Effects Of The Recombinant Sarcoptes Scabiei Translationally Controlled Tumor Protein(TCTP)on Degranulation Of KU812 Cells

Scabies is a stubborn,infectious zoonosis of the skin caused by Sarcoptes scabiei,which lives within the epidermis of host skin.The excretions of S.scabiei can cause pathological reactions in the host epidermal and dermal cells,leading to clinical symptoms such as itching,redness and swelling,and allergic inflammation.Translationally controlled tumor protein(TCTP)is a newly discovered parasite excretory-secretory protein.Previous studies have shown that this protein can participate in the host inflammatory response.It mainly affects mast cells,basophils degranulation and histamine release.The function of Sarcoptes scabiei TCTP(SsTCTP)has not been reported so far.In this study,the relative mRNA transcription levels of SsTCTP at different developmental stages of S.scabiei were analyzed,and the effects of rSsTCTP on skin vascular permeability and degranulation of KU812 cells were investigated.The results may provide new ideas for the pathogenesis,prevention and treatment of scabies.The main research work and results are as follows:1.The relative transcription level of SsTCTP mRNA and the effect of recombinant protein on skin vascular permeability in miceIn order to analyze the relative transcription level of SsTCTP mRNA at different stages,and to obtain the recombinant protein(rSsTCTP)to further investigate its effect on the host skin vascular permeability,the coding sequence of the SsTCTP gene was cloned from S.scabiei cDNA as a template.The relative transcription levels of SsTCTP mRNA at larva,nymph and adult stages were analyzed by qRT-PCR,and the recombinant protein rSsTCTP was expressed in E.coli.Finally,the effect of rSsTCTP on skin vascular permeability in mice was investigated by constructing an Evans blue vascular permeability model.The results showed that the length of the coding sequence of the SsTCTP gene was 522bp.The relative mRNA transcription level of the SsTCTP gene was the highest at larva,followed by nymph,and the lowest at adult.The result of Evans blue vascular permeability test showed,compared with the negative control groups,the skin samples injected with rSsTCTP showed significantly higher vascular permeability in situ and after quantitative determination with deionized formamide extraction dye.In conclusion,SsTCTP gene was transcribed in all three developmental stages,but the transcription level was relatively higher at larva.The rSsTCTP can increase skin vascular permeability and plasma leakage in mice after subcutaneous stimulation.2.Effects of rSsTCTP on degranulation of KU812 cellsTo further explore the effects of SsTCTP on the degranulation pathway and degranulation substances of host mast cells and basophils,a human basophilic leukemia cell line(KU812)degranulation model was constructed to simulate the degranulation mechanism of mast cells and basophils.Firstly,the effect of degranulation model construction was verified by β-HEX release test.Secondly,the expression and phosphorylation of major proteins in the degranulation pathway were detected by Western blot.Finally,the release characteristics of histamine and Th2-type cytokines after stimulation with the recombinant protein were analyzed by ELISA.The results showed that after rSsTCTP stimulation,the release rate of β-HEX increased,and the phosphorylation levels of Lyn,NF-κB and MAPK family proteins in the degranulation pathway increased,indicating that rSsTCTP could activate the Lyn mediated degranulation pathway and induce enhanced degranulation response in KU812 cells.ELISA results showed that the release of histamine and Th2-type cytokines IL-4,IL-6 and IL-13 were significantly increased after stimulation with the recombinant protein.In conclusion,rSsTCTP could activate the Lyn mediated degranulation pathway in KU812 cells,resulting in the increased release of histamine and Th2-type cytokines IL-4,IL-6 and IL-13.