Alpinetin attenuates inflammatory responses by interfering toll-like receptor 4/nuclear factor kappa B signaling pathway in lipopolysaccharide-induced mastitis in mice

https://doi.org/10.1016/j.intimp.2013.04.030Get rights and content

Highlights

  • Alpinetin markedly inhibited the LPS-induced TNF-α, IL-6 and IL-1β production.

  • Alpinetin inhibited the phosphorylation of IκB-α and p65 NF-κB, caused by LPS.

  • Alpinetin inhibited the expression of TLR4, caused by LPS.

  • Alpinetin attenuated mammary histopathologic changes in the mouse models.

Abstract

Alpinetin, a novel plant flavonoid derived from Alpinia katsumadai Hayata, has been reported to exhibit anti-inflammatory properties. However, the effect of alpinetin on mastitis has not been investigated. The aim of this study was to investigate the protective effect of alpinetin against lipopolysaccharide (LPS)-induced mastitis and to clarify the possible mechanism. In the present study, primary mouse mammary epithelial cells and an LPS-induced mouse mastitis model were used to investigate the effect of alpinetin on mastitis and the possible mechanism. In vivo, we observed that alpinetin significantly attenuated the infiltration of neutrophilic granulocytes, and the activation of myeloperoxidase; down-regulated the level of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6; inhibited the phosphorylation of IκB-α, NF-κB p65 and the expression of TLR4, caused by LPS. In vitro, we also observed that alpinetin inhibited the expression of TLR4 and the production of TNF-α, IL-1β and IL-6 in LPS-stimulated primary mouse mammary epithelial cells. However, alpinetin could not inhibit the production of IL-1β and IL-6 in TNF-α-stimulated primary mouse mammary epithelial cells. In conclusion, our results suggest that the anti-inflammatory effects of alpinetin against LPS-induced mastitis may be due to its ability to inhibit TLR4-mediated NF-κB signaling pathways. Alpinetin may be a promising potential therapeutic reagent for mastitis treatment.

Introduction

Mastitis, an infection of the mammary gland, is a highly prevalent and important infectious disease. The disease causes a decline in milk production and quality and results in impaired infant growth and development [1]. Some reports have shown that approximately 30% of women suffered at least one case of infectious mastitis [2]. Many microbial, host, and environmental factors can induce the development of mastitis [3], [4]. Lipopolysaccharide (LPS), a main component of the outer membrane of Gram-negative bacteria, has been identified as an important risk factor for mastitis [5]. LPS can activate the host receptor TLR4 and trigger an inflammatory response, resulting in mastitis. Injection of LPS through the duct of the mammary gland has gained wide acceptance as a clinically relevant model of mastitis [6], [7]. Several candidate therapy strategies have been applied to treat mastitis in the last decade. However, the incidence of mastitis remains high. Recently, treatments aimed at modulating TLR4 signaling and alleviating nonspecific inflammatory reactions may have potential therapeutic advantages for mastitis. Therefore, the development of novel therapies for mastitis is urgently needed.

Alpinetin (Fig. 1), a novel plant flavonoid isolated from Alpinia katsumadai Hayata, is responsible for the plant's pharmacological activities. Alpinetin has been reported to have antibacterial, anti-tumor and other important therapeutic activities [8], [9], [10]. Recently, it has been reported that alpinetin has an anti-inflammatory effect [11]. Alpinetin was found to inhibit inflammatory cytokine production in LPS-activated macrophages and to attenuate LPS-induced acute lung injury in mice [11]. Recently, it has been reported that the anti-inflammatory effects of alpinetin are mediated by blocking the activation of NF-κB and MAPKs signaling pathways [11]. Although a number of studies have addressed the therapeutic potential of alpinetin, its ability to protect against bacterial endotoxin-induced mastitis remains poorly understood. In this study, we sought to assess the preventive effects of alpinetin in an LPS-induced mouse mastitis model and elucidate the potential anti-inflammatory mechanism.

Section snippets

Animals

Seventy-two female and thirty-six male BALB/c mice were housed in microisolator cages and received food and water. The laboratory temperature was 24 ± 1 °C, and the relative humidity was 40–80%. All experimental protocols were approved by the regional Animal Ethics Committee.

Reagents

Alpinetin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Mouse TNF-α, IL-6 and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were purchased from

Effects of alpinetin on the MPO activity

PMNs are the major components of inflammatory and immunological reactions in an injured mammary gland. The increase of MPO activity reflects PMN accumulation in the mammary gland. Thus MPO plays an important role in the development of LPS-induced mastitis. The MPO activity was determined to assess the neutrophil accumulation within the mammary tissues. As shown in Fig. 2, LPS challenge resulted in a significant increase of MPO activity in the mammary gland compared with the control group (p < 

Discussion

Mastitis, the inflammation of the mammary gland, is characterized by the disruption of endothelial and epithelial integrity, the release of inflammatory mediators, and extensive neutrophil infiltration [15]. Mammary gland duct stimulation with LPS induces an ideal experimental model of mastitis, which is well suited for preliminary pharmacological studies of new drugs or other therapeutic agents. Alpinetin, a novel plant flavonoid derived from Alpinia katsumadai Hayata, has been reported to

Conflict of interest statement

All authors declare that they have no conflict of interest.

References (30)

  • J. Miao et al.

    The effect of taurine on the toll-like receptors/nuclear factor kappa B (TLRs/NF-kappaB) signaling pathway in Streptococcus uberis-induced mastitis in rats

    Int Immunopharmacol

    (2011)
  • G.A. Contreras et al.

    Mastitis: comparative etiology and epidemiology

    J Mammary Gland Biol Neoplasia

    (2011)
  • C. Burvenich et al.

    Severity of E-coli mastitis is mainly determined by cow factors

    Vet Res

    (2003)
  • S. Salminen et al.

    Microbial-host interactions: selecting the right probiotics and prebiotics for infants

    Nestle Nutr Workshop Ser Pediatr Program

    (2009)
  • D. Li et al.

    Salidroside attenuates inflammatory responses by suppressing nuclear factor-kappaB and mitogen activated protein kinases activation in lipopolysaccharide-induced mastitis in mice

    Inflamm Res

    (2013)
  • Cited by (0)

    View full text