The Mechanism Of Hmgb1 Mediating Neutrophil Autophagy To Regulate 1,3-β-glucan-induced Lung Inflammation

Objective: 1,3-β-glucan was a recognized biomarker of fungal exposure in occupational environments.Therefore,a model of lung inflammation induced by1,3-β-glucan was used to study lung inflammation caused by fungal exposure.1,3-β-glucan could recruit a large number of inflammatory cells,mainly neutrophils,to gather in the lung.Autophagy was an important protection and defense mechanism of the body,and HMGB1 could participate in the regulation of a variety of cellular functions.In this study,AAV-sh-HMGB1 and HMGB1 specific inhibitor EP were used to study the regulation of HMGB1 on 1,3-β-glucan-induced lung inflammation and the regulation mechanism of HMGB1 on neutrophil autophagy.Methods: The experimental animals were healthy male C57BL/6 mice.The mice were randomly divided into 4 groups according to their body weight.The alveolar lavage fluid and lung tissue were collected and the mouse bone marrow primary neutrophils were extracted.H&E staining was used to observe the pathological changes of lung tissue.ELISA and Realtime-PCR were used to determine the expression of HMGB1 and cytokines.Western blot and Realtime-PCR were used to determine the expression of HMGB1 and autophagy-related proteins.TUNEL method detected the level of apoptosis.Immunofluorescence was used to observe protein expression,and transmission electron microscopy was used to detect autophagosomes in lung tissues.Results: 1.1,3-β-glucan could induce the increase of HMGB1 secretion in mouse lung tissue and promote the translocation of HMGB1 from the nucleus to the cytoplasm.2.AAV-sh-HMGB1 could significantly reduce the expression of HMGB1 in lung tissue.3.Silencing HMGB1 aggravated the 1,3-β-glucan-induced lung inflammatory cell infiltration.4.1,3-β-glucan could induce the expression of inflammatory factors in mouse lung tissue.5.Silencing HMGB1 could reduce the number of autophagosomes in lung tissue induced by 1,3-β-glucan,and significantly reduce the expression of autophagy-related proteins LC3 and P62 induced by1,3-β-glucan;But silencing HMGB1 could not affect the fusion of autophagosomes and lysosomes.6.1,3-β-glucan could induce a significant increase in inflammatory cells,especially neutrophils in BALF in mice.Silencing HMGB1 further increased the total number of inflammatory cells and neutrophils in BALF,and HMGB1 could be expressed in neutrophils in lung tissues after exposed 1,3-β-glucan.7.EP could significantly inhibit the expression of neutrophils HMGB1 induced by 1,3-β-glucan.8.EP could inhibit the secretion of NETs by neutrophils,and autophagy could promote the release of NETs.9.Inhibition of HMGB1 could exacerbate the level of apoptosis in neutrophils induced by 1,3-β-glucan.10.Inhibition of HMGB1 significantly reduced the expression of autophagy-related proteins in 1,3-β-glucan-treated neutrophils,LC3 and P62,and increased BECN1 expression.11.The interaction between HMGB1 and BECN1 affected neutrophil autophagy.In the lung tissues of1,3-β-glucan-exposed mice,HMGB1 and BECN1 co-localize on neutrophils.Conclusion: 1.HMGB1 could inhibit the progression of 1,3-β-glucan-induced lung inflammation;2.HMGB1 could promote neutrophil autophagy and inhibit lung inflammation caused by 1,3-β-glucan;3.Cytoplasmic HMGB1 regulated 1,3-β-glucan inducing autophagy in neutrophils by combining with BECN1.