Reinforce The Healthy Qi And Dispel Toxin Formula Prevent Breast Cancer From Lung Metastasis In Vivo

Reinforce the Healthy Qi and Dispel Toxin Formula Prevent Breast Cancer from Lung Metastasis in Vivo.Background:Breast cancer is a leading cause of death in women, due primarily to ineffective treatment of metastasis. Taken together with the heterogeneous nature of solid tumors, this metastatic inefficiency suggests that only a subset of cells(cancer stem cells,CSC) can successfully navigate the metastatic cascade and eventually re-initiate tumor growth to form metastases. TCM can prevent lung metastasis to some extent. But the mechanism has not been clear. We guess that TCM can prevent lung metastasis to some extent by affecting CSC and internal environmentAim:To discuss the possible mechanism of lung metastasis caused by breast cancer through breast cancer stem cells, and to find the effect and mechanism of reinforce the healthy qi and dispel toxin formula (RHDT) that prevent lung metastasis of breast cancer to a certain extent.Methods:14T1-CSC caused lung metastasis by escaping from NK. cells1.1 separate and enrich 4T1-CSC and verify 4T1-CSC1.2 Compare the lung metastasis caused by 4T1-CSC with the lung metastasis caused by4T14T1 and 4T1-CSC were separately resuspended in PBS at 1×106 cells/ml. Using established models of experimental metastasis (tail vein injection) 100ul (1 ×105 cells) was injected into the lateral tail vein. On the 21th day, mice were killed,and compare the lung metastasis caused by the above two cells.TGF-βlin blood was tested by using ELISA, and NKG2D in blood was tested by using flow cytometry analysis.2 The effect of RHDT that prevent lung metastasis of breast cancer in vivo2.1 prepare RHDTHuangqi10g, Tiandong10g, KushenlOg, Jianghuang10g, Zhebeimu10g.2.2 Find the medication time and dose of RHDT:Find the effect of RHDT by using the models of experimentalmetastasis (tail vein injection with 4T1-luc) 50 Female BALB/c,40 Female BALB/c were used to establishUsing established models of experimental metastasis (tail vein injection), and divided into four groups (low-dose TCM group, medial-dose TCM group, high-dose TCM groupand the model group) randomly.Ten mice were taken as control group. After 24 hours from injection,medicine was given.In vivo bioluminescence imaging of tumor hypoxia dynamics was used to follow tumor progression on the 5th day、the 9th day and the 13th day.On the 21th day, mice were killed and assessed for metastatic burden. Compare the differences between the groups.2.3 Find the effect and mechanism of RHDT by using the models of experimental metastasis (tail vein injection with 4T1-CSC)40 Female BALB/c,30 Female BALB/c were divided into 3 groups (20x TCM group、 30x TCM group and positive control group) randomly.Ten mice were taken as negative control group.3 days before injection, medicine was given.4T1-CSC were resuspended in PBS at 1×106 cells/ml. Using established models of experimental metastasis (tail vein injection) 100u l(1 ×105cells) was injected into the lateral tail vein. On the 21th day, mice were killed and assessed for metastatic burden. Compare the difference between the groups. TGF-β1in blood was tested by using ELISA, and NKG2D in blood was tested by using flow cytometry analysis. Compare the MMP-9, VEGF及u-PA in lungs of different groups bylmmunohistochmeistry.Results:14T1-CSC caused lung metastasis by escaping from NK cells1.1 separate and enrich 4T1-CSC and verify 4T1-CSC4T1-CSCwas separated and enriched by using suspension culture, and 4T1-CSC was tested in vivo.1.2 Compare the lung metastasis caused by 4T1-CSC with the lung metastasis caused by4T1the lung metastasis caused by 4T1-CSC was more serious than the lung metastasis caused by 4T1, the differencebetween the two groups has statistical significance, P=0.009<0.05. CD314 of 4T1-CSC group was lower than CD314 of 4T1 group, the difference between the two groups has statistical significance. P=0.05. TGF-β1 of 4T1-CSC group was lower than TGF-β1 of 4T1 group,the difference between the two groups has statistical significance, P=0.016<0.05.2 The effect of RHDT that prevent lung metastasis of breast cancer in vivo2.1 prepared RHDT2.2 Find the medication time and dose of RHDT. Find the effect of RHDT by using the models of experimentalmetastasis (tail vein injection with 4T1-luc).The extent of lung metastasis had no statistical differences between thegroups, P=0.223>0.05.But The extent of lung metastasis of 20x TCM group was with a downward tendency.2.3 Find the effect and mechanism of RHDT by using the models of experimental metastasis (tail vein injection with 4T1-CSC)the lung metastasis of TCM group was fewer than the lung metastasis of model group, the differencebetween the two groups has statistical significance, P=0.001<0.05.In the respect of lung metastasis, there was no statistical differences between 20x TCM group and 30x TCM group,=0.737>0.05. In the respect of CD314, there was no statistical differences between 20x TCM group and model group, P=0.330>0.05.In the respect of TGF-β1, there was no statistical differences between 20x TCM group and model group, P=0.997>0.05.MMP-9、VEGF及u-PA in lungs of TCM group were fewer than the models group.Conclusions:14T1-CSC caused effectively lung metastasis by escaping from NK. cells2 Reinforce the healthy qi and dispel toxin formula (RHDT) can prevent metastasis of breast cancer to a certain extent.Maybe the mechanism of RHDT has no relationship with NK cells,the mechanism maybe that RHDT caused MMP-9、VEGF及u-PA in lungs fewer.