| Objective: To investigate the effects of recombinant Aedes Albopictus salivary 34k2 protein(abbreviated as rAlb-34k2 afterwards)on the activation of mast cells in mouse ears and on the expression of cytokines and chemokines in Raw264.7 cells in vivo and in vitro.Methods: 1)Balb/c mice were used as experimental objects and 0.6% acetic acid,heat-inactivited rAlb-34k2 and rAalb-CTL2 protein(another salivary secretory protein of Aedes albopictus expressed by prokaryotic recombination)was used as experimental control,and mouse ears were stimulated by subcutaneous injection of rAlb-34k2 protein(294.1pM).The mast cells degranulation ratio of mouse ear was observed and calculated by toluidine blue staining under the microscope.The changes of histamine release from mouse ears were detected by ELISA.2)Using mouse macrophage Raw264.7 cells as experimental objects and rAalb-CTL2 protein as experimental control,the cell was stimulated by rAlb-34k2 protein at different concentrations(29.41pM/mL and 294.1pM/mL)for.The expression of pro-inflammatory cytokines(IL-6,IL-1? and TNF-?),anti-inflammatory cytokines(IL-10),neutrophil-attracting chemokines(CXCL2)and monocytic chemoattractive chemokines(CCL2)in Raw264.7 cells was detected by qRT-PCR at 6h and 24 h.The protein expression of pro-inflammatory cytokines(IL-6,IL-1?)in Raw264.7 cell culture supernatant was analyzed by ELISA.Results: 1)Results of mast cell degranulation ratio: compared with the ratio in the N.S group(22.33%),the degranulation ratio of mast cells in the mouse ear are 22.33%(negative control),49.00%(rAlb-34k2 group),49.75%(positive control),40.40%(rAalb-CTL2 group),41.25%(inactivited rAlb-34k2 group),Significant difference was found between rAlb-34k2 group and negative group(P < 0.001)when stimulated by rAlb-34k2 protein in a manner of subcutaneous injection at a concentration of 294.1pM for 20 minutes.Results of histamine release: no significant changes in histamine release were detected when the ears were stimulated by rAlb-34k2 protein at a concentration of 294.1pM for 20 minutes.2)Results of cytokines and chemokines: compared with the control group,rAlb-34k2 protein at low concentration(29.41pM/mL)and high concentration(294.1pM/mL)could up-regulate cytokine IL-6(41 folds,P < 0.001 and 138 folds,P < 0.001,respectively),IL-1?(14 folds,P < 0.001 and 31 folds,P < 0.001,respectively),TNF-?(2 folds,P < 0.001,and 3 folds,P < 0.001,respectively)and IL-10(2.3 folds,P < 0.001 and 2.7 folds,P < 0.001,respectively).The expression of chemokine CXCL2(up-regulated by 4.7 folds,P < 0.001 and 10 folds,P < 0.001),CCL2(up-regulated by 6 folds,P < 0.001 and 9 folds,P < 0.001)were also up-regulated by quantitative real-time PCR.At 24 hours,rAlb-34k2 protein at a low concentration could up-regulate the expression of cytokine IL-6(187 folds,P < 0.001),IL-1?(26 folds,P < 0.001),TNF-?(2.2 folds,P < 0.001),IL-10(5.4 folds,P < 0.001)and chemokine CXCL2(11 folds,P < 0.001),CCL2(15 folds,P < 0.001)by quantitative real-time PCR.In addition,from the time dynamic observation,all factors(IL-6,IL-1?,TNF-?,IL-10,CXCL2,CCL2)in the low concentration group showed a continuous increasing from 6h to 24 h.Moreover,IL-6 and IL-1? secreted into the culture supernatants by Raw264.7 cells increased under the effect of high concentration of rAlb-34k2 protein.Conclusion: 1)rAlb-34k2 protein could activate mast cells in mouse ear.2)rAlb-34k2 protein could promote the expression of pro-inflammatory cytokines IL-6,IL-1? and chemokines CXCL2,CCL2 in Raw264.7 cells in vitro,and showed a dose-dependent pattern.These results suggested that rAlb-34k2 protein could promote inflammation and provide experimental basis for exploring the biological functions of 34k2 protein in the saliva of Aedes albopictus. |
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