Protective Effects of Lentinan Against Lipopolysaccharide-Induced Mastitis in Mice

Abstract

Mastitis is a worldwide production disease in dairy cows, which mainly affects milk yield, causing huge economic losses to dairy farmers. Lentinan is a kind of polysaccharide extracted from Lentinus edodes, which has no toxicity and possesses various pharmacological activities including antibacterial and immunomodulatory effects. Therefore, the anti-inflammatory function of lentinan on LPS-stimulated mastitis was carried out, and the mechanism involved was explored. In vivo, lentinan greatly reduced LPS-stimulated pathological injury, myeloperoxidase (MPO) activity, and the proinflammatory factor production (TNF-α and IL-1β) in mice. Further study was performed to determine the activation of the Wnt/β-catenin pathway during LPS stimulation. These results suggested that LPS-induced activation of the Wnt/β-catenin pathway was suppressed by lentinan administration. In vitro, we observed that the mouse mammary epithelial cell (mMEC) viability was not affected by lentinan treatment. As expected, LPS increased the TNF-α and IL-1β protein secretion and the activation of the Wnt/β-catenin pathway that was inhibited by lentinan administration in a dose-dependent manner in mMECs. Conclusively, lentinan exerts the anti-inflammatory function in LPS-stimulated mastitis via inhibiting the activation of the Wnt/β-catenin pathway. Thus, the results of our study also gave an insight that lentinan may serve as a potential treatment for mastitis.

Keywords: LPS; Wnt/β-catenin pathway; inflammation; lentinan; mastitis.

FIGURE 1

Effects of lentinan on LPS-stimulated histopathological changes. Histopathological changes in mammary gland tissues (H and E). (A) Control group, (B) LPS group, (C–E) lentinan (5, 10, and 20 mg/kg) groups, (F) dexamethasone group, and (G) histopathological grade score. The blue arrow indicates the mammary gland tissue lesion area. All data are represented as the mean ± S.E.M. of three replicates. ^# p < 0.05 vs the control group. *p < 0.05 vs. the LPS group. **p < 0.01 compared with the LPS group. ***p < 0.01 compared with the LPS group.

FIGURE 2

Effects of lentinan on MPO activity. (A,B) MPO activity assay in lentinan-treated mMECs. All data are represented as the mean ± S.E.M. of three replicates. *p < 0.05 vs. the LPS group. **p < 0.01 compared with the LPS group. ***p < 0.01 compared with the LPS group.

FIGURE 3

Lentinan inhibited the secretion of proinflammatory factors. The expression of TNF-α and IL-1β in LPS-stimulated mouse mastitis was detected using ELISA kits. All data are represented as the mean ± S.E.M. of three replicates. *p < 0.05 vs the LPS group. **p < 0.01 compared with the LPS group. ***p < 0.01 compared with the LPS group.

FIGURE 4

Effects of lentinan on the activation of the Wnt/β-catenin pathway. The activation of the Wnt/β-catenin pathway in LPS-stimulated mouse mastitis was determined by immunofluorescence assay. All data are represented as the mean ± S.E.M. of three replicates.

FIGURE 5

Cell biological detection. The nucleus was dyed blue. The cytoplasm was dyed green by CK-18.

FIGURE 6

Effects of lentinan on inflammatory response of LPS-induced mouse mammary epithelial cells. (A) The potential cytotoxicity of lentinan (5, 10, and 20 μg/ml) on mMECs was detected by MTT experiment. (B) The TNF-α and IL-1β protein levels were detected using the ELISA kit in LPS-stimulated mMECs. All data are represented as the mean ± S.E.M. of three replicates. *p < 0.05 vs. the LPS group. **p < 0.01 compared with the LPS group. ***p < 0.01 compared with the LPS group.

FIGURE 7

Effects of lentinan on the activation of the Wnt/β-catenin pathway in mMECs. (A,B) The activation of the Wnt/β-catenin signaling pathway in LPS-stimulated mMECs was also determined by the immunofluorescence technique. (C) The expression of Wnt/β-catenin proteins was detected by western blot. β-actin served as an internal control. All data are represented as the mean ± S.E.M. of three replicates.