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ORIGINAL ARTICLE Indirubin Inhibits LPS-Induced Inflammation via TLR4 Abrogation Mediated by the NF-kB and MAPK Signaling Pathways Jin-lun Lai,1 Yu-hui Liu,1 Chang Liu,1 Ming-pu Qi,1 Rui-ning Liu,1 Xi-fang Zhu,1 Qiu-ge Zhou,1 Ying-yu Chen,1,2 Ai-zhen Guo,1,2,3 and Chang-min Hu1,2,3 Abstract―Indirubin plays an important role in the treatment of many chronic diseases and exhibits st- rong anti-inflammatory activity.

However, the molecular mode of action during mastitis prophylaxis remains poorly understood. In this study, a lipopolysaccharide (LPS)-induced mastitis mouse model showed that indirubin attenuated histopathological changes in the mammary gland, local tissue necrosis, and neutrophil infiltration. Moreover, indirubin significantly downregulated the production of interleu- kin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α). We explored the mechanism whereby indirubin exerts protective effects against LPS-induced inflammation of mouse mammary epithelial cells (MMECs). The addition of different concentrations of indirubin before exposure of cells to LPS for

1 h significantly attenuated inflammation and reduced the concentrations of the three inflammatory cytokines in a dose-dependent manner. Indirubin downregulated LPS-induced cyclooxygenase-2 (COX- 2) and Toll-like receptor

4 (TLR4) expression, inhibited phosphorylation of the LPS-induced nuclear transcription factor-kappa B (NF-kB) P65 protein and its inhibitor IkBα of the NF-kB signaling pathway. Furthermore, indirubin suppressed phosphorylation of P38, extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK) of the mitogen-activated protein kinase (MAPK) signal pathways. Thus, indirubin effectively suppressed LPS-induced inflammation via TLR4 abroga- tion mediated by the NF-kB and MAPK signaling pathways and may be useful for mastitis prophylaxis. KEY WORDS: indirubin;

lipopolysaccharide (LPS);

TLR4;

NF-kB;

MAPK;

inflammation. INTRODUCTION Mastitis is one of the most costly diseases of dairy cattle [1]. Inflammation of the mammary gland has nega- tive physical, chemical, and biological consequences [2]. Many microorganisms, including the Gram-positive Staph- ylococcus aureus and the Gram-negative Escherichia coli, can cause mastitis [3C6]. E. coli is a major cause of bovine mastitis, particularly around the time of parturition or dur- ing early lactation [7]. Lipopolysaccharide (LPS), a princi- pal component of the outer membrane of Gram-negative bacteria, triggers mastitis in bovine and mouse models and induces the production of various pro-inflammatory cyto- kines. LPS is a key trigger of inflammation [8]. The innate immune system plays an important role in defense against invading pathogens. Mouse mammary epithelial cell (MMEC) cultures are widely used to study the capacity of cells to sense and respond to mastitis-causing bacteria [9]. Such MMECs are the first line of defense against Electronic supplementary material The online version of this article (doi:10.1007/s10753-016-0447-7) contains supplementary material, which is available to authorized users.

1 The Faculty of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China

2 State Key Laboratory of Agricultural Microbiology, Huazhong Agricul- tural University, Wuhan, 430070, China

3 To whom correspondence should be addressed at The Faculty of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China. E-mails: aizhen@mail.hzau.edu.cn;

hcm@mail.hzau.edu.cn Inflammation, Vol. 40, No. 1, February

2017 (# 2016) DOI: 10.1007/s10753-016-0447-7