The Material Basis And Molecular Mechanism Of Zeqi Enhancing Antitumor Immunity Of NK Cells

Objective:The purpose of this study was to identify the material basis of Zeqi(Euphorbia helioscopia L)promoting the anti-tumor immunity of NK cells,and elucidate its molecular mechanism.Methods: The luciferase chemiluminescence assay and luminescent assay was used to evaluate NK cell killing ability,and to guide the isolation and identification of components and compounds of Zeqi.The anti-tumor activity of the active compounds was evaluated in LLC subcutaneous tumor-bearing mice in vivo,and the proportions of NK cell and other immune cell in spleen was detected by flow cytometry.Then,the luciferase chemiluminescence assay and cytonote real-time kinetic label-free live-cell analysis system were used to further evaluat the effect of the active compond Eupho-A(euphohelioscopin A)on NK cell-mediated cell lysis.The activity of Eupho-A on NK cell degranulation was detected by flow cytometry,and the enhancement of Eupho-A on the productions of IFN-γ and TNF-α by NK cells were accessed by Real-time PCR and ELISA.The in vivo anti tumor activity by oral administration and the dependence of NK cells were evaluated in LLC subcutaneous tumor-bearing mice.The activity of Eupho-A in B16-F10 orthotopic tumor-bearing mice was also evaluated too,and the proportstion of lymphocytes in spleen and tumor tissues were detected by flow cytometry.Finally,the flow cytometry was used to analysis the regulation of Eupho-A on the expression of receptors on the NK cels surface and ligands on the tumor cell surface.Real-time PCR and ELISA were used to analysis the mechanism of Eupho-A enhancing IFN-γ and TNF-α through activating PKC.The effect of PKC was evaluated via luminescent assay and real time cellular analysis assay.The synergistic effect of Eupho-A,IFN-γ and TNF-α on the induction of apoptosis was evaluated by Annexin V and PI double staining assay.The effect of Eupho-A on the induction of pyroptosis was further confirmed by luminescent assay and Western blot,and the synergistic effect of Eupho-A and IFN-γ on GSDME expression was evaluated by Real-time PCR and Western blot.Results: 1.We have completed the screening of 51 components and 42 compounds in Zeqi,and confirmed Eupho-A(euphohelioscopin A),Eupho-C(euphoscopin C)and Eupho-noid A(euphoheliosnoid a)are the material basis of Zeqi.2.Eupho-A has a strong anti-tumor effect in LLC subcutaneous tumor-bearing mice,and can increase the proportion of NK cell in spleen.3.Eupho-A has a broad and substained activity on NK cell-mediated cell lysis.4.Eupho-A significantly enhance IFN-γ and TNF-α productions by NK cell,but has no effect on degranulation function.5.Eupho-A has a perfect in vivo anti-tumor activity by oral administration,and can up-regulate the proportion of NK cell in spleen and tumor tissues of B16-F10 orthotopic tumor-bearing mice;6.The in vivo anti-tumor activity of Eupho-A is depends on NK cells.7.The activity of Eupho-A on NK cell-mediated cell lysis is not related to regulation the expression of receptors and ligands.8.Eupho-A enhance IFN-γ and TNF-α productions of NK cell through activating PKC,and then enhance the cytotoxicity of NK cells;9.Eupho-A can induce pyroptosis which triggered by NK cells-derived Granzyme B.10.Eupho-A and IFN-γ can synergistically increase GSDME expression.Conclusion: 1.In this study,we identify that Eupho-A is the material basis of Zeqi promoting the anti-tumor immunity of NK cells for the first time.2.Enhancing IFN-γ and TNF-α productiosn of NK cell through PKC activating is one of the molecular mechanisms of Eupho-A.3.Inducing tumor cells pyroptosis through synergistically up-regulates GSDME expression with IFN-γ and increase NK cell-mediated GSDME cleavage is also molecular mechanism of Eupho-A.