Study On The Mechanism Of Huangqi Jiedu Decoction Intervening In Notch/JAG1 Signaling Pathway To Inhibit EMT And Prevent Lung Metastasis Of Breast Cance

Objective:The effect of Huangqi Jiedu Decoction on lung metastasis of breast cancer and Notch/JAG1 signaling pathway and key markers of EMT were observed in nude mice with lung metastasis of breast cancer.Methods:(1)Establishment of lung metastasis model of breast cancer in nude mice:4T1 cell suspension was injected into the skin of the right axillary breast of nude mice(0.2ml/mouse,cell concentration was 1×10~6/m L).The growth of orthotopic tumor was recorded to observe the proliferation of tumor cells,and the effectiveness of the model was evaluated by observing the himorphology of pathological sections.(2)Study on the inhibition of Huangqi Jiedu decoction on tumor formation and lung metastasis in nude mice:36 nude mice were randomly divided into 6 groups;Control group,model group,adriamycin group,low dose Huangqi Jiedu decoction group,middle dose Huangqi Jiedu decoction group,high dose Huangqi Jiedu decoction group.The blank group was not treated,and the tumor-bearing nude mouse models were constructed by injection in the other five groups.After successful modeling,the low-,medium-,and high-dose Huangqi Jiedu Decoction groups(17.82,35.64,and 71.28g/kg)were treated with traditional Chinese medicine by gavage,the adriamycin group was treated with adriamycin by intraperitoneal injection(0.05g/kg),and the model group was treated with the same dose of normal saline by gavage for 21 days.The length and short diameter of tumors were measured every 3 days,and the tumor volume was calculated.After the end of administration,the tumor tissue was separated,the tumor weight was measured,and the tumor inhibition rate was calculated.The lung tissues were separated,the number of pulmonary metastatic nodules was counted,and the lung metastasis inhibition rate was calculated.(3)The relationship between Huangqi Jiedu decoction inhibiting lung metastasis of breast cancer and Notch/JAG1 signaling pathway and EMT:Western Blot was used to detect JAG1 protein,EMT markers E-Cadherin and Vimentin protein expression in lung tissue.The expression of JAG1m RNA and EMT markers E-Cadherin and Vimentin m RNA in lung tissues were detected by Real-time fluorescent quantitative polymerase chain reaction(Real-time PCR).Results:(1)The tumor-bearing nude mice model was successfully established,and the tumor formation rate was 100%.Three days after the inoculation of nude mice,a small nodule was located under the right second nipple,with hard texture,clear boundary,regular shape,poor mobility,complete capsule,and partial adhesion.At the time of sampling,the incision of the orthotopic tumor was fish flesh.HE staining showed that the tumor cells in the tumor tissue of the model group were dense,and there was no obvious tumor necrosis.Tumor with hemorrhage,necrosis and hemosiderin deposition could be found in the lung tissue of the model group.(2)Compared with the model group,the low-,medium-and high-dose Huangqi Jiedu decoction groups could significantly reduce the volume of breast cancer(p<0.01),and the tumor weight was significantly reduced(p<0.01).The tumor inhibition rates of adriamycin group and Huangqi Jiedu decoction low-,medium-and high-dose groups were 47%,28%,40%and 50%,respectively.Compared with the model group,the number of lung metastatic nodules in the high-dose Huangqi Jiedu decoction group was significantly reduced(p<0.01),and the lung metastasis inhibition rates in the adriamycin group,low-dose Huangqi Jiedu decoction group,medium-dose Huangqi Jiedu decoction group and high-dose Huangqi Jiedu decoction group were 66%,33%,38%and 66%,respectively.(3)Compared with the model group,the adriamycin group,low dose Huangqi Jiedu decoction group,and medium dose Huangqi Jiedu decoction group had a significant reduction in JAG1m RNA(p<0.05),and the high-dose Huangqi Jiedu decoction group had a significant reduction in JAG1 mrna(p<0.01).E-cadherin mrna was significantly decreased in the high dose group of Huangqi Jiedu decoction(p<0.01).The expression of Vimentinm RNA in adriamycin group,Huangqi Jiedu decoction low dose group,Huangqi Jiedu decoction medium dose group and Huangqi Jiedu decoction high dose group were significantly decreased(p<0.01).(4)Compared with the model group,the expression of JAG1 and Vimentin protein in the adriamycin group and the low,medium and high dose Huangqi Jiedu decoction groups decreased significantly(p<0.01),and the expression of E-Cadherin protein increased significantly(p<0.01).Compared with the adriamycin group,the expression of JAG1 protein in the middle and high doses of traditional Chinese medicine was statistically significant(p<0.01).The expression of E-Cadherin and Vimentin protein in the three treatment groups of traditional Chinese medicine was statistically significant(p<0.01).The expression of JAG1,E-Cadherin and Vimentin protein in the three treatment groups were statistically significant(p<0.01).The high dose Huangqi Jiedu decoction group could significantly reduce the expression of JAG1 and Vimentin protein and increase the expression of E-Cadherin protein compared with the low dose group and the medium dose group.Conclusion:Huangqi Jiedu decoction can effectively inhibit the growth and lung metastasis of breast cancer xenografts in nude mice,which may be related to down-regulating the expression of key molecules in Notch/JAG1 signaling pathway and inhibiting the EMT process,so as to inhibit the lung metastasis of breast cancer.