Preliminary Study On The Efficacy And Mechanism Of Rapamycin Target Protein (mTOR) Inhibitor TAC In The Treatment Of Cystic Echinococcosis In Mice

Objective:Preliminarily studied the efficacy and mechanism of mTOR inhibitors for the treatment of cystic echinococcosis and to provide ideas for discovering new cystic echinococcosis drug targets.Methods:First,mTOR inhibitors with potent inhibitory activity against Echinococcus granulosus were screened from experiments in vitro seven kinds of mTOR inhibitors(Temsirolimus,Zotarolimus,CZ415,Deforolimus,Everolimus,Rapamycin,TAC)were applied,and setting the concentration of 100,50,25,12.5,6.25,3.125?mol/L to interfering with Echinococcus granulosus,using Albendazole Sulfoxide(ABZSO)as the positive drug group in vitro.After 4 days of intervention.Using eosin exclusion test to detecte the activity of protoscoleces(PSCs)in each experimental group,and the scanning electron microscope(SEM)and the transmission electron microscope(TEM)were used to detect the ultrastructural changes of PSCs,Comparing the effect of different mTOR inhibitors on the anti-cystic echinococcosis.Using four kinds of mTOR inhibitors(Deforolimus,Everolimus,Rapamycin,and TAC)with higher toxicity to PSCs,and selecting the concentration gradients of 100,25,and 6.25 ?mol/L,ABZSO as the positive drug.After 4 days of intervention vesicles in vitro,detected the rate of subsidence of vesicles at different times and TEM was used to detect ultrastructural changes.In order to screen more efficient mTOR inhibitors.Detected mRNA expression of genes(egfkbp,egraptor,egtor)after intervention of Everolimus,Rapamycin and TAC in vitro by fluorescence quantitative PCR.In the echinococcosis mouse model,we evaluated the anti-echinococcosis(protocephalus and cysts)efficacy of TAC.The dosage of TAC was 4,2 and 1mg/kg,once a day,with Albendazole(ABZ)as the positive control group.After 4 weeks of intervention,the vesicle diameter,vesicular wet weight,number of vesicles,and cystic inhibition rate of each experimental group were measured,liver and spleen index were calculated,and serum liver function(AST,ALT,ALP)and renal function(Urea,Crea)and other indicators were detected.HE staining was used to assess the effect of TAC on the pathological structure of mouse liver,kidney and vesicle.The changes of vesicles ultrastructure were detected by TEM.Measured the contents of glucose and ATP,Calcineurin(CaN)and Ca2+ concentration of vesicle tissue and cyst fluid in the experimental groups to evaluate the impact of TAC on glucose signaling pathway and target protein,in addition,related ions in PSCs.Results:Seven kinds of mTOR inhibitors had different degrees of toxicity to PSCs in vitro,among which Defolimus,Everolimus,TAC,and Rapamycin had higher toxicity.SEM and TEM results showed that the ultrastructure of the PSCs have a greater changes after after intervention of Deforlimus,the number of microvilli decreased,the suction cups deformed,and the inner cortex changed.Most of PSCs are invagination,the top mutant shape or absence,suction cup deformation is not easy to identify,the number of microvilli decreased,or even disappear.The cilia of the PSCs were significantly deformed or fused,and the collecting tube was enlarged and deformed,the cortex was changed.There were microbubbles of various sizes in the local cortex matrix,vacuoles in the cytoplasm,and swelling of the stratum comeum vesicle fusion.However,the LC50 of Defolimus was 1.53 ?mol/L,which was lower than the LC50 of TAC(10.37 ?mol/L),the LC50 of Everolimus(12.35 ?mol/L)and the LC50 of Rapamycin(12.60 ?mol/L).Vesicle experiments showed that the LC50 of TAC was 1.98?mol/L,lower than that of Rapamycin(3.44 ?mol/L),LC50 of Everolimus(5.70 ?mol/L),and LC50 of Defolimus(5.27 ?mol/L)in vitro.TAC has high activity against vesicles,after the vesicles were intervened by 100?mol/L TAC,TEM results showed that the cells in the germinal layer were destroyed,there was no intact germinal layer structure,the texture of the cuticle was rough.Transparent vesicles appear,the stratum comeum is separated from the germinal layer,the vesicle cortical area is uneven,the cortical matrix is blurred,and there are microbubbles of different sizes in the local cortical matrix.The qPCR results showed that 50?mol/L TAC could induce the expression of egfkbp,egraptor and egtor genes to be up-regulated by 3.08,1.98 and 2.15 times,respectively.12.5 ?mol/L Everolimus induced the expression of egfkbp,egraptor and egtor genes up-regulated by 8.13,1.80 and 2.02 folds,respectively.12.5 ?mol/L Rapamych induced the expression of egfkbp,egraptor and egtor genes up-regulated by 6.80,5.46 and 5.90 folds in vitro respectively.After one month of TAC intervention in vivo,the inhibition rate(58.83±39.12)%in the 4mg/kg TAC group was similar to that in the ABZ group(58.11±35.21)%.The number of vesicles in the 4 mg/kg TAC group was significantly lower than that in the solvent group(P<0.05).There was no obvious abnormal pathological structure in liver and kidney of TAC-administered mice.The AST value of the TAC group was significantly lower than that of the solvent group(P<0.05).The ALT value(69.90±26.14)in the 4 mg/kg TAC group was significantly lower than that in the solvent group(101.11±18.83)(P<0.05).The value of Crea in the TAC group was significantly lower than that in the solvent group(P<0.05).TEM results showed that the structure of mouse vesicles was incomplete after TAC administration,and cortical cells were necrotic.The glucose content in the cyst fluid of 4 mg/kg TAC group was significantly lower than that of the solvent group(P<0.05).The ATP content in vesicles of 4 mg/kg TAC mice was significantly lower than that of the solvent group(P<0.05).The content of CaN in cyst fluid of 4mg/kg TAC group was significantly lower than that of solvent group(P<0.05).There was no significant difference in calcium concentration between vesicles and cyst fluid:in the TAC group compared with the solvent group(P>0.05).Conclusion:This study found that the mTOR inhibitor TAC has efficacy in the treatment of cystic echinococcosis in vivo and in vitro,and TAC may inhibit the hydatid activity by affecting the glycometabolism of Echinococcus granulosus to achieve the treatment of cystic echinococcosis.