关键词: 弓形虫, 苯酰甲硝唑, 抗虫活性, 小鼠模型, 组织病理学

Abstract:

Objective To systematically evaluate the anti-Toxoplasma gondii activity and safety of benzoyl metronidazole (BMZ). Methods Vero cells were treated with BMZ at different concentrations (50, 100, 200, 400, 500, 600, 800, 1 000 μg/mL), and the cytotoxicity of BMZ to Vero cells was detected by cell counting kit-8 (CCK-8) assay. After inoculation with Toxoplasma tachyzoites, Vero cells were treated with BMZ at the above concentrations, and the killing effect of BMZ on Toxoplasma was determined by CCK-8 assay. Vero cells inoculated with Toxoplasma tachyzoites were treated with 50 μg/mL BMZ, and the quantitative and morphological changes of Toxoplasma tachyzoites were observed via Giemsa staining and scanning electron microscopy. Kunming mice were randomly divided into low-dose BMZ group (LD-BMZ group), medium-dose BMZ group (MD-BMZ group), high-dose BMZ group (HD-BMZ group), sulfadiazine sodium group (SD-Na group) and negative control group (NC group). All mice were intraperitoneally inoculated with 1 × 10⁴ Toxoplasma tachyzoites per mL. Four hours after infection, mice were intragastrically administered continuously for 5 days: the LD-BMZ, MD-BMZ and HD-BMZ groups received BMZ at final doses of 12.5, 25 and 50 mg/kg, respectively, the SD-Na group received SD-Na at a final dose of 50 mg/kg, and the NC group received an equal volume of corn oil. On day 7 post-infection, the survival rates of mice were recorded, and the parasite load in mice ascites was counted using a hemocytometer. The heart, liver, spleen, lung, kidney and brain tissues of mice in the MD-BMZ group and NC group were collected. After DNA extraction, qPCR was performed to detect the parasite load in each tissue, and the tissues were processed into pathological sections for HE staining to observe the histopathological changes. GraphPad Prism 9 software was used for data analysis and plotting, and independent sample t-test or one-way ANOVA was adopted for significance analysis. Results The cytotoxicity assay showed that the CC₅₀ of BMZ to Vero cells was 155.67 μg/mL; the cell viability was (108.78 ± 1.00)% when BMZ concentration was 50 μg/mL, and decreased to (81.22 ± 3.34)% at 100 μg/mL. Therefore, 50 μg/mL was determined as the maximum safe concentration of BMZ. The in vitro anti-parasitic assay showed that BMZ could inhibit the proliferation of intracellular Toxoplasma tachyzoites, and the inhibition rate increased with the elevation of BMZ concentration; the inhibition rate of BMZ against Toxoplasma reached 14.3% at 50 μg/mL. Giemsa staining results showed that the number of Toxoplasma tachyzoites in cells of the BMZ group was reduced. Scanning electron microscopy observation showed that normal Toxoplasma tachyzoites presented typical spindle or crescent structures with complete contours, plump morphology and smooth surface; while after BMZ treatment, the structural integrity of Toxoplasma was completely lost, with blurred contours and destroyed cell membrane. The in vivo anti-parasitic assay showed that on day 7 post Toxoplasma infection, the survival rates of mice in the LD-BMZ, MD-BMZ, HD-BMZ, SD-Na and NC groups were 2/10, 3/10, 2/10, 2/10 and 1/10, respectively, with no statistically significant difference (χ² = 3.19, P > 0.05); the ascites parasite loads were (15.00 ± 5.00) × 10⁴, (6.67 ± 2.89) × 10⁴, (6.67 ± 2.89) × 10⁴, (8.33 ± 2.89) × 10⁴ and (1 766.67 ± 2.33) × 10⁴ parasites per mL respectively, with statistically significant difference [F(4,10) = 123 320, P < 0.01]. The qPCR results showed that on day 7 post infection, the relative Toxoplasma parasite loads in the heart, liver, spleen, lung, kidney and brain of mice in the MD-BMZ group were 2.32 ± 0.85, 2.32 ± 0.76, 1.00 ± 0.45, 1.00 ± 0.52, 4.46 ± 0.98 and 1.59 ± 0.63 respectively, all significantly lower than 7.28 ± 1.25, 7.11 ± 1.18, 3.46 ± 0.95, 7.28 ± 1.22, 9.37 ± 1.36 and 8.67 ± 1.29 in the NC group (t = 6.56, 6.83, 4.68, 9.47, 5.86, 9.86, all P < 0.01). HE staining showed that BMZ treatment significantly reduced the inflammatory cell infiltration and granulomatous lesions in the spleen, kidney and liver, without causing obvious drug-related damage. Conclusion BMZ exhibited excellent anti-Toxoplasma activity both in vivo and in vitro, with superior efficacy to SD-Na and high safety at the medium dose.

Key words: Toxoplasma gondii, Benzoyl metronidazole, Anti-parasitic activity, Mouse model, Histopathology