NOX2在NOD2介导重症急性胰腺炎肠粘膜屏障损伤中的机制研究

Intestinal mucosal barrier injury in severe acute pancreatitis (SAP) is the central initiating factor in the development of turning to multiple organ dysfunction. Studies have shown that oxidative stress,inflammation and apoptosis play an important role in the intestinal mucosal barrier disorder, and NADPH oxidase and NOD2 may play upstream regulatory role in the activation of NF-κB pathway and in caspases in apoptosis signaling pathways,but yet to clarify the mechanism of the intestinal mucosal barrier injury in SAP. Accordingly we put forward the hypothesis: in SAP, intestinal NADPH oxidase overexpression and activation resulted to oxygen species increased production, would lead to a lot of NOD2 expression which further leading to activation of NF-κB and caspases, and subsequently a lot of mediators of inflammation would cause intestinal injury. To verify this hypothesis, the role of the NADPH oxidase would be studied during NOD2-mediated activation of NF-κB and caspases in SAP rats in vivo；By using the technology of gene silencing and over-expression of the NADPH oxidase in IEC-6 cell line in vivtro, would confirm that the NADPH oxidase could activate NOD2-mediated NF-κB inflammatory cascade reaction and caspases apoptotic signaling pathway, which lead to the intestinal mucosal barrier injury. This project is designed for the prevention and treatment of the intestinal mucosal barrier to provide a theoretical basis and experimental evidence in SAP.

重症急性胰腺炎(SAP)时肠粘膜屏障损伤是其向多器官功能不全发展的中心始动因素。研究表明，氧化应激、炎症反应和细胞凋亡在肠粘膜屏障障碍中发挥重要作用，而NADPH氧化酶2（NOX2）及NOD2在NF-κB炎症级联反应或caspases凋亡信号通路中起上游调控作用，但其在SAP肠粘膜屏障损伤中的作用机制仍未阐明。据此假说：SAP时肠道NOX2过表达引起氧自由基增加，导致NOD2大量表达、激活NF-κB和caspases通路，产生大量炎症介质，导致肠损伤。为此，本项目拟通过SAP大鼠模型体内实验，观察NOX2及其在NOD2介导NF-κB激活和caspases活化的作用；利用基因沉默和过表达NOX2技术的细胞株IEC-6体外实验，证实NOX2通过NOD2介导NF-κB炎症级联反应和caspases凋亡信号通路，引起肠粘膜屏障损伤。本项目可为SAP肠粘膜屏障损伤的防治提供理论基础和实验依据。

（1）按计划执行情况。.研究按照实验计划,建立了牛黄胆酸钠诱导的大鼠重症急性胰腺炎(SAP)模型,观察并评估了SAP模型中肠道组织的损伤以及NOX2、NOD2及其下游通路在SAP模型肠损伤中的变化特点与规律，同时应用NOX2抑制剂apocynin干预处理后，观察胰腺炎血清淀粉酶、脂肪酶、炎症因子水平的改变以及胰腺、肠组织结构和功能的变化和细胞凋亡水平，并对NOX2、NOD2及下游因子NF-κB、JNK、P38、Caspase信号凋亡通路、下游炎症因子等进行检测，探索NOX2在NOD2介导的NF-κB炎症级联反应和Caspase细胞凋亡信号通路在SAP肠黏膜屏障损伤中的作用。体外实验部分，我们采用大鼠小肠隐窝上皮细胞（IEC-6），加入胰腺炎血清模拟胰腺炎环境，并分别对NOX2进行沉默和过表达后，观察并检测NOX2、NOD2下游相关分子的表达水平，从细胞层面对本课题提出的科学假设进行进一步的炎症。.（2）研究目标完成情况。.本课题组通过体内实验和体外实验，证实了NOX2在NOD2介导的SAP肠黏膜损伤中发挥了重要的作用。动物实验中应用了NOX2抑制剂apocynin干预处理后，胰腺炎病情好转,血清各项指标好转，胰腺结构功能损伤减轻，同时肠道组织结构功能损伤减轻。apocynin抑制了肠道组织中NOX2的表达水平，下游因子NF-κB、p38、p-p38、caspase-3、caspase-9等水平下降，TUNEL法检测肠道组织凋亡水平降低。细胞实验部分由于人小肠粘膜上皮细胞获取和培养较困难等原因，我们更换为大鼠小肠隐窝上皮细胞（IEC-6），并分别对NOX2进行沉默和过表达后加入胰腺炎血清诱导造模，检测NOX2及下游因子的表达水平，从细胞水平验证了NOX2及其在NOD2介导的炎症级联反应通路和凋亡通路在SAP肠道损伤中的重要作用。本课题组建立了SAP肠损伤的动物模型和细胞模型，系统研究了NOX2及NOD2介导的症级联反应通路和凋亡信号通路导致SAP肠损伤及肠黏膜屏障障碍的发生，并从分子水平探讨及作用机制，基本按期完成了项目研究计划。