植物乳杆菌通过occludin蛋白磷酸化修饰调控仔猪肠上皮细胞屏障功能的机制研究

Our previous in vivo studies have demonstrated that Lactobacillus plantarum exerted protective effect against the damage to porcine intestinal barrier function induced by enterotoxigenic Escherichia coli (ETEC) K88 infection. And we found in vitro study that the protective effect of Lactobacillus plantarum may involve the upregulation of the occludin and zonula occluden-1(ZO-1) expression at both mRNA and protein levels. However, the molecular mechanism involved in the regulatory function of Lactobacillus plantarum in occludin remains poorly understood. The recent study has demonstrated that occludin phosphorylation at different sites may exert different role in the regulation of the intracellular tight junction barrier. Therefore, we hypothesize that the different phosphorylation site of occludin determines the capacity of occludin to bind ZO-1, in turn affecting the transmembrane location of occludin; and that the Lactobacillus plantarum could modulate the phosphorylation of occludin through activating TLR2 and PKC, in turn increasing transmembrane location of occludin and enhancing the tight junctions of intestinal epithelial cells. To test the hypothesis, firstly we are to evaluate the effects of Lactobacillus plantarum on the occludin phosphorylation and identify the occludin phosphosites in regulating the intestinal epithelial barrier functions using Mass Spectrometry, immunoprecipitation, and mutant assay of occludin. Then, a porcine intestinal epithelial cell line (IPEC-J2) with TLR2 gene knockout (IPEC-J2TLR2-/-) via CRISPR Cas9 gene editing technique or the activation/inhibition of PKC subfamily members will be used to study the potential signal pathway of Lactobacillus plantarum in mediating occludin phosphorylation modification. The results of this study will display the potential molecular mechanism at protein phosphorylation level by which Lactobacillus plantarum regulates the intestinal epithelial barrier function, thereby providing a theoretical basis for the practical application of Lactobacillus plantarum in swine production.

本课题组前期发现，饲粮中预添加植物乳杆菌能有效缓解大肠杆菌引起的仔猪肠道屏障功能损伤，进而体外验证其原因可能与肠上皮细胞occludin和ZO-1的调节有关，但其具体机制仍不明确。新的研究发现，occludin蛋白磷酸化修饰位点不同对细胞紧密连接可产生相反的调节作用。因此，我们提出，植物乳杆菌是否通过occludin特定位点磷酸化影响其与ZO-1的结合能力，进而促进occludin的肠上皮细胞跨膜表达。本项目拟采用免疫共沉淀、质谱分析、磷酸化位点突变等技术，明确植物乳杆菌作用于肠上皮细胞occludin特点位点磷酸化变化，及其对ZO-1的互作与细胞屏障功能的调节；并通过TLR2基因敲除、PKC亚家族特异性激活/抑制，研究介导植物乳杆菌对肠上皮细胞occludin磷酸化的信号机制。本研究将有望从蛋白修饰水平上揭示植物乳杆菌调控肠上皮细胞屏障功能的分子机制，为植物乳杆菌的生产应用提供理论依据。

本项目针对植物乳杆菌是否通过occludin蛋白特定位点磷酸化影响其与ZO-1的结合能力促进肠上皮屏障功能这一假设进行了科学研究，首先进行了植物乳杆菌对仔猪肠上皮细胞屏障功能及对紧密连接蛋白occludin磷酸化修饰的影响研究，结果发现，植物乳杆菌能够促进肠上皮细胞的屏障功能，但不是通过影响occludin蛋白特定位点磷酸化的途径。因此，我们调整了后续试验内容。首先，通过蛋白组学和磷酸化蛋白组学筛选仔猪肠上皮细胞屏障形成过程中的差异表达蛋白及磷酸化蛋白，结果表明，共有102个差异表达蛋白（57个上调，45个下调），主要富集在氧化磷酸化、非酒精性脂肪肝（NAFLD）等途径，同时有814个差异表达磷酸化肽段（623个上调，191个下调），主要富集在PI3K-Akt signaling pathway、MAPK signaling pathway、Cell cycle等途径；随后，我们利用蛋白组学和磷酸化蛋白组学筛选植物乳杆菌促进肠上皮细胞屏障功能的差异表达蛋白及磷酸化蛋白，结果发现，共有85个差异表达蛋白（50个上调，35个下调），主要富集在Apoptosis、multiple species、TNF signaling pathway、Sphingolipid signaling pathway等途径，同时，LP组与对照组相比，共有203个差异表达磷酸化蛋白（94个上调，109个下调）和296个磷酸化位点（129个上调，167个下调），主要富集在Adherens junction、Arrhythmogenic right ventricular cardiomyopathy (ARVC)、Lysine degradation等途径，CDK1、DYNC1I2、NVL、YWHAB和SRSF7等节点度较高，可能为植物乳杆菌促进肠上皮细胞屏障功能的核心磷酸化蛋白。本研究结果在蛋白组学和磷酸化蛋白组学上进一步明确了植物乳杆菌促进肠上皮细胞屏障功能的可能机制，为植物乳杆菌促屏障功能提供了理论基础。