GPR109A alleviate mastitis and enhances the blood milk barrier by activating AMPK/Nrf2 and autophagy

Abstract

Mastitis causes great psychological and physical pain among women. Our previous studies found that niacin has anti-inflammatory effect, and the realization of this function depends on GPR109A. However, there are no previous reports about the anti-inflammatory function of GPR109A in mastitis. In our study, we observed the effect of niacin on the WT and GPR109A^-/- mice mastitis model. The results showed that administration of niacin to WT mice reduced the damage, proinflammatory mediators and protected the integrity of the blood milk barrier in mammary gland. While in GPR109A^-/- mice, there was no effect on the above indexes. In mammary epithelial cells, GPR109A was able to promote autophagy and Nrf2 nuclear import through AMPK. In LPS-induced mammary epithelial cells, niacin inhibited the LPS-induced inflammatory response and downregulation of tight junction proteins, and these effects were eliminated by knocking down GPR109A, blocking autophagy or inhibiting Nrf2 nuclear import. These results indicate that in mastitis, GPR109A promotes autophagy and Nrf2 nuclear import through AMPK, thereby inhibiting inflammatory damage to the mammary gland and repairing the blood milk barrier. Our results suggested that GPR109A may be a potential target for the treatment of mastitis.

Keywords: AMPK/Nrf2; Autophagy; Blood milk barrier; GPR109A; Mastitis; Niacin.

Figure 1

Effects of GPR109A on mice mammary gland injury. (a) The H&E staining of mammary gland (original magnification 200 ×). (b) The injury score of mammary gland, this is based on the three points previously described.Values are presented as means ± SD (*** p<0.001, **** p<0.0001).

Figure 2

Activating GPR109A can reduce the release of cytokines. (a) Activity of myeloperoxidase (MPO). (b, c) mRNA levels of IL-6 and TNF-α. (d-f) The Protein contents of IL-1β, TNF-α and IL-6. Values are presented as means ± SD (n = 5) (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).

Figure 3

Penetration of FITC-albumin as it enters mammary acini through the blood milk barrier. Mammary glands from the eight groups were treated with previous description. (a) The color green represent FITC-albumin and the colot blue means nuclei (DAPI). After LPS injection, we can observe green FITC-albumin in mammary acini under fluorescence microscope. (b-e) The levels of Occludin, Claudin 3 and ZO-1. Values are presented as means ± SD (*** p<0.001, **** p<0.0001).

Figure 4

Immunofluorescence results of Claudin 3 and Occludin. Immunofluorescence staining was performed on 8 groups of mammary glands, and the colocalization and fluorescence intensity of Claudin 3 and Occludin in mammary glands were determined.

Figure 5

Immunofluorescence results of ZO-1 and Occludin. Immunofluorescence staining was performed on 8 groups of mammary glands, and the colocalization and fluorescence intensity of ZO-1 and Occludin in mammary glands were determined.

Figure 6

Effect of GPR109A on autophagy. (a-c) Protein levels of p62 and LC3B in the mammary gland. (d, e) Protein levels of p-APMK, AMPK, p-Beclin, Beclin, p-ULK1, ULK1, P62 and LC3B in niacin treated cells at 0, 3, 6, 12 and 24 hours. (f) After 24 hours of niacin treatment, the autophagy flow of EpH4-Ev increased significantly. (g) Autophagy lysosomes in EpH4-Ev. Values are presented as means ± SD (n = 3) (**** p<0.0001).

Figure 7

GPR109A inhibits inflammation by activating autophagy through p-AMPK, p-Beclin and p-ULK1. (a, b) The levels of p-AMPK, p-ULK1 and p-Beclin after knocking down GPR109A. (c, d) Interaction between p-ULK1, p-Beclin and p-AMPK after knocking down GPR109A. (e-g) Protein levels of p-ULK1 and p-Beclin after inhibition of AMPK. (h) Protein levels of the autophagy related pathway proteins after GPR109A was activated in LPS-induced MECs. (j) Autophagy flow in the LPS-induced MECs after GPR109A activation. Values are presented as means ± SD (n = 3) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 8

GPR109A promotes Nrf2 nuclear import to exert anti-inflammatory effect by phosphorylating AMPK. (a, b) Protein levels of T-Nrf2, C-Nrf2, HO-1 and N-Nrf2. (c) Immunofluorescence results of Nrf2 nuclear import after treating with niacin for 24 hours. (d, e) The levels of N-Nrf2 and T-Nrf2. (f) Immunofluorescence results of Nrf2 nuclear import after knocking down GPR109A. (g, h) The levels of N-Nrf2 and T-Nrf2. (i) Immunofluorescence results of Nrf2 nuclear import after treating with niacin, CC or niacin+CC for 24 hours. (j, k) Protein levels of T-Nrf2, HO-1 and N-Nrf2. (l) Immunofluorescence results of Nrf2 nuclear import after treating with LPS, niacin or niacin+LPS for 24 hours. Values are presented as means ± SD (n = 3) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 9

GPR109A could exert anti-inflammatory effect by activating autophagy and Nrf2 nuclear import through AMPK signal pathway. (a-e) The levels of TNF-α, IL-1β, IL-6, COX-2 and iNOS were detected using qRT-PCR in EpH-Ev (n=3). (f-j) Gene levels of TNF-α, iNOS, COX-2, IL-6 and IL-1β were detected using qRT-PCR in EpH-Ev (n=3). Values are presented as means ± SD (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 10

GPR109A enhances tight junction protein expression by activating autophagy and Nrf2. (a, b) The levels of ZO-1, Claudin 3 and Occludin after niacin treatment for 0, 3, 6, 12 and 24 hours. (c) Immunofluorescence results of Claudin 3, ZO-1 and Occludin after niacin treatment for 24 hours. (d, e) The levels of ZO-1, Claudin 3 and Occludin after knocking down GPR109A. (f) The results of electron microscopy show that the tight junction was strengthened after adding niacin. (g) Immunofluorescence results of Claudin 3, ZO-1 and Occludin after knocking down GPR109A. (h, i) The levels of Occludin, Claudin 3 and ZO-1 in EpH-Ev cells that were treated with niacin, niacin+3-MA, niacin+ML385 or niacin+CC. (j, k) The levels of Occludin, Claudin 3 and ZO-1 after the cells were treated with LPS, niacin or niacin+LPS. Values are presented as means ± SD (n=3) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 11

The mechanism of niacin in anti-mastitis and enhanced blood milk barrier. GPR109A can alleviate mastitis mainly through two ways. On the one hand, it can alleviate the inflammatory response of mammary epithelial cells and protect the integrity of blood milk barrier by activating AMPK/Nrf2 and autophagy. On the other hand, it can reduce the damage of mammary epithelial cells by inhibiting the release of pro-inflammatory mediators by macrophages.