRNA (2-△△Ct) were 1.46±0.38,1.77±0.65,1.73±0.50 vs.3.13±0.44,VCAM-1 mRNA (2-△△Ct) 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 significantly in middle,high dose Sheshang capsule groups compared with the content in model group (μg/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 injury by inhibiting the inflammatory response induced by Trimeresurus stejnegeri bites.