Peiminine Protects against Lipopolysaccharide-Induced Mastitis by Inhibiting the AKT/NF-κB, ERK1/2 and p38 Signaling Pathways

Abstract

Peiminine, an alkaloid extracted from Fritillaria plants, has been reported to have potent anti-inflammatory properties. However, the anti-inflammatory effect of peiminine on a mouse lipopolysaccharide (LPS)-induced mastitis model remains to be elucidated. The purpose of this experiment was to investigate the effect of peiminine on LPS-induced mastitis in mice. LPS was injected through the canals of the mammary gland to generate the mouse LPS-induced mastitis model. Peiminine was administered intraperitoneally 1 h before and 12 h after the LPS injection. In vitro, mouse mammary epithelial cells (mMECs) were pretreated with different concentrations of peiminine for 1 h and were then stimulated with LPS. The mechanism of peiminine on mastitis was studied by hematoxylin-eosin staining (H&E) staining, western blotting, and enzyme-linked immunosorbent assay (ELISA). The results showed that peiminine significantly decreased the histopathological impairment of the mammary gland in vivo and reduced the production of pro-inflammatory mediators in vivo and in vitro. Furthermore, peiminine inhibited the phosphorylation of the protein kinase B (AKT)/ nuclear factor-κB (NF-κB), extracellular regulated protein kinase (ERK1/2), and p38 signaling pathways both in vivo and in vitro. All the results suggested that peiminine exerted potent anti-inflammatory effects on LPS-induced mastitis in mice. Therefore, peiminine might be a potential therapeutic agent for mastitis.

Keywords: AKT/NF-κB; ERK1/2; LPS; mastitis; p38; peiminine.

Figure 1

Chemical structure of peiminine.

Figure 2

Histopathological sections of the mammary tissues (H&E, 100× and 400×). The mammary tissues (n = 6 in each group) from each group were processed for histological evaluation. Representative histological changes in the mammary tissues from each group were as follows: (A,F) no-treatment (NT) group; (B,G) lipopolysaccharide (LPS) group; (C,H) LPS + 1 mg/kg peiminine group; (D,I) LPS + 3 mg/kg peiminine group; and (E,J) LPS + 5 mg/kg peiminine group. Histologic grade of the mammary gland (K). The histological morphology and pathology results showed that peiminine treatment alleviated the LPS-induced pathological changes. The number sign (#) indicates a significant difference from the NT group at p < 0.0001. The tetrad asterisks (****) indicate p < 0.0001 and the double asterisks (**) indicate p < 0.01 vs. the LPS group.

Figure 3

Effect of peiminine on (A) the myeloperoxidase (MPO) activity in the mammary gland of LPS-induced mastitis, the levels of (B) tumor necrosis factor alpha (TNF-α), (C) interleukin-6 (IL-6), and (D) interleukin-1β (IL-1β) in the homogenate of the mouse mammary tissues, including the NT group, the LPS group, and the groups treated with peiminine (1, 3, and 5 mg/kg), and the protein levels of (E,F) COX-2 and (E,G) iNOS were measured by western blotting. The data are presented as the mean ± SD (n = 6). The number sign (#) indicates p < 0.0001, which was significantly different from the NT group. The tetrad asterisks (****) indicate p < 0.0001, the triple asterisks (***) indicate p < 0.0005, the double asterisks (**) indicate p < 0.01, and the single asterisk (*) indicates p < 0.05 vs. the LPS group.

Figure 4

Peiminine inhibits the phosphorylation of the AKT, nuclear factor-κB (NF-κB) p65, ERK1/2 and p38 signaling pathways in the mammary tissues. The results for the protein levels of (A,B) p-AKT, (A,C) p-NF-κB p65, (A,D) p-ERK1/2, and (A,E) p-p38 were measured by western blotting. The data are presented as the mean ± SD (n = 6). The number sign (#) indicates a significant difference from the NT group at p < 0.0001. The tetrad asterisks (****) indicate p < 0.0001, the triple asterisks (***) indicate p < 0.0005, and the double asterisks (**) indicate p < 0.01 vs. the LPS group.

Figure 5

Effect of peiminine on (A) cell viability. The cells were cultured with different concentrations of peiminine (30, 50, or 70 μg/mL) for 4 h. Cell viability was determined by the CCK8 assay. The levels of (B) TNF-α and (C) IL-6 in the mouse mammary epithelial cells (mMECs), including the NT group, the NT + peiminine 70 μg/mL group, the LPS group, and the groups treated with peiminine (30, 50, or 70 μg/mL), and the protein level of (D,E) COX-2 were measured by western blotting. The data are presented as the mean ± SD (n = 6). The number sign (#) indicates a significant difference from the NT group at p < 0.0001. The tetrad asterisks (****) indicate p < 0.0001, the triple asterisks (***) indicate p < 0.0005, the double asterisks (**) indicate p < 0.01, and the single asterisk (*) indicates p < 0.05 vs. the LPS group.

Figure 6

Peiminine inhibits the phosphorylation of the AKT, NF-κB p65, ERK1/2, and p38 signaling pathways in mMECs. The results for the protein levels of (A,B) p-AKT, (A,C) p-NF-κB p65, (A,D) p-ERK1/2, and (A,E) p-p38 were measured by western blotting. The data are presented as the mean ± SD (n = 6). The number sign (#) indicates a significant difference from the NT group at p < 0.0001. The tetrad asterisks (****) indicate p < 0.0001, the triple asterisks (***) indicate p < 0.0005, the double asterisks (**) indicate p < 0.01, and the single asterisk (*) indicates p < 0.05 vs. the LPS group.

Figure 7

Peiminine inhibits LPS-induced inflammation by suppressing the phosphorylation of the AKT, NF-κB p65, ERK1/2, and p38 signaling pathways.