香附酮抑制鸡致病性大肠杆菌侵袭肺泡Ⅱ型上皮细胞的分子机制研究

Avian pathogenic Eecherichia coli (APEC) cause colibacillosis, one of the most common and costly diseases in chickens, accounting for large financial losses in the worldwide poultry industry. The invasion of APEC into alveolar epithelial type II cell (AEC II) is the key step for APEC entering the blood circulation through breaking blood-air barrier to cause chicken septicemia. However, the mechanism of the invasion remains unknown. In the previous study, we found that cyperone significantly inhibited the injury of AEC II induced by strain O78 of APEC. Therefore, lightened by the interaction between bacterial pathogens and host cells, we will investigate the inhibitory effect of cyperone on the attaching and effacing (A/E) as well as inflammatory lesions of AEC II induced by APEC with the cellular and molecular techniques, such as confocal microscopy, Western blot and real time RT-PCR. Furthermore, we need to discover the key routes of signal transduction pathways for A/E (Rho and Nck2) and inflammation (MAPK and NF-κB) involved in the process to illuminate the molecular mechanism of cyperone on inhibiting the invasion of APEC into AEC II. These results not only provide the novel way to study the pathogenesis of APEC, but also supply the new putative targets for developing antibacterial drugs, as well as establish the theoretical basis and experimental data for the prevention and control of chicken colibacilosis with traditional Chinese herbal medicine.

鸡致病性大肠杆菌对肺泡Ⅱ型上皮细胞的侵袭损伤，是其突破"气血屏障"进入血液循环，导致败血症的首要环节，由于其相关机制尚不清楚，以致该病的临床预防和治疗迄今难以取得突破性进展。本课题组前期工作发现，香附酮能够显著抑制鸡致病性大肠杆菌O78株对肺泡Ⅱ型上皮细胞的损伤。基于此，本项目在前期建立鸡致病性大肠杆菌侵袭肺泡Ⅱ型上皮细胞模型基础上，采用激光共聚焦、Western blot及荧光定量RT-PCR等分子生物学技术，研究香附酮对细胞骨架重排、细菌黏附及炎症因子分泌的抑制作用；并进一步检测香附酮对黏附/擦拭损伤信号通路Rho、Nck2及炎性损伤信号通路MAPK、NF-κB中关键蛋白的影响，从而在细胞和分子水平上阐明香附酮抑制鸡致病性大肠杆菌侵袭肺泡Ⅱ型上皮细胞的信号转导机制，深入揭示鸡大肠杆菌的致病机制，为进一步研发抗菌类药物提供新的预测靶点，同时为鸡大肠杆菌病的中药防治奠定理论基础和实验依据。

本实验通过分离鸡肺II型上皮细胞，经过扫描电镜观察，碱性磷酸酶、嗜锇板层小体、流式细胞术、免疫荧光等方法鉴定证明提取的鸡肺II型上皮细胞纯度在98%左右，可以用于后续的实验。在构建的鸡致病性大肠杆菌侵袭鸡肺II型上皮细胞模型的基础上，探究了大肠杆菌对鸡肺II型上皮细胞转录组的影响，以及香附酮对该模型黏附/擦拭损伤和炎性损伤的作用。同时，研究了穿心莲内酯和芦丁对大肠杆菌群体感应系统的作用。转录组学分析发现，大肠杆菌侵袭鸡肺II型上皮细胞后，上皮细胞的差异基因在BP分类中与细胞内信号转导过程相关性最高，在MF分类中，促分裂原活化蛋白激酶结合相关性最高，在CC分类中与细胞核的相关性最高，并且通过信号通路分析发现，鸡肺Ⅱ型上皮细胞感染大肠杆菌时主要涉及到NF-kappa B信号通路、细胞凋亡和炎症通路等。香附酮对大肠杆菌侵袭鸡肺II型上皮细胞模型的作用研究发现，香附酮可以明显减少大肠杆菌的黏附数，增加鸡肺II型上皮细胞F-actin的表达，减轻上皮细胞骨架的重排，显著抑制细胞骨架重排相关通路Nck-2、CDC42和Rac1 mRNA的表达；同时香附酮可以降低TNF-α、IL-1β的分泌，增加IL-4、IL-10的分泌；能够显著降低炎性损伤MAPK信号通路ERK、P38的表达量。穿心莲内酯和芦丁对大肠杆菌群体感应的研究发现，穿心莲内酯和芦丁可以抑制群体感应信号分子AI-2的分泌，穿心莲内酯处理后，APEC-O78毒力相关及群体感应系统相关基因（papC，iucD，fuyA，vat）mRNA表达水平显著下降，控制AI-2分泌的luxS基因的表达量也明显下降。芦丁处理后，APEC-O78相关毒力基因（(Rpos、H-NS、iucD，fyuA，flic，CsgA、CsgB) mRNA分泌水平显著下降（p<0.05），控制AI-2分泌的luxS、pfs基因和负责AI-2在细菌内部转录的基因LsrB、LsrK的表达量在加入芦丁后，也受到明显的抑制作用。. 综上所述，香附酮通过细胞骨架重排等通路抑制大肠杆菌对鸡肺II型上皮细胞的黏附/擦拭损伤，通过MAPK等信号通路抑制大肠杆菌诱导的炎性损伤；穿心莲内酯和芦丁能够明显抑制大肠杆菌的群体感应系统和毒力基因的表达。这为鸡大肠杆菌病的中药防治提供了重要的理论基础和试验依据。