Ruyong Formula Improves Thymus Function In Breast Cancer Mice Stimulated By Chronic Unpredictable Mild Stress Via JAK2/STAT3/PI3K Pathway

Objective Breast cancer is the most common malignant tumor in women.Its occurrence and development are closely related to emotional factors,and the two influence each other to promote tumor growth and metastasis.In the late stage of breast cancer,the function of the thymus gland is affected,the immune function of the human body is reduced,and at the same time,it is accompanied by emotional disorders.Preliminary studies have shown that Ruyong Formula helps to repair the thymus function of the tumor-bearing body and plays the role of adjuvant therapy for breast cancer.In this experiment,a model of breast cancer mice stimulated by chronic unpredictable mild stress(CUMS)was constructed,and the Ruyong Formula was studied to explore the changes of thymus immune function in breast cancer organisms under emotional disorders such as depression/anxiety,and to clarify the mechanism of it.Methods Section One BALB/c mice orthotopically seeded with 4T1 breast cancer cells were stimulated with varying degrees of CUMS,the experiment divided into normal group,tumor-bearing group,CUMS-1,2,3 group(stimulated 1,2,3 times a day,respectively).The behavioral activities of mice were observed through open field test and forced swimming test;the content of 5-HT in mouse brain was detected by ELISA;the expressions of CK5 and CK8 of thymus epithelial cells(TECs)were detected by immunofluorescence;the proportion of mouse thymic T cell subsets including CD3,CD4,CD8 were analysis by flow cytometry;the function of thymic output was detected by TRECs method;Real-time qPCR was used to analysis mRNA levels of TSLP,STAT3,IL-1α,IL-7,Foxp3,TGFβ1 and TNFα;the protein levels of TSLP,STAT3,p-STAT3,JAK2,PI3K and p-PI3K were detected by western blotting.Section Two BALB/c mice bearing 4T1 breast cancer tumors were stimulated with CUMS 5 times a day,the experiment was divided into a low-dose Ruyong Formula group(RYF-L group,146.5 mg/kg),a middle-dose Ruyong Formula group(RYF-M group,293 mg/kg),a high-dose Ruyong Formula group(RYF-H group,586 mg/kg),cyclophosphamide group(CTX group,30 mg/kg),normal group and model group(0.9%saline 0.2 mL).The experimental methods and detection indicators used in this part are the same as those in the section one.Section Three The iTECs cells were treated with Astragaloside Ⅰ,Ⅱ,Ⅲ(0.001,0.01,0.1,1,10,100 μM);ursolic acid,quercetin,luteolin,ferulic acid(0.1,1,10,25,50,100μM)for 48h,the effect of monomers on the proliferation and function of iTECs was detected by MTT and crystal violet methods;TSLP,IL-7,IL-10,IL-12,IL-17,STAT3,Foxp3 and TNFαmRNA levels in iTECs were analyzed by qPCR;The 4T1 and MCF-7 cells treated with different concentrations of lactun prescription active ingredients monomeric ursolic acid,quercetin,luteolin,ferulic acid(0.1,1,10,50 μM),and the effect of monomer on cell proliferation was detected by MTT method;the mRNA expression of AKT1,JUN,NFKB1,PLCG1,GSK3B,IKBKG and TNFα in cells were analyzed by qPCR after administration.Results Section One Compared with the normal group,the structure of the thymus in the model group and CUMS groups was disordered,the levels of TGFβ1,IL-5,6,10,etc.in the thymus tissue of the model group were significantly increased,and the proportions of CD3+CD4-CD8-and CD3+CD4+CD8+T cells in the thymus were significantly increased,the positive selection process of thymic T cells was blocked,and p-STAT3 was abnormally elevated.Compared with the model group,the level of 5-HT in the brain of CUMS-3 group was significantly decreased,the lung metastasis was increased,the output function of the thymus was inhibited,the number of epithelial cells in the medulla was significantly decreased,the number of TECs in the medulla decreased,and IL-5,IL-7 and other cytokine mRNA levels were significantly decreased,TSLP mRNA levels were significantly increased,the expressions of p-JAK2 and p-PI3K p55 in thymus tissue were statistically decreased.Section Two Compared with the model group,the forced swimming immobility time of the mice in the RYF-M group was shortened,and the content of 5-HT in the brain was significantly increased;the output function of the thymus was improved,and the number of TECs in the medulla increased;the proportion of CD3+CD4-CD8-and CD3+CD4+CD8+T cells in the thymus increased,and the positive selection process of thymic T cells was restored;the expression level of TSLP mRNA in the thymus of mice was significantly decreased,and the expression level of IL-7 mRNA was significantly increased after administration of Ruyong Formula;the expression level of TSLP protein was significantly increased,and the expression of p-STAT3,p-JAK2,p-PI3K p55 protein was significantly increased.Section Three Compared with the control group,0.1 and 0.01 μM concentrations of astragalus saponins Ⅰ,Ⅱ,and Ⅲ promoted the.proliferation of iTECs;quercetin at concentrations above 1 μM inhibited the proliferation of iTECs,and luteolin at concentrations below 25 μM,ferulic acid and ursolic acid at various concentrations all inhibited the proliferation of iTECs,50 μM and 100μM luteolin promoted the proliferation of cells;except 10 μM quercetin significantly increased the expression level of IL-7,10 μM quercetin,various concentrations Luteolin,ferulic acid and ursolic acid all decreased the mRNA expression levels of TSLP,IL-7,10,12,17,STAT3,Foxp3 and TNFα.Ursolic acid,quercetin,luteolin and ferulic acid at concentrations below 10 μM can promote the proliferation of 4T1 and MCF-7 cells grow.Compared with the control group,the expressions of JUN and IKBKG in 4T1 and MCF-7 cells were decreased after ferulic acid administration,while the expression of NFKB1 was decreased in 4T1 cells and increased in MCF-7 cells;The expression of AKT1 and EGFR in 4T1 cells decreased after treated with quercetin,while the expression of AKT1 in MCF-7 cells increased,and the expression of EGFR decreased;after treated with luteolin,the expression of TNF mRNA in 4T1 cells was increased,and the expression of GSK3B was decreased,while the expressions of GSK3B and TNFα were decreased in MCF-7 cells;PLCG1 expression was decreased in 4T1 cells after ursolic acid administration,but increased in MCF-7 cells.Conclusion The thymus function of breast cancer mice is disordered,and CUMS may further inhibit the thymus function of breast cancer mice and promote lung metastasis by inhibiting the JAK2-PI3K pathway.Ruyong Formula can regulate the proteins expression of JAK2/STAT3/PI3K pathway by reducing the expression level of TSLP in model mice,increasing the level of IL-7,and enhancing the function of iTECs,inhibiting the proliferation of 4T1 and MCF-7 cells,and regulating the expression level of intracellular immune-related factors,to a certain extent,maintains the normal structure of the thymus,restores the function of the thymus,and inhibits the lung metastasis of breast cancer.