Abstract
Introduction
Acute respiratory distress syndrome (ARDS) is a life-threatening form of Acute Lung Injury (ALI) that affects (ARDS stats). Mechanical Ventilation is a life-saving intervention for ARDS, but the various stresses it puts on the lung can exacerbate lung injury, leading to ventilator-induced lung injury (VILI), for which pharmacologic therapies are limited. Endocannabinoids are a family of molecules that interact with cannabinoid receptors. The most abundant endocannabinoid, 2-arachidonoylglycerol (2-AG), is a potent regulator of inflammatory responses. The abundance of 2-AG is tightly regulated by an enzyme known as monoacylglycerol-lipase (MAGL). Degradation of 2-AG releases free-fatty acid and arachidonic acid (AA). AA is a precursor for the production of eicosanoids, another class of molecules that regulate inflammatory responses. Inhibition of MAGL has been demonstrated to confer protection against inflammatory and mechanical tissue injury. Based on this rationale, we tested the hypothesis that pharmacological inhibition of MAGL attenuates VILI by decreasing the abundance of eicosanoids.
Methods
A two-hit murine lung injury model was employed using intratracheal injection of lipopolysaccharide (LPS) followed by high tidal volume (30 ml/kg, 2h, 3cm H2O positive end-expiratory pressure, with room air) mechanical ventilation. C57BL/6J mice received peripherally restricted MAGL inhibitor LEI-515 (20 mg/kg, i.p) after LPS exposure and before ventilation. At the end of ventilation, the total cell number and protein content in the bronchoalveolar lavage fluid (BALF) were determined. Neutrophil population and CD63 expression in neutrophils were assessed by flow cytometry. The levels of IL-1β, CXCL2, and arachidonic acid (AA) in the BALF were determined by ELISA. Lung injury was assessed by histology, and inflammatory signaling in the lung was assessed by immunoblot analysis of lung homogenate. Neutrophil extracellular traps (NETS) release was assessed by immunoblot analysis of CitH3 and H2B in the BALF.
Results
MAGL inhibition with LEI-515 significantly reduced BALF total cell counts and protein content, which were increased in response to VILI. BALF levels of IL-1β, CXCL2, TNF-α, and AA were significantly reduced in LEI-515-treated mice. Flow cytometry analysis revealed that mice treated with LEI-515 had lower neutrophil populations and reduced neutrophil surface CD63 expression. Histological analysis showed that LEI-515 treated mice had lower tissue injury. Immunoblot analysis revealed lower expression of NOX4, p-IKB, 5-LOX, and COX2 in LEI-515 treated mice. In addition, immunoblot analysis of BALF samples revealed that CitH3 and H2B levels were significantly reduced in LEI-515 treated mice.
Conclusion
These findings suggest that MAGL inhibition exerts a protective effect against ventilator-induced lung in part by reducing arachidonic acid mediated inflammation, oxidative stress, and neutrophil activation.