MCC950 Alleviates Fat Embolism-Induced Acute Respiratory Distress Syndrome Through Dual Modulation of NLRP3 Inflammasome and ERK Pathways

Abstract

Fat embolism is a critical medical emergency often resulting from long bone fractures or amputations, leading to acute respiratory distress syndrome (ARDS). The NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome, a key regulator of innate immunity, is activated by reactive oxygen species and tissue damage, contributing to inflammatory responses. This study examines the role of NLRP3 in fat embolism-induced ARDS and evaluates the therapeutic potential of MCC950, a selective NLRP3 antagonist. Fat embolism was induced by fatty micelle injection into the tail vein of Sprague Dawley rats. Pulmonary injury was assessed through lung weight gain as an edema indicator, NLRP3 expression via Western blot, and IL-1β levels using ELISA. Histological damage and macrophage infiltration were evaluated with hematoxylin and eosin staining. Fat embolism significantly increased pulmonary NLRP3 expression, lipid peroxidation, IL-1β release, and macrophage infiltration within four hours, accompanied by severe pulmonary edema. NLRP3 was localized in type I alveolar cells, co-localizing with aquaporin 5. Administration of MCC950 significantly reduced inflammatory responses, lipid peroxidation, pulmonary edema, and histological damage, while attenuating MAPK cascade phosphorylation of ERK and Raf. These findings suggest that NLRP3 plays a critical role in fat embolism-induced acute respiratory distress syndrome, and its inhibition by MCC950 may offer a promising therapeutic approach.

Keywords: MCC950; NOD-like receptor pyrin domain-containing 3 (NLRP3); acute respiratory distress syndrome (ARDS); fat embolism; reactive oxygen species (ROS).

Figure 1

Fat embolism induces pulmonary damage and NLRP3 expression: (A) Quantification of lung weight gain (LWG) as a measure of pulmonary edema at 4 h post-fat embolism (FE) induction compared with sham controls. Data are presented as mean ± SEM (n = 6 per group). * p < 0.05 vs. sham group. (B) Measurement of lipid peroxidation levels using MDA assay in lung tissue 4 h after FE induction. Values are expressed as mean ± SEM (n = 6 per group). * p < 0.05 vs. sham group. (C) Representative hematoxylin and eosin (H&E) staining of lung sections showing alveolar damage 4 h after FE induction. Scale bar = 100 μm. (D) Western blot analysis and quantification of NLRP3 protein expression in lung tissue 4 h after FE induction. β-actin served as loading control. Data represent mean ± SEM (n = 5 per group). * p < 0.05 vs. sham group.

Figure 2

MCC950 attenuates FE-induced NLRP3 activation and pulmonary inflammation: (A) Effects of MCC950 (10 mg/kg) on FE-induced NLRP3 expression by Western blot analysis. Data are presented as mean ± SEM (n = 6 per group). (B) ELISA quantification of IL-1β levels in lung tissue after FE induction with or without MCC950 (10 mg/kg) treatment. Data are presented as mean ± SEM (n = 5 per group). (C) Effect of MCC950 (10 mg/kg) on FE-induced pulmonary edema measured by lung weight gain. Data are presented as mean ± SEM (n = 6 per group). (D) MDA levels in lung tissue comparing control, FE, and FE+MCC950 groups. Data are presented as mean ± SEM (n = 5 per group). ** p < 0.01, *** p < 0.001 were considered significantly different from sham values; # p < 0.05, ## p < 0.01 were considered significantly different from rats with FE treatment by the Mann–Whitney U-test.

Figure 3

Histological analysis of MCC950′s protective effects against FE-induced lung injury. Representative H&E staining of lung sections from (A) control, (B) vehicle, (C) FE-induced, and (D) FE+MCC950-treated groups. White arrows indicate alveolar damage and inflammatory cell infiltration. Scale bar = 100 μm.

Figure 4

Effects of MCC950 on the pulmonary phosphorylation of Raf (p-Raf) and ERK (p-ERK) in sham rats (sham), rats with vehicle (vehicle), rats with fat embolism (FE), or rats treated with MCC950 (FE + MCC950). Upper: Western blot analysis of sham rats (sham), rats with vehicle (vehicle), rats with fat embolism (FE), or rats treated with MCC950 (10 mg/kg) (FE + MCC950). Lower: Relative density presented as folds compared with the sham group. Data are presented as the mean ± SEM values (n = 3). ** p < 0.01 were considered significantly different from sham values; and # p< 0.05, ## p< 0.01 were considered significantly different from rats with FE treatment by the Mann–Whitney U-test.

Figure 5

Cellular localization of NLRP3 in FE-induced lung injury and the effects of MCC950 administration: Double immunofluorescence staining showing co-localization of NLRP3 (green) with aquaporin-5 (red, type I alveolar cell marker). Merged images display overlapping regions, indicated by the white arrow. Scale bar = 100 μm. After FE, the expression of NLRP3 significantly increased and co-localized with aquaporin-5, and administration of MCC950 attenuated the expression of NLRP3 in type I alveolar cells.

Figure 6

Proposed mechanisms of NLRP3 in ARDS.