Chrysin As An Adjuvant Enhances The Anti-tumor Immune Response Of B16F10 Melanoma Mice Through IL-12-STAT4 Signal Pathway

Objective:Chrysin(CHR),Formononetin(FOR),Vitexicarpin(VIT)and Eriodictyol(ERI)are all four flavonoid monomers,which are reported to have antioxidant,anti-tumor effects,and they are low production cost and easy degradation.In this study,CHR,which has the best function of activating antigen-presenting cells(APCs),was selected from four kinds of flavonoid monomers.Furthermore,B16F10 melanoma-bearing mice were used as a model to prove whether CHR as an immunogen adjuvant would enhance immunogenicity of protein antigen and enhance the immune perform of CD8~+T cells to kill tumor cells,and explore its immunologic mechanism.Methods:Low,middle and high concentrations of CHR,VIT,FOR and ERI,negative control and lipopolysaccharide(LPS)positive control were set up respectively.RAW264.7cells and DC2.4 cells were stimulated in vitro for 48 hours,and the expression levels of four surface markers CD80,CD86,MHCI and MHCII were detected.In order to determine whether CHR plays an adjuvant role by activating APCs,innate immunity experiments in vitro and in vivo were carried out.In vitro,chicken ovalbumin(OVA)at a working concentration of 10μg/ml was used as antigen peptide with 20μg/ml CHR to co-stimulate RAW264.7 and DC2.4 cells for 48 hours.The activation of four surface markers CD80,CD86,MHCI and MHCII were determined.In vivo,the soluble protein extracted from B16F10 cells was used as antigen to immunize mice with CHR as adjuvant.Three days after the primary immunization of mice,the expression levels of CD80,CD86,MHCI,MHCII and TNF-ɑin CD11b~+cells and CD11c~+cells were detected.Meanwhile,the typing of DC cells was also analyzed.After co-stimulating RAW264.7 cells with CHR and OVA for forty eight hours,the expression levels of cytokines IL-12 and IL-10 were detected,and then the changes of IL-12 downstream in splenocytes,STAT4 and p-STAT4,of were detected after IL-12 was up-regulated,so as to explore the immunological mechanism of CHR.In order to detect the effect of CHR on T cell function,mouse spleen cells were prepared into splenocyte suspension 7 days after twice immunization,and the cytokines secreted by CD4~+T cells and CD8~+T cells were detected.Next,in order to verify whether CHR can be used as an adjuvant for prophylactic vaccine,a tumor-bearing mouse model was established 7 days after twice immunization,and the survival curve and tumor size of mice were recorded.Finally,so as to verify the key role of CD8~+T cells in inhibiting the expansion of B16F10 malignant melanoma,we infused CD8~+T cells from immunized mice into the tail vein of B16F10 tumor-bearing mice,and recorded the survival curve and tumor size of mice.Results:By comparing the expression levels of four surface markers of four kinds of traditional Chinese medicine monomers activating RAW264.7 and DC2.4 cells in vitro,it was found that CHR had the best immune activation effect on APCs,that might be used as a candidate vaccine adjuvant for follow-up experiments.In the innate immunity experiment in vitro,CHR,mixed with OVA antigen peptide,can significantly up-regulate the expression of CD80,CD86,MHCI and MHCII surface markers.In the experiment of innate immunity in vivo,CHR,as an adjuvant mixed with B16F10 soluble protein antigen as vaccine,can effectively activate APCs and up-regulate the expression of CD80,CD86,MHCI,MHCII of CD11b~+cells as well as CD86,MHCI,MHCII,TNF-ɑof CD11c~+cells in mouse splenocytes.The immunological action of CHR may be to enhance the function of Th1 cells by activating macrophages to secrete IL-12,thus affecting the phosphorylation of STAT4 signal pathway,and then assist to boost the anti-tumor impact of cytotoxic T lymphocytes(CTL)in vivo.The effect of CHR as an adjuvant on T cell function was detected.Compared with the group immunized with B16F10 antigen alone,B16F10antigen+CHR group could effectively activate interferon-γ(IFN-γ)and interleukin-4(IL-4)secreted by CD4~+T cells in mouse spleen cells,as well as IFN-γand TNF-ɑsecreted by CD8~+T cells.In addition,B16F10 soluble protein antigen combined with CHR could inhibit the growth of tumor in mouse model,and the overall survival rate of mice was higher than that of B16F10 antigen alone.Conclusion:To sum up,CHR may be used as a possible adjuvant to boost the immunogenicity of antigen and enhance the response of CD4~+T and CD8~+T cells through IL-12-STAT4 signal pathway,inhibiting the expansion of B16F10 growth and improve the response to growth.