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Abstract One of a neglected parasitic disease is CE caused by E. granulosus. It has huge socio-economic impact with a worldwide geographical distribution.The available treatments are surgery and chemotherapy with ABZ, MBZ or other antihelmintic drugs. Chemotherapeutic options for echinococcosis are extremely limited and success with ABZ treatment (the drug of choice) is not guaranteed, therefore, new drugs are needed. Among several metal NPs, Ag NPs was found nontoxic substance with satisfactory antimicrobial efficacy against viruses, bacteria and parasites. Moreover, it was evaluated as a suitable carrier of various therapeutic drugs. Another way of treatment is the herbal extracts. One of these herbs is Punica granatum which is well-known for many therapeutic and pharmaceutical effects such as anti-cestoidal, antinematoidal and anti-protozoan, antioxidants and anti-inflammatory substance that contain polyphenols and tannins. The present study was conducted in order to evaluate the effects of Ag NPs and PGE in comparison with ABZ on experimentally infected mice with E. granulosus. The therapeutic effects of these drugs on hydatid cyst were assessed through parasitological, histopathological, TEM ultrastructural studies. Also, serological studies by detection of IFN-γ in mouse serum samples by ELISA and liver enzymes were assessed. Summary and Conclusion 149 The study was carried out on 125 pathogen-free laboratory bred female albino mice and the experimental animals were divided into the following seven groups: group I (GI): Five mice were served as uninfected and untreated group (negative control). group II (GII): Twenty mice served as infected untreated group (positive control). group III (GIII): Twenty mice served as ABZ-treated group. The treatment was started three months p.i. ABZ was given at a dose of 200mg/Kg/day for five consecutive days per week orally for two months. group IV (GIV): Twenty mice served as Ag NPs treated group. The treatment was started three months p.i. Ag NPs suspention was given at a dose of 25mg/Kg/day for five consecutive days per week orally for two months. group V (GV): Twenty mice served as ABZ coated with Ag NPs treated group. The treatment was started three months p.i. ABZ coated with Ag NPs was given at a dose of 100mg/Kg/day for five consecutive days per week orally for two months. group VI (GVI): Twenty mice served as PGE treated group. Treatment was started two days p.i and the extract was given at a dose of 0.65 g/kg/day orally for three months. group VII (GVII): Twenty mice served as combined ABZ with PGE- treated group. Treatment was started three month p.i. The drugs were given at a dose of 200mg/Kg/day of ABZ and 0.65 g/kg/day of PGE for five consecutive days per week orally for two months. Summary and Conclusion 150 The experiment was terminated five months p.i. All mice overcoming the infection and treatment period were subjected to evaluate parasitological, liver histopathological on hydatid cyst using TEM and also detection of IFN-γ in mouse serum samples by ELISA and assessment of ALT, AST and ALP liver enzymes activities were performed. Concerning mortality rates of experimental animals among the different studied groups, the highest mortality rate was reported in group II (infected not treated group) while the lowest mortality rate was recorded in all the treated groups. Concerning hydatid cyst sizes, weights and efficacy rates of treatments results, there were significant differences between the infected treated groups (GIII, GIV, GV, GVI and GVII) and the infected untreated group (GII) (P <0.001). The biggest hydatid cyst size and weight (5.14 ± 1.75 mm, 0.78 ± 0.08 g) were detected in the infected control group (GII). All the treated groups showed significant reductions; however, the smallest values were detected in ABZ-loaded nanoparticles-treated group (GV)(0.39 ± 0.48mm, 0.11 ± 0.14 g) followed by 0.48 ± 0.47 mm, 0.17 ± 0.18 g which were detected in PGE+ABZ (GVII). Accordingly, the least efficacy rate of 22.9% was obtained with ABZ while the highest efficacy rate of 63.9% was obtained with ABZ-loaded nanoparticles followed by the efficacy rate of PGE+ABZ (GVII) (56.2%). Summary and Conclusion 151 The histopathological study revealed the occurrence of granulomatous reactions (Table 5,oFigo34) associated with liver fibrosis (Table 6, Fig 35), inflammatory infiltration (Table 7, Fig 36), congestion (Table 8, Fig 37), liver cell necrosis (Table 9, Fig 38), apoptosis (Table 10, Fig 39) and steatosis (Table 11, Fig 40) mainly in the infected control group and variable degrees of improvements were found in the treated groups. The results revealed improvements of the granulomatous reactions sizes, fibrosis, congestion and the inflammatory infiltrates in all the treated groups. However, free nanosilver was slightly better than free ABZ and the best results were obtained with ABZ-loaded nanosilver treatment followed by PGE+ABZ treatment. Moreover, liver cell necrosis and steatosis were increased with free ABZ treatment denoting a hepatotoxic effect of this drug. Conversely, improvements were observed with PGE (GVI) due to its hepatoprotective effect followed by ABZ-loaded nanoparticles (GV) indicating the potential safety of AgNPs in vivo and decreasing ABZ toxic effects. Concerning the results of TEM, the ultrastructure of the hydatid cysts obtained from GII revealed intact germinal layer with microtriches projecting into the laminated layer. Undifferentiated cells with intact nuclear membranes and non-distorted nucleus were also seen. The cytoplasm of these cells was intact and rich in glycogen granules.The hydatid cysts from other treated groups showed slightly vacuolated cytoplasm, glycogen depletion and heterochromatin (pyknosis) in the nucleus. The hydatid cysts retrieved from AgNPs and ABZnanoparticle- treated groups showed destruction of the germinal layer with the loss of microtriches, separation from the laminated layer and depletion of glycogen. The nanosilver particles appeared as black rods inside the nuclei which showed fragmentations. Summary and Conclusion 152 The cytoplasm showed distortion of the mitochondria, glycogen depletion and autophagosomes in vacuolated cytoplasm.These changes were more obvious with ABZ-loaded nanoparticles and PGE+ABZ treatment. In the present study, there was a significant difference (P <0.0001) between the studied groups regarding serum values of IFN-γ. There were significant increase in the sera IFN-γ in all groups when they were compared with the normal control group (GI). The highest significant increase was in ABZ-treated group when it was compared with GI and GII. Interestingly, with nanosilver treatment either alone (GIV) or combined with ABZ (GV) and with PGE treatment either alone (GVI) or combined with ABZ (GVII), there were significant reductions in serum IFN-γ when they were compared with the infected control (GII) and ABZ-treated (GIII) groups. Moreover, the most significant decrease was in ABZ-loaded nanoparticles-treated group (GV) followed by PGE+ABZ treated group (GVII). Regarding liver enzymes, there were significant increases (P < 0.001) of the serum levels of ALT, AST and ALT between the studied groups. In ABZ-treated group, there were increases in the levels of ALT, AST and ALP when they were compared with GI. An obvious improvement of liver enzymes was found in ABZ-loaded Ag NPs (GV), PGE (GVI) and PGE+ABZ -treated groups (GVII) when it was compared with the infected control (GII); however, the values were still significantly higher than the normal control group (GI). Summary and Conclusion 153 In conclusion, this study demonstrated that ABZ-loaded nanoparticles showed the best drug efficacy on experimental echinococcosis with significant reduction of hydatid cyst size, weight and improvement of the histopathological changes with less hepatic injury in comparison with ABZ. This was followed by the results obtained with combined treatment (PGE+ABZ). Therefore, they could be considered as new potential therapeutic options against E. granulosus infection instead of using ABZ alone, however, further studies should be done to evaluate these treatments from other aspects. |