In Vivo Evaluation Of The Efficacy Of Sophora Moorcroftiana Alkaloids?E2a? In Combination With Bacillus Calmette–Guérin?BCG? And Nano Drug PLGA-Fe₃O₄-E2a Treatment For Echinococcosis In Mice

Background: Human cystic echinococcosis is a widespread,chronic,endemic,helminthic zoonosis caused by larval forms?metacestodes?of tapeworms of the genus Echinococcus and it is a major global health problem to date.Although surgical treatment is the primary option,all patients need drug treatment before or after surgery.For some patients for whom surgery is contraindicated,chemotherapy is the only modality of treatment.The main clinical drugs for echinococcosis are ABZ and MBZ.However,these drugs can cause severe adverse reactions.Therefore,the identification of novel and efficient treatment options is essential for the treatment of CE.Sophora moorcroftiana is a folk-medicinal shrub endemic to the wide valleys and middle reaches of several main tributaries of the Yalu Tsangbo River in Tibet,China.In Chinese folk medicine the decoction of the seeds is used as an anti-inflammatory,detoxicant,antiemetic and antibacterial medicine.Bacillus Calmette–Guérin?BCG?is composed of attenuated mycobacteria and is used as an effective vaccine to prevent tuberculosis.BCG contains many immune stimuli to improve the Th1-type cellular immune responses against tumours and parasites.However,few studies have evaluated combined therapy with S.moorcroftiana alkaloids and BCG against echinococcosis.In this study,we evaluated the efficacy of the combined treatment with water-soluble alkaloids and BCG in vivo.Methods: Each NIH mouse was inoculated intraperitoneally with approximately 5,000 Echinococcus granulosus progenitors to establish a secondary mouse hydatidosis model.After 20 weeks of secondary infection with protoscoleces,mice were randomly allocated to five groups and treated for 6 weeks by daily intragastric administration of albendazole?ABZ,100 mg/kg?,E2a?100 mg/kg?,BCG?abdominal subcutaneous injection at 5 × 106 CFU?,E2 a + BCG?100 mg/kg E2 a and 5 × 106 CFU BCG?or normal saline?untreated group?,respectively.After 6 weeks treatment,mice were euthanized and tested.The mice were cyst-weighed to calculate the inhibition rate of the drug on the growth of hydatid cysts,hematoxylin and eosin staining of the liver,kidneys and cysts.The microstructure of mouse cysts was observed under a scanning electron microscope.The level of IL-4 and IFN-? in serum was detected by ELISA.Flowcytometry was used to detect splenic T lymphocyte subsets.Results: After six weeks treatment,The efficacy was determined in vivo by measuring the wet weights of hydatid cysts in infected mice.The treatment with E2 a + BCG?the hydatid cyst weight: 2.7 ± 1.3 g,the inhibition rate of hydatid cysts: 76.1%?showed an additive effect in decreasing the weightof hydatid cysts.The results indicated a significant reduction in the weight of hydatid cysts in the E2 a + BCG group compared with the E2a?8.4±3.3g,25.7%?and BCG?8.3±2.5g,26.6%?groups,respectively.However,there was no significant difference in the weight of hydatid cysts between the ABZ and E2 a +BCG?p > 0.05?groups.The wall tissues of hydatid cysts after 6 weeks of treatment with E2 a + BCG mice were examined by microscopy.the stratum corneum of hydatid cysts in mice was thin and the germinal membrane was degenerated and obviously ruptured.The cells of the germinal membrane of hydatid cysts in mice treated with E2 a + BCG were condensed and disintegrated.Scanning electron microscopy revealed that the germinal layer cells of the cysts of mice in the E2 a + BCG group were severely detached,and the cell structure lost its integrity.Compared with the untreated control,the E2 a + BCG group showed a non-significant increase in serum interleukin-4?IL-4?.Furthermore,the serum levels of interferon-??IFN-??between the untreated and E2 a + BCG groups were not statistically different.Compared with the untreated group?14.74%±9.39%,50.33%±27.91%?,the frequency of CD3+ T cells and CD3+CD4+ T cells in the E2a+BCG group?41.71%±16.58%,72.17%±3.80%?were significantly higher?p=0.048,p=0.012?.There was no significant difference in the proportion of CD3+CD8+ T cells between the groups?p > 0.05?.Compared with the untreated group?27.51%±11.48%,12.59%±6.35%,2.77%±1.86%?,the frequency of PD-1+ T cells,CD4+PD-1+ T cells and CD8+PD-1+ T cells in E2a+BCG group?7.91%±4.10%,6.20%±2.93%,1.11%±0.86?