Apocynin, an NADPH Oxidase Enzyme Inhibitor, Prevents Amebic Liver Abscess in Hamster

Abstract

Amebiasis is an intestinal infection caused by Entamoeba histolytica. Amebic liver abscess (ALA) is the most common extraintestinal complication of amebiasis. In animal models of ALA, neutrophils have been shown to be the first cells to come into contact with Entamoeba histolytica during the initial phase of ALA. One of the multiple mechanisms by which neutrophils exhibit amebicidal activity is through reactive oxygen species (ROS) and the enzyme NADPH oxidase (NOX2), which generates and transports electrons to subsequently reduce molecular oxygen into superoxide anion. Previous reports have shown that ROS release in the susceptible animal species (hamster) is mainly stimulated by the pathogen, in turn provoking such an exacerbated inflammatory reaction that it is unable to be controlled and results in the death of the animal model. Apocynin is a natural inhibitor of NADPH oxidase. No information is available on the role of NOX in the evolution of ALA in the hamster, a susceptible model. Our study showed that administration of a selective NADPH oxidase 2 (NOX2) enzyme inhibitor significantly decreases the percentage of ALA, the size of inflammatory foci, the number of neutrophils, and NOX activity indicated by the reduction in superoxide anion (O₂^-) production. Moreover, in vitro, the apocynin damages amoebae. Our results showed that apocynin administration induces a decrease in the activity of NOX that could favor a decrease in ALA progression.

Keywords: Entamoeba histolytica; NADPH oxidase; NOX2; amebic liver abscess; apocynin; hamster; neutrophil.

Figure 1

Scheme of control and experimental groups to evaluate the effect of apocynin on ALA development in hamsters. Hamsters weighing approximately 100 g were divided into two groups, one inoculated intrahepatically with 1 × 10⁶ amoebae and the other group intrahepatically administered with apocynin at 1.64 mg/100 g of body weight every 8 h and subsequently inoculated with amoebas. Animals were sacrificed at 3, 6 or 12 h post-amoeba inoculation.

Figure 2

Effects of apocynin on the evolution of ALA in hamsters inoculated with E. histolytica trophozoites. Macroscopic aspect of ALA in hamsters untreated or treated with apocynin (1.64 mg/100 g) at 3, 6 and 12 h of ALA evolution. Arrowheads indicate the lesion.

Figure 3

The NOX inhibitor decreased the ALA percentage. Hamsters were treated with 1.64 mg/100 g of apocynin, followed by amoeba inoculation, and sacrificed at 3, 6 or 12 h post-inoculation. Percentage of lesion was obtained for untreated hamsters (black bar) and hamsters treated with apocynin (gray bar). Statistical analysis was performed using the one-way ANOVA test; the comparison between groups was performed according to the Bonferroni correction. Asterisks represent statistically significant differences between groups; **** p ≤ 0.0001. Data represent mean ± SD.

Figure 4

Histology of ALA in hamsters untreated or treated with apocynin. H&E staining of representative ALA at 3, 6 and 12 h of evolution. ALA of apocynin untreated hamsters at 3 h, with multiple inflammatory foci (arrows) and presence of amoebae (arrowheads) observed. At 6 h, a large inflammatory area with presence of acute cells is observed (arrows), and at 12 h, several inflammatory infiltrates are observed (arrows). ALA of apocynin-treated animals: at 3 h, scarce acute inflammatory cells (arrow), and at 6 and 12 h, small inflammatory infiltrate at hepatic parenchyma, are observed. Bar = 50 µm.

Figure 5

Size of inflammatory foci in ALA. ALA samples were processed to determine the sizes of inflammatory infiltrates at 3, 6 and 12 h post-inoculation in ALA of hamsters untreated or treated with apocynin. Statistical analysis was performed by one-way ANOVA test, and comparison between groups was performed according to the Bonferroni correction. Asterisks indicate statistically significant differences between groups; **** p ≤ 0.0001. Data are presented as mean ± SD.

Figure 6

Decrease in the number of AS-D-chloroacetate esterase-positive neutrophils in apocynin-treated and untreated hamsters. Neutrophils identified by AS-D chloroacetate esterase-positive staining were quantified at different times (3, 6 and 12 h post-inoculation) in the inflammatory infiltrates of animals inoculated with amoebae and apocynin-treated or untreated. Quantification was performed using NIS-Elements BR software version 4.3 and Nikon microscope (Nikon, Eclipse Ci-S, Tokyo, Japan) at 40× magnification. Statistical analysis was performed by one-way ANOVA test, and between-group comparison was performed according to Bonferroni correction. Asterisks indicate statistically significant differences between groups; **** p ≤ 0.0001. Data are presented as mean ± SD.

Figure 7

Liver tissue was processed by immunohistochemistry to detect the presence of NOX2 in hamsters inoculated with amoebae and untreated or treated with apocynin at 3, 6 and 12 h post-inoculation. Positive labeling for NOX2 was observed. Barr = 50 µm.

Figure 8

Determination of NADP/NADPH (pmol/μL) in ALA in hamsters treated and untreated with apocynin. Asterisks indicate statistically significant differences between the two groups; **** p ≤ 0.0001. Data represent mean ± SD.

Figure 9

Determination of O₂⁻ production (U/g) in the ALA from hamsters treated with apocynin and untreated. Data represent the mean ± SD of three independent experiments. p values were determined by one-way ANOVA (**** p < 0.0001), comparing ALA with the APO group.

Figure 10

Viability of E. histolytica trophozoites at different concentrations of apocynin. The number of damaged amoebae incubated at different concentrations of apocynin (30 and 300 µM) evaluated at 3, 6 and 12 h by trypan blue (0.4%) exclusion dye staining. Data represent mean ± SD (quadruplicate). p values were determined with ANOVA (**** p < 0.0001) compared to the control group (Ctrl).