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Bioactive Constituents, Metabolites, and Functions

Surface Layer Protein from Lactobacillus acidophilus NCFM Inhibits Lipopolysaccharide-Induced Inflammation through MAPK and NF-#B Signaling Pathways in RAW264.7 Cells Huifang Wang, Li Zhang, Shichen Xu, Jie Pan, Qiuxiang Zhang, and Rong-Rong Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02012 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Journal of Agricultural and Food Chemistry

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Surface Layer Protein from Lactobacillus acidophilus NCFM Inhibits

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Lipopolysaccharide-Induced Inflammation through MAPK and NF-κB

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Signaling Pathways in RAW264.7 Cells

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Huifang Wang a, Li Zhang b, Shichen Xu a,b, Jie Pan a, Qiuxiang Zhang a, Rongrong Lu *a

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a

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Jiangsu 214122, China.

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b

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School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi,

Jiangsu Institute of Nuclear Medicine, Key Laboratory of Nuclear Medicine, Ministry of

Health, 20 Qian Rong, Wuxi, Jiangsu 214063, China

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*Corresponding author

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Rongrong Lu

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Tel: +86 0510 85329061

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Fax: +86 0510 85329061

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E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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The objective of our research was to evaluate the molecular mechanism of the

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anti-inflammatory effects of surface layer protein (Slp) derived from Lactobacillus

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acidophilus NCFM in lipopolysaccharide-induced RAW264.7 cells. Our results presented that

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Slp, with an apparent size of 46 kDa, attenuated the production of TNF-α, IL-1β, and reactive

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oxygen species (ROS), by inhibiting the MAPK and NF-κB signaling pathways. In addition,

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10 µg mL-1 of Slp significantly inhibited NO and PGE2 production (P < 0.001) through

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down-regulating the expression levels of iNOS and COX-2 protein. Furthermore, Slp was

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found to inhibit NF-κB p65 translocation into the nucleus to activate inflammatory gene

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transcription. These findings suggest that Slp is a potential immune-modulating bioactive

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protein derived from probiotics and holds promise for use as an additive in functional foods.

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Keywords: Lactobacillus acidophilus NCFM, Surface layer protein, Anti-inflammatory,

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MAPK, NF-κB.

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INTRODUCTION

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Food-based interventions that help suppress inflammation and regulate immune function have

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received a great deal of attention in recent years. Lactobacillus acidophilus is a probiotic

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strain available from fermented food such as yogurt, cheese, and pickles.1 Its recognized

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probiotic properties offer numerous health benefits.2, 3 In vitro cytologic experiments have

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confirmed that L. acidophilus can regulate the human immune system by competing with

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adhesion sites in intestinal epithelial cells (IECs), interfering with the pathogens colonization,

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producing bacteriocin, and lowering pH.4-7 Several literatures have shown that surface layer

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protein (Slp) extracted from L. acidophilus is closely related to the human health protection

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via modulation of the immune system.8-10

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The immunomodulatory effects of Slp currently focus mostly on the immune protection

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of IECs. SlpA isolated from L. acidophilus NCFM was shown to alleviate colonic

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inflammation in mice by modulating Dendritic Cells (DCs) and T cell immune activity.11, 12

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SlpA derived from L. helvetius MIMLh5 strain reduced the activation of NF-κB and exerted

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anti-inflammatory reactions on intestinal epithelial Caco-2 cells.13 Gao et al.14 found that L.

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acidophilus GG and its crude Slp exerted anti-inflammatory actions by reducing the tumor

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necrosis factor-α (TNF-α) production in lipopolysaccharide (LPS) simulated porcine IECs to

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relieve cell inflammation. In addition, Houem et al.15 reported that Slp separated from

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Propionibacterium freudenreichii regulated the immune system by reducing the secretion of

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TNF-α on HT-29 cells.

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IECs and macrophages participate in immune regulation via different response

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mechanisms,16 among which macrophage-mediated inflammation plays a key role in

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intestinal diseases. To date, few studies have examined macrophage immune regulation with ACS Paragon Plus Environment


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Slp. Zhang et al.17 found that Slp from L. acidophilus CICC6074 promoted the proliferation

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of RAW264.7 cells and the secretion of lysosome. SlpA from L. helvetius MIMLh5 was

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shown to significantly inhibit the TNF-α secretion in LPS-stimulated human macrophage cell

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line U937.13 However, the precise mechanism of inflammation regulation by Slp on

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macrophages is still unclear.

