| ObjectiveIn this study,we investigated the effects of eupatilin on the biological behavior of hepatocellular carcinoma through in vitro and in vivo experiments.The aim of this study was to elucidate the anti-hepatocellular carcinoma mechanism of eupatilin.Method(1)The total flavonoids in the Artemisia argyi leaves were extracted by methanol impregnation method.The optimum experimental conditions were selected for the extraction of eupatilin from total flavonoids of Artemisia argyi by high performance liquid chromatography.(2)In this study,we used different concentration eupatilin act on SMMC-7721 cells.Subsequently,cytotoxicity test,cell clone formation assay,cell proliferation assay and flow cytometry were performed.And the expression of p53,TopoⅡα,bcl-2,caspase-3,p-IRE1,p-JNK,MCP-1 protein were detected by western blot.(3)In this study,nude mice transplanted tumor model was established by subcutaneous injection of SMMC-7721 cells.24 nude mice bearing tumor were injected intraperitoneally with different concentration eupatilin.The volume of transplanted tumor in nude mice was calculated at different time points,and the growth curve was drawn.The weight of tumors was weighed and the inhibition rate was calculated;the expression of p53,Topo Ⅱa and Bcl-2 protein in nude mice were detected by immunohistochemistry.Western blot was used to detect the expression of p-IRE1,p-JNK and MCP-1 in all groups of-nude mice.Results(1)The optimal combination of our HPLC was determined as follows:the column was TIANHE C18(250 mm × 4.6 mm,5 μm)and the mobile phase was acetonitrile:water:2%glacial acetic acid(64:36:2),methanol as extractant,ultrasonic time 60 min,column temperature 30 C,flow rate 1.0 mL/min,test sample solution injection volume 10 μL.(2)The results showed that with the increase of the concentration of eupatilin on SMMC-7721 cells,the toxicity of gradually increased,the number of clones gradually decreased,the proliferation inhibition rate and the apoptosis rate gradually increased(P<0.05).Western blot showed that the expression of p53,caspase-3,p-IRE1,p-JNK and MCP-1 protein in the cells of different concentrations of eupatilin group cells was significantly higher than that of DMSO group(P<0.05),and the expression of Topo Ⅱα and Bcl-2 protein was significantly lower than that of DMSO group(P<0.05),all of them were dose dependent.(3)In this study,24 nude mice with transplanted human hepatocellular carcinoma SMMC-7721 cells were all tumorigenic.The tumor volume of nude mice was significantly lower than that of the control group at each time point(P<0.05).Compared with the 0 mg/kg group,the expression levels of p53 protein in nude mice transplanted tumor cells were significantly increased(P<0.05)in a dose-dependent manner.Compared with the 0 mg/kg group,the expression levels of Topo Ⅱα and Bcl-2 protein in nude mice transplanted tumor cells were significantly decreased(P<0.05)in a dose-dependent manner.With the increase of eupatilin concentration,the expression of p-IRE1,p-JNK and MCP-1 protein in the transplanted tumors increased,and the contents of p-IRE1,p-JNK and MCP-1 protein in the transplanted tumor tissues at different concentrations of eupatilin were significantly higher than that of 0 mg/kg group(P<0.05),and it was dose-dependent.ConclusionEupatilin can inhibit the growth and promote apoptosis of hepatoma SMMC-7721 cells and xenografts in vitro and in vivo,its mechanism of action and inhibition of Bcl-2,Topo IIa expression,promote the expression of p53,caspase-3,activate IRE1JNK/MCP-1 signaling pathway is involved. |
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