Artemisia Argyi Essential Oil Inhibit The Metastasis And Invasion Of Hepatocellular Carcinoma And Its Mechanism

Objective:Hepatocellular carcinoma ranks the sixth among the most common malignant tumors in the world,the metastasis of HCC is the primary factor affecting the survival of liver cancer patients,and it is also an urgent problem to be solved.Human DEPDC1 is a tumor related gene discovered recent years.It has been reported that high expression of DEPDC1 can activate the Wnt/?-catenin signaling pathway to promote metastasis of cancers.Folium artemisiae argyi are the dry leaves of Artemisia argyi Levl.Et Vant.Artemisia argyi essential oil(AAEO)is one of the main active ingredients in Artemisia argyi leaves.Earlier studies confirmed that AAEO has the effect of anti-hepatitis B virus activity.Studies in vitro showed that AAEO could down-regulate the expression of DEPDC1.Our research will study the influence of AAEO on the migration and invasion of hepatocellular carcinoma cells and its mechanism,so as to pro vide a basis for further research on AAEO anti-tumor.Methods:1.The inhibitory effect of AAEO on HCC cells migration and invasion in vitro and its mechanismThe proliferation inhibition rate of AAEO on HepG2 and SMMC-7721 cells was detected by MTT assay.HepG2 and SMMC-7721 cells were stimulated with AAEO for24 h,48 h,cell invasion was determined using Transwell and scratch-wound assays.Quantitative real-time PCR(qPCR)was used to detect the effects of AAEO on the expression of DEPDC1 in HepG2 and SMMC-7721 cells.Western Blot was used to detect the expression changes of DEPDC1,?-catenin and GSK-3? proteins in HCC cell lines.2.Effect of knockdown DEPDC1 expression on Wnt/?-catenin pathway mediated by DEPDC1Target silencing of DEPDC1 in HepG2 cells.The expression changes of DEPDC1 and Wnt/?-catenin pathway target genes:WNt-1,MMP9 and ?-catenin were detected by qPCR.Western Blot was used to detect the expressions of DEPDC1,?-catenin and GSK-3? in ko-HepG2 cells.3.The inhibitory effect of AAEO on HCC cells metastasis in vivo and its mechanismFor the lung metastasis model,Luciferin-labeled HepG2 cell lines(Luc-HepG2)were injected through tail vein of male Balb/c nude mice.The animals were grouped as follows:solvent control group(10 mL/kg),AAEO high dose group(230 mg/kg),medium dose group(115 mg/kg),low dose group(57.5 mg/kg),and positive control group(sorafenib 40 mg/kg).QPCR was used to detect the effect of AAEO on the expression of DEPDC1 in the lung tissues of nude mice.Immunohistochemical method was used to detect the expression changes of ?-catenin and GSK-3? proteins in lung tissues.4.Tissue distribution of AAEO in ratsThe distribution of AAEO in heart,liver,spleen,lung,kidney and brain tissues was detected by GC.The mechanism of AAEO inhibiting liver cancer metastasis was elucidated from the perspective of pharmacokinetics.Results:1.HepG2 and SMMC-7721 cells were stimulated with AAEO for 72 h,and MTT results showed that AAEO significantly inhibited the proliferation of cells in vitro,with IC50 values of 309.76 ?g/mL and 364.85 ?g/mL,respectively.HepG2 and SMMC-7721 cells were stimulated with AAEO for 24 h,48 h,the scratch healing experiment and the transwell experiment showed that AAEO could effectively inhibit cells migration and invasion,and had obvious concentration dependence.The results of qPCR showed that AAEO down-regulated the expression of DEPDC1 in HCC cell lines.Western Blot results showed that AAEO significantly down-regulated the expression of DEPDC1 and(?-catenin protein,as well as the up-regulated expression of GSK-3? proteins in cells.2.HepG2 cells targeting silence of DEPDC1 expression were successfully constructed(ko-HepG2).QPCR results showed that silencing the expression of DEPDC1 significantly inhibited the expression of Wnt/?-catenin pathway target genes:Wnt-1,MMP9 and ?-catenin,while the protein expression of DEPDC1,?-catenin was also inhibited,and GSK-3? was up-regulated.The above experimental results showed that AAEO could down-regulate DEPDC1 expression in cells,thereby blocking Wnt/?-catenin pathway to inhibit the migration and invasion of HCC cells.3.Lung metastasis model of HCC in nude mice was successfully constructed.In vivo imaging of small animals intuitively results showed that the metastasis of cancer cells in AAEO group was significantly inhibited.The nude mice in the control group were found decreased activity and weight loss,there was no difference in body weight of AAEO group.The results of qPCR showed that AAEO had a significant inhibitory effect on DEPDC1 in mouse lung tissue.Immunohistochemistry showed that the expression of ?-catenin in the lung tissues of AAEO group was down-regulated.4.A method was established to determine the content of eucalyptus in the tissues of SD rats after gavage of AAEO,which was reasonable and feasible.The results of tissue distribution showed that the absorption of AAEO after gavage was better,and the content of AAEO was higher in lung,liver,brain and other tissues.Conclusion:1.AAEO has an obvious inhibitory effect on the migration and invasion of HCC cells in vivo and in vitro.2.AAEO was well absorbed after gavage and can be effectively distributed to the liver,lung and other metastatic tissues.3.By down-regulating the gene expression of DEPDC1,AAEO blocking the activation of the Wnt/?-catenin pathway and inhibiting the migration and invasion of HCC cells.