Methotrexate associated with nanoparticles decreases NLRC4 inflammasome expression in infarcted rat hearts

Abstract

Abstract   Abstract: Following myocardial infarction (MI), molecules released from necrotic myocytes sensitize nucleotide-binding receptors (NLRs), activate inflammasome complex, and initiate acute inflammatory process and interleukin (IL)-1β production, essential for tissue damage resolution. However, excessive IL-1β production impairs cardiac remodeling. After MI, IL-1β is released by the NLRP3 inflammasome; involvement of the NLRC4 inflammasome in post-MI remodeling has not been established. Methotrexate (MTX) carried by lipid core nanoparticles in low density emulsion (MTX-LDE) reduces post-MI inflammatory process and improves cardiac function in rodents. Purpose To evaluate the influence of early treatment with MTX-LDE on myocardial expression of NLRP3 and NLRC4 inflammasomes in infarcted rats. Methods Male Wistar rats were divided into three groups: control (Sham, n=6); MI (n=6); and MI treated with MTX-LDE (MI-MTX, n=6). MI was induced by ligation of the left anterior descending coronary artery. Twenty-four hours later, the MI-MTX group was treated with 1 mg/kg of MTX-LDE, intraperitoneally. Euthanasia was performed 72 hours after surgery under anaesthesia with ketamine and xylazine. Infarct size was assessed by histology. Myocardial expression of NLRC4, NLRP3, ASC, pro-caspase-1, caspase-1, pro-IL-1β and IL-1β was quantified by Western blotting. IL-1β concentration in serum and heart macerate supernatant was analyzed by ELISA. Statistical: ANOVA or Kruskal-Wallis; 5% significance level. Results Rats with MI lower than 30% of the left ventricle total area were excluded. Infarct size was larger in the MI-MTX group (43,8 ± 5,4) than MI (36,2 ± 6,22). NLRC4 and caspase-1 expression and IL-1β serum concentration were higher in MI than Sham and MI-MTX. Myocardial IL-1β concentration was higher in MI and MI-MTX than Sham and lower in MI-MTX than MI. NLRP3, ASC, pro-caspase-1, and IL-1β protein expression did not differ between groups. Conclusion Our study shows for the first time that NLRC4 inflammasome protein expression increases acutely after myocardial infarction in rats. Early treatment with nanoparticles-derived methotrexate reduces myocardial NLRC4 inflammasome expression and myocardial and serum IL-1β concentration.