iE-DAP Induced Inflammatory Response and Tight Junction Disruption in Bovine Mammary Epithelial Cells via NOD1-Dependent NF-κB and MLCK Signaling Pathway

Abstract

γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), a bacterial cell wall component, can trigger an inflammatory response. A mammary inflammatory response causes tight junction (TJ) dysfunction. This study aimed to explore the effects and involved mechanisms of iE-DAP-induced inflammatory response on the TJ integrity in bovine mammary epithelial cells (BMECs). The results showed that iE-DAP-induced inflammatory response and TJ disruption was associated with increased expression levels of inflammatory cytokines and decreased gene expression of ZO-1 and Occludin, as well as a reduction in transepithelial electrical resistance and elevation in paracellular dextran passage. While MLCK inhibitor ML-7 reversed the TJ disruption induced by iE-DAP. NF-κB inhibitor BAY 11-7085 hindered the activation of NF-κB and MLCK signaling pathways, the inflammatory response and TJ disruption induced by iE-DAP. NOD1-specific shRNA also inhibited the activation of the NOD1/NF-κB signaling pathway and reversed the inflammatory response and TJ injury in iE-DAP-treated BMECs. Above results suggest that iE-DAP activated the NF-κB and MLCK signaling pathway in NOD1-dependent manner, which promoted the transcription of inflammatory cytokines and altered the expression and distribution of tight junction proteins, finally caused inflammatory response and TJ disruption. This study might provide theoretical basis and scientific support for the prevention and treatment of mastitis.

Butyrate Protects against γ-d-Glutamyl-meso-diaminopimelic Acid-Induced Inflammatory Response and Tight Junction Disruption through Histone Deacetylase 3 Inhibition in Bovine Mammary Epithelial Cells

“…Previous studies have shown that NF-κB modulates the LPS-induced TJ injury by targeting the expression level of the TJ effector protein MLCK . Increased MLCK can phosphorylate downstream proteins, destroy the interaction between TJ proteins and the cytoskeleton, induce the redistribution of ZO-1 and occludin, and damage TJ integrity. , While MLCK inhibitor administration decreases MLCK expression and increases TJ protein expression, thus ameliorates barrier dysfunction. , The study has also shown that butyrate treatment leads to the inhibition of MLCK, which further restores TJ protein distribution and TJ barrier function in Caco-2 . Consistent with previous studies, we also found that butyrate significantly reduced the protein expression of MLCK in BMECs compared with the DAP group.…”

“…First of all, BMECs were exposed to a gradient concentration of SB (0.1, 0.5, 1, 2, and 4 mM) for 24 h to assay the effect of SB on cell viability. Based on our previous study and the result of cell viability, 22 cells were further exposed to 1000 ng/mL iE-DAP with various concentrations of SB (0.1, 0.5, 1, and 2 mM) for 12 h to detect the regulatory effects of SB on the iE-DAP-triggered inflammatory response and TJ injury. Next, cells were treated with 1000 ng/mL iE-DAP with or without 2 mM SB for 12 h to further assay the nuclear transfer of NF-κB p65, the cellular distribution of ZO-1, and the integrity of the TJ barrier.…”

“…The following experimental procedures were performed, as described. 22 In brief, RNA reverse transcription and RT-qPCR were conducted with Hifair II first Strand cDNA Synthesis SuperMix (Yeasen Biotechnology Co., Ltd., Shanghai, China) and ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China), respectively. Table S1 listed the specific primers used in RT-qPCR, which were synthesized by the company (Jierui Bioengineering Co., Ltd., Shanghai, China).…”

“…iE-DAP, a peptidoglycan constitute, can be found in the cell walls of most Gram-negative bacteria as well as Gram-positive bacteria, such as Escherichia coli (common pathogenic microorganisms of mastitis). , Similar to LPS, iE-DAP can also be specifically identified by its receptor, called nucleotide oligomerization domain binding protein 1 (NOD1). After detection of iE-DAP, NOD1 oligomerizes and subsequently initiates the activation of NF-κB, which facilitates the expression of inflammatory cytokines and finally triggers an inflammatory response. − Scientific investigations have shown that iE-DAP stimulation can trigger an inflammatory response, oxidation disorder, and TJ disruption in bovine mammary epithelial cells (BMECs). , …”

“…18 can trigger an inflammatory response, oxidation disorder, and TJ disruption in bovine mammary epithelial cells (BMECs). 21,22 Antibiotics have been employed as the main treatment of mastitis for a long time, but there are also many adverse effects, including increased resistance to antibiotics, a low cure rate, and antibiotic residues in milk. 23 Therefore, the investigation of novel, secure, and cost-effective alternative agents is indispensable for the treatment of bovine mastitis.…”