Glutamine pretreatment protects bovine mammary epithelial cells from inflammation and oxidative stress induced by γ-D-glutamylmeso-diaminopimelic acid (iE-DAP)

Glutamine (GLN) has many types of biological activity in rats, including anti-inflammatory, antioxidative stress, and anti-apoptosis effects. However, little is known about the effects of GLN on bovine mammary epithelial cells (BMEC). gamma-D-Glutamyl-meso-diaminopimelic acid (iE-DAP) is a cell wall peptidoglycan component of gram-negative bacteria that can be recognized by the intracellular receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and can cause bovine mastitis. The goal of the present study was to investigate whether GLN protects BMEC from iE-DAP-induced inflammation, oxidative stress, and apoptosis. We cultured BMEC in a GLN-free medium for 24 h and then separated them into 4 groups: cells treated with 1x PBS for 26 or 32 h (control); cells stimulated by 10 mu g/mL iE-DAP for 2 or 8 h (2- or 8-h iE-DAP); cells pretreated with 8 or 4 mM GLN for 24 h followed by 2 or 8 h of 1 x PBS treatment (8 or 4 mM GLN); and cells pretreated with 8 or 4 mM GLN for 24 h followed by 2 or 8 h of iE-DAP treatment (DG). In the 2-h iE-DAP group, when levels of inflammation peaked, iE-DAP treatment increased both the mRNA and protein expression of NOD1, inhibitor of nuclear factor-kappa B (NFKBIA-, I kappa B), and nuclear factor-kappa B subunit p65 (RELA, NF-kappa B p65), as well as the mRNA expression of IL6 and IL8 and levels of IL-6 and tumor necrosis factor-alpha in cell culture supernatants. In contrast, 8 mM GLN pretreatment inhibited the mRNA and protein expression of inflammatory-related factors by suppressing the NOD1/NF-kappa B pathway. In the 8-h iE-DAP group, iE-DAP treatment decreased the mRNA and protein expression of extracellular regu- lated kinase (Erk, ERK) and nuclear factor erythroid 2-associated factor2 (NFE2L2, Nrf2), as well as the mRNA expression of superoxide dismutase 1 (SOD1), catalase (CAT), coenzyme II oxidoreductase 1 (NQO1), and heme oxygenase 1 (HMOX1, HO1). In addition, iE-DAP treatment increased the expression of malondialdehyde in BMEC when oxidative stress levels peaked. Interestingly, 4 mM GLN pretreatment induced the mRNA and protein expression of antioxidative stress-related factors and inhibited the expression of reactive oxygen species in BMEC by promoting the ERK/Nrf2 pathway. Moreover, GLN reduced apoptosis caused by inflammation and oxidative stress in BMEC. This is the first report showing that GLN protects against iEDAP-induced inflammation and oxidative stress via the NOD1/NF-kappa B and ERK/Nrf2 pathways in BMEC.

Automated summary

This study found that GLN can protect BMEC from iEDAP-induced inflammation and oxidative stress through the NOD1/NF-kappa B and ERK/Nrf2 pathways, inhibiting cell apoptosis.