Effect Of JAK1/STAT6 On M2-type Macrophage Polarization Mediated By Ganoderma Lucidum Spore Power In Regulating The Immune Microenvironment Of Triple Negative Breast Cancer

Objevtive:This experiment explored the relationship between the regulating effect of Ganoderma lucidum spores on TNBC immune microenvironment and its regulating of JAK1/STAT6 signaling pathway mediating M2 macrophage polarization,and explored the anti-TNBC mechanism of ganoderma lucidum spores combined with PTX injection,which provided scientific basis for the adjuvant anti-tumor of Ganoderma lucidum spores and enriched the scientific connotation of clinical application of ganoderma lucidum spores.Metheds:(1)The M2-type macrophage model was established by stimulating RAW264.7 macrophages with IL-4(100 ng/m L).The effect of Ganoderma lucidum spores on the polarization of M2-type macrophages was evaluated by flow cytometry,ELISA and RT-PCR.The phosphorylation levels of JAK1 and STAT6 were detected by Western blot.(2)The mouse TNBC model was established,and the polarization effect of ganoderma lucidum spores on M2-type macrophages was evaluated by immunofluorescence and RT-PCR.The changes of JAK1/STAT6 signaling pathway expression in tumor tissues were detected by western blot analysis to elucidate the mechanism of ganoderma lucidum spores on the polarization of M2 macrophages mediated by JAK/STAT6 signal.After it was determined that ganoderma lucidum spores could inhibit the polarization of M2 macrophages through JAK1/STAT6 signal,the changes of immune cell groups(CD8~+T cells,CD4~+T cells,MDSCs cells and Tregs cells)in tumor tissues were further observed by immunofluorescence staining to explore whether ganoderma lucidum spores regulates TNBC immune microenvironment by inhibiting the polarization of M2 macrophages.(3)The mouse TNBC model was established.After combined treatment with Ganoderma lucidum spores and Paclitaxel(PTX)injection,tumor cell apoptosis was observed by monitoring tumor inhibition rate and TUNEL staining to evaluate whether ganoderma lucidum spores could enhance the anti-tumor efficiency of chemotherapy drug PTX injection.After it was clear that ganoderma lucidum spores could enhance the anti-tumor efficiency of PTX injection,the changes of cytokines in tumor tissues of each group were analyzed by RT-PCR,and the changes of immune cell population in tumor tissues were observed by immunofluorescence staining,so as to explore whether the enhancement of anti-tumor efficiency of PTX injection by Ganoderma lucidum spores was related to the improvement of TNBC immunosuppressed microenvironment.Results:(1)Compared with normal group,the expressions of M2-type polarization related markers CD206,TGF-β1,IL-10 and Arg-1 were significantly increased in IL-4group,and the levels of JAK1 and STAT6 phosphorylated proteins were significantly increased;After ganoderma lucidum spore powder intervention,the above M2-type polarization related markers and protein phosphorylation levels were significantly reduced.(2)The results of immunofluorescence,RT-PCR and western blot showed that Ganoderma lucidum spores could inhibit the polarization of M2-type macrophages by reducing the phosphorylation of JAK1 and STAT6 proteins,and the results of immunofluorescence showed that Ganoderma lucidum spores could inhibit the immune cell population of tumor tissue.(3)The results of tumor inhibition and TUNEL staining showed that Ganoderma spore powder could enhance the anti-tumor efficiency of PTX injection,and the results of RT-PCR and immunofluorescence showed that Ganoderma spore powder could improve the changes of related cytokines and immune cell groups in tumor tissue.Compared with ganoderma lucidum spore or PTX monotherapy group,the level of Th1 cytokines in the combined treatment group was significantly increased.In addition,the recruitment of CD4~+,CD8~+T cells and M1 macrophages in the tumor area was significantly increased in both the PTX treatment group and the combined treatment group.Moreover,the percentages of M2macrophages,MDSCs and Tregs in the combined treatment group were lower than those in the PTX group.Conclusion:(1)Ganoderma lucidum spore powder can regulate TNBC immune microenvironment through M2 macrophage polarization mediated by JAK1/STAT6signal.(2)Ganoderma lucidum spore powder can enhance the anti-tumor efficiency of chemotherapy drug PTX injection by regulating TNBC immune microenvironment.