Effects Of EGFR Inhibitors On Growth,invasion And Metastasis Of Hepatic Alveolar Echinococcosis In Mice

Objects:1.To establish an in vitro culture system of alveolar echinococcosis and investigate whether EGFR inhibitors have pharmacological activity against alveolar echinococcosis in vitro.2.Establish a mouse model of hepatic alveolar echinococcosis disease,verify the effect of EGFR inhibitor on the growth and invasion of hepatic alveolar echinococcosis in vivo,and expect to clarify the mechanism of this effect.Methods:1.Extraction and identification of alveolar echinococcoid protococcoid,and establishment of echinococcoid culture system in vitro.1.1 Blank control group,DMSO group and experimental group of Afatinib(BIBW2992)with different concentrations(5u M,10 u M,20 u M)were set.Eosin staining was used to calculate activity rate at different times.Caspase-3 enzyme activity of each group on the third day was detected by caspase-3 kit.1.2 Blank control group,DMSO group,Albendazole(SKF-62979)group,Trametinib(GSK1120212)group and afatinib group were set up,and eosin staining was used to calculate activity rate at different times.The caspase-3 activity of each group on the third day was detected by caspase-3 kit.2.Establish the mouse liver echinococcosis model.After 60 days,the rats were randomly divided into4 groups,namely blank control group,albendazole group,trametinib group and afatinib group,with 10 rats in each group.After 60 days of modeling,albendazole(20mg/ kg.wk),trimeitinib group(20mg/ kg.wk)and afatinib group(20mg/ kg.wk)were given intragastric treatment,while the blank control group was given the same volume of tap water.After 60 days of treatment,the mice were dissected and their liver and alveolar hydatid tissues were collected.The wet weight of the alveolar hydatid cyst in each group was compared,and the expressions of EGFR,P-EGFR,AKT,P-AKT,ERK,P-ERK,Bcl-2,Bax and Caspase-3in the hepatic hydatid cyst tissue of mice were detected by immunohistochemistry and Western blotting.Results:1.In vitro experiment:1.1 The activity of primary scolex in different doses of afatinib groups was significantly different from that in the control group(P<0.05);There were statistically significant differences among afatinib high-dose,medium-dose and low-dose groups(P<0.05).In the same concentration of afatinib treatment group,there were statistically significant differences between different measurement time points(P<0.05).Afatinib increased the activity of caspase-3 in echinococcus alveolaris,and the activity of Caspase-3 increased with the increase of action measurement and the prolongation of action time.1.2 Compared with the blank control group,there was statistical significance in the activity rate of primary cephalic segment after the same dose of albendazole,afatinib and trametinib(P<0.05).Inhibition of EGFR signaling pathway increased the activity of Caspase3 in echinococcus alveolaris.2.In vivo experiment: The mouse liver alveolar echinococcosis animal model was successfully established.Compared with the blank control group,the growth of alveolar echinococcosis lesions in albendazole group,trametinib group and afatinib group was limited.The wet weight of each experimental group was lower than that of blank control group(P<0.05).WB results indicated that the relative expression levels of P-EGFR,P-ERK and P-Akt in echinococcosis multilocular tissue in the afatinib group were lower than those in the blank control group 4 months after modeling.The expression of P-ERK in echinococcus multilocularis in trimeitinib group was lower than that in the control group(P<0.05),but there was no significant difference in the expression of P-EGFR and P-Akt(P>0.05).There were no differences in the expressions of non-phosphorylated EGFR,ERK and AKT among all groups(P > 0.05).Conclusion:1.EGFR inhibitors showed time-dependent and concentration-dependent anti-protoscoliae activity in vitro.EGFR inhibitors may induce apoptosis of echinococcus cellulosus cells through the Caspase3 pathway.2.In vivo studies have shown that EGFR signaling pathway mediates echinococcus alveolar growth and metastasis,and the mechanism may be related to down-regulation of EGFR pathway related molecules.