Anemoside A3 Inhibits Macrophage M2-Like Polarization To Against Triple-negetive Braest Cancer Metastasis

Objective:To explore the effect of Anemoside A3 on the metastasis of TNBC by inhibiting M2 type polarization of macrophages.Constructed the M2 type polarization of macrophages in vitro to investigate the effect and mechanism of Anemoside A3 on the M2 type polarization of macrophages;and the ability to inhibit the migration of TNBC cells.Constructed the 4T1-Luc intravenous model to investigate the effect of Anemoside A3 on the metastasis of TNBC,lays the foundation for subsequent experimental research.Part I Inhibitory Effect and Mchanism of Anemoside A3 on M2-type polarization of macrophagesMethod:Useing BMDM to Constructe the M2 type polarization of macrophages in vitro to evaluate the effect of Anemoside A3 on macrophage M2-type polarization.CCK8 was used to detect the effect of Anemoside A3 on the survival rate of BMDM cells;Flow cytometry was used to detect the effects of Anemoside A3 on the percentage of CD206 and F4/80;RT-PCR was used to detect the effect of Anemoside A3 on the m RNA expression of CD206,Fizz1,Ym1,and Arg-1;W-B was used to detect the expression of key proteins in JAK2/STAT3 signaling pathway.The molecular docking method was used to predicts the target of Anemoside A3 in inhibiting the M2 type polarization of macrophages;BMDM cells were transfected with STAT3-si RNA to knock down the STAT3 gene,and after the intervention of Anemoside A3,RT-PCR was used to detect the m RNA expression of CD206,Fizz1,Ym1,and Arg-1.Results:CCK8 results showed that Anemoside A3 had no notable effect on the survival rate of BMDM cells within the concentration range of 0-100μg/ml;Flow cytometry results showed that the percentage of CD206 positive cells was markedly increased by IL-4 from 2.92%±0.09% to 46.4%±1.72%(P <0.01).when treated with IL-4 and Anemoside A3,the percentage of CD206 positive cells was markedly decreased to28.2% ± 0.92 and 17.6% ± 0.34(P <0.01);RT-PCR results also showed that the m RNA expression of CD206,Fizz1,Ym1,and Arg-1 markedly increased by IL-4,when treated with IL-4 and Anemoside A3,the m RNA expression of CD206,Fizz1,Ym1,and Arg-1 were markedly reduced(P <0.01);W-B results showed that the expression level of STAT3 and JAK2 phosphorylation protein in BMDM cells was markedly increased by IL-4(P<0.01).when treated with IL-4 and Anemoside A3,the expression level of STAT3 phosphorylation protein in BMDM cells was markedly reduced(P<0.05).However,when treated with IL-4 and Anemoside A3,the expression level of JAK2 phosphorylation protein in BMDM cells has no significant changes.Molecular docking results showed that Anemoside A3 has better docking activity with STAT3 protein.STAT3 may be the target of Anemoside A3 to inhibit M2 type polarization of macrophages;After transfected STAT3-si RNA into BMDM cells,compared with IL-4-STAT3-si RNA group,the m RNA expression of CD206,Fizz1,Ym1,and Arg-1 was significantly down-regulated in IL-4+Anemoside A3-STAT3-si RNA group(P<0.01).PartⅡ Anti-metastasis Effect of Anemoside A3 on TNBC by inhibiting M2 type polarization of macrophagesMethods:Conducting vivo and vitro experiments to investigate the effect of Anemoside A3 on the metastasis of TNBC by inhibiting the M2-type polarization of macrophages.In vitro experiments,Collecting the conditioned medium of IL-4 and Anemoside A3 alone or together to incubate BMDM cells,then apply it to 4T1 cells;Transwell assay was performed to detect the migration ability of 4T1 cells cultivated with conditional medium of BMDM treated with IL-4 and/or Anemoside A3;Cell migration assay was performed to detect the migration ability of 4T1 cells cultivated with conditional medium of BMDM treated with IL-4 and/or Anemoside A3;Constructing the 4T1-Luc mouse lung metastasis model in vivo to investigate the effect of Anemoside A3 on the metastasis of TNBC;In vivo bioluminescent analysis was used to measure tumors fluorescence intensity changes;HE was used to observe the lung tumor metastasis in4T1-Luc mice.Immunohistochemical staining was used to determine the expression of CD206 in lung tissue.Results:Transwell assay results showed that IL-4 induced conditioned medium can markedly increase the number of 4T1 cells migrating to the bottom of the cell,from109±15 to 231±32 per field of view(P <0.01);when treated with IL-4 and high dose of Anemoside A3,the the number of 4T1 cells migrating to the bottom of the cell was markedly reduced to 187±34 per field of view(P <0.05).Cell migration assay results showed that IL-4 induced conditioned medium can markedly increase the wound healing ability of 4T1 cells(P <0.01);when treated with high dose of Anemoside A3,the wound healing ability of 4T1 cells was markedly reduced(P<0.05).Simultaneously,the direct effect of Anemoside A3 has no obvious effect on the migration of 4T1 cells.Its showed that the after the intervention with Anemoside A3,compared with model,the body weight of 4T1-Luc mice in the high-dose Anemoside A3 group was significantly increased(,P<0.01),and the fluorescence intensity in the 4T1-Luc mice in the high-dose Anemoside A3 group was significantly reduced(P<0.05,P<0.01);and HE results showed that the middle and high dose groups of Anemoside A3 can markedly reduce the number of lung metastases in 4T1-Luc mice;Immunohistochemical results showed that after the intervention of Anemoside A3,the proportion of CD206 in the lung tissue was significantly down-regulated(P<0.05).Conclusion:Anemoside A3 can inhibit the metastasis of TNBC by inhibiting the M2 polarization of macrophages in vivo and in vitro.The mechanism may is that Anemoside A3 targets the STAT3 protein and interferes with the STAT3 pathway to inhibit the M2 type of macrophages.Therefore,it plays an anti-metastasis effect on TNBC.