| Background:Malignant melanoma has the characteristics of high degree of malignancy,strong invasiveness and easy metastasis.In fact,immunotherapy using inhibitors targeting programmed cell death protein 1(PD-1)in combination with its ligand PD-L1 has become a hot topic in the treatment of melanoma in recent years.PD-1 is a kind of transmembrane immunoglobulin.PD-1 is normally expressed on the surface of T cells,and when it binds to PD-L1 on the surface of tumor cells,it can inhibit T cell activation and even induce T cell apoptosis.Thus,it weakens the anti-tumor effect of the body’s immune system and contributes to the occurrence of tumor immune escape.Evodiamine(EVO),an extract of traditional Chinese herbal medicine,has been proved to have obvious inhibitory effect on many kinds of tumor cells,but the anti-tumor immune effect of EVO has not been widely concerned.A large number of studies have confirmed that EVO can regulate PI3K/AKT signal pathway,while the expression of PD-L1 is regulated by PI3K/AKT pathway.Objective:To investigate the transformation of malignant phenotype of mouse melanoma cells B16-F10 cells after being treated with different concentrations of EVO.In addition,the anti-tumor effect of EVO and the changes of immune organs and immune cell subsets in mice were verified in the mouse model of melanoma allogeneic subcutaneous transplantation.Further,to research the effect of EVO on PD-L1 expression in mice melanoma through in vivo and in vitro experiments.To explore the role of PI3K/AKT signal pathway in it,and to provide new ideas for immunotherapy of melanoma.Methods:1.Different concentrations of EVO were used to treat mouse melanoma B16-F10cells.CCK-8,plate cloning,Annexin V-FITC/PI,Transwell and wound healing experiments were used to evaluate the effects of EVO on the proliferation,apoptosis,invasion and migration of B16-F10 cells.2.Melanoma allograft tumor model was established by subcutaneous injection of B16-F10 cells into the back of C57BL/6 mice.After the successful establishment of the model,the mice were randomly divided into two groups that treated with EVO and saline respectively.The survival status,tumor growth and spleen volume of mice were observed.The histopathological changes of tumor cells and spleen were observed by HE staining.The infiltration proportion of T cell subsets in spleen and bone marrow of mice was detected by flow cytometry to observe the effect of EVO on immune cells in mice.The infiltration of T cells in tumor and spleen was verified by immunohistochemistry.The histopathological changes of important organs(heart,liver,lung and kidney)of mice were observed by HE staining,and the biological safety of EVO in vivo was evaluated.3.qRT-PCR and Western Blot experiments were used to study the expression of PD-L1 in B16-F10 cells treated with EVO and subcutaneously transplanted tumor.In order to further explore the mechanism of EVO regulating PD-L1,the changes of PI3K/AKT signal pathway and its related factors were observed by Western Blot experiment.Results:1.The results of CCK-8,plate cloning,Annexin V-FITC/PI,Transwell and wound healing experiments showed that EVO could significantly inhibit the proliferation,invasion and migration of mouse melanoma B16-F10 cells and promote its apoptosis.2.After treatment with EVO,the volume and mass of tumor tissue in EVO group were remarkable lower than those in Model group(P<0.001).The results of HE showed that the shape of tumor cells in Model group was different.The heteromorphism was obvious.The mitosis was increased and the ratio of nucleus to cytoplasm was abnormal.In EVO group,the nucleus of tumor decreased and the ratio of nucleus to cytoplasm decreased,suggesting cell apoptosis.3.EVO could significantly inhibit the spleen coefficient of melanoma-bearing mice,and the results of HE showed that compared with Model group,the margin of white pulp and red pulp of EVO group was clear.The structure was regular,and dense lymphocyte infiltration could be seen in white pulp.The results of flow cytometry showed that the number of CD4~+and CD8~+T cells in spleen and bone marrow of EVO group was significantly higher than that of Model group(P<0.05).The positive expression of CD4 and CD8 in spleen and tumor tissue of EVO group was stronger than that of Model group(P<0.05).The HE results showed that compared with the Control group,the structure of each organ in the EVO group was intact and the edge was clear.No tissue degeneration,necrosis and other obvious pathological changes were observed under the microscope.4.The results of qRT-PCR,Western Blot,and immunohistochemistry showed that the expression of PD-L1 in B16-F10 cells and transplanted tumor tissue treated with EVO was significantly lower than that in the control group(P<0.01).The expression of P-PI3K and P-AKT was decreased in EVO group(P<0.05).After B16-F10 cells were treated with AKT agonist SC79 combined with EVO,the expressions of P-PI3K,P-AKT and PD-L1 were higher than those treated with EVO only(P<0.05).Conclusions:EVO has excellent anti-tumor effect on mouse melanoma in vivo and in vitro.It may inhibit the expression of PD-L1 by down-regulating PI3K/AKT signal pathway,so as to improve the tumor immune microenvironment and play a certain anti-tumor effect. |
