Fig 1.
Arbutin has potent anti-T. gondii activities both in vitro and in vivo.
(A–C) Relative intracellular parasite numbers in mouse WT BMDMs (A), human THP-1-derived macrophages (B), or human HFF cells (C) that pretreated with vehicle control or arbutin at the indicated concentrations were quantified by qPCR at the indicated time points post-infection. n = 3 samples per time point. (D) The relative intracellular parasite numbers in WT BMDMs that were either pre-treated with arbutin for 12 h before Tg infection, or post-treated with arbutin 30 min after infection, were monitored by qPCR 24 h post-infection. n = 3 samples/group. (E) Representative immunofluorescence images of RH tachyzoites in the presence (+) or absence (-) of arbutin pretreatment in the indicated cell types. White arrows indicate the intracellular parasitophorous vacuoles of Toxoplasma. Red: TgSAG1; Green: F-actin; Blue: nuclei. Scale bar = 20 μm. n = 3 samples/group. (F and G) C57BL/6 WT mice were provided with either vehicle or arbutin-supplemented drinking water three days prior to intraperitoneal infection with Tg RH strain tachyzoites, and they continued to receive the same type of water thereafter. (F) The survival rate post infection over time. n = 6 mice/group. (G) The number of RH tachyzoites in the peritoneal fluid was quantified by microscopic examination using Giemsa staining eight days post-infection. The inset provides an enlarged view of a portion of the graph, highlighting typical tachyzoites (indicated by arrows). Scale bar = 50 μm. n = 5 mice/group. All of the experiments were repeated at least 2 times. Data are presented as the mean ± SEM. Statistical analysis was performed using one-way ANOVA (D), two-way ANOVA (A–C), Log-rank test for (F), and two-sided Student’s t-test for (G). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 2.
Arbutin attenuates T. gondii infection-induced inflammatory responses.
(A–D) WT BMDMs were pretreated with vehicle or 20 mM arbutin for 12 hours, followed by Tg RH infection or left uninfected. At 4 hours post-infection, cells were harvested for RNA sequencing. “Ctrl”: vehicle, no infection; “Arbutin”: arbutin, no infection; “RH”: vehicle + infection; “RH+Arbutin”: arbutin + infection. n = 3 biological replicates. (A and C) KEGG pathway enrichment analysis (adjusted p < 0.05) of (A) upregulated and (C) downregulated pathways in RH + Arbutin vs. RH groups. Top 20 KEGG pathways ranked by enrichment score are displayed. Red arrows highlight the pathways that are related to glutathione metabolism, iron homeostasis, or immune pathways against toxoplasmosis. (B) Heatmap visualization of arbutin-induced upregulated genes associated with iron homeostasis and glutathione metabolism. (D) Heatmap depicting arbutin-mediated downregulated genes involved in host immune responses against toxoplasmosis. (E) Relative mRNA transcription levels of Il-1β, Il-6, and Nos2 in WT BMDMs that were pretreated with vehicle control or 5 mM arbutin for 12 h before infection with Tg RH. n = 3 biological replicates per group. (F) C57BL/6 WT mice were administered with either vehicle control or arbutin-supplemented drinking water for consecutive days prior to intraperitoneal challenge with Tg RH strain tachyzoites. Serum levels of IFN-γ, CCL2/MCP-1, and IL-6 were measured at 1- and 4-day post-infection. n = 6 mice/group. (G and H) Secondary challenge experiment: (G) Schematic diagram of the experimental timeline, including arbutin treatment, primary infection, and secondary challenge phases; (H) Survival rates were monitored daily for 10 days post-secondary infection. n = 5 mice/group. Data were shown as the mean ± SEM. Statistical analysis with two-way ANOVA analysis (E, F), and Log-rank test for (H). **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no statistical significance.
Fig 3.
The anti-T. gondii effect of arbutin is associated with its capacity to restrict LIP.
