LPS靶向抗菌肽-壳聚糖纳米微粒抑制断奶仔猪肠道炎症反应研究

Intestinal inflammation induced by pathogen infection during the post-weaning period is one of the most important reasons causing diarrhea in piglets. Antimicrobial peptides are able to kill pathogens and inhibit inflammation. However, the antibacterial activity of antimicrobial peptides is limited by LPS, which served as a defensive barrier on the outer membrane of gram-negative bacteria. Additionally, the application of antimicrobial peptides in animal industry is hindered by their poor stability in the gastrointestinal tract. In this study, LPS-targeting antimicrobial peptides will, therefore, be designed to enhance the antimicrobial activity of antimicrobial peptides. The interaction between antimicrobial peptides and LPS will be elucidated. The in vitro biological activities of the peptides will be assessed including antimicrobial activity, cytotoxicity, LPS binding affinity, etc, to screen the most promising LPS-targeting antimicrobial peptide. The LPS-targeting antimicrobial peptide-chitosan nanoparticles will be constructed by using nanotechnology to increase the stability of the peptide through the ionotropic gelation process by coating the screened peptide with chitosan as the wall materials. The inhibitory effects of the LPS-targeting antimicrobial peptide-chitosan nanoparticles on intestinal inflammation in weaned piglets will be evaluated by real-time fluorescent quantitative PCR, Western-blot, and immunohistochemical. The objective of this project is to explore the high efficient utilization method of antimicrobial peptides using nanotechnology and provide the theoretical basis and technical support for their application in animal production to alleviate diarrhea in weaned piglets.

断奶仔猪易受病原菌感染引发肠道炎症反应是导致仔猪腹泻的重要原因之一。抗菌肽可杀灭病原菌，抑制炎症反应。但LPS作为革兰氏阴性菌细胞外膜的防御屏障，限制了抗菌肽抑菌活性的发挥。此外，抗菌肽消化道稳定性差也制约了其在动物生产中的应用。因此，本研究拟设计LPS靶向抗菌肽，以提高抗菌肽的抗菌活性，解析抗菌肽与LPS的相互作用，通过抗菌活性、细胞毒性、LPS绑定能力等体外生物学活性评价，筛选优异LPS靶向抗菌肽；借助纳米技术，以壳聚糖为纳米壁材，采用离子交联法包被筛选的抗菌肽，构建LPS靶向抗菌肽-壳聚糖纳米微粒，以提高抗菌肽稳定性；结合实时荧光定量PCR、Western-blot、免疫组化等方法，探明LPS靶向抗菌肽-壳聚糖纳米微粒对断奶仔猪肠道炎症反应的抑制作用。本研究旨在运用纳米技术探索抗菌肽的高效利用模式，为其在动物生产中应用减轻断奶仔猪腹泻提供理论依据和技术支持。

畜禽细菌性腹泻是制约畜牧业发展的重要原因之一。本项目从细菌的致病机制出发，开展了饲用抗生素替代物抗菌肽的研究。通过非完美两亲性抗菌肽和杂合肽的设计与生物学活性评价，揭示了抗菌肽的构效关系，在此基础上通过LPS结合基序杂合设计得到对LPS具有高结合能力的抗菌肽分子，并揭示了抗菌肽通过破坏细菌细胞膜完整性发挥抗菌作用的机制。为了提高抗菌肽的消化道稳定性，以壳聚糖为壁材，应用纳米技术进行抗菌肽包被，成功构建了抗菌肽-壳聚糖纳米微粒PR-2-CS-NPs和3W-2-CS-NPs，提高了抗菌肽在蛋白酶和小肠液中的稳定性。建立细菌性腹泻模型，并通过灌服抗菌肽-壳聚糖纳米微粒评价其体内应用效果。抗菌肽-壳聚糖纳米微粒可以减轻动物腹泻症状，并对细菌性肠道炎症具有缓解作用，证实了抗菌肽-壳聚糖纳米微粒在动物生产中应用减轻细菌性腹泻的可行性。本项目将纳米技术应用于抗菌肽研究，为抗菌肽应用于动物生产奠定了理论基础。