Study on the efficacy and mechanism of pseudolaric acid B against Echinococcus multilocularis
Objective To investigate the in vitro effect of pseudolaric acid B(PAB)on apoptosis of protoscolece cells and its regulatory effects on angiogenesis and cell apoptosis in the the lesion-host microenvironment tissue in vivo,as well as its possible mechanisms,in order to provide a basis for the clinical development of new alternative drugs for Echinococcus multilocularis.Methods In vitro experiments:the protoscoleces,vesicles,germinal cells,human foreskin fibroblasts(HFFs)and normal human liver cells were treated with different concentrations of PAB(0,2.5,5,10,20,40,80,160 and 320 μmol/L)for 7,5,5,5 and 5 days,then evaluated the survival rate of the protoscoleces,the release level of phosphoglucose isomerase(PGI)from the vesicles,the viability of the germinal cells,as well as the viability of HFFs and normal human liver cells.The protoscoleces and vesicles were fixed with 2.5％glutaraldehyde and used for scanning electron microscopy and transmission electron microscopy observation.Animal experiments:the protoscoleces were isolated from the abdominal lesions of the protected gerbils,and then infected 18 C57BL/6J mice by intraperitoneal injection to establish models,dividing into 3 groups with 6 mice in each group.The model group was given 0.3 mL of PBS by gavage daily,the albendazole(ABZ)group was given 0.3 mL ABZ(100 mg/kg)daily by gavage,the PAB group was given 0.3 mL of PAB(40 mg/kg)by gavage daily.After continuous gavage for 6 weeks,the lesion host microenvironment tissue was taken and ELISA was used to detect the expression levels of vascular endothelial growth factor(VEGF),endothelial nitric oxide synthase(eNOS)and cysteinyl aspartate specific proteinase3(caspase3),the expression levels of nitric oxide(NO)was detected using a biochemical detection kit,Western blot was used to detect the expression levels of BCL2-associated X protein(Bax),B-cell lymphoma-2(Bc12),caspase3,cleaved-caspase3,VEGF,vascular endothelial growth factor receptor 2(VEGFR2),phosphatidylinositol 3 kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(AKT)and phosphorylated AKI(p-AKT)protein.Results In vitro experiments:the protoscoleces of Echinococcus multilocularis were cultured with different concentrations of PAB for 7 days in vitro,the protoscoleces of 40,80,160 and 320 μmol/L group all died after 6,4,2 and 1 day,respectively;PAB exhibited a certain time and concentration dependence on the protoscoleces of Echinococcus multilocularis.After PAB treatment,the release of PGI in culture supernatant of Echinococcus multilocularis gradually increased with the increase of PAB concentration[concentration for 50％of maximal effect value was(24.40±1.42)μmol/L],the vitality of germinal cells was significantly inhibited[half maximal inhibitory concentration value was(15.94±2.55)μmol/L].PAB had no significant toxicity to mammalian cells.When 20 μmol/L PAB intervention in the protoscoleces for 3 days,the expression levels of Bax and caspase3 proteins were upregulated,while the expression level of Bcl2 protein was downregulated.Animal experiments:compared with the model group,the wet weight of lesions in the PAB and ABZ groups decreased(P＜0.01),and the inhibition rates of lesion growth in the PAB and ABZ groups were 91.03％and 74.44％,respectively.The expression of proliferation and angiogenesis indicators(Ki67,CD34,VEGF,VEGFR2,eNOS,NO)were downregulated in the lesion host microenvironment tissues of mice in the ABZ and PAB groups(P＜0.05),while the expression of apoptosis related proteins(caspase3,cleaved-caspase3 and Bax)were upregulated and the expression of PI3K/AKT signaling pathway related proteins(p-PI3K and p-AKT)were downregulated(P＜0.05).Conclusion PAB has a strong in vitro and in vivo effect against Echinococcus multilocularis,and its mechanism may be related to the inhibition of PI3K/AKT signaling pathway,leading to increased apoptosis and decreased angiogenesis.
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