Nlrc5异常表达在急性肺损伤中的作用及靶向机制研究

Respiratory failure is the biggest cause of highest mortality rate of Paraquat (PQ) poisoning. So how to clarify the main pathogenic mechanism of ALI is the key to improve the success rate of rescue. Whole profiling of gene expression and transcription of lung tissue in PQ poisoning mice was detected previously using a newly whole-genome transcription of gene chips, and then the profiling of differential gene expression was determined. Bioinformatics analysis established: ① Nlrc5 is the most important core regulatory gene in intracellular signaling network of differential gene-co-expressed cells in lung injury of PQ poisoning mice. ②Nlrc5 significantly increased after 6h of lung tissue in PQ poisoning mice and largely reduced after 24h using quantitative PCR and Western Blot. In vitro studies confirmed that Nlrc5 was able to significantly inhibit both NF-kB and type I IFN pathway. Therefore, we believe that sustained NF-kB and type I IFN pathway activation induced by downregulation of Nlrc5 is a key mechanism to PQ-induced pulmonary injury. To validate our conclusion, combining the data from bioinformatics analysis and reverse phase protein microarray, we will systematically study the role of the core regulatory gene, Nlrc5, in the PQ induced lung injury, and explore its critical signaling pathways. Not only will this study deepen the understanding of gene function in Nlrc5, but it provides new clues for the next specific interventions.

急性肺损伤(ALI)致死率高，故阐明ALI致病机理是提高抢救成功率的关键。百草枯(PQ)诱导小鼠急性肺损伤是典型急性氧化应激损伤模型。前期采用新型全转录本基因芯片检测了PQ致小鼠肺损伤全基因转录表达谱,经生物信息学分析确立：Nlrc5是PQ致肺损伤差异基因共表达细胞内信号传递网络最重要的核心调控基因；Western Blot证实小鼠6h肺组织Nlrc5增高，24h则显著降低；茶多酚(EGCG)可显著减低PQ中毒小鼠的死亡率。体外研究证实Nlrc5能同时显著抑制NF-kB和I型IFN通路。故认为：Nlrc5分子下调致NF-kB和I型IFN通路活化是氧化应激肺损伤的关键机制。为此，采用反相蛋白微阵列技术和PQ中毒患者样本，系统研究Nlrc5在氧化应激肺损伤中的调控机制和EGCG的干预作用及其与NF-kB的关系。为探索基于特异干预Nlrc5 分子和茶多酚联合治疗氧化应激肺损伤新方案提供实验依据。

百草枯中毒死亡率高，主要是百草枯诱导的氧化应激器官损伤。但是百草枯如何诱导氧化应激的机理不清。本研究以证实Nlrc5参与百草枯中毒诱导的肺、肾损伤为切入点，系统研究了Nlrc5异常表达在急性肺损伤中作用的靶向机制。通过临床患者资料、PQ诱导小鼠模型和Nlrc5基因敲出小鼠验证了Nlrc5的致氧化应激作用；随后发现该作用与Nlrc5 基因通过调控NK-KB通路过度活化的有关。免疫组织化学染色发现PQ中毒小鼠肺组织CD68细胞数显著增高，Nlrc5 基因敲出小鼠显著减低，同时伴有肺肾损伤的减轻。这些实验证实了单核细胞的异常活化是PQ致器官损伤的关键环节。为此，在构建了斑马鱼百草枯中毒模型并进行系统评估模型的可靠性后，发现Rho和JNK-MMP13是斑马鱼巨噬细胞活化的主要通路。采用转基因斑马鱼，筛选具有抑制单核细胞的异常活化的新化合物分子，筛选出还原型谷胱甘肽（Lgr），半胱氨酸（NAG），ZCL278（Cdc42抑制剂）和NSC（鸟氨酸核苷交换因子诱导的Rac GTPase 特异Rac 激活物）可显著减低斑马鱼的死亡率。同时我们还验证了前期百草枯中毒患者的代谢组学检测的分析结果，确定了与百草枯中毒死亡相关的重要关键代谢分子，为百草枯中毒的分子分型提供了基础。这些研究结果为我们下一步研究新型抗氧化应激干预药物用于抢救百草枯中毒提供了新的线索。