Mechanism Of Nur77 Mediated Extracellular Matrix Remodeling Of ARDS By Regulating TGF-β1/Smad3/MMP9 Signaling Axis

Acute respiratory distress syndrome(ARDS)is a common critical illness in clinic,which is characterized by intense neutrophilic lung inflammation and increased alveolar-capillary barrier permeability leading to severe hypoxemia and respiratory failure.Despite the significant advances have been made in the treatment strategies of ARDS include pulmonary protection ventilation,prone position and oxygenation by extracorporeal membrane,there are patients with pathologic remodeling of extracellular matrix(ECM)for persistent lung damage and aberrant tissue repair,which may trigger persistent fibrosis that can result in reduced quality of life and a poor prognosis for ARDS patients.Therefore,to study ECM remodeling and suggests new molecular targets for treatments are essential for preventing and treating pulmonary fibrosis post ARDS.Nur77 is a transcription factors occurs widely in various tissue cells regulated by inflammatory factors,growth factors and hormones and involved in a diverse variety of biological processes through regulating the expression of target genes in the post-transcriptional level.The present study found that Nur77 and its downstream products can attenuate inflammation and lung injury in ARDS.Moreover,Nur77 has shown antifibrotic effects in various fibrotic diseases.Transforming growth factor-β1(TGF-β1)is an important regulator of lung development,repair from injury and fibrosis.TGF-β1 is activated accompanying with injured tissues in early ARDS.In response to ligand binding,the TGF-β1receptor induces collagen production via Smad signaling pathway.Furthermore,our previous research found that TGF-β1 up-regulated MMP9 involved in ARDS lung ECM remodeling.Prior studies denoted that Nur77 contributed to TGF-β1signaling,conversely,it plays diametrically opposing roles.To date,the mechanism of Nur77 involved ECM remodeling is largely unknown,and its role of TGF-β/Smad signaling regulation in ARDS remains to be determined.In the present study,we established an ARDS rat model and cultured A549 cells in vitro,to investigate the role and mechanism of Nur77 in ARDS lung ECM remodeling and provide valuable insights into new diagnostic targets and therapeutic strategies for clinical intervention in patients with ARDS.The research contents of this paper include:1.Inflammatory ARDS rat model was established by tail vein injection of lipopolysaccharides(LPS)and treated with Nur77 agonists Cytosporone B(Csn B).Thirty-two adult male SD rats were divided into four groups randomly:the control group,LPS group,Csn B group,LPS + Csn B group.Myeloperoxidase(MPO)activities,leukocyte count,hydroxyproline content were tested.HE staining was used to observe the pathological changes of inflammation and lung tissue damage.Masson staining is used to evaluate ECM deposition.The q PCR,western blot,immunohistochemical assay were performed to determine the expression levels of MMP9 and TIMP1.2.Plasmids construction and lentivirus production stable knockdown and overexpression Nur77 in A549 cell lines were generated by using the lentiviral system and verified using q PCR and western blot.Wild type,knockdown and overexpression Nur77 were treated with PBS and LPS for 24 hours.Protein expression levels of MMP9 were measured by western blot.3.The dual-luciferase reporter assay system was applied to analyze the effects of overexpressed Nur77 on TGF-β/Smad signaling pathway.Wild type and knockdown Nur77 were treated with PBS and TGF-β1.The expressions of MMP9,p-Smad3,Smad3 at various time points were measured using western blot.A immunofluorescence technique was used to locate the subcellular position of Smad3 in A549 cells.The interaction between Nur77 and Smad was confirmed by co-immunoprecipitation.4.Nur77-binding sites of the MMP9 promoter region sites were predicted by JASPAR database.To test whether Nur77 binded the MMP9 promoter region,chromatin immunoprecipitation(Ch IP)assays were performed.A mutant plasmid in MMP9 promoter region binding site was also established.To investigate whether Nur77 could directly bind to the promoter region of MMP9 and its regulatory effects on MMP9 transcriptional activity,dual-luciferase reporter assay was performed in vitro.The results showed that: 1.Leukocyte count and lung tissue MPO activity were significantly higher in the LPS group,compared to the control group.And lung tissue structures were ambiguous,a large number of inflammatory cells and red blood cells infiltration were observed.The alveolar septa enlarged,with collagen fibrils.The Csn B pretreatment significantly decreased the collagen fibre deposition and thickness of the adhesion layers.The expression levels of MMP9 protein and m RNA in LPS + Csn B group were lower than those in LPS group,and TIMP1 was not affected by Csn B pretreatment.2.q PCR and western blot results showed that A549 cell line with stable knockdown and overexpression of Nur77 was successfully constructed.Compared with wild type A549 cells,overexpression of Nur77 inhibited LPSinduced MMP9 protein expression of A549 cells,and deletion of Nur77 significantly increased LPS-induced MMP9 protein expression.3.Dual luciferase reporting assay showed that overexpression of Nur77 inhibited the transcriptional activity of TGF-β/Smad signaling pathway.Knockout Nur77 increased TGF-β1-induced MMP9 and P-Smad3 expression.Overexpression of Nur77 inhibited Smad3 entry into the nucleus.Immunoprecipitation results showed that Nur77 and Smad3 had an interaction.4.JASPAR database predicted that there were Nur77 binding sites at-999bp～-990 bp upstream of MMP9 transcription start site.Chromatin immunoprecipitation and dual luciferase reporting assay confirmed that Nur77 directly binds to this site and inhibits MMP9 transcriptional activity.Conclusion: 1.Nur77 alleviates LPS-induced lung inflammation in ARDS rats,reduces lung collagen deposition,and plays a protective role in ARDS inflammation and ECM remodeling.2.Nur77 is an upstream transcription factor of MMP9 and participates in ECM remodeling through negative regulation of MMP9 expression.3.Nur77 inhibits Smad3 phosphorylation and nuclear transfer by binding to Smad3,thereby blocking TGF-β1/Smad3/MMP9 signal transduction.