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Several studies in our group have confirmed that Slp derived from L. acidophilus NCFM

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exhibits various biological functions, such as adherence to IECs,18 complete binding to

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adhesion sites to prevent the invasion of pathogens,19 repair of oxidative damage in

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H2O2-induced HT-29 cells, and inhibition of apoptosis in pathogen-invaded cells.20,

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Whether Slp can activate signaling pathways to exert an anti-inflammatory effect is unclear.

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In this research, we aimed at establishing a LPS-induced RAW264.7 cell model in vitro to

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examine the inflammatory response and investigating the molecular mechanism of Slp on

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macrophages to provide useful information on the use of active proteins from probiotics.

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MATERIALS AND METHODS

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Chemicals and Reagents

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide

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(DMSO), 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), and LPS from Escherichia

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coli 0111:B4 were purchased from Sigma Co. (St. Louis, MO). Mouse TNF-α enzyme-linked

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immunosorbent (ELISA) kits (PT512) total NO-detection kit (S0024), and mouse interleukin

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(IL)-1β ELISA kits (PI301) were provided by the Beyotime Biotechnology (Shanghai, China).

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Detection kits for prostaglandin E2 (PGE2) was purchased from Jiancheng Bioengineering

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Institute (Nanjing, China). Antibodies in this research, including iNOS, COX-2, JNK,


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phosphor-JNK (p-JNK), ERK, p38, phosphor-ERK (p-ERK), phosphor-p38 (p-p38), IκBα,

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phosphor-IκBα (p-IκBα), HRP conjugated anti-rabbit or anti-goat IgG, GAPDH were

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obtained from Cell Signaling Technology (Beverly, MA). β-Actin was purchased from Santa

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Cruz Biotechnology Inc. (Santa Cruz, CA). Other chemicals in this research were of

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analytical grade and obtained from China National Pharmaceutical Group Corporation

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(Beijing, China).

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Bacterial Strain Culture, Extraction, and Purification of Slp

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L. acidophilus NCFM was offered by Dansico (DuPont, USA) and cultivated in

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deMan-Rogosa-Sharpe medium at 37 °C until the logarithmic phase. Slp was extracted and

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purified according to the previous description of Meng et al.19 In brief, the isolation of Slp

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from L. acidophilus NCFM was fulfilled using LiCl (5 mol L-1) and purified by fast protein

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liquid chromatography (AKTA Protein Purification System, GE Healthcare Life Science,

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Uppsala, Sweden) on a HiPrep SP Sepharose FF column (GE Healthcare Life Science). The

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crude Slp was eluted with 8 mol L-1 Urea-Tris-HCl (pH = 7.0) and stepwise NaCl aqueous

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solutions (pH = 7.0) from 0.2 mol L-1 to 1.0 mol L-1 with the 1.0 ml min-1 flow rate. The

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purified Slp was obtained after lyophilization (Labconco Corp., Kansas City, MO).

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Bicinchoninic acid (BCA) assay was employed for the protein content measurement, and the

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purity was confirmed by SDS-PAGE using 12% (w/v) acrylamide gel.22

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Cell Culture Condition and Viability Assay

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RAW264.7 macrophages were obtained from the American Type Collection, and maintained

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in high-glucose DMEM medium contained 10% fetal bovine serum (FBS) at 37 °C in a 5%

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CO2 humidified incubator. The cell viability was measured by MTT assay. Briefly, 2 × 104



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RAW264.7 cells were planted into 96-well plates, cultured overnight. After pretreatment with

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varying doses of Slp (1, 5, 10 µg mL-1) for 4 h, cells were exposed to LPS (1 µg mL-1) for 20

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h. 20 µL MTT solution (0.5 mg mL-1 dissolved in PBS) incubated cells for 4 h without light.

