Probucol-Induced α-Tocopherol Deficiency Protects Mice against Malaria Infection

Abstract

The emergence of malaria pathogens having resistance against antimalarials implies the necessity for the development of new drugs. Recently, we have demonstrated a resistance against malaria infection of α-tocopherol transfer protein knockout mice showing undetectable plasma levels of α-tocopherol, a lipid-soluble antioxidant. However, dietary restriction induced α-tocopherol deficiency is difficult to be applied as a clinical antimalarial therapy. Here, we report on a new strategy to potentially treat malaria by using probucol, a drug that can reduce the plasma α-tocopherol concentration. Probucol pre-treatment for 2 weeks and treatment throughout the infection rescued from death of mice infected with Plasmodium yoelii XL-17 or P. berghei ANKA. In addition, survival was extended when the treatment started immediately after parasite inoculation. The ratio of lipid peroxidation products to parent lipids increased in plasma after 2 weeks treatment of probucol. This indicates that the protective effect of probucol might be mediated by the oxidative stressful environment induced by α-tocopherol deficiency. Probucol in combination with dihydroartemisin suppressed the proliferation of P. yoelii XL-17. These results indicated that probucol might be a candidate for a drug against malaria infection by inducing α-tocopherol deficiency without dietary α-tocopherol restriction.

Fig 1. Probucol treatment confers protection to mice against Plasmodium yoelii XL-17 infection.

Six-week-old C57BL/6J mice were treated with a standard diet (Std) or 1% w/w probucol in the diet for 2 weeks and then infected with 0.2 mL of 1 × 10⁵ erythrocytes /mL infected with P. yoelii XL17. Survival (A), mean parasitemia (B) and the erythrocyte count (C) of probucol-treated mice (n = 4) and Std diet-fed mice (n = 5) was monitored. Probucol treatment extended the survival rate after infection with P. berghei ANKA. Six-week-old C57BL/6J mice were treated with Std diet or 1% w/w probucol in diet for 2 weeks and then infected with P. berghei ANKA through mosquito bite. Survival (D) and mean parasitemia (E) of probucol-treated mice (n = 11) and Std diet-fed mice (n = 10). Parasitemia was monitored every 2 days and the erythrocyte count was determined (F). All data are expressed as mean ± standard error (SE). Statistical analysis was carried out by analysis of variance (ANOVA). For the survival rate analysis, the Kaplan–Meier long-rank method was performed; *p < 0.05, **p < 0.025, and ***p < 0.001 compared to standard diet-fed mice.

Fig 2. Plasma concentrations of α-tocopherol and cholesterol decreased after probucol treatment.

A, Experimental design for α-tocopherol deficiency induction using 1% w/w probucol in the diet. Six-week-old C57BL/6J mice were treated with 1% w/w probucol for 2 weeks. Then, mice were withdrawn from the probucol diet and changed to a standard diet for 2 weeks. Plasma and erythrocyte samples were obtained at day 0, 1, 2, 4, 7, and 14 after starting probucol treatment (n = 5 per group) and 1 or 2 weeks post-withdrawal (n = 3 per group). In addition, the plasma of eight-week-old α-tocopherol transfer protein knockout (α-ttp^Δ) mice fed a standard diet was obtained (n = 3). Plasma α-tocopherol concentrations were measured after probucol treatment (B) and after withdrawal (C). The levels of α-tocopherol in erythrocytes were normalized with the protein concentration (n = 5 per group) (D). Plasma cholesterol concentrations were measured by using the cholesterol E-test after 2 weeks of probucol treatment and after withdrawal (n = 3) (E). All data are expressed as mean ± SE. Statistical analysis was carried out by analysis of variance (ANOVA; multiple comparisons Tukey’s test). *p < 0.05.

Fig 3. Effect of simultaneous probucol treatment and infection and effect of probucol in combination with antimalarial.

