Mammary inflammatory gene expression was associated with reproductive stage and regulated by docosahexenoic acid: in vitro and in vivo studies

Sen Lin, Yalin Zhang, Yanrong Long, Haifeng Wan, Lianqiang Che, Yan Lin, Shengyu Xu, Bin Feng, Jian Li, De Wu, Zhengfeng Fang

Periparturient mastitis is the most prevalent disease affecting lactating animals. However, it has long been relied on antibiotics to deal with mastitis, leading to a potential threat to food safety. This study was aimed to investigate the expression of pro-inflammatory cytokines in mammary glands of sows around parturition when mastitis and oxidative stress usually occur, and evaluate the anti-inflammatory effect of docosahexenoic acid (DHA) in porcine mammary epithelial cells (PMEC) challenged by lipopolysaccharide (LPS). Mammary tissues and blood samples were collected from seven pregnant sows at different reproductive stages. Primarily cultured PMEC at passage 4 were assigned to four treatments: basal medium (control), basal medium with LPS (10 μg/mL) (LPS treatment), basal medium with LPS (10 μg/mL) and DHA (100 or 200 μM) (LPS + DHA treatments), and cell samples were harvested after 24 h incubation. The measurements included oxidative stress markers in blood samples and gene expression in mammary tissues and PMEC samples. Serum α-tocopherol concentration was lower at parturition than at day 90 of gestation and day 28 post parturition, while serum malondialdehyde concentration was higher at day 28 post parturition than at day 90 of gestation. Higher interleukin (IL)-1β mRNA abundance while lower LPS binding protein mRNA abundance in mammary tissues were observed at day 90 of gestation compared with that at parturition and at day 28 and 35 post parturition. Mammary tumor necrosis factor (TNF)-α mRNA abundance were lower at parturition than at day 90 of gestation and day 28 and 35 post parturition, whereas mammary IL-8 mRNA abundance were lower at parturition than at day 35 post parturition. In the PMEC experiment, compared with the control, increased mRNA abundances of Toll-like receptor (TLR)-4 downstream target, myeloid differentiation factor 88 (MyD88), IL-6 and IL-8 were observed in LPS treatment, whereas DHA appeared to decrease mRNA abundances of MyD88, IL-6 and IL-8 induced by LPS. The down-regulated expression of pro-inflammatory cytokines in mammary tissues and aggravated systemic oxidative stress at parturition suggest that sows are in a vulnerable status during periparturient period. DHA appears to attenuate inflammatory responses in LPS-challenged PMEC through modulation of TLR4 signalling pathway.