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DMSO was then added for the crystals dissolution, absorbances were detected at 490 nm

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through a microplate reader (Bio-Rad, Hercules, CA). The cell morphology was observed

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through an inverted light microscope (Nikon, Tokyo, Japan).

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Intracellular ROS Detection

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The intracellular ROS formation was analyzed with DCFH-DA following the procedures

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described by Joung et al. 23 Briefly, 1 × 105 RAW264.7 cells were planted in 6-well plates

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and cultured overnight. Cells were pretreated with varying concentrations (1, 5, 10 µg mL-1)

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of Slp for 4 h and then LPS (1 µg mL-1) stimulated them for 20 h. DCFH-DA (10 µM

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dissolved in serum-free media) was loaded for 30 min at 37 °C in the dark. After incubation,

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cells were harvested and ROS formation was carried out by flow cytometry (FACSCalibur,

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Becton Dickinson, USA), and the DCFH-DA fluorescence morphology was examined under

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an inverted fluorescence microscope (Olympus, Japan).

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Determination of NO Production

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NO released from RAW264.7 cells during stimulation was assessed using a total

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NO-detection kit. 1 × 105 cells were seeded in 6-well plates and cultured overnight. After 4

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h of pretreatment with various doses of Slp (1, 5, 10 µg mL-1), cells were incubated with LPS

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(1 µg mL-1) for 20 h. NO production was measured following the processing instructions, the

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absorbances were measured at 540 nm using a microplate reader.

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TNF-α, PGE2, and IL-1β Production Detection ACS Paragon Plus Environment

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The inhibitory effects of Slp on the production of TNF-α, PGE2, and IL-1β were determined

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using ELISA kits, according to the operating instructions.

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Western Blotting

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2 × 104 cells were planted in a 6-well plates, cultured overnight, pretreated with various

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concentrations (1, 5, 10 µg mL-1) of Slp for 4 h and subsequently stimulated by LPS (1 µg

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mL-1) for 20 h. After harvesting by iced-PBS, cells were lysed with RIPA lysis buffer

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involving protease inhibitors. The lysate protein concentration was measured by Bradford

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assay. Then an equivalent protein was loaded, resolved on 15 or 10% SDS-PAGE, and

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transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk

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buffer (dissolved in TBST) for 1 h, incubated with the appropriate primary antibodies at 4 °C

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overnight and then matching second antibodies for 1 h. A chemiluminescence kit (Thermo

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Fisher Scientific, 32109) was used for visualizing the protein bands.

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Immunofluorescence Staining

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The NF-κB nuclear translocation was detected by following the procedures of a NF-κB

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activation-translocation kit (Beyotime Biotechnology, SN368). 1 × 103 RAW 264.7 cells

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were planted in sterile coverslips placed in 6-well plates, cultured overnight at 37 °C, and

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pretreated with various concentrations (1, 5, 10 µg mL-1) of Slp for 2 h before stimulation by

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LPS (1 µg mL-1) for 1 h. Cells were fixed for 10 min with fixation buffer and blocked for 1 h

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with a specific buffer. Then cells were incubated with the primary NF-κB antibody at 4 °C

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overnight, subsequently, incubated with Cy3 conjugated secondary antibody for 1 h at room

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temperature, and stained with 4,6-diamidino-2 phenylindole (DAPI) for 5 min. Finally, the

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coverslips were removed and observed under a fluorescence microscope (Olympus).



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Statistical Analysis

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All results were expressed as the means ± SD. One-way analysis of variance followed by

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Tukey’s multiple tests was applied for assessing the differences. P values of less than 0.05

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were considered to indicate statistical significance.

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RESULTS

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Extraction and Purification of Slp

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The crude Slp from L. acidophilus NCFM was extracted using LiCl (5 mol L-1). The

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SDS-PAGE results showed that two dominant bands and a few faint bands were visible

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(Figure S1A in the Supporting Information, Lane 1). After purification through a HiPrep SP

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Sepharose Fast Flow column, one independent peak appeared (Figure S1B), and a unique

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protein band with an apparent size of 46 kDa was observed (Figure S1A, Lane 2), thus

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confirming the purification of the Slp.

