Screening Of Traditional Chinese Medicine Monomers As Vaccine Adjuvants And Studies On Hesperetin Enhancing The Body's Antitumor Immune Response

Objective:Through the study of the characteristics of four chinese medicine monomers:hesperetin(HES),scutellaria(SCU),ziyuglycoside?(ZIY?)and polydatin(PD),the potential of chinese medicine monomers as immune adjuvants of tumor vaccine was preliminarily evaluated,and the best chinese medicine monomers hesperidine was selected as an adjuvant research object.Hesperetin was used as a vaccine adjuvant in combination with inactivated B16F10 melanoma cells in tumor-bearing mice,and the mechanism of enhancing the immunity of tumor-bearing mice was explored,which provides a reference for further research and development of new tumor vaccine adjuvants.Methods:HES,SCU,ZIY?and PD were used to stimulate the RAW264.7 and DC2.4cell lines according to the different concentration.After co-culture for 48 hours,the expression of MHC-?,MHC-?,CD80 and CD86 was detected by flow cytometry.In order to explore the immune activation effect of different traditional Chinese medicine monomers on antigen-presenting cells(APC).HES was selected as the main research object through cell experiments.RAW264.7 cell line and DC2.4 cell line were stimulated with 125?g/ml HES,RNA and protein were extracted from the cells for analysis of APC-activated signaling pathways.125?g/ml HES was used alone or mixed with inactivated melanoma,and it was tested in vivo and in vitro to determine whether it could stimulate APC activation.In vitro experiments LPS was used as the positive control,and the expression of surface markers MHC-?,MHC-?,CD80 and CD86 on RAW264.7and DC2.4 cell lines were detected by flow cytometry;In vivo experiments,a vaccine prepared by mixing the inactivated B16F10 melanoma cells and Freund's complete adjuvant was set as a positive group,a vaccine prepared by containing only inactivated melanoma and a vaccine prepared by mixing the inactivated B16F10 melanoma cells and hesperetin was set as an experimental group,a vaccine prepared by containing only the culture medium group was set as negative group,and mice were immunized by intramuscular injection.Spleen cells of immunized mice were collected,and the expression of MHC-?,MHC-?,CD80 and CD86,the expression of cytokines IL-4,IFN-?,TNF-?secreted by T cells and T Cell killing ability were analyzed by flow cytometry.Results:We found that hesperetin can activate the PI3K-Akt pathway in APC,enhance CD8~+T cell response,and reduce Treg cell activity.We also observed that the combination of inactivated melanoma and hesperetin inhibited tumor growth in tumor-bearing mice,and that overall survival was significantly improved compared to vaccines containing only inactivated melanoma.In order to verify the important role of CD8~+T cells in inhibiting the growth of melanoma,CD8~+T cells were screened from the spleens of immunized mice and returned to the model of tumor-bearing mice through the tail vein of the mice.We found that the survival rate of tumor bearing mice was significantly prolonged.Conclusions:HES can be used as a potential adjuvant to improve the body's anti-tumor immune response and the immunogenicity of antigens,providing a valuable reference for the study of new vaccine adjuvants.