Inhibitory Effects Of Iridoid Glycosides From Boschniakia Rossica Combined With 5-fluorouracil On Epithelial-mesenchymal Transition Of Human Hepatocellular Carcinoma HepG2、SK-Hep1 And SMMC-7721 Cells

Objective:To study the effect of Iridoid glycosides from Boschniakia rossica(IGBR)combined with 5-fluorouracil on epithelial-mesenchymal transition of human hepatoma HepG2,SK-Hepl and SMMC-7721 cells and its mechanism.Methods:The logarithmic growth-stage human hepatoma HepG2,SK-Hepl and SMMC-7721 cells were randomly divided into 5 groups,control group,model group,IGBR group,5-FU group,and combined group.The control group was treated with DMEM for 48 h;The model group was used 10ng/ml of TGF-β1 for 48 h;The IGBR group was given 500 mg/mL of IGBR and 10 ng/mL of TGF-β1 for 48 h;The 5-FU group was given 75μg/mL of 5-FU and 10 ng/mL of TGF-β1 for 48 h;The combination group was given 500 mg/mL of IGBR,75 μg/mL of 5-FU,and 10 ng/ml of TGF-β1 for 48 h.Morphological changes was observed by the microscope.Survival ability and adhesion rate was detected by MTT method.The expression of matrix metalloproteinase(MMP)MMP2,MMP7,MMP9,Snail,Slug was detected by Western blot.The localization and expression intensity of E-cadherin and vimentin was detected by immunofluorescence.Result:1.Compared with the control group,the model group cells exhibit the long fusiform,fibrous and spindle shape.Compared with the model group,5-FU group,IGBR group and combined group cell morphology changes was decreased.Compared with the combined group,5-FU group and IGBR group cell of morphological changes was increased.2.Compared with the model group,5-FU group,IGBR group and combination group cell survival ability and adhesion rate was decreased(P<0.01).Compared with 5-FU group and IGBR group,the combination group survival ability and adhesion rate were significantly reduced(P<0.05).3.Western Blot shows that compared with the control group,the expression of MMP2,MMP7,MMP9,Snail,Slug were up-regulation(P<0.01)in the model group.Compared with the model group,the expression of MMP2,MMP7,MMP9,Snail and Slug were down-regulated(P<0.05)in the 5-FU group,IGBR group and combined group.Compared with the combined group,the expressions of MMP2,MMP7,MMP9,Snail and Slug were up-regulated(P<0.05)in 5-FU group and IGBR group.4.Immunofluorescence shows that E-cadherin and Vimentin were located in the cell membrane and cytoplasm.Compared with the control group,the E-cadherin fluorescence intensity was decreased in the model group,but the Vimentin fluorescence intensity was increased.Compared with the model group,the E-cadherin fluorescence intensity was increased in 5-FU group,IGBR group and combined group,but the Vimentin fluorescence intensity was decreased.Conclusion:1.IGBR combined with 5-Fu can reverse the human hepatoma EMT model,inhibit the proliferation and reduce adhesion rate.2.IGBR combined with 5-Fu decreased the expression of MMP2,MMP7,MMP9,Snail,Slug,enhanced E-cadherin fluorescence intensity,reduced Vimentin fluorescence intensity and inhibited EMT of human hepatoma,which may be a EMT mechanism.3.IGBR combined with 5-Fu can inhibit EMT of hepatocellular carcinoma HepG2,SK-Hep1,SMMC-7721 cells,which has a stronger effect than IGBR and 5-Fu single drugs,and can be used as an auxiliary anti-tumor drug.