Jiedu Formula (Jin-Mao Jiedu Granule) Inhibits Metastasis Through Regulating The Crosstalk Between Cancer Cells And Macrophages In Hepatocellular Carcinoma Under Hypoxia Condition

Objectives:1.To explore the relationship between liver cancer cells and macrophages under hypoxia condition.2.To clarify whether JDF could inhibit macrophages recruitment and M₂ polarization induced by liver cancer cells.On the basis,relative mechanism was studied.Methods:1.MTT method was performed to explore the effect of JDF on the proliferation of liver cancer cells and macrophages;2.Protein expression level was detected by WB,q RT-PCR method was used to detect the m RNA expression level;3.Cell migration ability was validated by transwell chamber migration assay;4.The gene expression was downloaded in TCGA database,Pearson correlation analysis was performed to explore the relationship between different genes in HCC patients;5.The effect of JDF on the growth of tumor was observed by subcutaneous transplanted tumor model;6.The growth of tumor was observed by in vivo imaging system;7.The protein expression in tumor was detected by immunohistochemistry.Results:1.JDF could significantly reduce the viability of hepatoma cells in a dose and time dependent manner;2.JDF could inhibit the expression of EMT related proteins which were induced by hypoxia;3.High dose JDF（2mg/m L）has no obvious toxicity to macrophages;4.HIF-1αknockdown and JDF could significantly inhibit the recruitment of macrophages induced by hypoxia stimulated hepatoma cells;5.JDF could significantly reduce expression level of hypoxia-induced HIF-1αand VEGF-A in hepatoma cells;6.Macrophages which stimulated by conditioned medium of hepatoma cells under hypoxia,the M₂ macrophage related gene expression is up regulated;7 In turn,Macrophages which stimulated by conditioned medium of hepatoma cells under hypoxia can promote the migration of hepatoma cells;8.Macrophages which stimulated by conditioned medium of hepatoma cells under hypoxia can up-regulate the protein level of N-cadherin and Vimentin in hepatoma cells;9.The m RNA expression of HIF-1α,TGFβ1,CD68,MRC-1 and CD163 had positively correlated each other in HCC;10.TGFβ1 could induce macrophages to M₂ polarization;11.HIF-1αknockdown and JDF could significantly inhibit the hypoxia-upregulated TGFβ1 expression in hepatoma cells;12.JDF could significantly inhibit the growth of tumor and inhibit the expression of HIF-1α,VEGF-A,F4/80,CD163,β-catenin,Vimentin in vivo.Conclusions:1.JDF could directly inhibit the EMT of hepatoma cells induced by hypoxia;2.JDF could inhibit macrophages recruitment by regulating HIF1α/VEGF-A pathway under hypoxia;3.JDF could inhibit M₂ polarization of macrophages through HIF1α/TGFβ1 pathway,and thereby suppress the migration and EMT process of hepatoma cells.