N-acetyl-cysteine inhibits liver oxidative stress markers in BALB/c mice infected with Leishmania amazonensis

Abstract

Background: Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells.

Objectives: To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters.

Methods: Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed.

Findings: In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1β, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters.

Main conclusions: L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function.

Fig. 1. : oxidative stress parameters in organs of mice infected with Leishmania amazonensis. Liver, kidney, spleen, heart and lung tissues were isolated, homogenised and the activities of the antioxidant enzymes CAT (A) and SOD (B) were assessed. Free sulfhydryl (-SH) groups (C) were measured and protein carbonylation (D) was determined. Values represent mean ± standard error of the mean (SEM) of samples obtained from five mice of two distinct groups and two different infections. Student’s t test (two-tail) was applied for all data; * denotes p value < 0.05 and *** denotes p value < 0.0001.

Fig. 2. : liver parameters of oxidative stress in mice infected with Leishmania amazonensis. Total radical antioxidant parameter (TRAP) kinetic assay was employed with liver tissue samples (A). The luminescence emission was recorded during time; data points represent mean ± standard error of the mean (SEM) values of repeated measures. Lipoperoxidation was estimated by assessing TBARS levels (B). Western blot analysis for carbonyl groups was performed using an antibody against DNP (C, representative blot) and quantification relative to b-actin levels was performed (D). Detection of nitrotyrosine in samples was performed by ELISA (E). Values represent mean ± SEM of samples obtained from five mice of two distinct groups (two different infections). Student’s t test (two-tail) was applied for all data; * denotes p value < 0.05 and ** denotes p value < 0.005.

Fig. 3. : effect of N-acetyl-cysteine (NAC) on parameters of liver oxidative stress. Animals infected with Leishmania amazonensis were subjected to five consecutive days of NAC (NAC, 20 mg/kg b.w.) i.p. injection before euthanasia and tissue collection. Western blot analyses were performed for SOD1 (A) and SOD2 (B) isoforms; b-actin was used as internal marker control. Free sulfhyfryl groups (C) and nitrotyrosine levels (D) were assessed as described earlier. Values represent mean ± standard error of the mean (SEM) of samples obtained from five mice of two distinct groups (two different infections). Data were analysed by ANOVA. Tukey’s multiple comparison was applied to detect significant differences between all groups; *p < 0.05 and ***p < 0.0001 compared to control group; &p < 0.05 compared to infected group.

Fig. 4. : effect of N-acetyl-cysteine (NAC) on molecular markers of pro-inflammatory activation in liver. Animals infected with Leishmania amazonensis were subjected to five consecutive days of NAC (NAC, 20 mg/kg b.w.) i.p. injection before euthanasia and tissue collection. Tissue levels of IL-1β (A), IL-6 (B), TNF-α (C) and IL-10 (D) were assessed by ELISA. The content of RAGE (E) and the total fraction of NF-kB subunit p65 (F) were assessed by western blot (representative blots depicted above graphs; b-actin was used as internal marker control). Values represent mean ± standard error of the mean (SEM) of samples obtained from five mice of two distinct groups (two different infections). Data were analysed by ANOVA. Bonferroni’s multiple comparison test was applied to confirm efficacy of experiment (control vs. infected groups, #p < 0.05). Tukey’s multiple comparison was applied to detect significant differences between all groups; *p < 0.05 and **p < 0.0001 compared to control group. No significant differences (p < 0.05) were detected between “infected” and “infected + NAC” groups.