Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

Abstract

Mastitis is one of three bovine diseases recognized as a cause of substantial economic losses every year throughout the world. Niacin is an important feed additive that is used extensively for dairy cow nutrition. However, the mechanism by which niacin acts on mastitis is not clear. The aim of this study is to investigate the mechanism of niacin in alleviating the inflammatory response of mammary epithelial cells and in anti-mastitis. Mammary glands, milk, and blood samples were collected from mastitis cows not treated with niacin (n = 3) and treated with niacin (30 g/d, n = 3) and healthy cows (n = 3). The expression of GPR109A, IL-6, IL-1β, and TNF-α in the mammary glands of the dairy cows with mastitis was significantly higher than it was in the glands of the healthy dairy cows. We also conducted animal experiments in vivo by feeding rumen-bypassed niacin. Compared with those in the untreated mastitis group, the somatic cell counts (SCCs) and the expression of IL-6, IL-1β, and TNF-α in the blood and milk were lower. In vitro, we isolated the primary bovine mammary epithelial cells (BMECs) from the mammary glands of the healthy cows. The mRNA levels of IL-6, IL-1β, TNF-α, and autophagy-related genes were detected after adding niacin, shRNA, compound C, trans retinoic acid, 3-methyladenine to BMECs. Then GPR109A, AMPK, NRF-2, and autophagy-related proteins were detected by Western blot. We found that niacin can activate GPR109A and phosphorylate AMPK, and promote NRF-2 nuclear import and autophagy to alleviate LPS-induced inflammatory response in BMECs. In summary, we found that niacin can reduce the inflammatory response of BMECs through GPR109A/AMPK/NRF-2/autophagy. We also preliminarily explored the alleviative effect of niacin on mastitis in dairy cows.

Keywords: AMPK/NRF-2; BMECs; GPR109A; autophagy; mastitis; niacin.

Figure 1

The relative expression of GPR109A, IL-6, TNF-α, and IL-1β. The mammary glands were collected from healthy dairy cows and mastitis dairy cows (n = 6). (a,b) The results of hematoxylin and eosin (H&E) staining and immunohistochemistry assays in the control and mastitis dairy cows. (c) The gene levels of GPR109A in the control and mastitis dairy cows. (d–f) The expression of pro-inflammatory factors in the control and mastitis dairy cows. The mRNA levels of GPR109A (g), IL-6 (d), TNF-α (e), and IL-1β (f) were normalized to the level of β-actin. The values are presented as the means ± SD (∗ p < 0.05 and ∗∗ p < 0.01).

Figure 2

Effects of autophagy, GPR109A, and NRF2 on BMECs inflammation. (a–i) The bovine mammary epithelial cells (BMECs) were pre-treated with niacin and NC shRNA, shRNA, retinoic acid (RA), or 3-methyladenine (3-MA) for 1 h and then stimulated with LPS for 24 h. The mRNA levels of IL-6, IL-1β, and TNF-α were determined by qRT-PCR. The mRNA levels of IL-6, IL-1β, and TNF-α were normalized to the level of β-actin. The values are presented as the means ± SD (∗ p < 0.05 and ∗∗ p < 0.01).

Figure 3

Niacin can activate the GPR109A/NRF2/autophagy signal pathway. The cells were collected at 0, 3, 6, 12, and 24 h to extract the total protein. The total protein was prepared and subjected to Western blotting using GPR109A, NRF-2, HO-1, and β-tubulin antibodies. T-NRF-2 means total NRF-2. (a–d) The protein levels of GPR109A, NRF-2, and HO-1. The cells from different experimental groups were treated with niacin or shRNA+niacin for 24 h, and then, the total protein was collected. N-NRF-2 means NRF-2 in the nucleus. C-NRF-2 means NRF-2 in the cytoplasm. (e–h) The protein levels of GPR109A, C-NRF-2, N-NRF-2, and HO-1. Each immunoreactive band was digitized and expressed as a ratio of the β-tubulin level. (i) The immunofluorescence results of the assay for NRF-2. The scale length in the figure is 200 μM. (j) The relative fluorescence intensity of NRF-2/ARE. The mRNA levels were determined by qRT-PCR. (k) The mRNA levels of ATG12, ATG4D, p62, ATG5, ULK1, ATG4B, Beclin, LC3B, and ATG7 were normalized to the level of β-actin. The values are presented as the means ± SD (∗ p < 0.05 and ∗∗ p < 0.01).

Figure 4

GPR109A regulates energy metabolism, AMPK phosphorylation, and P62 interact with Keap-1 in BMECs. The cells were collected at 0, 3, 6, 12, and 24 h to extract the total protein. (a,b) The protein levels of p-AMPK. The cells from different experimental groups were treated with niacin or shRNA + niacin for 24 h, and then, the total protein was collected. The cell lysates were prepared and subjected to Western blotting using AMPK and p-AMPK antibodies. (c,d) The protein levels of p-AMPK. Each immunoreactive band was digitized and expressed as a ratio of the β-tubulin. (e) The BMECs were pre-treated with niacin, niacin+shRNA, or shRNA for 24 h. The cells were then collected, and the ATP, ADP, and AMP levels were detected by liquid chromatography (n = 3). (f–h) of the ATP, ADP, and AMP content in the BMECs after adding niacin, niacin + shRNA or shRNA. (i) The ratio of (AMP + ADP)/ATP. The values are presented as the means ± SD (ns means no difference, ∗ p < 0.05 and ∗∗ p < 0.01).

Figure 5

GPR109A alleviates inflammation of BMECs by regulating autophagy. Cells from different experimental groups were treated with niacin, LPS, or niacin+LPS for 24 h, and then, the total protein was collected. The cell lysates were prepared and subjected to Western blotting using β-tubulin (a), LC3B (a,b), and P62 (a,c) antibodies. Each immunoreactive band was digitized and expressed as a ratio of the β-tubulin. (d) The left panel shows 6000 × 120 V; the right panel shows is 12,000 × 120 V. The results revealed by the electron microscopy showed that niacin could induce autophagy in the non-treated BMECs and LPS-treated BMECs. (e–m) The mRNA levels were determined by qRT-PCR. The mRNA levels of ATG12, ATG4D, p62, ATG5, ULK1, ATG4B, Beclin, LC3B, and ATG7 were normalized to the level of β-actin. Values are presented as the means ± SD (∗ p < 0.05 and ∗∗ p < 0.001).

Figure 6

Effect of GPR109A on the AMPK/NRF-2/HO-1, P38, JNK1/2, and ERK1/2 signaling pathways. The cells from different experimental groups were treated with niacin, niacin, or LPS for 24 h, and then, the total protein was collected. N-P65 indicates P65 in the nucleus. N-NRF-2 means NRF-2 in the nucleus. The cell lysates were prepared and subjected to Western blotting using antibodies for P38 (a,d), p-P38 (a,d), JNK1/2 (a,b), p-JNK1/2 (a,b), ERK1/2 (a,c), p-ERK1/2 (a,c), AMPK (a,f), p-AMPK (a,f), HO-1 (a,g), β-tubulin (e), N-P65 (a,h), N-NRF-2 (a,i–k), and laminin B (e).