Elafin通过NE/EGFR-PI3K-Akt抑制NF-κB活化对炎症性肠病肠屏障保护性机制研究

Intestinal mucosal inflammation and dysfunctional intestinal barrier have been implicated as pathogenic factors in IBD, which are remarkably associated with the activation of NF-кB signaling induced by neutrophil elastase (NE). Elafin is a specific inhibitor of NE, and we previously found that Elafin effectively suppressed the development of colitis, which suggested that its anti-inflammation effect was partly mediated by protect intestinal barrier, the underlying mechanisms have not been clarified. In addition, elafin potently inhibited NF-кB signaling and the activity of EGFR,PI3K,Akt in Caco2 cells stimulated by NE. We therefore speculate that elafin inhibits colitis and protects intestinal barrier by inhibiting NF-кB activity via NE/EGFR-PI3K-Akt signaling. In this study, we will investigate the effects of elafin on the recruitment, activation of neutrophils and NE released by neutrophils, with a particular focus on its ability to inhibit the activation of NF-кB via NE/EGFR-PI3K-Akt signaling in intestinal epithelial cell line models and mouse model of DSS-induced colitis, and clarify the mechanism by which elafin attenuates the activation of NF-кB to inhibit colitis and protect intestinal barrier. The insight gained from this study will facilitate the development of novel therapeutic approaches for IBD.

肠黏膜炎症及屏障受损是炎症性肠病（IBD）的关键致病机制，与中性粒细胞弹性蛋白酶（NE）大量释放并诱导NF-κB活化密切相关。Elafin是NE特异性抑制剂，我们前期研究发现Elafin可抑制结肠炎发展，与对肠屏障的保护有关，具体机制不明。深入研究发现Elafin还可抑制炎症性结肠上皮细胞EGFR,PI3K,Akt及NF-κB活性。我们提出假设：Elafin通过抑制NE及其介导的EGFR-PI3K-Akt通路减弱NF-κB活性，发挥抗炎和肠屏障保护作用。本研究拟在体内外探讨Elafin对中性粒细胞活化及NE产生的直接调控作用；在肠上皮细胞系模型和DSS诱导的小鼠急慢性结肠炎模型上，深入研究Elafin对NE及其介导的EGFR-PI3K-Akt作用于NF-κB的分子调控机制及与炎症发生进展的关系；阐明Elafin减弱NF-κB活性，抗炎和保护肠屏障的分子机制，为IBD的防治新策略提供依据。

肠粘膜炎症和上皮屏障功能障碍被认为是炎症性肠病（IBD）的病理因素，这些因素与中性粒细胞弹性蛋白酶（NE）诱导的NF-κB信号激活显著相关。Elafin是NE的特异性抑制剂，我们之前发现Elafin有效抑制结肠炎的发展。本研究通过构建能够表达Elafin的EcN以探讨EcN-Elafin对结肠炎的保护机制及其对肠道微生态的作用。.构建携带Elafin基因的重组质粒并将该质粒导入野生型EcN（EcN-WT），Western blot验证该基因工程菌在体外Elafin的表达。通过葡聚糖硫酸钠构建小鼠急慢性结肠炎模型，研究EcN-Elafin对小鼠体重、排便情况及疾病活动度指数（DAI）、结肠长度的影响。H-E染色进行组织病理学评分。流式细胞术检测结肠固有层浸润中性粒细胞和巨噬细胞的比例。RT-qPCR和ELISA定量小鼠结肠组织中的促炎细胞因子TNF-α和IL-6以及趋化因子CXCL-1的mRNA及蛋白质表达水平。免疫组化检测结肠组织中的MPO和ZO-1蛋白表达。通过16S rDNA测序和GC–MS靶向代谢组学方法检测肠道菌群和短链脂肪酸含量。.基因工程菌EcN-Elafin体外成功表达Elafin。EcN-Elafin灌胃于小鼠显著改善急慢性结肠炎小鼠体重减轻、结肠缩短及DAI评分；降低小鼠结肠组织病理学评分；下调结肠组织中促炎细胞因子TNF-α、IL-6和中性粒细胞特异性趋化因子CXCL-1的表达水平；增强结肠黏膜上皮细胞紧密连接蛋白ZO-1的表达。同时，EcN-Elafin还减少了结肠炎状态下结肠黏膜固有层中性粒细胞和巨噬细胞浸润的比例。在体外，EcN-Elafin细菌提取物显著下调Caco-2细胞中NF-κB通路的活性。最后，EcN-Elafin显著改善DSS所致的小鼠结肠内容物微生态失衡，尤其是增加对肠道有益的Alloprevotella，Clostridium ⅩⅣa和Prevotella的相对丰度，同时显著上调小鼠肠内容物丁酸和戊酸的含量。.表达Elafin的基因工程菌EcN-Elafin显著减轻DSS诱导的急慢性小鼠结肠炎，其保护性作用可能是通过下调肠黏膜上皮细胞中NF-κB通路、增强肠屏障功能、改善肠道菌群失衡和增加肠道短链脂肪酸的含量实现的。本研究结果有望成为治疗炎症性肠病的一种新的方法。

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