Dimethyl itaconate protects against lippolysacchride-induced mastitis in mice by activating MAPKs and Nrf2 and inhibiting NF-κB signaling pathways

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Highlights

  • DI has protective effects against LPS-induced mastitis in mice.

  • DI significantly decreases the secretion of TNF-α and IL-1β.

  • DI inhibits LPS-induced TLR4 expression and NF-κB activation.

  • DI up-regulated the MAPKs and Nrf2 actiation.

Abstract

Mastitis, as the main disease to affect the dry dairy cow with the characterized by increasing number of somatic cells in milk and reducing milk production, has been known as one of the most serious expensive disease for the dairy industry. Escherichia coli (E.coli), a gram negative bacterial, have normally been considered to be an opportunistic pathogen that can invade the mammary gland sometimes to cause inflammatory diseases. Lippolysacchride (LPS), as the co-cell wall component of the Escherichia coli (E.coli), is the main virulence factors to induce acute inflammation. Itaconate is an endogenous metabolite which has recently been reported to regulate the macrophage function and has the ability to reduce the secretion of pro-inflammatory cytokines, such as IL-6 and IL-12. Here, the aim of this study is to investigate the protective role of dimethyl itaconate (DI)-the membranepermeable derivative of itaconate, on LPS-induced mastitis in mice. To establish the model of mastitis, mice 5–7 day after delivery were utilized by nipple duct injection of LPS, while DI was treated 24h intraperitoneally before LPS injection. Further, the hematoxylin-eosin (H&E) staining was used to evaluate the pathological changes of the mammary gland, the inflammatory cytokines of TNF-α and IL-1β and the myeloperoxidase (MPO) activity were also measured respectively by enzyme-linked immunosorbent assay (ELISA) and MPO assay kit. To clarify the underling mechanisms of the protective role of DI on mastitis, the MAPKs, NF-κB and Nrf2 signaling pathways were detected via western blotting. The results demonstrated that DI markedly decreased the pathological injury of mammary, and considerably reduced the production of TNF-α and IL-1β, as well as up-regulated the Nrf2, HO-1, phosphorylation of p38 and ERK, but down-regulated TLR4 and phosphorylation of p65 NF-κB. Our research recommended that DI ameliorated LPS-induced mastitis which highlights itaconate may as a potential candidate to protect against mastitis.

Introduction

Bovine mastitis, which often induced the declining of milk production and the increase of somatic cells in the milk, is a serious disease to cause a large economic loss to the dairy industry and threat all the dairy animals [1]. Generally, mastitis is caused by various causes such as the infection of pathogens, including gram-positive (mainly staphylococcus aureus and streptococcus) [2] and gram-negative bacterial (mainly Escherichia coli) [3]. The previous has reported that Escherichia coli often induced the clinical mastitis which is distinguished by acute inflammation of breast tissue companied decresed milk production [4]. Lipopolysaccharide (LPS), the co-component of the cell wall of Escherichia coli, has been considered as the major causative factor to induce acute inflammation by activation toll like receptor 4 (TLR4)-mediated nuclear transcription factor kappa B (NF-κB) [5]. Further, antibiotics, the traditional treat strategy for mastitis, have made people to worry about the quality and safety of the milk and dairy products due to the drug residues and resistance [6]. Therefore, it is important and urgent to seek effective and safe therapeutic methods for mastitis.

Itaconate was first recognized as a product of citric acid and used for production of industrial raw materials [7]. Recently, itaconate regulated by immunoresponsive gene 1 (Irg1) has been identified as an endogenous metabolite during macrophage activation, with the ability to alleviate inflammation by inhibiting IL-1β, IL-6, IL-12, NO and HIF-1α via Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) so that being considered as a crucial anti-inflammatory agent [8]. The transcription factor Nrf2 has been shown to alleviate inflammation through inducing heme oxygenase-1 (HO-1) which can increase Irg1 and down-regulate TNF-α production [9]. Further, findings from in vitro studies have demonstrated that itaconate has the ability to inhibit succinate, a metabolite which could prompt O2 consumption and increase the secretion of pro-inflammatory cytokines, via suppressing succinate dehydrogenase (SDH) [10]. On the other, the TLR4-mediated NF-κB could be depressed by the activation of HO-1 [11]. Interestingly, the phosphorylation of mitogen-activated protein kinase (MAPK) signal pathways, including p38, ERK1/2 and JNK, may contribute to raise HO-1 by activating Nrf2 [11], despite the phosphorylation of MAPKs has been shown to increase during LPS stimulated in some studies. Given all the above, it is necessary to investigate whether DI holds the influence on LPS-induced mastitis and the fundamental mechanism remains elucidate.

In our study, we investigated the protective effect of DI on LPS-induced mastitis by injection DI intraperitoneally 24h before LPS treated. The results illustrated that DI reduced the pathological damage of mammary gland, decreased the production of TNF-α and IL-1β and up-regulated MAPKs/Nrf2 and down-regulated NF-κB signal pathways. Hence, the endogenous metabolite DI may have the possibility to serve as a potential and effective candidate for the treatment of mastitis.

Section snippets

Animals

A total of 80 BALB/c mice, 60 female and 20 male aged 6–8 week and the weight is 35–40g, were purchased from Experimental Animal Center of Baiqiuem Medical College, Jilin University. Females and males were mixed of 3 to 1 in each cage with enough water and food until pregnancy. The model of mouse mastitis was built according to the previous studies 5–7 days after delivery, complying with the experimental animal manual. The present study was completed consistent with the care and use of

DI attenuates the histopathological damages of in LPS-induced mastitis

The histopathological results demonstrated that the control group had normal structure with integrity breast acinus and almost no inflammatory cell infiltration (Fig. 2A). On the contrary, mammary gland structure destruction, and inflammatory cell infiltration (mainly neutrophils and macrophages) was showed in the LPS group (Fig. 2B). After DI treated, the integrity of mammary structure has increasingly restored and the neutrophils have reduced (Fig.2C and D). However, there was no markedly

Discussion

Mastitis, as an important inflammatory disease of the dairy industry, has caused a large economic consumption and generally characterized by mammary gland tissues injury with the increased level of pro-inflammatory cytokines (mainly TNF-α and IL-1β) [15]. LPS, the main virulence factor of E.coli, has been used to establish the model of acute mouse mastitis [16]. The previous findings have demonstrated that TLR4-mediated MyD88/NF-κB is the main signaling pathway to induce inflammatory cytokines

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

This work was supported by a grant from the National Natural Science Foundation of China (No. 31772812).

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These anthors contributed equally to this article.

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