Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica

Abstract

Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound) on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC50 was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca2+ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis.

Fig 1. Effect of resveratrol in E. histolytica trophozoites.

A) Trophozoites growth was measured at different times by spectrophotometry using the WST-1 reagent. 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.

Fig 2. Effect of resveratrol in trophozoites morphology.

A) Light microscopy images of untreated and IC₅₀ resveratrol-treated trophozoites. B) Size of trophozoites during different incubation times measured in images obtained by light microscopy. Boxes represent 50% of population containing the median of three independent experiments. Bars indicate the maximum and minimum sizes of the other 50%. □ Untreated trophozoites. ■ 0.4% ethanol-treated trophozoites. ■ IC₅₀ resveratrol-treated trophozoites. **p<0.01, ***p<0.001. C) Flow cytometry of size (forward scatter) and granularity (side scatter) of trophozoites incubated 48 h with IC₅₀ resveratrol. R1 represents the gate of trophozoites population selected for these experiments. Untreated and 0.4% ethanol-treated trophozoites were used as controls.

Fig 3. Ultrastructure of resveratrol-treated trophozoites.

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

Fig 4. Oxidative stress produced by resveratrol in trophozoites.

A) ROS production in trophozoites untreated or treated with IC₅₀ resveratrol. Quantification was performed by flow cytometry using the DCFDA reagent. 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.

Fig 5. PS externalization produced by resveratrol in trophozoites.

A) Confocal microscopy of untreated or IC₅₀ resveratrol-treated trophozoites incubated with Annexin V-FITC and PI. Merge: fluorescence channels and phase contrast. 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.

Fig 6. TUNEL assays in resveratrol-treated trophozoites.

A) 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) Flow cytometry analysis of dUTP-FITC-treated trophozoites. 0.4% ethanol- and 0.5 mM H₂O₂-treated trophozoites were used as controls.

Fig 7. Biochemical changes in resveratrol-treated trophozoites.

A) Cytosolic Ca²⁺ was measured in extracts of untreated or IC₅₀ resveratrol-treated trophozoites, using Fluo-4 AM. B) Calpain activity of trophozoites extracts incubated with Suc-Leu-Leu-Val-Tyr-AMC was measured by fluorescence spectroscopy. C) 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.

Fig 8. Effect of resveratrol in in vitro virulence of E. histolytica trophozoites and in E. invadens encystment.

A) 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) Representative images of trophozoites with ingested erythrocytes at different incubation times. C) 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. D) 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.

Fig 9. Hepatic abscess formation by resveratrol-treated and untreated trophozoites.

A) 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) 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. C) 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.

Fig 10. Effect of resveratrol administration in hamsters intraportally inoculated with virulent trophozoites.

A) 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) 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.

Fig 11. Histopathology of amoebic liver abscesses.

0.4 μm sections of hamster’s livers treated as described in Fig 10, were processed for hematoxylin-eosin staining and observed under light microscope. A) Animals not inoculated with trophozoites and orally treated with pathogens-free water. B, C) Hamsters intraportally inoculated with 3 x 10⁶ trophozoites and treated with 50 μl of ethanol each 8 h (2 days before and 10 days after inoculation). B) Granulomatous reaction representative of damage produced. C) Magnification of a granuloma. D, E) Hamsters intraportally inoculated with 3 x 10⁶ trophozoites and treated with 100 mg/Kg of resveratrol (diluted in 50 μL of ethanol) each 8 h (2 days before and 10 days after inoculation). D) Liver without damage after resveratrol-treatment of animals. E) Liver with small abscesses. Square: magnified area showing a trophozoite. Arrows: granulomas. F) Animals intraportally inoculated with 3 x 10⁶ trophozoites and treated with 20 mg/Kg of metronidazole each 8 h (2 days before and 10 days after inoculation). LP: liver parenchyma. CV: centrolobulillar vein. H: hepatocytes. N: necrosis area. IR: inflammatory reaction. PV: a branch of the portal vein. Arrowheads: trophozoites. Arrows: epithelioid cells.

Fig 12. Immunochemistry of livers from hamsters inoculated with virulent trophozoites.

Paraffin sections of livers from hamsters treated as in Fig 10. A) Non-challenged hamsters (healthy liver). B) Challenged hamsters treated with ethanol. C) Challenged hamsters treated with resveratrol that did no develop hepatic abscesses. D) Challenged hamsters treated with resveratrol that develop small abscesses. E) Challenged hamsters treated with metronidazole. F) Challenged hamsters treated with ethanol and developed only with the pre-immune serum. G) Parasitic burden quantified in 15 sections of livers from hamsters treated as above.