Melatonin Alleviates Sepsis-induced ARDS By Inhibiting NLRP3 Inflammasome Activation In Alveolar Macrophages Via ROS/HIF-1α/GLUT1 Axis

Background Sepsis-induced acute respiratory distress syndrome（ARDS）is a common acute and critical clinical condition with a high mortality rate and poor prognosis,and no reliable and effect treatment is available.Melatonin（MEL）,an endogenous neurohormone,has been shown to be effective in alleviating sepsis-induced ARDS in several studies because of its strong anti-inflammatory and immunomodulatory effects,but the mechanism is not yet clear.Previous Studies have shown that lipopolysaccharide（LPS）stimulation of alveolar macrophages（AMs）lead to intracellular NLRP3inflammasome activation,which promotes the release of inflammatory factors and further aggravates the cascade expansion of the inflammatory response in the lung,ultimately leading to the development of ARDS lung injury.In addition,recent studies have shown that down-regulation of macrophage glycolysis levels can significantly inhibit NLRP3 inflammasome activation,thereby reducing the inflammatory response.Therefore,the aim of this study was to investigate whether MEL could inhibit NLRP3inflammasome activation by regulating alveolar macrophage glycolysis levels,thereby alleviating sepsis-induced ARDS,with the aim of finding new targets and providing a theoretical basis for the clinical treatment of ARDS.Methods In this study,we used LPS to stimulate mouse alveolar macrophage（MH-S）model and intraperitoneal injection of LPS to construct an animal model of ARDS in mice.STF-31,an inhibitor of GLUT1,was used to inhibit the GLUT1 expression in cells and lung tissues.GLUT1 in MH-S cells was overexpressed by lentivirus.PX-478was used to inhibit the expression of HIF-1αin cells and DMOG was used to promote the expression of HIF-1αin cells and tissues.H₂O₂was used as ROS donor to promote cellular ROS production.The expression of MT2 receptor was inhibited by 4-P-PDOT and Luzindole.The expression of MT1 receptor was inhibited by small interfering RNA technique;the levels of MT1,HIF-1α,GLUT1,NLRP3,pro IL-1β,mature IL-1βand cleaved caspased-1 were detected by western blot.The level of GLUT1 in mouse lung tissues was also detected by immunohistochemistry.The content of IL-1βin cell culture medium and bronchoalveolar lavage fluid（BALF）of mice was determined by ELISA kit.The lactate（LA）kit measured the level of lactic acid,the end product of glycolysis.Glucose levels in the culture medium and serum were measured using a glucose（Glu）assay kit to assess the glucose uptake function of cells and tissues.In addition,the transport function of glucose by alveolar macrophages was measured using a 2-NBDG fluorescent labeling assay.The level of cellular ROS was evaluated under fluorescence microscopy.Histopathological tests were performed to assess lung tissue damage.Results We found that in LPS-induced MH-S cells,both MEL and STF-31 significantly inhibited the upregulation of GLUT1,NLRP3,pro IL-1β,mature IL-1βand cleaved caspase-1,and significantly inhibited the upregulation of LPS-induced lactic acid release and increased glucose uptake.Lentivirus was further used to overexpress GLUT1 in MH-S cells,and the inhibitory effect of MEL on the upregulation of the above indicators was weakened.Further research using an inhibitor（PX-478）and agonist（DMOG）of HIF-1α,respectively,revealed that both MEL and PX-478 inhibited the expression of HIF-1α,GLUT1,NLRP3,pro IL-1β,mature IL-1β,cleaved caspase-1in MH-S cells,and suppressed the release of lactic acid in LPS-treated MH-S cells.However,after DMOG pretreatment of MH-S cells,the inhibitory effect of MEL on the above indices was significantly weakened.H₂O₂pretreatment of MH-S cells significantly increased the level of cellular ROS and inhibited the regulatory effect of MEL on the HIF-1α/GLUT1 axis,resulting in a diminished inhabitation of NLRP3inflammasome activation in alveolar macrophages by MEL.Interestingly,we found that the use of MT2 receptor inhibitors did not affect the regulation of MEL on the above indicators,while the protective effect of MEL was weakened after downregulation of MT1 expression in MH-S cells using small interfering RNA.In addition,we confirmed that MEL significantly ameliorated LPS-induced lung tissue injury and improved mouse survival in a mouse model of intraperitoneal LPS injection.The GLUT1 inhibitor STF-31 and the agonist of HIF-1α,DMOG,were used at the animal level,respectively,and the alterations of these indices in mouse tissue specimens were examined.Thus,it was confirmed in vivo that MEL could inhibit LPS-induced NLRP3 inflammasome activation in lung tissue by modulating the HIF-1α/GLUT1 pathway,thereby attenuating LPS-induced ARDS.Conclusion MEL can downregulate glycolysis levels through modulation of the ROS/HIF-1α/GLUT1 axis,which in turn inhibits LPS-induced NLRP3 inflammasome activation in alveolar macrophages and ultimately alleviates sepsis-induced ARDS.in addition,we found that MEL inhibits NLRP3 inflammasome activation in alveolar macrophages through the ROS/HIF-1α/GLUT1 axis associated with its MT1 receptor.This study may provide a new experimental basis and theoretical basis for the prevention and treatment of ARDS.