Vitamin A affects the expression of antioxidant genes in bovine mammary epithelial cells with oxidative stress induced by diethylene triamine-nitric oxide

Abstract

The considerable increase in oxygen requirements due to the high metabolic rate of the bovine mammary epithelial cells (BMEC) during lactation results in an augmented production of reactive nitrogen species (RNS), such as nitric oxide (NO), which may expose cows to increased oxidative stress. Vitamin A (VA) has been shown in several studies to enhance the antioxidant defence system against oxidative stress, but whether the reason is related to a reduced NO production remains unclear. Diethylene triamine-nitric oxide polymer (NOp) is a type of NO-generating compound, which is safe, efficacious, and releases NO over a long period. The current study was conducted to investigate the effect of VA on the antioxidant function in BMEC and the underlying mechanism by discussing the protection of VA on NO-induced oxidative stress of BMEC. The experiment was conducted using a single-factor completely randomized arrangement. Primary BMEC were isolated from the mammary glands of Holstein dairy cows. The third generation cells were randomly divided into four equal groups with six replicates each. Each group received different combinations of VA and NOp treatment as follows: controls (without VA and NOp), NOp treatment alone, VA treatment alone, and VA and NOp treatment together. The lysates were collected to evaluate the activities of glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) and the contents of reactive oxygen species (ROS) and malondialdehyde (MDA), and the cell-free supernatants were collected to analyze selenoprotein P (SelP) content, inducible nitric oxide synthase (iNOS) activities and nitric oxide (NO), interleukin-1 (IL-1), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) contents. The results suggested that compared to the control, the cell proliferation, the activity of the antioxidants GPx and TrxR, the content of SelP and the antioxidant gene expressions of GPx1, GPx4, and TrxR1 were significantly decreased (P < 0.05), and the contents of ROS and MDA, the activity of iNOS, the contents of NO and IL-1, IL-6, TNF-α, and their mRNA expressions were increased dramatically in the NOp treatment alone group (P < 0.05), but the opposite changes were observed in the VA treatment alone group. Compared to the NOp treatment alone, the VA and NOp treatment together significantly improved cell proliferation, the activities of the antioxidants GPx and TrxR, and the gene expressions of GPx1 and TrxR1, and dramatically decreased the contents of ROS and MDA, the activity of iNOS, the contents of NO and IL-1, IL-6, TNF-α and their mRNA expression levels (P < 0.05). The present research suggests that VA can improve the antioxidant function of BMEC and protect the cells from experiencing the NOp-induced oxidative stress by regulating antioxidant gene expression. The probable mechanism is that VA can reduce the activity of iNOS and its mRNA expression by down-regulating of the expression of IL-1, IL-6, and TNF-α to reduce NO production. However, the exact mechanism warrants future exploration.