Li Qiao, Li Linyan, Chen Yanhong, Yu Changning, Azevedo Paula, Gong Joshua, Yang Chengbo
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.
Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada.
J Anim Sci Biotechnol. 2022 Jul 8;13(1):86. doi: 10.1186/s40104-022-00746-8.
Enterotoxigenic Escherichia coli (ETEC) F4 commonly colonizes the small intestine and releases enterotoxins that impair the intestinal barrier function and trigger inflammatory responses. Although Bacillus licheniformis (B. licheniformis) has been reported to enhance intestinal health, it remains to be seen whether there is a functional role of B. licheniformis in intestinal inflammatory response in intestinal porcine epithelial cell line (IPEC-J2) when stimulated with ETEC F4.
In the present study, the effects of B. licheniformis PF9 on the release of pro-inflammation cytokines, cell integrity and nuclear factor-κB (NF-κB) activation were evaluated in ETEC F4-induced IPEC-J2 cells.
B. licheniformis PF9 treatment was capable of remarkably attenuating the expression levels of inflammation cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-8, and IL-6 during ETEC F4 infection. Furthermore, the gene expression of Toll-like receptor 4 (TLR4)-mediated upstream related genes of NF-κB signaling pathway has been significantly inhibited. These changes were accompanied by significantly decreased phosphorylation of p65 NF-κB during ETEC F4 infection with B. licheniformis PF9 treatment. The immunofluorescence and western blotting analysis revealed that B. licheniformis PF9 increased the expression levels of zona occludens 1 (ZO-1) and occludin (OCLN) in ETEC F4-infected IPEC-J2 cells. Meanwhile, the B. licheniformis PF9 could alleviate the injury of epithelial barrier function assessed by the trans-epithelial electrical resistance (TEER) and cell permeability assay. Interestingly, B. licheniformis PF9 protect IPEC-J2 cells against ETEC F4 infection by decreasing the gene expressions of virulence-related factors (including luxS, estA, estB, and elt) in ETEC F4.
Collectively, our results suggest that B. licheniformis PF9 might reduce inflammation-related cytokines through blocking the NF-κB signaling pathways. Besides, B. licheniformis PF9 displayed a significant role in the enhancement of IPEC-J2 cell integrity.
产肠毒素大肠杆菌（ETEC）F4通常定殖于小肠并释放肠毒素，这些肠毒素会损害肠道屏障功能并引发炎症反应。尽管地衣芽孢杆菌已被报道可促进肠道健康，但在用ETEC F4刺激的猪肠道上皮细胞系（IPEC-J2）中，地衣芽孢杆菌在肠道炎症反应中是否具有功能作用仍有待观察。
在本研究中，评估了地衣芽孢杆菌PF9对ETEC F4诱导的IPEC-J2细胞中促炎细胞因子释放、细胞完整性和核因子-κB（NF-κB）激活的影响。
地衣芽孢杆菌PF9处理能够显著降低ETEC F4感染期间炎症细胞因子肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-8和IL-6的表达水平。此外，Toll样受体4（TLR4）介导的NF-κB信号通路上游相关基因的基因表达也受到显著抑制。这些变化伴随着在用PF9处理的ETEC F4感染期间p65 NF-κB磷酸化的显著降低。免疫荧光和蛋白质印迹分析表明，地衣芽孢杆菌PF9增加了ETEC F4感染的IPEC-J2细胞中紧密连接蛋白1（ZO-1）和闭合蛋白（OCLN）的表达水平。同时，地衣芽孢杆菌PF9可通过跨上皮电阻（TEER）和细胞通透性测定减轻上皮屏障功能的损伤。有趣的是，地衣芽孢杆菌PF9通过降低ETEC F4中毒力相关因子（包括luxS、estA、estB和elt）的基因表达来保护IPEC-J2细胞免受ETEC F4感染。
总体而言，我们的结果表明，地衣芽孢杆菌PF9可能通过阻断NF-κB信号通路来减少炎症相关细胞因子。此外，地衣芽孢杆菌PF9在增强IPEC-J2细胞完整性方面发挥了重要作用。
