京尼平抑制NLRC4炎性体的活化

发布时间：2018-07-07 19:04

本文选题：京尼平 + 炎性体　；参考：《吉林大学》2015年硕士论文

【摘要】：炎性体是天然免疫模式识别受体（PPPs）识别病原相关分子模式（PAMPs）或损伤相关分子模式（DAMPs）后在胞浆内形成的一种多蛋白复合物。炎性体主要由模式识别受体、ASC和caspase-1构成，在病原感染、炎症以及自身免疫性疾病等过程中发挥重要作用。现已发现多种炎性体，尤以对NLRC4、NLRP3、AIM2等炎性体的研究较为深入，，但对其具体的活化和负调控机制仍不十分清楚，亟待进一步阐明。京尼平（Genipin）是栀子苷经β-葡萄糖苷酶水解后的产物，广泛存在于栀子、杜仲等植物中。已有研究表明，京尼平在抗感染、抗炎以及治疗肝脏疾病和Ⅱ型糖尿病等自身免疫性疾病过程中具有一定的效果。尽管对于京尼平的功效及机制有一定认识，但其调节炎症反应的机制并不十分清楚，因而限制了京尼平在疾病治疗中的应用。京尼平是否参与调控炎性体信号，进而对其所介导的炎症具有调节作用，是本课题热切关注的科学问题。有研究表明，在绿脓假单胞杆菌、军团菌、肺炎克雷伯菌等病原感染过程中通过活化NLRC4炎性体，诱导肺炎的发生。因此，本课题在体实验以沙门氏菌重组鞭毛蛋白诱导的小鼠肺炎模型为研究对象，探究京尼平对NLRC4炎性体介导的炎症结局的影响。用ELISA和免疫组织化学方法检测肺部炎性因子分泌和炎性细胞浸润，并评价京尼平对细菌鞭毛蛋白引起的肺组织病理变化的作用。结果表明，京尼平可以显著抑制肺组织中IL-1β与KC的分泌和中性粒细胞的浸润，同时缓解鞭毛蛋白导致的肺泡出血，肺泡间质增厚。由此表明京尼平能够显著抑制细菌鞭毛蛋白引起的小鼠肺炎。体外实验以小鼠原代巨噬细胞为研究模型，探究京尼平对鼠伤寒沙门氏菌及沙门氏菌重组鞭毛蛋白诱导的NLRC4炎性体活化的负调控机制。应用免疫蛋白印迹的方法检测NLRC4、UCP2等蛋白的表达以及Caspase-1、IL-1β的活化片段和ASC寡聚化；流式细胞术检测细胞死亡及ROS的产生；免疫荧光分析ASC斑点的形成和LC3蛋白的表达；ELISA检测IL-1β和TNFα细胞因子的分泌。结果表明，京尼平显著抑制鼠伤寒沙门氏菌及沙门氏菌重组鞭毛蛋白诱导的Caspase-1剪切成熟和IL-1β分泌，并且通过抑制细胞自噬以及NLRC4蛋白的表达，实现对NLRC4炎性体的负调控。综上所述，本研究确证京尼平通过抑制NLRC4蛋白的表达和细胞自噬抑制NLRC4炎性体的活化，进而有效缓解和治疗沙门氏菌重组鞭毛蛋白引起的小鼠肺炎。
[Abstract]:Inflammatory body is a polyprotein complex formed in cytoplasm by innate immune pattern recognition receptors (PPPs) which recognize pathogen-associated molecular patterns (PAMPs) or damage related molecular patterns (DAMPs). Inflammatory bodies are mainly composed of pattern recognition receptors ASC and caspase-1, which play an important role in pathogenic infection, inflammation and autoimmune diseases. Many kinds of inflammatory bodies have been found, especially NLRC4NLRP3AIM2, but their specific activation and negative regulation mechanisms are still unclear and need to be further elucidated. Genipin is a product of geniposide hydrolyzed by 尾-glucosidase. It is widely found in Gardenia jasminoides and Eucommia ulmoides. Studies have shown that geniapine has a certain effect in the process of anti-infection, anti-inflammation and the treatment of autoimmune diseases such as liver disease and type II diabetes. Although there is a certain understanding of the efficacy and mechanism of geniapine, its mechanism of regulating inflammatory response is not very clear, which limits the application of genipin in the treatment of diseases. Whether geniapine is involved in regulating inflammatory signal and then regulating inflammation mediated by genistein is a scientific issue which has been paid close attention to. Some studies have shown that in the process of infection of Pseudomonas aeruginosa, Legionella pneumoniae and Klebsiella pneumoniae, pneumonia is induced by activating the inflammatory body of NLRC4. Therefore, in vivo, we investigated the effect of geniapine on inflammatory outcome mediated by NLRC4 in mice pneumonia model induced by recombinant flagellin of Salmonella. Lung inflammatory factor secretion and inflammatory cell infiltration were detected by Elisa and immunohistochemistry, and the effect of geniapine on pathological changes of lung tissue induced by bacterial flagellin was evaluated. The results showed that geniapine could significantly inhibit the secretion of IL-1 尾 and KC and the infiltration of neutrophils in lung tissue, and alleviate alveolar hemorrhage caused by flagellin and the thickening of alveolar interstitial. It is suggested that geniapine can significantly inhibit bacterial flagellin-induced pneumonia in mice. In vitro, the mouse primary macrophages were used to investigate the negative regulation mechanism of geniapine on the activation of NLRC4 inflammatory body induced by recombinant flagellin of Salmonella typhimurium and Salmonella typhimurium. Western blot was used to detect the expression of NLRC4UCP2, the activated fragment of Caspase-1 and IL-1 尾 and ASC oligodepolymerization; flow cytometry was used to detect cell death and Ros production; immunofluorescence was used to detect the formation of ASC spots and the expression of LC3 protein. The secretion of IL-1 尾 and TNF 伪 cytokines was detected by Elisa. The results showed that geniapine significantly inhibited Caspase-1 shearing maturation and IL-1 尾 secretion induced by recombinant flagellin of Salmonella typhimurium and Salmonella typhimurium, and negatively regulated the inflammatory body of NLRC4 by inhibiting autophagy and the expression of NLRC4 protein. In conclusion, this study confirmed that geniapine can effectively relieve and treat pneumonia induced by recombinant flagellin of Salmonella by inhibiting the expression of NLRC4 protein and inhibiting the activation of NLRC4 inflammatory body by autophagy.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7

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