(A) The levels of LIP in BMDMs at 24 hours post-infection (hpi) and in HFF cells at 72 hpi were determined using QIP analysis. “Ctrl” (vehicle, no infection), “Arbutin” (arbutin, no infection), “RH” (vehicle + infection), and “RH+Arbutin” (arbutin + infection). n = 3 samples/group. The experiments were repeated at least 2 times. (B and C) The levels of LIP (B) and the relative intracellular parasite numbers (C) were determined in THP-1 cells at 24 hpi and in HFF cells at 72 hpi. Both cell types were pretreated with vehicle control or the FPN inhibitor VIT-2763 for 2 h in the presence of arbutin before infection. n = 4 samples/group. The experiments were repeated at least 2 times. (D and E) Western blot analysis and densitometry quantification were performed to assess the protein expression levels of FPN1 and TFR1 in THP-1 cells (D) at 24 hpi and in HFF cells (E) at 72 hpi. n = 3 samples/group. (F and G) The parasite loads were determined in THP-1 cells at 24 hpi (F) and in HFF cells at 48 and 72 hpi (G). The cells were pre-treated with either culture medium or arbutin in the presence of FAC (10 mM, for THP-1 cells) or FeSO4 (50 μM, for HFF cells), respectively, for 12 hours, followed by exposure to Tg RH tachyzoites. n = 3 samples/group. The experiments were repeated at least 2 times. (H) The survival rate post infection was monitored daily in C57BL/6 WT mice that were fed a high-iron (1000 ppm) or a normal iron control diet (40 ppm) starting on day -7 before infection. n = 6 mice/group. Data were shown as the mean ± SEM. Statistical analysis with two-sided unpaired t-test (B, C), one-way ANOVA analysis (A, D-F), two-way ANOVA analysis (G) and Log-rank test for (H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no statistical significance.
Fig 4.
Biliverdin functions as an effector molecule against T. gondii.
(A–C) The expression levels of HMOX1 before or after infection in the presence or absence of arbutin were monitored by immunoblot at 24 hpi for BMDMs, at 12 and 24 hpi for THP-1, and at 48 and 72 hpi for HFFs. n = 3 samples/group. (D) The relative intracellular parasite numbers in the presence of hemin with or without arbutin treatment were quantified by qPCR at 24 h post-infection in THP-1 cells. n = 3 samples/group. (E-G) The relative intracellular parasite numbers in the presence of CORM-3 (E), bilirubin (F), or biliverdin (G) with or without arbutin treatment were quantified by qPCR at 24 h post-infection in THP-1 cells. n = 3 samples/group. (H) The relative intracellular parasite numbers in the presence of vehicle or biliverdin treatment were quantified by qPCR at 72 h post-infection in HFF cells. n = 3 samples/group. (I and J) HMOX1 gene expression was knockdown by siRNA in THP1 cells with or without Tg RH infection and in the presence or absence of biliverdin. (I) The effect of HMOX1 gene silencing on HMOX1 protein levels was evaluated using immunoblot. (J) The relative parasite loads were determined 24 hpi using qPCR. n = 3 samples/group. siRNA-NC: negative control (NC) scramble siRNA; si-HOMX1: HOMX1-specific siRNA. (K) THP-1 cells were incubated with vehicle or biliverdin (50 μM) 2 h before infection with Tg RH. The levels of LIP at 24 hpi were determined using QIP analysis. (L) C57BL/6 WT mice were fed a normal control diet (“RH” group) or biliverdin-supplemented diet (20 mg/kg) (“RH + biliverdin” group) starting on day -7. On day 0, the mice were intraperitoneally infected with Tg RH tachyzoites. The survival rate post infection was monitored daily. n = 6 mice/group. The experiments in (A-K) were repeated at least 2 times. Data were shown as the mean ± SEM. Statistical analysis with two-sided unpaired t-test (H), one-way ANOVA analysis (D-G, J, K), and Log-rank test for (L). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no statistical significance.
Fig 5.
Arbutin intervention reduced infection-related mortality in immunocompromised mice.
(A) Schematic diagram of the experimental timeline, including cyclophosphamide (CTX) treatment, arbutin supplementation, and primary infection. (B) Flow cytometry analysis of the percentage of CD4, CD8, CD19 positive cells among CD45 positive cells in the blood in the indicated groups 7 days post infection. n = 3 (RH group), and 6 (‘RH + CTX’ and ‘RH + CTX + arbutin’ group, respectively). (C) Survival rates were monitored daily for 15 days post infection. n = 10 mice/group. Data were shown as the mean ± SEM. Statistical analysis with one-way ANOVA analysis (B), and Log-rank test for (C). ***P < 0.001; ****P < 0.0001.