基于NF-κB/MLCK通路研究大肠杆菌O157:H7介导肠上皮屏障功能损伤的分子机制

大肠杆菌O157:H7感染极易引发肠屏障损伤，进而导致出血性肠炎和血性腹泻等严重疾病，但其分子机制尚不清楚。前期，课题组发现大肠杆菌O157:H7毒力蛋白EspF能与宿主ANXA6蛋白互作，进而激活肌球蛋白轻链激酶（MLCK），导致肌球蛋白轻链（MLC）磷酸化，破坏肠上皮紧密连接。我们还发现，EspF蛋白可改变ANXA6蛋白表达与定位，增加NF-κB炎症通路蛋白p65磷酸化水平，上调TNF-α、IL-1β水平。因此猜测：EspF-ANXA6可能通过调控NF-κB/MLCK通路介导肠上皮屏障功能损伤。本项目拟通过动物及细胞实验验证EspF-ANXA6对NF-κB通路蛋白IKKα、p65、IκBα等的调控作用；明确NF-κB通路激活在MLCK依赖性紧密连接破坏、细菌易位中的作用。以期揭示大肠杆菌O157:H7感染导致肠屏障功能损伤的分子机制，为探索临床治疗肠炎和严重腹泻的新方法提供理论依据。

Escherichia coli (E. coli) O157:H7 infection can easily cause intestinal barrier damage, leading to severe diseases such as hemorrhagic enteritis and bloody diarrhea. However, the molecular mechanism of E. coli O157:H7-mediated intestinal barrier damage is still unclear. In previous studies, we reported that the E. coli O157:H7 effector protein EspF interacts with host ANXA6 protein to activate myosin light chain kinase (MLCK)，which leads to the phosphorylation of myosin light chain (MLC) and to destroy tight junctions. We also found that EspF protein can change the expression and localization of ANXA6 protein, which increases the phosphorylation level of NF-κB inflammation pathway protein p65, and up-regulates the levels of TNF-α and IL-1β. Thus we speculate that: EspF protein interacts with and modulates ANXA6 protein (EspF-ANXA6) to regulate the NF-κB/MLCK pathway to mediate intestinal barrier damage. To verify this hypothesis, we intend to utilize animal and cell experiments, to study further the regulation effect of EspF-ANXA6 on the NF-κB pathway proteins IKKα, P65, IκBα, etc.; to clarify the role of NF-κB pathway activation in the process of MLCK-dependent tight junction destruction and bacterial translocation. This project aims to reveal the molecular mechanism of the intestinal barrier function damage caused by E. coli O157:H7 infection and provide a theoretical basis for exploring new clinical treatment methods of enteritis and severe diarrhea.