NOD2调控内质网应激-自噬在肠外营养致肠屏障损伤中的机制研究

Parenteral nutrition (PN) is an essential life-maintaining treatment for premature infants. However, long-term PN may induce intestinal barrier injury and bacterial translocation, which consequently increases gut derived infection and PN-associated liver disease. Currently, the pathogenesis is still unclear. Our previous studies suggested that various fatty acids took different effects on endoplasmic reticulum stress (ERS)- autophagy pathway in Caco-2 cells. ERS-autophagy pathway was demonstrated to be important in the maintenance of intestinal homeostasis and regulated by intracellular bacterial sensing mediated by nucleotide-binding oligomerization domain containing 2 (NOD2).Thus, we proposed a hypothesis that NOD2 probably regulated ERS-autophagy pathway and participated in intestinal barrier injury induced by PN. To test this hypothesis, we will explore the roles of NOD2 in ERS-autophagy in intestinal epithelial cells by using CRISPR/Cas9, 3D spheroids culture, organoids culture, electron microscopy. Furthermore, we will illustrate the interaction of NOD2 with molecules involved in ERS-autophagy pathway by using pull-down assay and laser confocal microscope. This study will be helpful in revealing the mechanism and providing potential targets for the prevention and treatment of intestinal barrier injury induced by PN from a new perspective.

肠外营养（parenteral nutrition,PN）是维持早产儿生命的重要手段，但长期应用可导致肠屏障损伤，增加肠源性感染和肝损害的机率，其机制目前尚不明确。我们前期研究发现：不同脂肪酸可引起Caco-2细胞内质网应激（endoplasmic reticulum stress,ERS）和自噬通路发生改变。ERS-自噬对维持肠上皮细胞稳态有重要意义，而NOD2是调控ERS-自噬通路的重要机制之一。由此提出科学假设：NOD2调控肠上皮细胞ERS-自噬通路在PN致肠屏障损伤的发生机制中发挥重要作用。本项目拟采用CRISPR/Cas9基因编辑、细胞三维球体培养、离体类器官培养、电镜等技术，研究NOD2对肠屏障功能及肠上皮细胞ERS-自噬通路的影响；并采用pull-down、激光共聚焦共定位技术探索NOD2与ERS-自噬通路相关分子的相互作用。以期为临床上防治PN致肠屏障损伤提供新的靶点。

背景与目的：肠外营养（parenteral nutrition，PN）长期使用会导致肠上皮屏障损伤，进而增加与其相关的多种并发症发生风险。核苷酸结合寡聚化结构域2（nucleotide binding oligomerization domain 2，NOD2）作为胞浆模式识别受体参与肠免疫反应，并且是调控肠上皮细胞内质网应激（endoplasmic reticulum stress，ERS）和自噬通路非常重要的分子。因此，在本研究中，我们研究了NOD2在PN致肠屏障功能损伤的发生机制中的作用。方法：中长链脂肪乳剂处理Caco2细胞，采用胞壁酰二肽(Muramyl Dipeptide，MDP)作为NOD2的激动剂，上调NOD2的表达。使用乳酸脱氢酶（LDH）活性检测、transwell单层细胞模型和三维球体细胞模型来检测细胞毒性和肠屏障损伤的变化。RT-qPCR比较Caco2细胞内质网应激-自噬相关分子的mRNA表达水平变化。结果：NOD2预激活Caco2细胞后，可使中长链脂肪乳剂导致的Caco2细胞毒性相较于对照组有所减轻。Transwell单层细胞模型同样显示了NOD2上调的细胞能够维持更好的上皮细胞屏障功能。三维球体培养的Caco2细胞在乳剂处理相比于对照组能够维持更好的囊泡形态和生长速率。RNA分子水平和蛋白水平上，内质网应激相关分子XBP1和ATF4以及自噬相关分子ATG16L1、Beclin1 转录水平随着NOD2的上调而增高，下调而降低。结论：NOD2上调在Caco2细胞模型中可以减弱脂肪乳剂导致的肠上皮屏障损伤，其机制可能其与进一步激活的内质网应激-自噬通路有关，尚需进一步深入研究。