Farrerol Relieve Lipopolysaccharide (LPS)-Induced Mastitis by Inhibiting AKT/NF-κB p65, ERK1/2 and P38 Signaling Pathway

Abstract

Farrerol has been proved to have an anti-inflammatory effect. However, the effects of farrerol on mastitis have not been investigated. This study was aimed to investigate the effect and mechanism of farrerol in lipopolysaccharide (LPS)-induced mouse mastitis and LPS-induced inflammatory response of mouse mammary epithelial cells (mMECs). In vivo, LPS were injected to the tetrad pair of nipples for establishing mouse mastitis, and then tested the effect of farrerol on histopathological changes, inflammatory response and activation degree of protein kinase B (AKT), nuclear factor-kappa B p65 (NF-κB p65), p38, extracellular regulated protein kinase (ERK1/2). In vitro, the mMECs were incubated by farrerol for 1 h following by stimulating with LPS, and then the inflammatory response and the related signaling pathways were detected. The in vivo results found that farrerol could improve pathological injury of mammary gland, attenuate the activity of myeloperoxidase (MPO), inhibit the production of pro-inflammatory mediators and the phosphorylation of AKT, NF-κB p65, p38 and ERK1/2. The in vitro results also found farrerol inhibited inflammatory response and the related signaling pathways. Collectively, this study revealed that farrerol inhibits the further development of LPS-induced mastitis by inhibiting inflammatory response via down regulating phosphorylation of AKT, NF-κB p65, p38, and ERK1/2. These findings suggest that farrerol may be used as an anti-inflammatory drug for mastitis.

Keywords: LPS; Mitogen-activated protein kinase (MAPK); NF-κB; farrerol; mastitis.

Figure 1

Chemical structure of farrerol.

Figure 2

Effects of farrerol on the pathological changes of mammary gland ((A–F), ×100 and (G–L), ×400). Mammary glands were collected after lipopolysaccharide (LPS) injection 24 h. The fixed tissue blocks were fixed, dehydrated, transparent, dipped wax, embedded, sliced, made of the paraffin sections and then stained by means of hematoxylin–eosin (HE) staining. Representative histopathological changes of mammary tissues from each group: no treatment (NT) group (A,G); LPS group (B,H); LPS + farrerol (20 mg/kg) group (C,I); LPS + farrerol (30 mg/kg) group (D,J); LPS + farrerol (40 mg/kg) group (E,K); LPS + dexamethasone (5 mg/kg) group (F,L). Histomorphology and pathology showed that treatment with farrerol can alleviated LPS-induced pathological changes.

Figure 3

Effects of farrerol on the myeloperoxidase (MPO) activity in mammary gland. The values were presented as the means ± SEM of three independent experiments (n = 3). # p < 0.05 vs. NT group; ** p < 0.01 and *** p < 0.001 vs. LPS group.

Figure 4

The levels of TNF-α (A); IL-6 (B) and IL-1β (C) in the homogenate of mouse mammary glands including NT group, LPS group and LPS + farrerol (20, 30, 40 mg/kg) group; The protein levels of iNOS (D,E) and COX-2 (D,F) were measured by Western blot. Data are presented as mean ± SEM (n = 3). # p < 0.05 vs. NT group; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. LPS group.

Figure 5

Farrerol inhibited the phosphorylation of protein kinase B (AKT) and NF-κB signal pathways protein in LPS-induced mouse mastitis. The protein levels of p-AKT (A,B) and p-NF-κB p65 (A,C) were measured by Western blot. Data were presented as mean ± SEM (n = 3). # p < 0.05 vs. NT group; **** p < 0.0001 vs. LPS group.

Figure 6

Farrerol inhibited the phosphorylation of ERK1/2, p38 in LPS-induced mouse mastitis. The phosphorylation of p38 (A,B) and ERK1/2 (A,C) were measured by Western blot. Data are presented as mean ± SEM (n = 3). # p < 0.05 vs. NT group; **** p < 0.0,001 vs. LPS group.

Figure 7

Effects of farrerol on the cell viability of mouse mammary epithelial cells (mMECs) cultured with different concentrations of farrerol (70, 90, 110 and 130 μM). MMECs viability were determined by CCK-8 assay. Data are presented as mean ± SEM (n = 6).

Figure 8

The effect of farrerol on pro-inflammatory mediator’s production in LPS-stimulated mMECs. MMECs were divided into six groups: NT group, Farrerol group, LPS group, LPS + farrerol (90, 110, 130 μM) group. The mRNA levels of TNF-α (A); IL-6 (B) and IL-1β (C) were measured by real-time PCR. The protein levels of iNOS (D,E) and COX-2 (D,F) were measured by Western blot. Data are presented as mean ± SEM (n = 3). # p < 0.05 vs. NT group; ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. LPS group.

Figure 9

Farrerol inhibited the phosphorylation of AKT, NF-κB, p38 and ERK1/2 in LPS-stimulated mMECs. The phosphorylation AKT (A,B); NF-κB p65 (A,C); p38 (A,D); and ERK1/2 (A,E) were measured by Western blot. Data were presented as mean ± SEM (n = 3). # p < 0.05 vs. NT group; ** p < 0.01 and **** p < 0.0,001 vs. LPS group.