Accepted Manuscript Inhibitory effects of Astragalin on LPS-Induced Inflammatory Response in Mouse Mammary Epithelial Cells Fengyang Li , M.D. Wei Wang , Ph.D. Yongguo Cao , Ph.D. Dejie Liang , M.D. Wenlong Zhang , M.D. Zecai Zhang , M.D. Haichao Jiang , M.D. Mengyao Guo , Ph.D. Naisheng Zhang , M.D. PII:

S0022-4804(14)00528-9

DOI:

10.1016/j.jss.2014.05.059

Reference:

YJSRE 12761

To appear in:

Journal of Surgical Research

Received Date: 12 February 2014 Revised Date:

2 May 2014

Accepted Date: 19 May 2014

Please cite this article as: Li F, Wang W, Cao Y, Liang D, Zhang W, Zhang Z, Jiang H, Guo M, Zhang N, Inhibitory effects of Astragalin on LPS-Induced Inflammatory Response in Mouse Mammary Epithelial Cells, Journal of Surgical Research (2014), doi: 10.1016/j.jss.2014.05.059. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Revised

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May 2, 2014

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Fengyang Li1, M.D., Wei Wang1, Ph.D., Yongguo Cao1, Ph.D., Dejie Liang, M.D., Wenlong Zhang, M.D., Zecai Zhang, M.D., Haichao Jiang, M.D., Mengyao Guo, Ph.D., Naisheng Zhang*, M.D.

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Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province 130062, Peoples Republic of China

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Running title: Astragalin inhibits inflammatory response in mouse mammary epithelial cells Author’s contributions: Fengyang Li wrote the article, and designed the study with Dejie Liang; the revised 1 manuscript was mainly altered and edited by Dr. Yongguo Cao and Wei Wang; these authors contributed equally to this work. The experiment was performed by Wenlong Zhang, Zecai Zhang, and Haichao Jiang; data was collected and analyzed by Dr. Yongguo Cao; literature search of the revised manuscript was performed by Mengyao Guo; the whole study was kindly founded by Prof. Naisheng Zhang. All authors declared that they had read and approved the final version of the manuscript.

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Corresponding author *Prof. Dr. Naisheng Zhang, Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province 130062, Peoples Republic of China. E-mail: [email protected]. Phone: +86 431 87836181 Fax: +86 0431 87836181.

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ABSTRACT

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Background: Tea brewed from the leaves of persimmon or Rosa agrestis have several medical functions including treating allergy, anti-atopic dermatitis and anti-inflammatory effects. The objective of this study was to investigate the molecular mechanisms of astragalin, a main flavonoid component isolated from these herbs, in modifying lipopolysaccharide (LPS)-induced signaling pathways in primary cultured mouse mammary epithelial cells (mMECs). Materials and Methods: The mMECs were treated with LPS in the absence or presence of different concentrations of astragalin. The expression of pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-6, as well as NO production were determined by enzyme-linked immunosorbent assay (ELISA) and Greiss reaction respectively. Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), inhibitor protein of NF-κB (IκBα), P38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase were measured by Western blot. Results: The results showed that astragalin suppressed the expression of TNF-α, IL-6, and NO in a dose-dependent manner in mMECs. Western blot results showed that the expression of iNOS and COX-2 was inhibited by astragalin. Besides, astragalin efficiently decreased LPS-induced TLR4 expression, NF-κB activation, IκBα degradation, and the phosphorylation of p38, ERK in bMECs. Conclusions: Our results indicated that astragalin exerts anti-inflammatory properties possibly via the inactivation of TLR4-mediated NF-κB and MAPKs signaling pathways in LPS-stimulated mMECs. Thus, astragalin may be a potential therapeutic agent for bovine mastitis.

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Keywords: Mastitis; Astragalin; LPS; Cytokine; TLR4; NF-κB;

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Inhibitory effects of Astragalin on LPS-Induced Inflammatory Response in Mouse Mammary Epithelial Cells

