The Activity And Mechanisms Of Niclosamide And Myrislignan Against Toxoplasma Gondii

Toxoplasma gondii?T.gondii?is an obligate intracellular parasite,which can infect all the warm blood animals including human beings.To date,the current treatment drugs have many limitations,safe and effective drugs for the treatment of toxoplasmosis are urgently needed.In this study,a series of potential compounds which may have anti-parasite activity were selected to screen the anti-T.gondii activity in vitro,and the in vivo anti-T.gondii activity of the active compounds was also studied,and the mechanisms of action were preliminarily explored.Test of in vitro anti-T.gondii activity and screening of effective candidate compounds were included in this research.The cytotoxicity effects of the compounds against human foreskin fibroblast?HFF?were evaluated by MTT test.Meanwhile,the anti-T.gondii activities of the compounds were screened in vitro at the maximum safe concentrations.The results showed that niclosamide and myrislignan had strong anti-T.gondii activity and could be used as an effective candidate compound for further study.Study on the activity and mechanism of niclosamide against T.gondii.The in vitro anti-T.gondii activity of niclosamide was evaluated by real-time quantitative polymerase chain reaction?qPCR?and Giemsa staining.The results showed that niclosamide inhibited the invasion and proliferation of T.gondii,and the 50%effective concentration(EC₅₀)of niclosamide was 45.3 ng/mL.The acute infection model of BALB/c mice infected with T.gondii was established and administered orally by niclosamide.Under the concentration of 240 mg/kg,niclosamide could protected 50%of the mice from death and reduced the parasite load in the mice whether in the tissues or blood.The effect of niclosamide on the ultrastructure of T.gondii was observed by scanning electron microscopy?SEM?and transmission electron microscopy?TEM?.It was found that niclosamide caused the surface shrinkage and mitochondria swelling of T.gondii.The mitochondrial membrane potential was detected to decrease using JC-1 staining.Meanwhile,ATP level of T.gondii was also significantly decreased.In order to further explore the mechanism of action of niclosamide,transcriptome and surface plasmon resonance?SPR?analysis showed that niclosamide may affect the proton transport of T.gondii vacuolar type H⁺ATPase?V-ATPase?.Acridine orange staining confirmed that niclosamide caused the imbalance of pH in T.gondii.Flow cytometry analysis showed that niclosamide caused T.gondii death in a dose-dependent manner.Study on the activity and mechanism of myrislignan against T.gondii.The cytotoxicity of myrislignan against African green monkey kidney cell?Vero?was determined using CCK-8 assay.Myrislignan could significantly reduce the invasion and proliferation of T.gondii at the concentration ranging from 20 to 90?g/ml with the EC₅₀ of 32.41?g/mL.In the acute infection model of BALB/c mice infected with T.gondii,myrislignan was injected intraperitoneally and significantly?P<0.01?reduced the parasite load in the tissues of BALB/c mice.The effects of myrislignan on the ultrastructure of T.gondii were observed by SEM and TEM.After incubation,myrislignan induced the surface deformation,mitochondria swelling,ridged structured formation and autophagic double membrane structure in T.gondii tachyzoites.MitoTracker Red CMXRos probe was used to verify the decrease of mitochondrial membrane potential after incubation with myrislignan.At the same time,ATP level also decreased.MDC staining showed that myrislignan induced autophagy of T.gondii.Furthermore,the expression of autophagy gene increased significantly?P<0.01?at mRNA and protein levels.Studies have shown that niclosamide and myrislignan have strong activities against T.gondii tachyzoites in vitro and in vivo.Niclosamide may damage the pH homeostasis of T.gondii and cause the death of T.gondii by affecting the proton transport of V-ATPase.Myrislignan may damage the mitochondrial function of T.gondii,cause autophagy of T.gondii,and eventually lead to death of T.gondii.Hence,niclosamide and myrislignan could be considered as potential candidate lead compounds for developing new therapeutic drugs against T.gondii.