Ministry of Education Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
J Dairy Sci. 2021 Feb;104(2):2123-2139. doi: 10.3168/jds.2020-18402. Epub 2020 Dec 23.
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-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 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 regulated 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 iE-DAP-induced inflammation and oxidative stress via the NOD1/NF-κB and ERK/Nrf2 pathways in BMEC.
谷氨酰胺（GLN）在大鼠中具有多种生物学活性，包括抗炎、抗氧化应激和抗细胞凋亡作用。然而，人们对 GLN 对牛乳腺上皮细胞（BMEC）的影响知之甚少。γ-d-谷氨酰基-meso-二氨基庚二酸（iE-DAP）是革兰氏阴性细菌细胞壁肽聚糖的组成部分，可被细胞内受体核苷酸结合寡聚结构域蛋白 1（NOD1）识别，并可引起牛乳腺炎。本研究的目的是研究 GLN 是否能保护 BMEC 免受 iE-DAP 诱导的炎症、氧化应激和细胞凋亡。我们将 BMEC 在不含 GLN 的培养基中培养 24 h，然后将其分为 4 组：用 1×PBS 处理 26 或 32 h 的细胞（对照组）；用 10 μg/mL iE-DAP 处理 2 或 8 h 的细胞（2 或 8 h iE-DAP 组）；用 8 或 4 mM GLN 预处理 24 h 后用 1×PBS 处理 2 或 8 h 的细胞（8 或 4 mM GLN 组）；以及用 8 或 4 mM GLN 预处理 24 h 后用 iE-DAP 处理 2 或 8 h 的细胞（DG 组）。在 2 h iE-DAP 组中，当炎症水平达到峰值时，iE-DAP 处理增加了 NOD1、核因子-κB 抑制剂 IκB（NFKBIA）和核因子-κB 亚基 p65（RELA，NF-κB p65）的 mRNA 和蛋白表达，以及细胞培养上清液中 IL6 和 IL8 的 mRNA 表达和 IL-6 和肿瘤坏死因子-α的水平。相比之下，8 mM GLN 预处理通过抑制 NOD1/NF-κB 通路抑制炎症相关因子的 mRNA 和蛋白表达。在 8 h iE-DAP 组中，iE-DAP 处理降低了细胞外调节激酶（Erk，ERK）和核因子红细胞 2 相关因子 2（NFE2L2，Nrf2）的 mRNA 和蛋白表达，以及超氧化物歧化酶 1（SOD1）、过氧化氢酶（CAT）、辅酶 II 氧化还原酶 1（NQO1）和血红素加氧酶 1（HMOX1，HO1）的 mRNA 表达。此外，当氧化应激水平达到峰值时，iE-DAP 处理增加了 BMEC 中丙二醛的表达。有趣的是，4 mM GLN 预处理通过促进 ERK/Nrf2 通路诱导抗氧化应激相关因子的 mRNA 和蛋白表达，并抑制 BMEC 中活性氧的表达，从而诱导抗氧化应激。此外，GLN 减少了炎症和氧化应激引起的 BMEC 凋亡。这是第一项表明 GLN 通过 NOD1/NF-κB 和 ERK/Nrf2 通路在 BMEC 中保护细胞免受 iE-DAP 诱导的炎症和氧化应激的研究。
