Study On The Mechanism Of Modified Yangying Huoxue Decoctionon Primary Liver Cancer Based On Wnt Signaling Pathway

Objective In this experiment,the effects of Yangying Huoxue Decoction on H22 liver cancer cells and tumor-bearing mice were observed in vivo and in vitro,and the mechanism of action of the prescription on regulating the Wnt signaling pathway of liver cancer cell signal transduction pathway was explored.Methods In vitro cell experiments,CCK-8 assay was used to detect the proliferation of liver cancer H22 cells treated with different concentrations;the cell cycle arrest of H22 liver cancer cells was detected by flow cytometry.Flow cytometry apoptosis assay was used to detect the apoptosis of cells after drug-containing serum intervention in each group.The m RNA and protein expressions of β-catenin,TGF-β,E-cadherin,Cyclin D1,c-myc and Survivin were detected by RT-PCR and Western blot.In vivo animal experiments,BALB/c mice were randomly divided into model control group,modified Yangying Huoxue Decoctiongroup,fluorouracil group(hereinafter referred to as 5-FU group),modified Yangying Huoxue Decoctioncombined with 5-FU group.After the mouse model of subcutaneous tumor of liver cancer was prepared,the mice were continuously intervened for10 days to observe and record the basic situation of the mice and measure the size of the subcutaneous tumor.After the last administration,the blood was centrifuged and the liver function indexes of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and serum alkaline phosphatase(ALP)were detected.The m RNA and protein expressions ofβ-catenin,TGF-β,E-cadherin,Cyclin D1,c-myc and Survivin were detected by RT-PCR and Western blot,respectively.Results 1.In vitro cell experiments showed that : CCK8 method was used to determine the effect of each group of drug-containing serum on the proliferation of liver cancer H22 cells.It was found that 10% modified Yangying Huoxue Decoctiongroup and 10% 5-FU group were treated with H22 cells for 24,48 hours,compared with 10% negative serum control group,the difference was statistically significant and the drug concentration was closest to IC50;The modified Yangying Huoxue Decoctiongroup,5-FU group,The modified Yangying Huoxue Decoctioncombined with 5-FU group can induce H22 liver cancer cell cycle arrest in S phase;compared with the control group,the apoptosis rate of liver cancer H22 cells in the modified Yangying Huoxue Decoctiongroup increased significantly(P<0.01).Compared with 5-FU group,the apoptosis rate of liver cancer H22 cells in modified Yangying Huoxue Decoctioncombined with 5-FU group increased significantly(P<0.01).Compared with the model group,the expression levels of E-cadherin m m RNA and protein in modified Yangying Huoxue Decoctiongroup,5-FU group and modified Yangying Huoxue Decoctioncombined with 5-FU group increased(P<0.01),and the expression levels ofβ-catenin,TGF-β,Cyclin D1,c-myc and Survivin m RNA and protein decreased significantly(P<0.01).Compared with 5-FU group,the expression of E-cadherin m RNA and protein in Yangying Huoxue Decoction combined with 5-FU group increased(P<0.05).The expression levels of β-catenin,TGF-β,Cyclin D1,c-myc and Survivin m RNA and protein were significantly decreased(P<0.05).2.In vivo animal experiments showed that compared with the model group,the serum ALT,AST and ALP levels in the modified Yangying Huoxue Decoctiongroup,5-FU group,and modified Yangying Huoxue Decoctioncombined with 5-FU group were significantly lower(P<0.05);compared with the 5-FU group,the serum ALT,AST and ALP levels in the modified Yangying Huoxue Decoctioncombined with 5-FU group were significantly lower(P<0.01);compared with the model group,the expression levels of E-cadherin m RNA and protein in modified Yangying Huoxue Decoctiongroup,5-FU group and modified Yangying Huoxue Decoctioncombined with 5-FU group increased(P<0.01),and the expression levels of β-catenin,TGF-β,Cyclin D1,c-myc and Survivin m RNA and protein decreased(P<0.01).Compared with 5-FU group,the expression of E-cadherin m RNA and protein in modified Yangying Huoxue Decoctioncombined with 5-FU group increased(P<0.05),and the expression of β-catenin,TGF-β,Cyclin D1,c-myc and m RNA and protein decreased significantly(P<0.05).Conclusion The modified Yangying Huoxue Decoction can inhibit the proliferation of liver cancer cells,induce cell cycle arrest in S phase,and promote the apoptosis of H22 liver cancer cells.The modified Yangying Huoxue Decoctioncan reduce the content of ALT,AST and ALP in serum of tumor-bearing mice and improve the level of liver function.The inhibitory effect of modified Yangying Huoxue Decoctionon the proliferation of liver cancer cells and subcutaneous tumors in tumor-bearing mice may be related to the increase of E-cadherin m RNA protein expression,the decrease of β-catenin,TGF-β,Cyclin D1 and c-myc m RNA protein expression,and the regulation of Wnt/β-catenin signaling pathway in liver cancer.