%)significantly decreased?p=0.000,p = 0.018,p = 0.019?.Conclusion: The water-soluble alkaloid E2 a combined with BCG from the seeds of sand pods has the effect of treating secondary hydatid disease in mice.E2 a combined with BCG anti-enkephalin can damage vesicle tissue,induce CD4+ T cell increase,and down-regulate T cell surface negative.Modulates the expression of the molecule PD-1 and promotes cellular immune function in mice so as to achieve the purpose of treating hydatidosis.Background: Magnetic targeting therapeutic agents?either drugs or genes?are attached to magnetic particles,which are then injected directly into the bloodstream.A strong,permanent?generally rare earth?magnet is positioned above the target site outside the body.As the particles,carrying the therapeutic agent,pass below the magnet,the magnetic field captures them and pulls them towards the field source.The unique features of magnetic Fe₃O₄ nanoparticles?eg,superparamagnetic and good biocompatibility?and low toxicity,as well as proper surface modification and conjugated targeting of ligands/molecules have become the main research focus in drug delivery applications.Sophora moorcroftiana is a folk-medicinal shrub endemic to the wide valleys and middle reaches of several main tributaries of the Yalu Tsangbo River in Tibet,China.In Chinese folk medicine the decoction of the seeds is used as an anti-inflammatory,detoxicant,antiemetic and antibacterial medicine.Therefore,in this study,we applied the magnetic targeting properties of magnetic Fe₃O₄ nanoparticles and the water-soluble alkaloid?E2a?to prepare PLGA-Fe₃O₄-E2 a medicines and establish secondary disease model mouse of AE.Preliminary evaluation of nanoparticle PLGA-Fe₃O₄-E2 a drug against alveolar echinococcosis.Methods: Polylactic-co-glycolic acid?PLGA?was used as carrier material and loaded with magnetic Fe₃O₄ nanoparticles and water-soluble alkaloid?E2a?.The PLGA-Fe₃O₄-E2 a particles were prepared via single emulsion/solvent evaporation method.We characterized the physical properties of the PLGA-Fe₃O₄-E2 a carrier,such as size,morphology,entrapment rate,drug loading rate,drug release and targeting etc.BALB/c mice were intraperitoneally inoculated with echinococcus multilocularis to establish a AE model.After 14 weeks secondary infection with protoscoleces,mice were treated for 6 weeks by daily intragastric administration of albendazole?ABZ,100 mg/kg?,E2a?100 mg/kg?,PLGA?Intraperitoneal injection 617 mg/kg/3day?,E2a?Intraperitoneal injection 50mg/kg/3day?,PLGA-E2a?Intraperitoneal injection 667mg/kg/3day?PLGA-Fe₃O₄-E2a?Intraperitoneal injection 1163 mg /kg /3day?or normal saline?untreated group?,respectively.After 6 weeks treatment,mice were euthanized and tested.The mice were cyst-weighed to calculate the inhibition rate of the drug on the growth of hydatid cysts,hematoxylin and eosin staining of the liver,kidneys and cysts.Results: Characterization of PLGA-Fe₃O₄-E2 a particles: The PLGA-Fe₃O₄-E2 a particleswere spherical and well-dispersed particles,with a diameter of 2315 nm.Entrapment rate and drug-loading rate of the PLGA-Fe₃O₄-E2 a particles was 58.1% and 4.3%.The preparation method was proved duplicable.Magnetic drug PLGA-Fe₃O₄-E2 a microspheres in PBS solution release rate of 7.08 % at 10 h.The drug release rate was 50% after 168 hours and stabilized at 53%afterwards.Under the action of external magnetic field,the magnetic nano drug PLGA-Fe₃O₄-E2 a can be affected by the magnetic field to generate the migration.After six weeks treatment,The efficacy was determined in vivo by measuring the wet weights of hydatid cysts in infected mice.Compared with the wet weight of mice in the untreated group?5.1±0.6g?,E2a?3.6±0.2g?,PLGA-E2a?2.9±0.5g?,PLGA-Fe₃O₄-E2a?2.7±0.2?and Albendazole treatment groups?2.6±0.3?showed an additive effect in decreasing the weightof hydatid cysts?p<0.05?.There was no difference in the wet weight between mice in each treatment group?p> 0.05?.The inhibitory rates of hydatid cysts in the treatment group were 29.4%,43.1 %,47.1% and 49.0% in the E2 a,PLGA-E2 a,PLGA-Fe₃O₄-E2 a and ABZ groups,respectively.In the treatment group,the stratum corneum of the mice's cyst wall became loose and fractured,the cells in the germinal layer decreased,the structure of the cyst changed seriously and showed a pathological change in morphology.Conclusion: The successful acquisition of magnetic PLGA-Fe₃O₄-E2 a particles provides an effective carrier for the study of the active alkaloid E2 a,a targeted drug in the in vivo and in vitro studies.The drug can damage secondary AE by injuring cysts.