[Ruyiping Inhibited Pulmonary Metastasis of Breast Cancer by Regulating the Formation of Pre-metastasis Microenvironment].

Objective To observe the effect of Ruyiping (RYP, a recipe for fighting against re- currence and metastasis of breast cancer) on pre-metastatic microenvironment, and to study its possi- ble mechanism. Methods The experiment was divided into two parts. The 1st part lies in setting the pre- cancerous transfer, and the 2nd part lies in the effect of RYP on pre-metastatic microenvironment. There were 24 BALB/c mice in the 1st part. Logarithmic phase 4T1 cells were dispensed into cell suspension. Blood cells were counted by blood cell counter. Then they were injected into the 4th mammary fat pad of the 24 BALB/c mice under aseptic condition (1 x 10⁶ cells/mL, 0.1 mL for each mouse). There were 60 BALB/c mice in the 2nd part. They were divided into the blank group, the model group, low, middle, high dose RYP groups by random digit table, 12 in each group. The modeling method was the same as men- tioned above. Medication was started from the 2nd day of inoculation. Mice in low, middle, high dose RYP groups were administered with 5. 13, 10. 26, 20. 52 g/kg RYP crude drugs per day by gastrogavage, once per day for 14 successive days. Equal volume of normal saline was administered by gastrogavage to mice in the blank group and the model group. Six mice were sacrificed at day 10, 14, 18, and 22, respectively in the 1 st part of the experiment. The pulmonary metastasis was observed. The histology and mi- cromorphology of lung tissues were observed under light microscope and electron microscope/transmission electron microscopy (TEM) in the 2nd part of the experiment. The relative pulmonary vascular per- meability was determined by Evans blue. The effect of RYP on the formation of pre-metastatic microenvironment was observed. The levels of angiogenin2 (Angpt2), vascular endothelial growth factor (VEGF) , IL6 and IL1 β were detected by Western blot and Real time PCR. Results The period from day 0 to day 14 was considered to be the pre-metastatic phase. Compared with the model group, significant inhibition on the tumor weight and tumor volume were shown in middle and high dose RYP groups (P <0. 05,P <0. 01). RYP dose-dependently inhibited the tumor weight and tumor volume (P <0. 05,P <0. 01). Infiltration of lymphocytes occurred in the model group and the low dose RYP group. But there was no statistical difference in the morphology of lung tissue in light microscopic results between middle/high dose RYP groups and the blank group. The pulmonary blood vessel net was consisted of continuously densely capillaries. The structure of pulmonary capillaries was normal in the blank group. The blood vessel walls were not regular and even in the model group, with obviously distended capillaries. After treated by RYP, the injury was improved, with normal basic morphology of blood vessels. Compared with the blank group, the exudate in Evans blue was obviously increased, protein and mRNA expressions of Angpt2, VEGF, IL6, and IL1β were increased in the model group (P <0. 05,P <0. 01). Compared with the model group, the exu- date in Evans blue was obviously decreased in each YRP group. The reduction of the exudate was dose- dependently with the dose of YRP (P <0. 01). Protein and mRNA expressions of VEGF in the middle dose RYP group, protein and mRNA expressions of Angpt2, VEGF, IL6, and ILI1β were decreased in middle and high dose RYP groups (P <0. 05,P <0. 01). Protein expressions of IL6 were decreased in the middle dose RYP group (P <0. 01). Conclusions RYP had favorable regulation in the tumor growth and the formation of pre-metastatic microenvironment. It could protect the integrity of vascular system, inhibit the formation of pre-metastatic microenvironment possibly through inhibiting the expressions of Angpt2, VEGF, IL6, and IL11β, and finally inhibiting the occurrence of pulmonary metastasis of breast cancer.