Base On Tumor Associated Macrophages Polarization,Explore The Effect Of Dihydromyricetin On The Proliferation And Invasiveness In Osteosarcoma Cell

OBJECTIVETo investigate the effect of pretreatment of tumor associated macrophages?TAMs?with dihydromyricetin?DMY?on the proliferation and invasiveness of osteosarcoma cell induced by TAMs,to explore the possible mechanism from macrophages polarization and to provide new ideas and strategies for inhibiting the metastasis of osteosarcoma.METHODSOsteosarcoma cell line HOS cells were cultured in DMEM culture medium containing 10%fetal bovine serum in 24 well plates.Mouse macrophage RAW 264.7cells were treated with 20 ng/mL interleukin-4?IL-4?for 24 h to induce M2 type polarization and the culture medium was replaced by fresh culture medium.RAW264.7 cells were treated with 60 mg/L DMY for 24 h and the culture medium was replaced by fresh culture medium.The cell suspension was preparated and the cells were inoculated in the upper chamber of Transwell with membrane pore size of 0.4?m.After RAW 264.7 cells were attached,Transwell was placed in 24 well plates which HOS cells were pre inoculated and RAW 264.7 cells and HOS cells were co-cultured in no contact manner for 48 h.The methyl thiazolyl tetrazolium?MTT?colorimetric assay was used to detect cell viability and cell proliferation inhibition rate was calculated.Flow cytometry was used to detect the expression of M2 specific surface protein CD206.The levels of tumor necrosis factor-??TNF-??,interferon gamma?IFN-??,transforming growth factor beta?TGF-??and interleukin-10?IL-10?in supernatant of culture medium were measured by enzyme linked immunosorbent assay?ELISA?.Scratch test and Transwell test were used to detect the invasiveness of HOS cells.The expressions of M2 type macrophage molecular marker arginine enzyme-1?Arg-1?and invasion associated protein matrix metalloproteinases-2?MMP-2?,matrix metalloproteinases-9?MMP-9?,culsert of differentiation 44 variant type 6?CD44v6?and nm23-h1 were measure by Western Blot.RESULTS?1?Effect of DMY on the cell proliferation in RAW264.7 cellsThe growth of RAW264.7 cells was not significantly inhibited by less than 60mg/L DMY,but the growth of RAW264.7 cells was obviously inhibited in concentration-dependent manner by DMY?60²00 mg/L?.IC₅₀ that DMY inhibited the proliferation of RAW264.7 cells was 171.25 mg/L.The concentration of DMY was selected as 60 mg/L for subsequent experiments.?2?Identification of M2 type polarization of macrophages in RAW264.7RAW264.7 cells were treated with 20 ng/mL IL-4 to induce M2 type polarization and IL-4 did not affect the cell morphology.Compared with the control group,the protein expression of Arg1,the number of CD206 positive cells and the levels of TGF-?and IL-10 in supernatant of cell culture medium were significantly increased in IL-4 group?all P<0.05?.?3?Effect of M2 type macrophages pretreated with DMY on the proliferation of HOS cells induced by M2 type macrophagesThere was no significant difference about the proliferation of HOS cell between HOS group and HOS+RAW group?P>0.05?.Compared with HOS group or HOS+RAW group,the proliferation of HOS cell in HOS+M2 type RAW group was significantly increased?P<0.05?.Compared with HOS+M2 type RAW group,the proliferation of HOS cell in HOS+M2 type RAW pretreated with DMY group was significantly decreased?P<0.05?.?4?Effect of M2 type macrophages pretreated with DMY on the proliferation of HOS cells induced by M2 type macrophagesThere was no significant difference about motion migration velocity and size of scratch of HOS cells between HOS group and HOS+RAW group?P>0.05?.Compared with HOS group or HOS+RAW group,the motion migration velocity of HOS cells was obviously increased and size of scratch of HOS cells was significantly decreased in HOS+M2 type RAW group?P<0.05?.Compared with HOS+M2 type RAW group,the motion migration velocity of HOS cells was significantly decreased and size of scratch of HOS cells was significantly increased in HOS+M2 type RAW pretreated with DMY group?P<0.05?.There was no significant difference about the number of HOS cells of damage matrix and invasiveness between HOS group and HOS+RAW group?P>0.05?.Compared with HOS group or HOS+RAW group,the number of HOS cells of damage matrix and invasiveness was significantly increased in HOS+M2 type RAW group?P<0.05?.Compared with HOS+M2 type RAW group,the number of HOS cells of damage matrix and invasiveness was significantly decreased in HOS+M2 type RAW pretreated with DMY group?P<0.05?.?5?Effect of M2 type macrophages pretreated with DMY on the expression of invasion associated protein in HOS cells induced by M2 type macrophagesThere was no significant difference about the expressions of MMP2,MMP9,CD44v6 and nm23-h1 between HOS group and HOS+RAW group?P>0.05?.Compared with HOS group or HOS+RAW group,the expressions of MMP2,MMP9and CD44v6 was significantly increased and the expression of nm23-h1 was significantly decreased in HOS+M2 type RAW group?all P<0.05?.Compared with HOS+M2 type RAW group,the expressions of MMP2,MMP9 and CD44v6 was significantly decreased and the expression of nm23-h1 was significantly increased in HOS+M2 type RAW pretreated with DMY group?P<0.05?.?6?DMY inhibited macrophage M2 type polarization60 mg/L DMY did not affect the morphology of RAW 264.7 cells.Compared with the M2 type RAW group,DMY decreased significantly the protein expression of Arg1,the number of CD206 positive cells and the levels of TGF-?and IL-10 in supernatant of cell culture medium?all P<0.05?.CONCLUSIONSDMY inhibits the proliferation and invasion of osteosarcoma cells induced by tumor associated macrophages through inhibiting the M2 type polarization of tumor associated macrophages.