Effect Of NR4A1-mediated Autophagy On NLRP3 Inflammasome Signaling Pathway In Adipocytes

Objective: The orphan nuclear receptor NR4A1 belongs to the nuclear receptor NR4 A superfamily.It can recognize specific receptors and regulate gene expression in biological processes such as mitophagy and apoptosis at the transcriptional level,and play an important role in life activities such as inflammatory response,metabolic process and energy balance.Obesity is an important risk factor for metabolic diseases such as insulin resistance and type 2 diabetes.It is mainly characterized by low-grade chronic inflammation and macrophage infiltration in adipose tissue,which is manifested by the increase of systemic inflammatory markers.During the development of obesity,the dangerous molecular signal generated by overnutrition will activate nucleotidebinding oligomerization domain-like receptor protein 3(NLRP3)and associate with apoptosis-associated speck-like protein contain a caspase activation and recruitment domain(ASC)combined to recruit the precursor of cysteinyl aspartate specific proteinase(Caspase-1)to form the NLRP3 inflammasome.At the same time,it promotes the maturation of Caspase-1,and then promotes the release of proinflammatory factors such as interleukin-1β(IL-1β),leading to inflammatory response and cell pyroptosis.Autophagy is a process of recycling necrotic substances in cells in eukaryotes,and it is a self-protection mechanism to maintain the homeostasis of the body.Many studies have shown that autophagy can reduce the inflammatory response by inhibiting the activation of NLRP3 inflammosomes by removing damaged mitochondria or by directly degrading inflammosome components.The orphan nuclear receptor NR4A1 is inextricably linked with autophagy,and can play a role in inflammatory diseases by participating in the regulation of autophagy.The first part of this study will establish the adipocyte autophagy model and pyroptosis model,and explore the expression of NR4A1 in the adipocyte autophagy model and pyroptosis model;In the second part,by inducing pyroptosis in adipocytes transfected with overexpressed NR4A1 virus and empty virus,we will explore whether NR4A1 can mediate autophagy to resist the activation of the NLRP3 inflammasome signaling pathway in adipocytes,thereby delaying the obesity-related inflammatory response.Methods: Part 1: 1.1 The differentiated and mature mouse adipocytes(3T3-F442A)were treated with different concentrations of rapamycin for 12 hours to establish the autophagy model of adipocytes,and the expression of autophagy marker proteins LC3Ⅱ and NR4A1 were detected by Western blot.1.2.Mature 3T3-F442 A cells were treated with lipopolysaccharide(LPS)for 24 hours to establish the pyroptosis model of adipocytes,and the expression of NR4A1 and NLRP3,Caspase-1,and IL-1β proteins related to the NLRP3 inflammasome signaling pathway were detected by Western blot.Part 2:We screened the transfection of 3T3-F442 A cells overexpressing NR4A1 virus and empty virus.In this experiment,we named the 3T3-F442 A cells transfected with empty virus as NC cells and the 3T3-F442 A cells transfected with overexpressed NR4A1 virus as OV cells.LPS was used to treat differentiated mature NC cells and OV cells for 24 hours.The experimental cells were divided into four groups: NC cells,OV cells,NC cells + LPS and OV cells + LPS.The expressions of NLRP3,IL-1 β in NLRP3 inflammatory body signaling pathway and autophagy marker protein LC3 II were detected by Western blot.Results: Part 1: 1.1 The differentiated mature 3T3-F442 A cells were treated with rapamycin to establish adipocyte autophagy model,and the expression levels of NR4A1 and autophagy marker proteins LC3Ⅱ were found to be up-regulated,indicating that NR4A1 may be involved in the process of autophagy.1.2 The differentiated mature 3T3-F442 A cells were treated with LPS to establish adipocyte pyroptosis model,and the expression levels of NLRP3,IL-1β and NR4A1 were found to be up-regulated,indicating that NR4A1 may be involved in the process of cell pyroptosis.Part 2: After treating differentiated mature NC cells and OV cells with LPS for 24 hours,we found that compared with NC cells,the expression level of NLRP3 and IL-1β in OV cells were down-regulated,and the expression level of LC3Ⅱ was up-regulated,indicating that NR4A1 could inhibit NLRP3 in 3T3F442 A cells.The activation of inflammasome signaling pathway also activated autophagy,and NR4A1 may resist the inflammatory response of NLRP3 inflammasome signaling pathway in 3T3-F442 A cells by mediating autophagy.Conclusion: NR4A1 could participate in the regulation of autophagy and pyroptosis in 3T3-F442 A cells,and NR4A1 could inhibit the activation of NLRP3 inflammasome signaling pathway in 3T3-F442 A cells,as well as the activation of autophagy,indicating that NR4A1 may mediate autophagy through autophagy.NR4A1 may play a role in resisting the inflammatory response of NLRP3 inflammasome signaling pathway in 3T3-F442 A cells by mediating autophagy.