Experimental Study On The Effect Of Lichong Decoction Plus 5-fluorouracil On Epithelial-mesenchymal Transformation Of Hepatocellular Carcinoma

Objective:1.At the animal level,observe the effects of Lichong Decoction plus 5-FU or alone on the growth of subcutaneous xenografts and lung metastases in H22 tumor-bearing mice,and further observe the effects of Lichong Decoction plus 5-FU or alone on the expression of emt-related markers in mouse transplanted tumors2.At the cellular level,observe the effect of Lichong Decoction plus 5-FU or alone on the proliferation of human hepatoma cell HepG2,and evaluate the effect of the two drugs by Medium-effect Principle Method,and further observe the effect of Lichong Decoction plus 5-FU or alone on the migration,invasion and adhesion of human hepatoma cells HepG2,and to explore its mechanism of inhibiting liver cancer metastasis from EMT.Methods:1.Establish H22 tumor-bearing mouse model,and divide the mice into blank group,5-fluorouracil group(5-FU group),Lichong decoction+5-FU group(Chinese medicine+5-FU group),Lichong Decoction group(Chinese medicine group),after 14 days of administration,observe the effect of Lichong Decoction plus 5-FU or alone on mouse body weight,subcutaneous xenograft of liver cancer and lung metastasis.2.RT-PCR and Western Blot were used to detect the effects of Lichong Decoction plus 5-FU or alone on EMT-related markers in mouse liver cancer subcutaneous xenografts.3.Using human hepatoma cell HepG2.MTT assay was used to detect the Lichong Decoction plus 5-FU or alone on its value-added.The medium-effect principle was used to evaluate the interaction effect between Lichong Decoction and 5-FU4.According to the principle of medium-effect principle,a reasonable drug regimen is obtained,and the concentration of' Lichong Decoction plus 5-FU or alone is determined.The effect of-Lichong Decoction plus 5-FU or alone on migration'invasion,and adhesion of HepG2 cells ai the concentration of the drug is further observed.5.RT-PCR and Western Blot were used to detect the effects of Lichong Decoction plus 5-FU or alone on EMT-related markers in HepG2 cells.Results:1.Establish H22 tumor-bearing mouse model.By comparing the body weight changes of each group of mice,it was found that the mice in the 5-FU group had the most weight loss,while the combination of Lichong Decoction plus 5-FU can alleviate the trend of weight loss;by calculating the tumor inhibition rate of each group of mice,compared with the blank group,the anti-tumor effect of Lichong Decoction alone on H22 tumor-bearing mice is not obvious(P>0.05).The combination of Lichong Decoction and 5-FU and 5-FU can inhibit the growth of subcutaneous xenografts in H22 tumor-bearing mice(P<0.05),but the combination of Lichong Decoction and 5-FU on the anti-tumor effect was greater than 5-FU alone(p<0.05);by calculating the combined drug index,it was shown that the combination of Lichong Decoction and 5-FU can enhance the anti-tumor effect of chemotherapy drugs;It was found that in the inhibition of liver cancer metastasis,Lichong Decoction plus 5-FU>5-FU>Lichong Decoction alone.2.RT-PCR and Western Blot showed that Lichong Decoction plus or 5-FU alone could up-regulate E-cadherin(P<0.05)and down-regulate N-cadherin,Snail,Twist and Vimentin(P<0.05)in mice with liver cancer subcutaneously transplanted,and its regulating ability was Lichong Decoction plus 5-FU>5-FU>Lichong Decoction.3.Using human hepatoma cell HepG2 as the research object,it was found by MTT method that the combination of Richong Decoction and 5-FU can inhibit the proliferation of HepG2 cells in a concentration-dependent and time-dependent manner.The principle of effect and discovery that Lichong Decoction combined with 5-FU can exert low-dose synergistic anti-tumor proliferation effect on HepG2 cells for 24h4.Through cell scratch test,invasive experiment and adhesion experiments,it was found that Lichong Decoction combined with 5-FU and single use can inhibit the migration,invasion and adhesion of HepG2 cells(P<0.05).but inhibit ability of migration,invasion and the adhesion was LiChong Decoction+5-FU>5-FU>LiChong Decoction(P<0.05).5.RT-PCR and Western Blot showed that Lichong Decoction plus or 5-FU alone could up-regulate E-cadherin(P<0.05)in HepG2 cells and down-regulate N-cadherin,Snail,Twist and Vimentin(P<0.05),and its regulating ability was Lichong Decoction plus 5-FU>5-FU>Lichong Decoction.Conclusion:Lichong Decoction plus 5-FU or alone can inhibit the growth of subcutaneous xenografts and the number of lung metastases in mice,and the combination of Lichong Decoction and 5-FU is better than 5-FU is used alone.Lichong Decoction plus 5-FU or alone can inhibit the expression of EMT-related molecular markers in subcutaneous xenografts in mice,and the effect of Lichong Decoction plus 5-FU is better than that of 5-FU Single use.Lichong Decoction plus 5-FU or alone can inhibit the proliferation,migration and adhesion of human hepatoma cell line HepG2,and the combination of Lichong Decoction and 5-FU is synergistic.Lichong Decoction plus 5-FU or alone can inhibit the expression of EMT-related markers in hepatocellular carcinoma cells,and the effect of Lichong Decoction plus 5-FU is better than that of 5-FU alone.In this study,the in vitro and in vivo experiments showed that Lichong Decoction combined with 5-fu has synergistically inhibited the growth and metastasis of HCC,and its mechanism may be related to the inhibition of liver cancer.