| Objective:In vitro and in vivo effects of ethyl acetate extract from Cremastra appendiculata(Cr Ap)on 4T1 breast cancer and its immune mechanism in mice bearing 4T1 breast cancer.Methods:1.MTT assay was used to measure the inhibition of proliferation of Cr Ap with different concentrations(1250ug/m L,1250 ug/ml,125ug/m L,12.5ug/m L,1.25ug/m L,0.125ug/m L)on 4T1 breast cancer cells at 24h,48h,72h.2.100 BABL/c female mice were implanted with tumor.After successful implantation,they were randomly divided into 5groups:blank control group,model group,Adriamycin(ADM)positive control group,Cr Ap group and Cr Ap+ADM group,with 20 mice in each group.The administration concentration of Cr Ap group was given 96mg/kg once a day,the positive control group was given 2mg/kg ADM once every other day,Cr Ap+ADM group was given the same dose of Cr Ap and ADM,the rest groups were given normal saline once a day,all intraperitoneal injection for 21 days.The mice were weighed every 7 days to draw the weight change curve;the tumor diameter and short diameter were measured every 3 days to draw the tumor curve;the tumor inhibition rate was calculated by peeling off the tumor of mice;some cancer tissues were stained with HE;the coefficients of thymus and spleen were calculated by stripping thymus and spleen;part of thymus and spleen were stained with HE;take peripheral blood and put it into anticoagulant tube,the changes of CD4~+T,CD8~+T,regulatory T cells(Tregs),B-lymphocyte(B)classification and the ratio of CD4~+/CD8~+were detected by Flow Cytometry(FCM);preparing tissue suspension from the rest spleen and cancer tissue,the levels of Interleukin-2(IL-2),Tumor necrosis factor-?(TNF-?),Interferon-?(IFN-?)and i Interleukin-10(IL-10)were detected by Enzyme-linked immunosorbent assay(ELISA).Results:In MTT experiment,the inhibitory rate of Cr Ap with different concentrations ranged from 13.48%to 74.47%,of which Cr Ap(125?g/m L)had the strongest inhibitory effect at 72h,with the inhibitory rate of 74.74%.2.1 Weight curve:the weight of model group was significantly lower than that of blank group(P<0.01);at the same time,the weight of ADM group and Cr Ap+ADM group were significantly lower than that of model group(P<0.01),but the weight of Cr Ap group was significantly increased(P<0.01);in addition,the weight of Cr Ap group and Cr Ap+ADM group were significantly higher than that of ADM group(P<0.01).2.2 Tumor curve:compared with the model group,the tumor volume of all drug groups were significantly reduced(P<0.01).2.3 Tumor inhibition rate:all drug groups have inhibitory effect on tumor of tumor-bearing mice,of which the tumor inhibition rate of Cr Ap+ADM group was 53.67%,with the strongest inhibitory effect.2.4 HE staining of cancer tissue:in the model group,the cancer cells were in good shape,with large numbers of cancer cells,and few necrotic areas;compared with the model group,the tumor cells of Cr Ap group and Cr Ap+ADM group showed cytoplasmic rupture and nucleolysis,and cell density decreased,and there are necrotic areas of different degrees.2.5 The organ index of immune organs:compared with the blank group,the thymus coefficient in the model group was decreased(P<0.01),but the spleen coefficient was significantly increased(P<0.01);compared with model group,the thymus coefficient in Cr Ap group had no statistical significance(P>0.05),the spleen coefficient of Cr Ap group was significantly increased(P<0.01);compared with ADM group,the thymus coefficient in Cr Ap group was significantly increased(P<0.01),the spleen coefficient of Cr Ap group and Cr Ap+ADM group were significantly increased(P<0.01).2.6 The pathological sections of immune organs:thymus pathology chart showed that compared with the blank group,the number of thymic lymphocytes in the model group was reduced and the boundary between cortex and medulla was fuzzy;compared with the model group,the thymus morphology in the Cr Ap group was relatively complete,with a large number of lymphocytes and a clear boundary between cortex and medulla,the thymus morphology in the Cr Ap+ADM group was improved to some extent.The spleen pathology showed that compared with the blank group,the boundary between white pulp and red pulp in the model group was unclear,and the contents of lymphocytes and macrophages in white pulp were less;Cr Ap group Compared with the model group,the boundary of red and white pulp was relatively clear,and the contents of lymphocytes and macrophages in white pulp were increased;the changes of Cr Ap+ADM group were more than in ADM group,but less than in Cr Ap groups.2.7 FCM Results:compared with the blank group,the expression of CD4~+T,CD19~+B and the ratio of CD4~+/CD8~+in the model group were significantly decreased(P<0.01),CD8~+T was decreased(P<0.01),while the expression of CD4~+CD25~+Tregs was significantly increased(P<0.01);However,compared with the model group,the expression of CD4~+T in each drug group was significantly increased(P<0.01),while the expression of CD4~+CD25~+Tregs was significantly decreased(P<0.01),the expression of CD8~+T in ADM group and Cr Ap+ADM group was significantly increased(P<0.01),and the ratio of CD4~+/CD8~+and the expression of CD19~+B in Cr Ap group and Cr Ap+ADM group were significantly increased(P<0.01);in addition,compared with ADM group,the expression of CD4~+T in Cr Ap+ADM group was significantly increased(P<0.01),while the ratio of CD4~+/CD8~+and the expression of CD19~+in Cr Ap group and Cr Ap+ADM group were significantly increased(P<0.01).2.8 ELISA results:in spleen tissue,the contents of IL-2,TNF-?and IFN-?in the model group were significantly lower than those in the blank group,while the content of IL-10 was significantly higher(P<0.01);however,compared with the model group,the contents of IL-2 and TNF-?were significantly increased in all drug groups(P<0.01),and the content of IL-10 was significantly decreased(P<0.01),meanwhile,the content of IFN-?in the Cr Ap group was significantly increased(P<0.01).At the same time,in cancer tissues,compared with the model group,the content of IL-2 in each drug group was significantly increased(P<0.01),and the content of TNF-?was significantly decreased(P<0.01),the content of IFN-?in the Cr Ap group and Cr Ap+ADM group was significantly increased(P<0.01),and the content of IL-10 was significantly decreased(P<0.01).In addition,compared with ADM group,the content of IFN-?in spleen tissue was significantly increased in Cr Ap group(P<0.01),the content of IL-2 in spleen tissue was increased in Cr Ap+ADM group(P<0.05),and the content of TNF-?in cancer tissue was significantly decreased in Cr Ap group(P<0.01),while the content of IFN-?in cancer tissue was significantly increased in Cr Ap group and Cr Ap+ADM group(P<0.01).Conclusion:In vitro,Cr AP can inhibit the proliferation of 4T1 breast cancer cells.In vivo,Cr AP can inhibit the growth of 4T1 breast cancer.The mechanism of Cr Ap against TNBC breast cancer may be realized by regulating tumor microenvironment and systemic immune function.In addition,Cr Ap can enhance ADM's anti-4T1 breast cancer and immunoregulation effects in Cr Ap and ADM combined medication. |
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