Wogonin Inhibits M2 Macrophage-mediated Of Breast Cancer Lung Metastasis After Myocardial Infarction

Background:Breast cancer is the most prevalent cancer in women in the world.More than 90%of the mortality of breast cancer patients is related to metastasis.Among breast cancers,triple-negative breast cancer has the highest mortality rate and the most difficult to cure.It is currently targeted at triple-negative breast cancer.There is no better treatment other than chemotherapy.Patients with triple-negative breast cancer are more likely to develop lung metastasis.A large number of clinical and experimental data show that M2 type macrophages play a significant role in the metastasis of breast cancer and other tumors.Therefore,inhibit M2 type macrophages to become breast cancer And other important targets for tumor metastasis therapy.Recent studies have shown that mice of breast cancer after myocardial infarction can lead to the conversion of monocytes in the body to immunosuppressive M2 type macrophages,which makes patients with myocardial infarction more susceptible to breast cancer and breast cancer lung metastasis.Wogonin is derived from the traditional Chinese medicine Scutellaria baicalensis.It has anti-inflammatory,anti-tumor and anti-metastasis effects.This study found that wogonin can inhibit the polarization of macrophages to M2 type.On the basis of the results,we further explored whether wogonin can inhibit the lung metastasis of breast cancer after myocardial infarction by regulating the polarization of macrophages and their functions.AIM:To explore whether Wogonin can inhibit the lung metastasis of breast cancer after myocardial infarction by regulating the polarization of macrophages andtheir functionsMethod:RAW264.7,M0 macrophages obtained from THP-1 stimulated by PMA for 24 h and bone marrow-derived macrophages(BMDM)were induced to M2 macrophages as the research objects,and in vitro M2 macrophages(IL-4/IL-13)model.Add different concentrations of Wogonin for intervention.RT-PCR method was used to detect the influence of the expression of key functional genes Arg-1,MR,IGF-1 and TGF-β1 in M2 type macrophages.Western-blot method was used to detect the effect of Wogonin on the expression of macrophage polarization-related protein p-STAT6 in M0 macrophages and bone marrow-derived macrophages(BMDM)obtained from RAW264.7 and THP-1 stimulated by PMA for 24 h.Select female BALB/c mice(6 weeks old,weight 20 g-22 g,SPF grade),orthotopic transplantation of breast cancer cells 4T1,3 days after successful inoculation,according to the method established by the previous research group,the left anterior coronary artery was ligated The LAD is used to make myocardial ischemia model,and the changes in EF and FS values are used as evaluation indicators.The experiment was divided into 3 groups: the control group,the MI model group(0.5% CMC-Na),and the hanflavin group(50 mg/kg).The intragastric administration was continued for 14 days.The changes of lung breast cancer metastasis in mice were observed with a microscope;the changes in heart function of each group were observed with echocardiography after 7 days of administration;RT-PCR method was used to detect the inflammation-related genes INOS,IL-6,and IL-6 in heart tissue after 14 days of administration.The effect of IL-1β expression changes;immunohistochemical method was usedto observe the effect of M2 macrophage marker CD206 in tumor tissue 14 days after administration;immunofluorescence staining to detect the effect of Wogonin on M2 in mouse tumor tissue 14 days after surgery The influence of thenumber of type macrophage marker CD206;RT-PCR method was used to detect the effect of the expression changes of M2 type macrophage related gene indicators Arg-1,MR,IGF-1,TGF-β1 in tumor tissue 14 days after administration.Taking breast cancer cell MCF-7 and breast cancer cell MDA-MB-231 as the research objects,an in vitro breast cancer cell migration model was constructed,and the supernatant of M2 macrophage treated with different concentrations of wogonin and wogonin was added to intervene.The Incu Cyte method was used to detect the effect of wogonin on the migration of breast cancer cells mediated by M2 macrophages.ELISA method was used to detect the effect ofwogonin on CXCL1 and TNF-α secreted by M2 macrophagesResults:After M0 macrophages induced by RAW264.7 and THP-1 were stimulated by PMA for 24 h and bone marrow-derived mononuclear macrophages were treated with Wogonin at the same concentration(10 μ M,20 μ M and 40 μ M),it was found that Wogonin could reduce the m RNA expression of key functional genes Arg-1,MR,IGF-1 and TGF-β1 in M2 macrophages of the three cell lines.In addition,Wogonin could inhibit the expression of p-STAT6 in M2 macrophages of three cell lines.The lung metastasis map showed that Wogonin could significantly reduce the lung metastasis of breast cancer enhanced by MI,and the echocardiographic results showed that the left ventricular short axis shortening rate LEVES(μL),LEVDS(μL),ejection fraction EF(%),FS(%)and ventricular structure of the model group changed significantly with the prolongation of ischemia time.After drug treatment,left ventricular short axis shortening LEVES(μL),LEVDS(μL),ejection fraction EF(%),FS(%)and ventricular structure were significantly improved,RT-PCR results showed that the expression of inflammation-related genes INOS,IL-6 and IL-1 β increased significantly in model group,while the expression of inflammation-related genes INOS,IL-6 and IL-1 β decreased significantly in Wogonin group.The model group increased the number of M2 macrophage marker CD206 in tumor tissue in immunohistochemistry and immunofluorescence staining,while the Wogonin group decreased the number of M2 macrophage marker CD206 in tumor tissue in immunohistochemistry and immunofluorescence staining.The results of RT-PCR showed that the expressions of key functional genes Arg-1,MR,IGF-1 and IL-10 of M2 macrophages in model group were significantly increased,while those of Arg-1,MR,IGF-1 and IL-10 in Wogonin group were significantly decreased.The migration of breast cancer cells in vitro showed that Wogonin could inhibit the migration of breast cancer cells mediated by M2 macrophages.ELISA results showed that Wogonin could reduce the secretion of CXCL1 and TNF-α factors by M2 macrophages.fter treated with different concentrations of Wogonin(10 μM,20 μM,40μM)of RAW264.7,M0 macrophages induced by PMA for 24 h and bonemarrow-derived mononuclear macrophages for 24 h,it was found that Wogonin could reduce the m RNA expression of Arg-1,MR,IGF-1,TGF-β1 of M2 macrophages in three cell lines.In addition,Wogonin could inhibit the expression of p-STAT6 in M2 macrophages of three cell lines.Conclusion:Wogonin inhibited the macrophages polarization and functions to inhibit breast cancer lung metastasis after myocardial infarction.This study provides an important basis for the application of wogonin in the treatment of myocardial infarction and breast cancer.