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Cell Cytotoxicity

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To evaluate the cytotoxic effect of Slp, cell viability was analyzed using MTT assay. As

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shown in Figure 1A, compared with the control group, the cell viability was not significantly

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changed (P ≥ 0.05) from 1 to 10 µg mL-1 of Slp, so the concentrations of Slp (1, 5, 10 µg mL-1)

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were selected for follow-up experiments. To determine the LPS stimulation time, the cells

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were processed by LPS (1 µg mL-1) alone for varying times (0 h, 4 h, 12 h, 16 h, 20 h). After

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observing the morphological changes, cells processed at 20 h showed large numbers of cells

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died, the cell became quite irregular, and there was almost no connection between cells (result

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not shown). So stimulation time for 20 h was determined for further research. Cells that were

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pre-incubated by Slp (1, 5, 10 µg mL-1) for 4 h and then exposed to LPS (1 µg mL-1) for 20 h ACS Paragon Plus Environment

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showed more than 50% (P < 0.05) greater cell viability than the LPS group (Figure 1B).

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Figure 1C illustrated that the cell morphology in the LPS group had markedly changed from

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circular to stretched (see the black arrows), whereas the morphology with Slp pre-incubation

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was gradually restored.

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Slp Inhibit LPS-Stimulated ROS Production

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As shown in Figure 2A, the DCFH-DA fluorescence intensity was visibly increased after LPS

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stimulation alone for 20 h. When pretreated with 1 to 10 µg mL-1 Slp for 4 h, the cell

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fluorescence was reduced relative to the LPS group. Flow cytometry further quantified the

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fluorescence intensity and changes in intracellular ROS. As shown in Figures 2B and 2C, the

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proportion of fluorescence cells markedly increased from 2.71 ± 2.10% to 33.71 ± 2.61%, and

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indicating that the intracellular ROS content significantly increased (P < 0.001). After

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pre-incubation of cells with various concentrations of Slp for 4 h, the intracellular ROS

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content significantly decreased relative to the LPS group. The proportion of fluorescence cells

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decreased to 19.53 ± 2.84% at 10 µg mL-1 of Slp pretreated cells, which was observably lower

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than the LPS group (P < 0.01).

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Slp Inhibit LPS-Stimulated NO and PGE2 Production, COX-2 and iNOS Proteins

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Expression

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As shown in Figures 3A and 3B, LPS alone markedly increased NO and PGE2 production

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relative to the control group (P < 0.001). When 5 to 10 µg mL-1 Slp pretreated cells, the NO

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and PGE2 production was significantly inhibited relative to the LPS group (P < 0.001). To

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validate whether the effects of Slp involved modulation of the COX-2 and iNOS proteins,

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which are correlated with NO and PGE2 production, Western blot analysis was used to



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examine their expression. As depicted in Figures 3C and 3D, after LPS stimulation, the

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expression levels of COX-2 and iNOS proteins were markedly increased (P < 0.05) relative to

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the control group, whereas pretreatment with 1 to 10 µg mL-1 Slp gradually decreased their

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expression, especially Slp was 10 µg mL-1, iNOS protein expression was significantly

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down-regulated (P < 0.05). These results suggested that Slp dose-dependently inhibited NO

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and PGE2 production by down-regulating the COX-2 and iNOS proteins expression.

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Slp Inhibit LPS-Stimulated TNF-α and IL-1β Formation

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In this study, we select TNF-α and IL-1β as the representatives to evaluate the effects of Slp

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on pro-inflammatory cytokines in LPS-stimulated RAW264.7 cells. As depicted in Figures 4A

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and 4B, the formation of TNF-α and IL-1β were relatively low in the control group but was

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significantly increased by LPS stimulation alone (P < 0.001). When cells were pretreated with

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5 to 10 µg mL-1 Slp, the levels of TNF-α and IL-1β were markedly reduced (P < 0.05).

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Specifically, the inhibitory activities of 10 µg mL-1 of Slp on TNF-α and IL-1β were 21.93%

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and 76.21%, respectively. These values demonstrated that the Slp could dose-dependently

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inhibit the excretion of pro-inflammatory cytokines in LPS-stimulated RAW264.7 cells.