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1. Introduction

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Mastitis is defined as infection and inflammation of the mammary gland (1). It not only poses a threat to women health, but also to dairy cows, resulting in great economic loses in dairy cattle industries. Bovine mastitis is caused by a wide array of microorganisms, including Gram-positive pathogen such as Staphylococcus aureus (S.aureus) and Gram-negative pathogen such as Escherichia coli (E.Coli) (2). These bacteria can invade the mammary gland by penetration through the teat canal and lead to mammary gland inflammation (3). Lipopolysaccharides (LPS) or endotoxin, which is possessed by E.Coli in the outer membrane of bacterial cell wall, is identified as a vital virulence factor of mastitis (4). Toll-like receptors (TLRs), which are differentially expressed on myeloid lineage cells, such as dendritic cells, monocytes or macrophages and non-immune cells, including endothelial cells, fibroblasts, adipocytes and epithelial cells, are critical pattern recognition receptors that localized as the first line of innate defense by recognizing pathogen-associated molecular patterns (PMAPs) leading to innate and adaptive immune response (5, 6). Up to now, there are 13 mammalian TLRs that have been reported (7). The expression of TLR4 on the cell surface can be activated by several bacterial surface molecules, including the conserved LPS of Gram-negative bacteria. LPS-activated TLR4/ NF-κB triggers the formation of signaling complexes and leads to the release of various pro-inflammatory cytokines and mediators such as TNF-α, IL-6 and IL-1β (8). Previous reports have shown that iNOS and COX-2 were also up-regulated by binding of LPS or TNF-α in mammary epithelial cells and further aggravating the inflammatory immune response during infection (9-11).

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When an intramammary E.Coli infection occurs, bacteria that enter the mammary gland release LPS. As a reaction, a host immune response is initiated where the polymorphonuclear neutrophil leukocytes (PMNs) play a pivotal role in clearing the mammary gland by phagocytizing invading bacteria (12). Besides, other cell types in the udder, such as mammary alveolar epithelial cells also come in direct contact with the bacteria invasion and act as more than a physical barrier for pathogens, but also participate in the immune-regulation during mastitis via producing inflammatory mediators (6, 13). Therefore, it is of importance to investigate the inflammatory and immune response in mammary epithelial cells during LPS infection.

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Nowadays, despite various therapeutic strategies have been developed to prevent mastitis, there are still no reliable and effective therapies against the disease (14). In the meantime, many researchers reported that many compounds that extracted from natural plants exerting beneficial effects against mastitis both in vitro and in vivo. Astragalin (shown in Figure 1) is a flavonoid that isolated from leaves of persimmon or Rosa agrestis, and it was widely distributed in tea and has been used for treating many diseases for a long time as a traditional Chinese medicine (15, 16). Several studies have been confirmed that astragalin exhibited a number of biological properties, including anti-inflammatory, antioxidant, and antiatopic dermatitis effects (16-19). It has been reported that astragalin exerted anti-inflammatory effects and showed to suppress the production of inflammatory cytokines in mouse peritoneal macrophages and acute lung injury through down-regulating NF-κB signaling pathway (16, 17). Nevertheless, as expounded above, the effect of astragalin on inflammatory response in LPS-induced mammary epithelial cells still

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ACCEPTED MANUSCRIPT need to be further understood. Thus, in this study we used primary cultured mouse epithelial cells (mMECs) to further investigate the anti-inflammatory effect of astragalin and the underlying mechanisms.

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2. Materials and methods

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2.1. Chemicals and reagents

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Astragalin (purity >99.9%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) (Fig. 1) and dissolved in dimethyl sulfoxide (DMSO, Sigma) before use. The DMSO concentration in the working solutions was less than 0.1%, which had no effect on present study. LPS (E.coli, 055:B5) and phenylmethyl sulfonylfluoride (PMSF) was purchased from Sigma Chemical CO (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was provided by Dojindo Laboratories (Kumamoto, Japan). Dulbecco’s modified Eagle’s medium/nutrient mixture F12 Ham (DMEM: F12/1:1), fetal bovine serum (FBS) and trypsin/EDTA were obtained from Hyclone (Logan, UT, USA). Collagenase I and collagenase II were provided by Invitrogen (Carlsbad, California, USA). Epidermal growth factor (EGF), transferrin and T3 were purchased from PeproTech Inc. (Rockville, MD, USA). Mouse TNF-α, IL-6 enzyme-linked immunosorbent assay (ELISA) kits was obtained from Biolegend (San Diego, CA, USA). Rabbit mAb IκBα, mouse mAb p65, p38, ERK, JNK, phosphor-IκBα, phosphor-p65, phosphor-p38, phosphor-ERK, and phosphor-JNK antibodies, together with mouse specific iNOS and COX-2 antibodies were purchased from Cell Signaling Technology Inc. Mouse mAb TLR4 were purchased from GeneTex. (Beverly, MA, USA). HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies, as well as β-actin were provided by Tianjin Sungene Biotech Co., Ltd (Tianjin, China). All other chemicals were of reagent grade, and endotoxin was free.