The survival was improved in mice infected with Plasmodium yoelii XL-17 and subjected to simultaneous probucol treatment. Eight-week-old mice fed a standard (Std) diet were infected with P. yoelii XL-17. Probucol (1% w/w) started simultaneously with the infection (n = 12). The control group fed Std diet (n = 11). Survival (A) and parasitemia (B) were monitored. Parasite proliferation was inhibited by combination therapy with probucol and DHA. Six-week-old mice pre-treated with Std or probucol diet for 2 weeks were infected with P. yoelii XL-17. Thereafter, mice were treated with DHA (30 mg/kg) or PBS on day 3, 4, and 5 post-infection. Survival (C) and parasitemia (D) of mice treated with Std diet with/without DHA and probucol with/without DHA, (n = 6 per group) were monitored. Data are expressed as mean ± SE. Statistical analysis was performed by ANOVA and Kaplan-Meier log-rank method. *p < 0.05, **p < 0.025.

Fig 4. The ratios of lipid peroxidation products to parent lipids in plasma increased after probucol pre-treatment.

Six-week-old C57BL/6J mice were treated with 1% w/w probucol in the diet for 2 weeks and then infected with 0.2 mL of 1 × 10⁵ erythrocytes /mL infected with Plasmodium yoelii XL-17. Plasma samples were obtained at day 0, 1, 2, 4, 7, and 14 after starting the probucol diet (n = 5 per group) and at day 0, 4, 7, 12, 19, and 22 post-infection (n = 2 to 7). The ratio of total hydroxyoctadecadienoic acid (HODE), a peroxidation product of linoleic acid (LA), to linoleic acid (tHODE/LA) in plasma (A) and the ratio of 7β-hydroxycholesterol (7β-OHCh), a peroxidation product of cholesterol, to total cholesterol (7β-OHCh/tCh) in plasma (B) were measured. The concentration of LA (C) and tCh (D) were measured by using gas chromatography-mass spectrometry (GC-MS). All data are expressed as mean ± SE. Statistical analysis was carried out by analysis of variance (ANOVA). *p < 0.05, **p < 0.025, and ***p < 0.001. The solid barsindicate the significant changes in probucol-treated groups and the dotted bars indicate the significant changes in standard (Std) diet-fed mice.

Fig 5. Probucol pre-treatment did not increase the lipid peroxidation products in erythrocytes.

Six-week-old C57BL/6J mice were treated with 1% w/w probucol in the diet for 2 weeks and then infected with 0.2 mL of 1 × 10⁵ erythrocytes /mL infected with Plasmodium yoelii XL-17. Erythrocyte samples were obtained from 0, 1, 2, 4, 7, and 14 days after starting the probucol diet (n = 5 per group). The ratio of total hydroxyoctadecadienoic acid (HODE), a peroxidation product of linoleic acid (LA), to linoleic acid (tHODE/LA) in erythrocytes (A) and the ratio of 7β-hydroxycholesterol (7β-OHCh), a peroxidation product of cholesterol (Ch), to total cholesterol (7β-OHCh/tCh) in erythrocytes (B) were measured. Moreover, the concentrations of LA (C) and Ch (D) in erythrocytes were determined by normalizing by the protein concentration. Statistical analysis was carried out by analysis of variance (ANOVA). *p < 0.05 and **p < 0.025.

Fig 6. Oxidative response of mice and parasites to α-tocopherol deficiency after infection.

Six-week-old C57BL/6J mice were treated with 1% w/w probucol in the diet for 2 weeks and then infected with 0.2 mL of 1 × 10⁵ erythrocytes infected with Plasmodium yoelii XL-17. Plasma, total blood, and liver samples were obtained on day 0, 4, 7, 12, 19, and 22 post-infection (n = 2 to 7). The plasma level of α-tocopherol (A) and the mRNA expression of α-ttp in liver (B) were analyzed. The mRNA expression of the parasite antioxidant enzymes, P.y 1-Cys Prx (C) and P.y Tpx-1 (D), and the oxidative stress response protein P.y Hsp-70 (E) were analyzed using parasite-specific primers and probes. All data are expressed as mean ± SE. Statistical analysis was carried out by analysis of variance (ANOVA). *p < 0.05.