Avaliação da atividade anti-inflamátória do nitrito de sódio (NaNO2) e do 2-nitrato-1,3-dibutoxipropano (NDBP) na aterosclerose experimental

Abstract

Coronary ischemic diseases and cerebrovascular accidents rank among the leading causes global mortality and mantain a close association with atherosclerosis, a chronic inflammatory vascular disease characterized by intense innate and adaptive immune activity. In the atherogenic process, a reduction in the bioavailability of nitric oxide (NO) and an increase in reactive oxygen species (ROS) are observed. In this context, an enhanced understanding of new therapeutic alternatives to improve NO bioavailability is necessary, including the study of new classes of NO-donating drugs and the nitrate-nitrite-NO pathway. This study aimed to evaluate the anti-inflammatory activity of sodium nitrite (NaNO2) and 2-nitrate-1,3dibutoxypropane (NDBP) in an experimental atherosclerosis model. Acute effects: C57BL/6 mice (controls) and ApoE-/- mice were treated with NaNO2 (dose 0.1 mmol/kg/day) or NDBP (dose 40 mg/kg) for three days. One hour after the last treatment, the animals were stimulated with zymosan (2 mg/mL) to induce peritoneal inflammation. After 4 hours, peritoneal fluid was collected and used for total and differential cell counts by optical microscopy and for the quantification of cytokines (IL-6, IL-10, TNF-α, and MCP-1) by ELISA. Chronic effects: ApoE-/- mice, upon reaching 8 weeks of age, received a Western-type atherogenic diet for 12 weeks, while their controls received a standard rodent diet. During the last 3 weeks, they were treated by gavage with NaNO2 (0.1 mmol/kg/day), NDBP (40 mg/kg/day), or saline. At the end of the treatment, blood samples were collected for lipid profile analysis, lipid peroxidation, plasma nitrite, and cytokines (IL-6, IL-10, TNF-α, and MCP-1). The aortas were removed and processed for histological assessment of atherosclerotic plaque deposition (stained with Oil Red), production of ROS (stained with dihydroethidium - DHE), and NO (stained with diaminofluorescein – DAF). The data were expressed as mean ± standard error of the mean, and statistical comparisons were made using ANOVA, followed by Tukey's post hoc test, considering p<0.05. In the peritonitis model, zymosan, as expected, induced an increase in cell migration and pro-inflammatory cytokine levels in the peritoneum. Treatment with NaNO2 reduced the total number of cells in the peritoneal cavity by reducing the migration of polymorphonuclear cells in C57BL/6 mice, and in ApoE-/- mice, it reduced plasma levels of IL10, TNF-α, and MCP-1. On the other hand, NDBP increased cell migration in both lineages. Regarding chronic effects, treatment with NaNO2 reduced atherosclerotic plaque deposition, plasma levels of MCP-1, systemic and in situ oxidative stress in the aorta, regardless of changes in NO levels, plasma nitrite, and lipid profile. NDBP differed from NaNO2 increased MCP-1 levels in plasma. These results suggest that NaNO2 has acute and chronic antiinflammatory effects, capable of reducing atherosclerotic plaque deposition through mechanisms involving the reduction of oxidative stress and MCP-1. They also suggest that NDBP has a dual effect, with pro-inflammatory action in the peritonitis model and antiatherosclerotic action when administred chronically.