State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Hubei Provincial Institute of Veterinary Drug Control, Wuhan, China.
J Appl Microbiol. 2022 Jan;132(1):605-617. doi: 10.1111/jam.15167. Epub 2021 Sep 22.
Zona occludens-1 (ZO-1) is a key regulatory tight junction protein that plays an important role in maintaining gastrointestinal health. In this study, we investigated the protective effect and regulation mechanism of the probiotic Enterococcus faecium HDRsEf1 on tight junction protein ZO-1 at the cellular and molecular levels.
We established lipopolysaccharide (LPS)-induced intestinal epithelial cell injury model and detected the protective effect of HDRsEf1 on ZO-1 in IPEC-J2 cells by real-time polymerase chain reaction and Western blot. The results showed that HDRsEf1 inhibited the downregulation of ZO-1 expression induced by LPS. HDRsEf1 stabilized the destruction of the ZO-1 structure caused by LPS in an immunofluorescence assay. Through gene overexpression and siRNA interference tests, we found that transcription factor activator protein 1 (AP-1) inhibited the level of ZO-1 expression. Silencing experiment further supported that the protective effect of HDRSEF1 might be mediated by suppression of LPS-provoked activation of apoptosis signal-regulating kinase 1 (ASK1)/mitogen-activated protein kinase kinase 7 (MKK7)/c-Jun N-terminal kinase (JNK) signalling pathways. In addition, HDRsEf1 could stabilize ZO-1 expression by increasing toll-like receptor 2 (TLR2) expression and competing with LPS for the TLR4 binding site. More interestingly, we also found that HDRsEf1 could stabilize ZO-1 expression through inhibiting the production of tumour necrosis factor-α (TNF-α) induced by LPS.
HDRsEf1 could protect the IPEC-J2 cell against LPS induced downregulation of ZO-1 expression by inhibiting the activation of TLR2/4-mediated JNK-AP-1 and signalling cascade and the production of TNF-α.
This study can provide a theoretical basis for probiotics to regulate the expression of intestinal tight junction proteins, and supply technical support for probiotics to prevent and treat animal intestinal infectious diseases.
封闭蛋白-1（ZO-1）是一种关键的调节紧密连接蛋白，在维持胃肠道健康方面发挥着重要作用。在本研究中，我们在细胞和分子水平上研究了益生菌屎肠球菌 HDRsEf1 对紧密连接蛋白 ZO-1 的保护作用及其调节机制。
我们建立了脂多糖（LPS）诱导的肠上皮细胞损伤模型，并通过实时聚合酶链反应和 Western blot 检测 HDRsEf1 对 IPEC-J2 细胞中 ZO-1 的保护作用。结果表明，HDRsEf1 抑制了 LPS 诱导的 ZO-1 表达下调。免疫荧光试验显示，HDRsEf1 稳定了 LPS 引起的 ZO-1 结构破坏。通过基因过表达和 siRNA 干扰试验，我们发现转录因子激活蛋白 1（AP-1）抑制了 ZO-1 表达水平。沉默实验进一步支持 HDRSEF1 的保护作用可能是通过抑制 LPS 诱导的凋亡信号调节激酶 1（ASK1）/丝裂原活化蛋白激酶激酶 7（MKK7）/c-Jun N-末端激酶（JNK）信号通路的激活来介导的。此外，HDRsEf1 可以通过增加 Toll 样受体 2（TLR2）的表达并与 LPS 竞争 TLR4 结合位点来稳定 ZO-1 的表达。更有趣的是，我们还发现 HDRsEf1 可以通过抑制 LPS 诱导的肿瘤坏死因子-α（TNF-α）的产生来稳定 ZO-1 的表达。
HDRsEf1 通过抑制 TLR2/4 介导的 JNK-AP-1 信号级联和 TNF-α的产生，抑制 LPS 诱导的 IPEC-J2 细胞 ZO-1 表达下调，从而保护 IPEC-J2 细胞。
这项研究可为益生菌调节肠道紧密连接蛋白的表达提供理论依据，为益生菌预防和治疗动物肠道感染性疾病提供技术支持。
