鼠李糖乳杆菌肽聚糖对鸡β-防御素9基因表达的调节作用及其信号通路

Our previous studies have shown that probiotic Lactobacillus rhamnosus and its cell components stimulate avian β-defensin 9 (AvBD9) gene expression in varying degrees in primary cultured chicken small intestinal epithelial cells, and intact peptidoglycan, one of the components from Lactobacillus rhamnosus induces AvBD9 mRNA expression through the TLR2－NF-κB/AP-1 signalling pathways. However, further research is needed to investigate whether intact peptidoglycan from Lactobacillus rhamnosus MLGA can systemically stimulate AvBD9 expression in other tissues of chicken and thus improve anti-infection ability, and to explore the underlying molecular mechanism. Based on recent advance in probiotic and defensin fields and our privious results, this project aims to demonstrate modulatory effects of peptidoglycan from Lactobacillus rhamnosus on AvBD9 expression in chicken tissues and to clarify the corelationship between AvBD9 expression and its ability to protect chicken from pathogenic bacteria infection in cellular and molecular, local and systemic levels by in vitro and in vivo experiments. In addition, efforts will being focused on the elucidation of molecular action mechamism of AvBD9 expression induction by peptidoglycan from the NOD-like receptors NOD1- and NALP3-mediated signal transduction pathways. The new manner and mechanism of action by which the special cell components from probiotics exert their and immunomodulatory and anti-infection effects will be illustrated from the β-defensin expression point of view in this project, which would contribute to the screening, modification of probiotics with higher efficacy and then their application and popularization.

申请者结题的国家青年基金项目研究发现：鸡源益生鼠李糖乳杆菌及其细胞组分能够不同程度地促进体外培养的鸡小肠上皮细胞β-防御素9（AvBD9）基因表达且鼠李糖乳杆菌完整肽聚糖通过TLR2－NF-κB/AP-1信号通路调控上皮细胞AvBD9的表达。鼠李糖乳杆菌肽聚糖能否在系统水平上提高鸡不同组织细胞的AvBD9表达并提高机体抗感染能力？其分子作用机制如何？基于本领域最新进展和申请者前期研究结果，本项目通过体外和体内实验，从分子和细胞、局部和整体水平上阐明鼠李糖乳杆菌肽聚糖对鸡组织AvBD9表达的调节作用及其与抗病力的关系，并着重从NOD样受体NOD1和NALP3介导的信号通路探讨肽聚糖调节AvBD9表达的分子机理。本研究从β-防御素角度进一步解析益生菌特定细胞组分发挥免疫调节和抗感染作用的新途径和机制，研究结果将为筛选高效益生菌、益生菌的遗传修饰和推广应用提供更加坚实的理论基础和依据。

我们前期研究发现，鸡源益生鼠李糖乳杆菌及其细胞组分能够不同程度地促进体外培养的鸡小肠上皮细胞β-防御素9（avian β-defensin 9，AvBD9）基因表达且鼠李糖乳杆菌细胞壁完整肽聚糖（whole peptidoglycan，WPG）通过Toll样受体2（Toll-like receptor 2，TLR2）－NF-κB/AP-1 信号通路调控上皮细胞AvBD9 的表达。鼠李糖乳杆菌肽聚糖能否在系统水平上提高鸡不同组织细胞的AvBD9表达并提高机体抗感染能力？其分子作用机制如何？针对这一科学问题，本项目从细胞和分子水平探讨了鼠李糖乳杆菌MLGA及其肽聚糖对健康和感染条件下鸡不同组织（细胞）AvBD9 表达的调节作用、鼠李糖乳杆菌MLGA及其肽聚糖对健康和感染条件下鸡组织TLR2、NOD1 和NALP3 基因表达的影响以及鼠李糖乳杆菌MLGA 肽聚糖调控鸡AvBD9 基因表达的分子机制。研究发现，WPG可剂量依赖性地提高鸡外周血单个核细胞（peripheral blood mononuclear cells，PBMCs）、外周血单核细胞、脾细胞、胸腺细胞、肝细胞及鸡胚盲肠、回肠和空肠AvBD9 mRNA的表达，且AvBD9的表达在不同免疫细胞之间、不同肠组织之间存在差异。相反，病原菌金黄色葡萄球菌来源的肽聚糖显著降低鸡PBMCs和脾细胞中AvBD9 mRNA的表达。鼠李糖乳杆菌MLGA来源的WPG通过促进PBMCs或脾细胞AvBD9的表达而发挥抑制肠炎沙门氏菌生长的作用。WPG可通过TLR2或NOD1介导的NF-κB和MAPK信号途径上调鸡外周血单核细胞宿主防御肽AvBD9、cathelicidin-B1的表达而发挥益生作用，且WPG及其溶菌酶酶解产物对AvBD9基因表达的促进作用是炎症细胞因子非依赖性的。体内试验发现，鼠李糖乳杆菌MLGA可不同程度地提高肠炎沙门氏菌感染条件下肉鸡PBMCs、肝脏、胸腺、脾脏、法氏囊、空肠和盲肠等组织中AvBD9和cathelicidin-B1 mRNA的表达，提高肉鸡抗肠炎沙门氏感染免疫功能。本项目从宿主防御肽如β-防御素9和cathelicidin-B1的角度进一步解析了益生菌特定细胞组分发挥免疫调节和抗感染作用的新途径和机制，研究结果为筛选高效益生菌、益生菌的遗传修饰和推广应用提供了更加坚实的理论基础和依据。