The Regulatory Effects Of STY39 On The Expression Of Inflammatory Cytokines In Human Lung Epithelial Cell Line A549 Stimulated With Lipopolysaccharide

α-melanocyte stimulating hormone (α-MSH), which is a tridecapeptide derived from pro-opiomelanocortin, has been recognized as having multifunctions of pyretolysis, inhibiting food intake, protecting the skin from UV radiation-induced damage, anti-inflammation and immunomodulation.α-MSH plays critical roles in the regulation of immune and inflammatory reactions, which is always one of hotspots in the field of neuroendocrinology and have been researched at the holistic or local and at the cell or molecule levels.a-MSH has been shown to influence various early inflammatory cytokines and immunological conditions by downregulating either the production or the action of the proinflammatory cytokines and upregulating the anti-inflammatory cytokines. Abroad and our researches have confirmed thatα-MSH can reduce several experimental models of inflammation, such as endotoxic shock and acute respiratory distress syndrome (ARDS) and so on. Scientists found thatα-MSH level decreased in the serum of endotoxic shock patients and increased during convalescence.Our previous researches also had identified thatα-MSH had a significant inhibition to late inflammatory mediators such as HMGB1.α-MSH functions specifically via melanocortin receptors(MCRs), which include five subtypes (MC1R to MC5R). They belong to type I G- protein-coupled receptors(GPCRs), also known as seven transmembrance receptors. a-MSH exerts the actions of anti-inflammation and immunoregulation mainly through MC1R and MC5R. MC3R and MC4R principally involved in the regulation of food, weight and energy metabolism. Butα-MSH has its own disadvantages. It has MC1 and MC3 receptor-selective and low affinities for MC4 and MC5 receptors. [Nle4, D-Phe7]α-MSH (NDP-MSH) is an analogue ofα-MSH and a strongly agonistic peptide, which has more powerful anti-inflammatory ability thanα-MSH and is not susceptible to degradation. But it does not show receptor-selective. Moreover, it has a stronger effect on skin pigmentation. Based on these disadvantages, we have already been designed a new peptide (named STY39) by InsightⅡsoftware, which is a a-MSH analogue and has MC1 and MC5 receptor-selective, it's property of binding MCRs and inducing cyclic AMP were already been identified. Furthermore, the effects of STY39 were appraised in the mouse model for endotoxic shock and ARDS.In the past decades, the investigation for the effects and mechanisms of a-MSH in anti-inflammation and immunoregulation mainly focused on neutrophils, monocytes, dendritic cells and lymphocytes, we had been confirmed that STY39 had brilliant protection from the mouse model for endotoxic shock and ARDS, but rarely research focused on the effects and mechanisms in lung parenchymal cells such as lung alveolar epithelial cells (AEC) especially on alveolar epithelial cell typeⅡ(AECⅡor ATⅡ) associated with STY39. Acute lung injury (ALⅠ) is caused by any stimulus of local or systemic inflammation, principally sepsis, trauma, shock and so on. ALI is a diffuse heterogeneous lung injury characterized by hypoxemia, non-cardiogenic pulmonary edema, low lung compliance and widespread capillary leakage, and the core pathology is disruption of the capillary-endothelial interface. ARDS is the severe stage of ALI. Clinical treatment difficulties associated with ALI is the sequelae pulmonary fibrosis. AEC is composed of two main cell types, the alveolar typeⅠcell (ATⅠ) and the alveolar typeⅡcell (ATⅡ). ATⅡare the progenitor cells for the alveolar epithelium and responsible for reforming the alveolar epithelium after damage of the very sensitive ATⅠ. ATⅡalso produce pulmonary surfactant and transport sodium and fluid from the apical surface into the interstitium under the physiological and pathological circumstances. ATⅡcan secrete a variety of substances to limit inflammation and clear microorganisms in the normal lung and play an critical role in pulmonary immune regulation and defense. Moreover, as immunoregulatory cells, ATⅡplay important roles in innate and adaptive immunity. Studies have showed that ATⅡexpress costimulatory molecules such as CD54,CD80 and are capable of delivering costimulatory signals for T cells, providing