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

Abstract

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). γ-d-Glutamyl-mesodiaminopimelic acid (iE-DAP) is a cell wall peptidoglycan component of gram-negative bacteria that can be recognized by the intracellular receptor nucleotidebinding 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 GLNfree medium for 24 h and then separated them into 4 groups: cells treated with 1× PBS for 26 or 32 h (control); cells stimulated by 10 μg/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× 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-κB (NFKBIA, IκB), and nuclear factor-κB subunit p65 (RELA, NF-κB p65), as well as the mRNA expression of IL6 and IL8 and levels of IL-6 and tumor necrosis factor-α 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-κB 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 stressrelated 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 iE-DAP-induced inflammation and oxidative stress via the NOD1/NF-κB and ERK/Nrf2 pathways in BMEC.

STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

“…LPS, treatment at low concentrations can only induce expression at the gene level but cannot increase protein expression, which requires transcriptional modification. There was a similar result in Cheng's study [24].…”

“…Previous studies have shown that LPS binds to receptors on the cell membrane to activate the monocyte-macrophage system inside the liver [23]. LPS induces the release of TNF, IL-6, IL-8, and other inflammatory mediators in the cell through the mitogen-activated protein kinase pathway, the PKC pathway, the NF-κB pathway, and other inflammatory signaling pathways, resulting in inflammatory reactions [21,24]. It has been reported that upregulation of STIM1/Orai1 can promote inflammation and ERS in mammary tissue [21], and this can inhibit the LPS-induced inflammation by regulating ERS in BHEC [20,25].…”

Sodium Butyrate More Effectively Mitigates the Negative Effects of High-Concentrate Diet in Dairy Cows than Sodium β-Hydroxybutyrate via Reducing Free Bacterial Cell Wall Components in Rumen Fluid and Plasma

“…The present study showed that the HC diet significantly increased the amounts of immune cells in the blood, which further demonstrated that the increase of PAMPs caused by the HC diet resulted in systemic inflammation in dairy cows. When the PAMPs are transported into the udder through blood circulation, they can induce inflammatory responses of mammary gland cells by activation of corresponding PRRs [ 8 , 9 , 46 ]. Both TLR4 and TLR2 are PRRs, which mainly are activated by LPS and LTA [ 8 , 47 ], respectively.…”

Cis-9, Trans-11 CLA Alleviates Lipopolysaccharide-Induced Depression of Fatty Acid Synthesis by Inhibiting Oxidative Stress and Autophagy in Bovine Mammary Epithelial Cells

“…Bovine mammary epithelial cells (BMECs) were purchased from Tongpai Biological Technology Co., Ltd. (Shanghai, China), which was established and characterized by Ke Zhao et al [ 31 ]. BMECs were well cultured in our experiment and used as a perfect in vitro model in several papers [ 32 , 33 , 34 ]. BMECs were cultured in complete medium, which contains 90% Roswell Park Memorial Institute (RPMI) medium 1640 basic (1×), 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin and placed in a 37 °C humidified cell incubator with 5% CO 2 .…”