Study On The Anti-4T1 Breast Cancer Effect Of Spleen Lymphocytes Induced By The Ethyl Acetate Extract Of Cremastra Appendiculata

Objective:To explore the immunomodulatory effect of spleen lymphocytes induced by ethyl acetate extract of Cremastra appendiculata(CrApE)on 4T1 breast cancer,and to provide some experimental basis for immunopharmacology for the treatment of triple-negative breast cancer.Methods:1.Kunming mice were implanted with tumor,after successful implantation,they were randomly divided into model group and CrApE group,with 20 mice in each group,and 20 mice were selected as a control group.The CrApE group was administered with 96 mg/kg of CrApE,and the control group and model group were injected with the same volume of normal saline once a day,for 10 consecutive days.After the tumor regression was felt,the mice were inoculated with 4T1 cells at the same concentration again.The mice were weighed every 3 days to draw the weight change curve,the spleen was taken for spleen index determination;Mice spleen and peripheral blood lymphocytes were collected,the proportion of T cells,B cells,NK cells,CD4+T cells,CD8+T cells,CD107a+CD8+T cells and memory T lymphocytes in spleen of mice were detected by flow cytometry.The levels of IL-2,TNF-α,and IFN-γ cytokines in cell supernatant were detected by ELISA to evaluate the effect of CrApE on spleen lymphocytes of mice with 4T1 immune rejection.2.CCK-8 assay was used to detect the killing effect of splenic lymphocytes on 4T1 cells.A Transwell chamber was used to establish the co-culture system of spleen lymphocytes and 4T1 cells.Cell cycle and apoptosis were detected using flow cytometry.Tumor cell invasion and metastasis were detected by cell scratch test and Transwell cell migration test,and the effect of CrApE-induced splenic lymphocytes on the characteristics of 4T1 cell was evaluated.3.The proportion of CD4+T cells,CD8+T cells,CD 107a+CD8+T cells,and memory T lymphocytes in the spleen after co-culture with 4T1 cells was detected by flow cytometry.The levels of IL-2,TNF-α,and IFN-γ cytokines in the co-culture supernatant were detected by ELISA to evaluate the immunomodulatory effect of CrApE induced splenic lymphocytes against 4T1 cells.Results:1.Effect of CrApE on spleen lymphocytes of mice with 4T1 immune rejection:at the end of the experiment,compared with the model group,the bodyweight of mice in the CrApE group was significantly increased(P<0.05),and the spleen index of mice in the CrApE group was significantly increased(P<0.05).Flow cytometry showed that CrApE could increase the proportion of T cells,NK cells,CD8+T cells,CD107a+CD8+T cells,CD4+TEM,and CD8+TCM cells in the spleen and CD4+T,CD8+T,CD107a+CD8+T cells in peripheral blood compared with the model group(P<0.05).ELISA results showed that CrApE could increase the contents of TNF-α and IFN-γ in cell supernatant(P<0.05).2.Effect of CrApE-induced splenic lymphocytes against 4T1 cells:CrApE can enhance the killing rate of 4T1 cells to 4T1 cells,block the 4T1 cell cycle by increasing the proportion of G0/G1 cells and decreasing the proportion of S cells,induce apoptosis of 4T1 cells,and inhibit migration and invasion of 4T1 cells(P<0.05).3.Immunomodulatory effect of CrAPe-induced splenic lymphocytes against 4T1 cells:Flow cytometry results showed that:Compared with the Model group,CrApE significantly decreased the proportion of CD4+T cells and CD107a+CD8+T cells in splenic lymphocytes after co-culture(P<0.05),and increased the proportion of CD4+TCM cells(P<0.05).Compared with a single culture,the proportion of CD4+T cells,CD8+T cells,and CD8+TEM cells in the CrApE group was significantly increased(P<0.05),and the proportion of CD4+TCM and CD8+TCM cells was significantly decreased(P<0.05).ELISA results showed that:Compared with the Model group,CrApE significantly increased the contents of TNF-α and IFN-γ in the supernatant of co-cultured cells(P<0.05),and significantly increased the contents of TNF-α and IFN-γ in the supernatant of co-cultured cells(P<0.05),while significantly decreased the content of IL-2(P<0.05).Conclusion:1.CrApE increased the body weight and spleen index of Kunming mice inoculated with 4T1 cells,and increased the proportion of NK cells,T cells,killer T cells,and memory T cells in spleen and peripheral blood lymphocytes of mice,promoting the secretion of IL-2,TNF-α,and IFN-γ cytokines,and enhanced the rejection of 4T1 cells by the immune system.It plays an immunomodulatory role in anti-4T1 breast cancer.2.In the co-culture system,CrApE enhanced the killing of 4T1 cells by splenocytes,blocked 4T1 cells in G0/G1 phase,and promoted the apoptosis of 4T1 cells,inhibited the invasion and migration of 4T1 cells,and played an anti-TNBC role.3.In the co-culture system,CrApE increased the proportion of killer T cells and memory T cells in splenic lymphocytes,promoted the secretion of IFN-γ and TNF-α cytokines,enhanced the specific cellular immune response and immune memory against TNBC,and played an immunomodulatory role against TNBC.