evidence that ATⅡare capable of acting as antigen-presenting and immunoregulatory cells of the lung. They have intimately association with the pathological processes of ALI, ARDS and pulmonary fibrosis. So we chose the human lung typeⅡepithelial cell line (A549 cells) as the experimental cell model to investigate the effects of STY39 on the expression of inflammatory cytokines by LPS-stimulated A549 cells and to reveal the protection mechanisms of STY39 in ALI. PartⅠThe regulatory effects of STY39 on the expression of inflammatory cytokines in human lung epithelial cell line A549 stimulated with lipopolysaccharideObjective: To observe effects of STY39 on the expression of inflammatory cytokines in human lung epithelial cell line A549 stimulated with LPS. Methods:The A549 human lung type II epithelial cell line was utilized as the experimental cell model and divided into control group, LPS-stimulated group, STY39 treated group (10-6mol/L,10-8mol/L, 10"10mol/L,10-12mol/L) and a-MSH treated group (10-6mol/L,10-8mol/L,10-10 mol/L, 10'12mol/L). At the various time points post treatments, the production of early inflammatory cytokines including IL-1β,IL-4,IL-6,IL-8 and TNF-a was detected by quantitative real time PCR and ELISA. The expression of late mediator HMGB1 was examined by quantitative real time PCR and Western Blot, and the secretion of HMGB1 was examined by ELISA. The human leukemic monocyte lymphoma cell line U937 was used to observe the effect of STY39 on the adhesion of monocytes to A549 cells induced by LPS. The expression of ICAM-1 was analyzed by flow cytometry and quantitative real time PCR. The production of SP-D was detected by ELISA. Results:The gene expression of early inflammatory cytokines, IL-1β,IL-6 IL-8 and TNF-a, were significantly decreased, but the gene expression of IL-4 was upregulated in STY39-treated group compared with LPS-stimulated group, which was further confirmed by the data obtained in ELISA. STY39 prevented LPS-induced HMGB1 release, reduced the expression of ICAM-1 and the adhesion of U937 cells to LPS-stimulated A549 cells. STY39 enhanced the level of SP-D produced by A549 cells, which is an important molecule in immune defense and the maintenace of pulmonary surface tension. Conclusion: These results indicate that STY39 markedly reduce the production of early proinflammatory cytokines and late mediator HMGB1, but increase the expression of IL-4 and SP-D.PartⅡThe molecular mechanisms of the suppressive effects of STY39 on inflammatory cytokine expression in human lung epithelial cell line A549 stimulated with lipopolysaccharideObjective: To investigate the molecular mechanisms of the suppressive effects of STY39 on the produce of inflammatory cytokines in LPS stimulated A549 cells. Methods:A549 cells cultured in vitro were divided into control group, LPS stimulated group, STY39 treated group (10-8mol/L), a-MSH treated group (10"8mol/L). The constitutive expression of MC1 to MC5 receptors by A549 cells was detected using semi-quantitative RT-PCR. After the stimulation of LPS with or without STY39 or a-MSH treatment, the expression of MC1 to MC5R mRNA was detected by quantitative real-time PCR, respectively. Then the dual-luciferase reporter assay was used to analyze the transcriptional activity of nuclear factor-kappa B (NF-κB), and the expression of phospho-Erk, phospho-JNK, phospho-P38, NF-κB p65 and IκBαwas detected by Western Blot. Results:The constitutive expression of MC1 to MC5 receptors in A549 cells was found. The expression of MC1 receptor was the highest, and MC5 receptor expression was lower than MC1 receptor. MC2 to MC4 receptors were weak compared with MC1-R and MC5-R. LPS enhanced the mRNA expressions of MC1 and MC5 receptors, but had no obviously effects on MC2-MC4 receptors. STY39 partially inhibited the phosphorylation of Erk, JNK, p38 in A549 cells stimulated by LPS. STY39 also partially suppressed the expression of NF-κB p65 and the degradation of IκBαin LPS-stimulated A549 cells. Dual-luciferase reporter assay identified that STY39 reduced LPS-induced increase of the transcriptional activity of NF-κB in A549 cells. Conclusion:The suppressive effects of STY39 on inflammatory cytokine expression in A549 lung epithelial cells may be through MCRs-mediated MAPKs and NF-kB signal transduction pathways.