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2.2. Primary culture and treatment of mouse mammary epithelial cells

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All procedures involving animals were approved by the Animal Welfare and Research Ethics Committee at Jilin University (approval ID: 20111106-2). MMECs were isolated and prepared for culture as previously described (20, 21). In brief, mammary glands from mid-term pregnant BALB/c mice were removed, minced, and subjected to collagenase I/II/trypsin mixture digestion. After enzymatic dissociation, the tissue suspensions were centrifuged at 250× g for 5 min. The fatty top layer and pellets were collected in DMEM/F12 with 10% FBS and then centrifuged, resuspended and filtered through 40 μm pore-size filter. The trapped cells were collected in growth media (DMEM/F12, 10% FBS, 0.5% transferrin, 0.1% T3, and 0.5% EGF), counted by phase microscopy, and plated in culture plates at 37°C and 5% CO2 in a humidified atmosphere. The primary cultured mMECs were divided into different indicated groups and allowed to acclimate for 24 h before any treatments in all experiments.

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2.3. CCK-8 analysis for cell viability

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A CCK-8 assay was used to analyze the effect of astragalin on mMECs viability. Briefly, mMECs were plated at a density of 1×104 cells/ml in a 96-well plate in a 37°C, 5% CO2 incubator for 4 h; then the cells were treated with different concentrations of astragalin (0-100 μg/ml) for another

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ACCEPTED MANUSCRIPT 1 h, followed by stimulation with 20 μl/well LPS. After incubated for additional 24 h, 10 μl CCK-8 solutions was added to each well, and the mMECs were incubated for 3 h in the dark. The optical density was measured at 450 nm on a microplate spectrophotometer (TECAN, Australia). MMECs cultured in medium alone and medium without cells were served as control.

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2.4. Nitric oxide analysis

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In order to analyze the nitric oxide (NO) production of mMECs, accumulation of nitrite (NO2-) in the culture supernatants was measured by using Greiss reaction. In brief, mMECs was treated with various concentrations of astragalin with or without astragalin stimulation for 30 min. Cellular NO production was induced by adding 1 μg/ml LPS for 18 h. Then the culture supernatant was collected and mixed with the same volume of Greiss reagent I (10% sulfanilamide, 40% phosphoric acid, Sigma) and Greiss reagent II (1% N -(1-naphthyl) -ethylenediamine -dihydrochloride, Sigma) to detect nitrite. The absorbance at 570 nm was determined using a microplate spectrophotometer. Nitrite concentration was calculated with reference to a standard curve of sodium nitrite.

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2.5. Cytokine analysis

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The mMECs were seeded in 24-well plates (4×105 cells/well) and incubated in the presence or absent of astragalin 25, 50, and 100 μg/ml for 1 h and then were challenged with LPS (1 μg/ml) for 18 h. After that, the culture supernatants were harvested and the amount of cytokine release of TNF-α and IL-6 in it were determined by ELISA according to the manufacturer’s instructions.

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2.6. Western blot analysis

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Primary cultured mMECs were untreated or pretreated with indicated concentrations of astragalin for 1 h; followed by stimulation with LPS (1 μg/ml) for 1 h (for NF-κB and MAPKs), or 6 h (for TLR4,) or 24 h (for iNOS and COX-2). After collected and washed by ice-cold PBS, total proteins in the supernatants were extracted using the M-PER Mammalian Protein Extraction Reagent (Thermal Scientific, USA) which contains a 1:100 dilution of PMSF as protease and protein phosphatase inhibitors. The presence of proteins was measured by a BCA protein assay kit (Pierce, Rockford, IL, USA). Lysate samples containing equal amounts of proteins (40 μg) were fractionated by SDS-PAGE using Tris-HCL Precast Gels and then transferred onto a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 h, and probed with primary antibodies (1:1000 dilutions in TBST) at 4°C overnight. Subsequently, the membrane was incubated with peroxidase-conjugated secondary antibody at room temperature for 1 h. The targeted proteins were visualized with Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific, USA). The β-actin protein served as an internal control.

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2.7. Statistical analysis

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Data are presented as mean ± SEM with three independent experiments. Differences between the mean values of normally distributed data were assessed with a one-way ANOVA (Dunnett’s t test) and the two-tailed Student’s t test. The analysis was performed by using GraphPad Prism

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Inhibitory effects of astragalin on lipopolysaccharide-induced inflammatory response in mouse mammary epithelial cells.

Tea brewed from the leaves of persimmon or Rosa agrestis have several medical functions including treating allergy, antiatopic dermatitis, and anti-in...
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