Zinc oxide nanoparticles produced by Zingiber officinale ameliorates acute toxoplasmosis-induced pathological and biochemical alterations and reduced parasite burden in mice model

Abstract

Background: Although, approximately 30% of the world's population is estimated to be infected with Toxoplasma gondii (T. gondii) with serious manifestations in immunocompromised patients and pregnant females, the available treatment options for toxoplasmosis are limited with serious side effects. Therefore, it is of great importance to identify novel potent, well tolerated candidates for treatment of toxoplasmosis. The present study aimed to evaluate the effect of Zinc oxide nanoparticles (ZnO NPs) synthesized using Zingiber officinale against acute toxoplasmosis in experimentally infected mice.

Methods: The ethanolic extract of ginger was used to prepare ZnO NPs. The produced ZnO NPs were characterized in terms of structure and morphology using Fourier Transformed Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD), UV- spectroscopy and scanning electron microscopy (SEM). The prepared formula was used in treatment of T. gondii RH virulent strain. Forty animals were divided into four groups, with ten mice per group. The first group was the uninfected, control group. The second group was infected but untreated. The third and the fourth groups received ZnO NPs and Spiramycin orally in a dose of 10 mg/kg and 200 mg/kg/day respectively. The effect of the used formulas on the animals survival rate, parasite burden, liver enzymes -including Alanine transaminase (ALT) and aspartate transaminase (AST)-, nitric oxide (NO) and Catalase antioxidant enzyme (CAT) activity was measured. Moreover, the effect of treatment on histopathological alterations associated with toxoplasmosis was examined.

Results: Mice treated with ZnO NPs showed the longest survival time with significant reduction in the parasite load in the livers and peritoneal fluids of the same group. Moreover, ZnO NPs treatment was associated with a significant reduction in the level of liver enzymes (ALT, AST) and NO and a significant increase in the antioxidant activity of CAT enzyme. SEM examination of tachyzoites from the peritoneal fluid showed marked distortion of T. gondii tachyzoites isolated from mice treated with ZnO NPs in comparison to untreated group. T. gondii induced histopathological alterations in the liver and brain were reversed by ZnO NPs treatment with restoration of normal tissue morphology.

Conclusion: The produced formula showed a good therapeutic potential in treatment of murine toxoplasmosis as demonstrated by prolonged survival rate, reduced parasite burden, improved T. gondii associated liver injury and histopathological alterations. Thus, we assume that the protective effect observed in the current research is attributed to the antioxidant capability of NPs. Based on the results obtained from the current work, we suggest greenly produced ZnO NPs as a chemotherapeutic agent with good therapeutic potential and high levels of safety in the treatment of toxoplasmosis.

Fig 1. Schematic representation for green synthesis of ZnO NPs using ginger ethanolic extract.

Fig 2. UV–Visible spectrum of ZnO NPs prepared using ginger.

Fig 3. FT-IR spectrum of ZnO NPs prepared using ginger.

Fig 4. XRD pattern of the ZnO NPs prepared using ginger.

Fig 5. SEM images of ZnO NPs prepared using ginger showing particle size ranging from 43.3 to 111.1 nm (X 60,000).

Fig 6. Kaplan-Meier overall survival curve for all groups showing the different survival time of untreated and treated animal groups.

Animals treated with ZnO NPs have the longest survival time (10^th dpi) followed by animals treated with Spiramycin (9^th dpi).

Fig 7. SEM examination of Toxoplasma tachyzoites.

(A) Tachyzoites from infected untreated control, revealing crescent shape with apparent conoid (X 15,000). (B) Tachyzoites from ZnO NPs-treated group, showing shrunken tachyzoites losing normal crescent shape with evident loss of its smooth surface, erosion and protrusion of the surface, (X 15,000). (C) Tachyzoites from Spiramycin-treated group, showing shrunken organism losing its smooth conoid shape with furrows and ridges (X 15,000).

Fig 8. ZnO NPs treatment significantly improved liver enzymes in infected mice.

Liver enzymes (AST and ALT) were measured in blood of normal, infected untreated, ZnO NPs treated and Spiramycin treated mice (10 mice/group). Data are expressed as means with error bars representing SD and were analyzed using ANOVA. Asterisk (*) indicates a significant difference in treated groups compared to the infected untreated group (p = 0.001).

Fig 9. ZnO NPs treatment significantly reduced the oxidative stress as indicated by NO level and increased the antioxidant activity of CAT enzyme in infected mice.

NO and CAT were measured in blood of normal, infected untreated, ZnO NPs- and Spiramycin-treated mice (10 mice/group). Data are expressed as means with error bars representing SD and were analyzed using ANOVA. Asterisk (*) indicates a significant difference in the mean level of NO in treated groups compared to the infected untreated group (p = 0.001), and “ns” indicates non-significant difference.

Fig 10. Photomicrograph of H & E stained liver tissue sections of all groups.

(A) Liver section of normal mice showing normal central vein area with normal lining endothelium (circle); normal hepatic cords (thick arrow) consisting of polygonal hepatocytes with large round central light vesicular Uni or binuclear (wave arrow), and Kupffer cells (thin arrow) between hepatic cords and sinusoids. (B, C, D & E) Liver sections of infected untreated group showing serious degenerative and hydropic changes with loss of hepatic tissue normal assembly. The central vein showing severe congestion (circle) with deteriorated endothelium (curved arrow). Hepatocytes disclosed vacuolations (thick arrow), apoptotic nucleus (wave arrow) and lost nucleus (arrow with tail). Hepatic sinusoids showed obvious dilatation (thin arrow), necrotic changes (arrowheads) and edema leading to dispersion between hepatic cords (cube). Aggregated T. gondii tachyzoites are recognized (stars). (F) Liver section of Spiramycin treated group showing central vein congestion and activated macrophages with brown coloration (circle) and deteriorated endothelium (curved arrow). Edema (cube), necrosis (arrowhead), dilated hepatic sinusoids (wave arrow) as well as vacuolated hepatocytes (thick arrow) are still recognized. (G) Liver section of ZnO NPs—treated group demonstrated normal assembly of central vein (circle), hepatocytes (thick arrow), and hepatic sinusoids (wave arrow) with few necrotic foci (arrowhead).

Fig 11. Photomicrograph of H & E stained brain tissue sections of all groups.

(A) Brain section of normal group showing normal architecture of neuron (thick arrow), neuroglia cells (wave arrow), blood capillaries (circle) as well as neuropil (thin arrow). (B, C, D & E) Brain sections of infected untreated group showing severe degenerative changes with aggregated T. gondii tachyzoites under meninges (cube), neurons with vacuolated apoptotic nucleus (thick arrow) or lost nucleus (wave arrow), apoptotic and vacuolated neuroglia cells (curved arrow), dilated capillaries (circle), gliosis, edema leading to dispersion between neurofibrils (star), and vacuolations along neuropil (arrowhead). (F) Brain section of Spiramycin treated group showing apoptotic vacuolated neurons (thick arrow), apoptotic neuroglia cells (curved arrows), edema (wave arrow), necrotic areas (arrowhead), and dilated capillaries (circle). (G) Brain section of ZnO NPs treated group showing marked improvement with normal appearance of neurons (thick arrow) and blood capillaries (thick arrow). Neuroglia cells detected in regular form (arrowhead) and vacuolated apoptotic one (thin arrow). Edema and vacuolated neuropil (wave arrow) were still observed.