TUNEL assays in resveratrol-treated trophozoites.

<p><b>A)</b> TUNEL assays of untreated or IC₅₀ resveratrol-treated trophozoites using dUTP-FITC and visualized under the laser confocal microscope. Nuclei were stained with DAPI. Squares in merge images were magnified in the zoom panels. <b>B)</b> Flow cytometry analysis of dUTP-FITC-treated trophozoites. 0.4% ethanol- and 0.5 mM H₂O₂-treated trophozoites were used as controls.</p

Effect of resveratrol administration in hamsters intraportally inoculated with virulent trophozoites.

<p><b>A)</b> Healthy livers: Liver of animals not inoculated with trophozoites B) Ethanol: Livers of intraportally inoculated hamsters (3 x 10⁶ virulent trophozoites), treated with 50 μl of ethanol. Damage after 4 days: Livers of animals inoculated with virulent trophozoites and examined four days after challenge. Resveratrol 2d before and 10 d after challenge: Livers of animals treated with resveratrol (100 mg/Kg diluted in 50 μl of ethanol) each 8 h (2 days before and 10 days after inoculation) and examined ten days after challenge. Resveratrol after 4 d challenge: Liver of animals treated with resveratrol, as above, for ten days, starting four days after challenge when abscesses were already formed <b>B)</b> Damage was evaluated as the weight of the abscesses formed divided by the weight of the whole liver, before the injured areas were removed. As a negative control, animals were not inoculated with trophozoites (healthy liver). As a pharmacological control hamsters were treated with 20 mg/Kg of metronidazole. Values represent the mean ± standard error of liver damage in inoculated animals. n = 7. ***p<0.001.</p

Immunochemistry of livers from hamsters inoculated with virulent trophozoites.

<p>Paraffin sections of livers from hamsters treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146287#pone.0146287.g010" target="_blank">Fig 10</a>. <b>A)</b> Non-challenged hamsters (healthy liver). <b>B)</b> Challenged hamsters treated with ethanol. <b>C)</b> Challenged hamsters treated with resveratrol that did no develop hepatic abscesses. <b>D)</b> Challenged hamsters treated with resveratrol that develop small abscesses. <b>E)</b> Challenged hamsters treated with metronidazole. <b>F)</b> Challenged hamsters treated with ethanol and developed only with the pre-immune serum. <b>G)</b> Parasitic burden quantified in 15 sections of livers from hamsters treated as above.</p

PS externalization produced by resveratrol in trophozoites.

<p><b>A)</b> Confocal microscopy of untreated or IC₅₀ resveratrol-treated trophozoites incubated with Annexin V-FITC and PI. Merge: fluorescence channels and phase contrast. <b>B)</b> Flow cytometry analysis of trophozoites incubated with Annexin V-FITC and PI. 0.4% ethanol- and 0.5 mM H₂O₂-treated trophozoites were used as controls. Q1: Trophozoite scheme with nucleus stained (red) representing entrance of PI. Q2: Trophozoite scheme representing PI stained (nucleus) and Annexin V plasma membrane stained. Q3: Trophozoite scheme representing plasma membrane integrity. Q4: Trophozoite scheme representing Annexin V (loss of plasma membrane asymmetry) stained without nucleus stained.</p

Ultrastructure of resveratrol-treated trophozoites.

<p>TEM of untreated, 0.4% ethanol-, IC₅₀ resveratrol- and 0.5 mM H₂O₂-treated trophozoites.</p

Hepatic abscess formation by resveratrol-treated and untreated trophozoites.

<p><b>A)</b> Trophozoites were incubated with 110 μM resveratrol for 12 h, then they were changed to fresh TYI-S-33 medium without resveratrol and cell proliferation was measured at different times by spectrophotometry using the WST-1 reagent. -•- Untreated trophozoites. -○- Trophozoites pretreated with 110 μM resveratrol for 12 h and then incubated in fresh TYI-S-33 medium. -▲- Trophozoites cultured in the presence of 110 μM resveratrol during 72 h. <b>B)</b> Hamsters were intraportally inoculated with 3 x 10⁶ untreated or resveratrol-treated (110 μM, 12 h, drug removal) trophozoites. After seven days, livers were examined. <b>C)</b> Damage was evaluated as the weight of the abscesses formed divided by the weight of the whole liver, before the injured areas were removed. Values represent the mean ± standard error of liver damage in inoculated animals. n = 8. ***p<0.001.</p

Effect of resveratrol in <i>E</i>. <i>histolytica</i> trophozoites.

<p><b>A)</b> Trophozoites growth was measured at different times by spectrophotometry using the WST-1 reagent. <b>B)</b> Viability of trophozoites after 48 h incubation with different concentrations of resveratrol was evaluated under light microscope by Trypan blue exclusion. IC₅₀ was calculated as described in Materials and Methods. Values represent the mean ± standard error of three independent experiments.</p

Effect of resveratrol in <i>in vitro</i> virulence of <i>E</i>. <i>histolytica</i> trophozoites and in <i>E</i>. <i>invadens</i> encystment.

<p><b>A)</b> Ingested erythrocytes after different incubation times with untreated or resveratrol -treated (110 μM_, 12 h and washing out) trophozoites. Values represent the mean ± standard error of three independent experiments. **p<0.01, ***p<0.001. <b>B)</b> Representative images of trophozoites with ingested erythrocytes at different incubation times. <b>C)</b> Destruction of MDCK cell monolayers incubated with untreated or resveratrol-treated (110 μM_, 12 h and washing out) trophozoites. Bottom: representative images of MDCK monolayers incubated with trophozoites and stained with methylene blue. C: MDCK cells that were not in contact with trophozoites. 0.4% ethanol-treated trophozoites were used as positive controls. Values represent the mean ± standard error of three independent experiments. *p<0.05,** p<0.01,*** p<0.001. <b>D)</b> Cysts formed were counted under the epifluorescence microscope and number of cysts in untreated cells was taken as 100%. Values represent the mean ± standard error of three independent experiments., ** p<0.01,***p<0.001.</p

Oxidative stress produced by resveratrol in trophozoites.

<p><b>A)</b> ROS production in trophozoites untreated or treated with IC₅₀ resveratrol. Quantification was performed by flow cytometry using the DCFDA reagent. <b>B)</b> Lipid peroxides in total lipids of trophozoites. 0.4% ethanol- and 0.5 mM H₂O₂-treated trophozoites were used as controls. Values represent the mean ± standard error of three independent experiments. ***p<0.001.</p

Biochemical changes in resveratrol-treated trophozoites.

<p><b>A)</b> Cytosolic Ca²⁺ was measured in extracts of untreated or IC₅₀ resveratrol-treated trophozoites, using Fluo-4 AM. <b>B)</b> Calpain activity of trophozoites extracts incubated with Suc-Leu-Leu-Val-Tyr-AMC was measured by fluorescence spectroscopy. <b>C)</b> SOD activity of trophozoite extracts was monitored by spectrophotometry using the SOD kit. 0.4% ethanol- and 0.5 mM H₂O₂-treated trophozoites were used as controls. Values represent the mean ± standard error of three independent experiments. ***p<0.001.</p