| Background:Alveolar Echinococcosis（AE）is a zoonotic parasitic disease caused by the larval stage of Echinococcus multilocularis（E.multilocularis）.The lesion of AE presents tumor-like invasion in the liver,which to be called"worm cancer".In AE patients,the Th1 immune response induced by metacestodes is beneficial to inhibit the proliferation of parasites.However,the Th2/Treg（Regulatory T cell,Treg）-dominant immune response existed in the chronic infection of AE,which becomes an important obstacle to parasite clearance.In recent years,the incidence of AE is increasing in China,but only albendazole and mebendazole have been approved for clinical use in humans,which led to the drug resistance and adverse side effects.Therefore,it is urgent to develop new drugs.Sophora moorcroftiana（S.moorcroftiana）is a unique plant in Tibet Plateau,which is characteristic of bitter and cold,and used to treat parasitic diseases,diphtheria and dyspepsia.In our previous studies,we found that the crude lipophilic alkaloids from S.moorcroftiana seeds can kill protoscolex in vitro and inhibit the growth of hydatid cyst in Echinococcus granulosus-infected mice,suggesting that the alkaloids contain the anthelmintic components.Due to the poor water-solubility as well as the weak anti-insect effect of lipophilic alkaloids,we extracted the water-soluble alkaloids from S.moorcorftiana seeds by refluxed extraction and the effective anti-insect component E2-a screened out.The aim of the research is to investigate the therapeutic effects of E2-a against alveolar echinococcosis,and explore the underlying molecular mechanism of downregulating Treg in vitro.Methods:In this study,metacestodes from E.multilocularis-infected mice were taken out under axenic conditions,and used to establish the system for the long-term in vitro cultivation of E.multilocularis metacestodes,that in the presence of Human hepatocarcinoma（Hep G2）cells.Up to 2 months,E.multilocularis metacestode vesicles were generated by the system and harvested.The vesicles were finally distributed to 24-well plate as 10 per well and E2-a treatments were performed.The following experiments were carried out.The morphology of vesicles was observed daily by stereomicroscope,and the treated vesciles were collected for routine H＆E staining.The ultrastructural changes of vesicles were observed by scanning electron microscopy（SEM）.The supernatant of vesicle culture was collected to detect alkaline phosphatase（ALP）activity.The treated vesicles were injected into BALB/c mice,which were routinely fed for 17 and 25 weeks to count the number of cysts.The emras,emmpk1 and emsmad D m RNA levels of the germinal layer cells were detected by RT-PCR.Two hundred vesicles,which collected after 12 months,cultured without feeder cells for five days to remove the cells.At the end of culture,the supernatant was collected as Excretory/secretory products（ES）,and the liquid in the vesicle was drawn as Vesicle Fluid（VF）,which were stored at-80℃.The spleen cells separated from BALB/c mice,and stimulated with anti-CD3,anti-CD28,IL-2 and TGF-βto induce the Treg cells.The groups were set as SP group:only spleen cells;SP+STIM（Stimulation,STIM）group:splenocytes+anti-CD3+anti-CD28+IL-2+TGF-β;SP+STIM（TGF-βnegative）group:splenocytes+anti-CD3+anti-CD28+IL-2.On the basis of SP+STIM（TGF-βnegative）group,the different concentrations of ES and VF were added respectively,and the expression of CD4,CD25 and Foxp3 was detected by flow cytometry（FCM）to screen out the optimal intervention concentration of ES and VF during the differentiation of spleen cells into Treg cells.According to the above results,the optimal concentration of ES and VF was added to SP+STIM group,respectively.The ES+E2-a group,VF+E2-a gruop and E2-a group were set,to evaluate the effects of E2-a on the differentiation of spleen cells into CD4+CD25+Foxp3+Treg cells under the stimulation of ES or VF.Based on the results of FCM,the protein expression levels of p-Smad2,p-STAT3and p-m TOR was detected by Western Blot,and ELISA was used to detect the level of IL-10 in the supernatent of the culture.Results:1.The effects and mechanism of water-soluble alkaloid E2-a from S.moorcorftiana seeds against E.multilocularis in vitro:（1）Stereomicroscope results showed that the vesicles in untreated group were no wrinkle or rupture during the experimental process,and the culture medium was clear.At the end of culture,the culture medium of 1 mg/m L E2-a group was slightly cloudy compared with untreated group,and most of the vesicles were slightly shrinke.In 2 mg/m L E2-a group and 4mg/m L E2-a group,the vesicles were shrink severely,releasing the contents.The vesicles were observed by light microscope after H＆E staining.The untreated group showed a typical structure:continuous and complete germinal layer,which tightly attached to the laminated layer.In E2-a（4 mg/m L）group,the germinal layer was detached from laminated layer partly,accompanying the reduction of cell number.SEM showed that the untreated group exhibited an intact typical structure with complete and smooth germinal cells,without missing or shrinking;the germinal cells were found shrinked,arranged irregularly and missing mostly in E2-a（4 mg/m L）group.（2）ALP activity showed the ALP in supernatant was no significant difference between untreated group and E2-a group（P>0.05）.（3）In viability test,at week 17,a large number of vesicles can be observsed in the livers and intestines of untreated group.In E2-a group,vesicles could be observed in intestines of mice;the total number of vesicles was lower in E2-a group（4.4±2.0）than untreated group（11.2±3.7）（P<0.01）.At week 25,compared with the total number of vesicles in untreated group（8.0±2.67）,there was no significant difference in E2-a group（10.2±1.7）（P>0.05）.（4）The emras,emmpk1 and emsmad D m RNA levels had no significant difference between untreated group and E2-a group（P>0.05）.2.The effects and molecular mechanism of water-soluble alkaloid E2-a from S.moorcorftiana seeds to downregulate Treg cells in vitro:（1）FCM was used to detect the CD4+CD25+Foxp3+Treg cells.The results showed that（1）The percentage of Treg cells in both SP+STIM（TGF-βnegative）+100μg/m L ES group and SP+STIM（TGF-βnegative）+100μg/m L VF group decreased significantly,compared to those in the SP+STIM（TGF-βnegative）group（P<0.001）.（2）The percentage of Treg cells in SP+STIM+ES group and SP+STIM+VF group decreased significantly when comparing with SP+STIM group（P<0.05）;Compared with SP+STIM+ES group,the percentage of Treg cells in SP+STIM+ES+E2-a group was significantly decreased（P<0.05）.The percentage of Treg cells in SP+STIM+VF+E2-a group showed a decreased trend,when comparing with SP+STIM+VF group,but there was no statistical significance（P>0.05）.（2）Western Blot results showed that the spleen cells treated E2-a after the stimulation of 100μg/m L ES,compared with SP+STIM group,the protein expression levels of p-Smad2（P<0.001）,p-STAT3（P<0.001）and p-m TOR（P<0.05）all decreased in SP+STIM+ES+E2-a group.The level of IL-10 in the supernatent of the culture was significantly decreased in SP+STIM+ES+E2-a group,comparing with SP+STIM group（P<0.001）.Conclusions:1.The water-soluble alkaloid E2-a from S.moorcroftiana seeds has an obvious anti-hydatid effect,without down-regulating the emras and emmpk1on Ras Raf-MEK-ERK MAP kinase cascade signaling pathway and emsmad D on TGF-β/BMP signaling pathway of germinal cells.2.The water-soluble alkaloid E2-a from S.moorcroftiana seeds treated spleen cells after the stimulation of 100μg/m L ES could inhibit the percentage of Treg cells in vitro,which maybe associated with down-regulating the protein expression levels of p-Smad2,p-STAT3 and p-m TOR. |
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