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Original Article

4-Hydroxybenzaldehyde Restricts the Intracellular Growth of Toxoplasma gondii by Inducing SIRT1-Mediated Autophagy in Macrophages

Jina Lee1,2, Jae-Won Choi1,2, Hye Young Han1, Woo Sik Kim3, Ha-Yeon Song3, Eui-Baek Byun3, Eui-Hong Byun4, Young-Ha Lee1, Jae-Min Yuk1,2,*
The Korean Journal of Parasitology 2020;58(1):7-14.
Published online: February 29, 2020

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*Corresponding author (yjaemin0@cnu.ac.kr)
• Received: September 2, 2019   • Revised: October 21, 2019   • Accepted: November 4, 2019

Copyright © 2020 by The Korean Society for Parasitology and Tropical Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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4-Hydroxybenzaldehyde Restricts the Intracellular Growth of Toxoplasma gondii by Inducing SIRT1-Mediated Autophagy in Macrophages
Korean J Parasitol. 2020;58(1):7-14.   Published online February 29, 2020
DOI: https://doi.org/10.3347/kjp.2020.58.1.7
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4-Hydroxybenzaldehyde Restricts the Intracellular Growth of Toxoplasma gondii by Inducing SIRT1-Mediated Autophagy in Macrophages
Korean J Parasitol. 2020;58(1):7-14.   Published online February 29, 2020
DOI: https://doi.org/10.3347/kjp.2020.58.1.7
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4-Hydroxybenzaldehyde Restricts the Intracellular Growth of Toxoplasma gondii by Inducing SIRT1-Mediated Autophagy in Macrophages
Image Image Image Image Image
Fig. 1 Cytotoxic effect of 4-HBA on BMDMs by CCK8 assays. (A, B) Evaluation of cell viability at 18 hr (A) or 48 hr (B) after 4-HBA treatment in BMDMs. **P<0.01, ***P<0.001. SC, solvent control.
Fig. 2 4-HBA increased antiparasitic activity in T. gondii-infected macrophages. (A–D) BMDMs were infected with GFP-RH strain (A, B, and C) or RH strain (D) for 2 hr (moi=1) and then treated with 4-HBA for 18 hr. (A) Representative images showing T. gondii invasion into and growth in BMDMs. Scale bar=10 μm. (B) Proportion of cells infected with T. gondii to total cells. (C) Number of parasites per vacuole. (D) Quantitative rt-PCR on SAG1 mRNA. *P<0.05, **P<0.01, ***P<0.001, U, untreated; SC, solvent control; Tg, T. gondii; ND, not detected.
Fig. 3 4-HBA-induced autophagy was required for the intracellular restriction of T. gondii in BMDMs. (A) Immunofluorescence microscopy on LC3 puncta formation in BMDMs treated with 4-HBA. Representative images (top; Scale bar=10 μm). LC3 punctate foci per cell (Lower panel). (B) SAG1 mRNA level guantifed by rt-PCR. BMDMs were infected with T. gondii RH strain, treated with 3-MA (10 μM) or WM (100 nM), and then incubated with 4-HBA (10 μM) for 18 hr. **P<0.01, ***P<0.001.
Fig. 4 Sirt1 was involved in 4-HBA-mediated activation of antiparasitic responses in BMDMs. (A) Immunoblot of SIRT1 and β-tubulin in BMDMs treated with 4-HBA. (B, C) SAG1 mRNA level. BMDMs were infected with RH strain in the presence of EX-527 (B) or sirtinol (C) and incubated with 4-HBA for 18 hr. *P<0.05, **P<0.01. SC, solvent control; Tg, T. gondii.
Fig. 5 Sirt1 contributed 4-HBA to induce formation and maturation of autophagosome and led activation of antiparasitic responses to T. gondii infection. (A) Immunofluorescence micrographs showing the formation of LC3 puncta in BMDMs treated with 4-HBA (10 μM; 18hr) in SC, EX-527 (4 μM), or sirtinol (30 μM). Left panel, representative images. Right panel, LC3 punctate foci in the cells. Scale bar=10 μm. (B, C) BMDMs were infected with GFP-RH strain, followed by treatments of SC, EX-527 (4 μM), or sirtinol (30 μM), and then incubated with 4-HBA (10 μM) for 18 hr. Cells were stained with Alexa 594-conjugated LC3 (red) and DAPI (blue), (B) Immunofluorescence micrographs showing co-localization of GFP-RH strain (green) and LC3 (red). Scale bar=10 μm. (C) Co-locol of LC3 with T. gondii. ****P<0.001. SC, solvent control; Tg, T. gondii.

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4-Hydroxybenzaldehyde Restricts the Intracellular Growth of Toxoplasma gondii by Inducing SIRT1-Mediated Autophagy in Macrophages
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