Experimental infection with P. berghei ANKA was carried out using C57BL/6J male mice (Japan CLEA, Tokyo, Japan) at biosafety level 2 in a specific pathogen-free facility. The room temperature (24 ± 1°C) and humidity (50 ± 10%) were adjusted, and the lighting was controlled (lights on from 7 AM to 7 PM). The mice were allowed free access to water and a diet (CA-1; CLEA Japan, Tokyo, Japan). The animals used in this study were handled and cared for in accordance with the “Guiding Principles for the Care and Use of Research Animals” established by Obihiro University of Agriculture and Veterinary Medicine, Japan. All animal experimental procedures were approved by the Obihiro University of Agriculture and Veterinary Medicine Institutional Animal Ethics Committee (ethical approval number #29-86, April 14, 2017; #18-112, May 16, 2018; #19-130, May 23, 2019).
Alpha-TEA (molecular weight 488.75 g/mol) distributed by Eisai (Eisai Co., Ltd., Tokyo, Japan) was mixed with the diet [0.75% and 1.5% (w/w) of α-TEA] and fed to 8-weeks old C57BL/6J male mice (23-25 g body weight) for 14 days after infection. The day of infection was defined as Day 0. The mixed diet was outsourced to Oriental Yeast Co. Ltd. (Tokyo, Japan).
For parasite infection, 4 × 104 P. berghei ANKA-infected red blood cells (iRBCs) were intraperitoneally injected into mice and their survival rates and parasitemia were monitored. On day 4 post-infection, 2 µL of blood was collected from the tip of the tail, smeared on a glass slide, and stained with Giemsa (Sigma-Aldrich, Tokyo, Japan). The percentage of infected erythrocytes to total erythrocytes (parasitemia) was determined using a phase contrast microscope (DIAPHOTO-TMD300, Nikon, Tokyo, Japan). More than 1,000 red blood cells (RBCs) were counted in each mouse to assess parasitemia. On day 7 after infection, the permeability of the blood-brain barrier (BBB) was evaluated by Evans blue staining as follows: 0.1 mL of 2% (w/v) Evans blue (056-04061; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) dissolved in PBS was injected into infected mice with or without treatment with α-TEA through the tail vein. Two hours after injection the mice were sacrificed, and brain samples were collected.
Survival rates were analyzed using log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests implemented in GraphPad Prism 5. Parasitemia was analyzed using a one-way analysis of variance and Tukey’s test. A value of P < 0.05was considered statistically significant.
Experimental in vitro infections with P. falciparum 3D7 and K1 were performed using human O+ RBCs (Japanese Red Cross Society, Hokkaido, Japan). Both parasites were maintained in complete Roswell Park Memorial Institute-1640 medium (RPMI-1640, Sigma-Aldrich, MO, USA), containing, per liter, 6 g HEPES (Sigma-Aldrich, MO, USA), 25 mg hypoxanthine (Wako, Osaka, Japan), 2 g NaHCO3 (Wako, Osaka, Japan), 250 µL of 50 mg/ml gentamicin solution (Gibco, CA, USA), and 5 g AlbuMaxTM II Lipid-Rich BSA (Gibco, CA, USA). The medium was changed daily and parasitemia was monitored using Giemsa-stained thin blood smears (Merck, Darmstadt, Germany). Malarial parasite culture in human blood was approved by the ethical committee of Obihiro University of Agriculture and Veterinary Medicine (#2013-04-3).
In vitro growth inhibition of P. falciparum 3D7 and K1 was performed using the SYBR Green I-based fluorescence assay (SYBR® Green I Nucleic Acid Stain 10,000×, ME, USA) as previously described [28, 29]. Briefly, test compounds were prepared in complete media at eight final concentrations (two-fold serial dilution) ranging from 100 to 0.78 µM. Chloroquine diphosphate (molecular weight 515.86 g/mol; Sigma-Aldrich, MO, USA) was used as the reference drug. Before use, the parasites’ life cycle was synchronized by treatment with 5% D-sorbitol to obtain ≥ 90% ring-stage parasites. Synchronous parasites (50 µL) at 0.5% parasitemia and 2% hematocrit were seeded in a 96-well plate containing 50 µL of the test compounds. The 96-well plates were incubated for 72 h at 37°C in 5% CO2, 5% O2, and 90% N2. Next, 100 µL of lysis buffer containing 1× SYBR Green I was added to each well, mixed by pipetting, and then incubated in the dark at room temperature for 2 h. Fluorescence intensities were measured using a Fluoroskan Ascent instrument (Thermo Scientific, MA, USA) at excitation and emission wavelengths of 485 and 518 nm, respectively. Cells treated with 1% (v/v) DMSO were used as a negative control, and wells containing only test compounds and erythrocytes were used to correct the background signals. The inhibition assays were performed in quadruplicate for each concentration and repeated three independent times. The half-maximal inhibitory concentration (IC50) values were analyzed using nonlinear regression analysis implemented in GraphPad Prism 8.
Human foreskin fibroblast cells (HFFs) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, MO, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (×100, Wako, Osaka, Japan) at 37°C and 5% CO2 in air. A cell viability assay was used to evaluate the cytotoxic effects of the tested compounds, as previously described . Briefly, the cell suspension was seeded in a 96-well plate at a density of 1×104 cells/well in DMEM with 10% FBS and incubated for 48 h at 37°C and 5% CO2 in air. Then, two-fold dilutions (total eight concentrations) of the test compounds in DMEM were added in quadruplicate to each well and incubated for an additional 72 h. Cell Counting Kit-8 (CCK-8) was added and incubated for an additional 3 h at 37°C and 5% CO2 in air, and the absorbance was measured at 450 nm. The tests were independently repeated three times. The half-maximal cytotoxic concentration (CC50) values were analyzed using nonlinear regression analysis implemented in GraphPad Prism 8.
The human RBC hemolysis assay was performed as previously described . Briefly, each test compound was prepared in 1× phosphate-buffered saline (PBS) at a desired concentration in a 96-well plate, and 3% RBCs suspension in PBS was added. The plate was incubated for 3 h at 37°C in 5% CO2, 5% O2, and 90% N2 and then centrifuged at 1,300 × g for 5 min. Finally, 100 µL of the supernatant of each mixture was transferred to a new 96-well plate, and the absorbance was recorded at 540 nm. PBS (with 1% DMSO) and RBC lysis buffer (0.83% NH4Cl; 0.01 M Tris-HCl, pH 7.2) were used as negative and positive controls, respectively. The hemolysis rate of RBCs was calculated using the following formula: \(Hemolysis rate=\)[(A sample – A negative control) / (A positive control – A negative control) × 100], where A stands for absorbance. The experiments were performed in technical quadruplicates, and independently repeated three times.