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Slp Inhibits LPS-induced Inflammatory Effects through MAPK and NF-κB Pathways in

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RAW264.7 Cells

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MAPK plays a crucial role in the regulation of cell growth, the differentiation and control of

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cellular responses to foreign and environmental stressors. As shown in Figure 5A, LPS

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induced phosphorylation of JNK, ERK, and p38 (P < 0.05) compared with the control group.

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Intriguingly, the pretreatment with Slp for 4 h mainly inhibited the phosphorylation of JNK

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and ERK, whereas the expression level of p-p38 was not dramatically reduced in our research.


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Cells pretreated with 10 µg mL-1 of Slp showed a highly significant (P < 0.01) decrease in

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p-JNK protein expression and a significant (P < 0.05) decrease in p-ERK protein expression.

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The regulatory effects of Slp on the NF-κB signaling pathway were also explored by

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Western blot analysis (Figure 5B). The LPS (1 µg mL-1) group showed IκBα phosphorylation

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together with IκBα degradation. Figure 5B also displayed that the phosphorylation of IκBα

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was inhibited by pretreatment with Slp, especially with Slp presence at 10 µg mL-1 (P < 0.01),

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and the degradation of IκBα was consequently obstructed. All above results indicated that

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pretreatment with Slp could inhibit LPS-induced inflammatory actions in RAW264.7 cells,

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partly as a result of the inhibition of the MAPK and NF-κB signaling pathways.

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Slp Inhibit LPS-Stimulated the NF-κB p65 Nuclear Translocation

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The inhibition effect of Slp on the NF-κB p65 activation was investigated with a fluorescence

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microscope. Figure 6 presented the immunofluorescence staining assay result. Original

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RAW264.7 cells of NF-κB p65 (red) were distributed in the cytoplasm, whereas NF-κB p65

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mostly appeared in the nucleus after LPS stimulation for 1 h, which was reversed by Slp

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pretreatment at concentrations of 1 to 10 µg mL-1. Together with the above analysis in Figure

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5B, these results illustrated that Slp could inhibit NF-κB p65 translocation into nucleus to

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activate inflammatory gene transcription by blocking IκBα phosphorylation.

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DISCUSSION

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Macrophages are essential for repairing intestinal inflammation, promoting remission of

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inflammation, and protecting the stability of the intestinal mucosal system.24 Normally,

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macrophages in the intestine cannot mediate acute inflammatory reactions in time. When

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exogenous substances (e.g., LPS) stimulate, cells show extracellular molecular degradation by



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releasing cytotoxic and inflammatory cytokines, for instance, ROS, PGE2 and NO, and

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secreting pro-inflammatory cytokines (TNF-α and IL-1β) into the intestinal mucosa.25 After

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stimulation by LPS, the MAPK and NF-κB signaling pathways are activated through

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intracellular cascade to produce inflammatory cytokines and regulate the macrophages

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inflammatory response.26 Slp extracted from L. acidophilus NCFM using LiCl is a type of

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crude protein. After purification by fast protein liquid chromatography, purified Slp becomes

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an exact protein with an apparent size of 46 kDa. Slp exhibits various biological functions,

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which is confirmed by several studies. To explore the precise inflammatory regulatory

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mechanism of Slp, the protein expressions of the MAPK and NF-κB signaling pathways were

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investigated in LPS-induced murine macrophages.

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NO and PGE2 are the important signal transduction molecule of inflammatory

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responses.27 Oh et al.28 reported that excessive NO could cause tissue damage, whereas a

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smaller amount of NO could promote wound healing. iNOS and COX-2, as essential enzymes

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in the inflammatory response, have a regulatory effect on the production of NO and PGE2 .29

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In this study, it was observed that Slp reduced NO and PGE2 secretion (Figures 3A and 3B) to

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inhibit the inflammatory response through the iNOS and COX-2 down-regulation expression.

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(Figures 3C and 3D).

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ROS play a key role in immune regulation. Excessive ROS are produced in macrophages

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cell, which results in immune response.30 TNF-α and IL-1β, considerable pro-inflammatory

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cytokines, mediate inflammatory responses produced by macrophages infected with bacteria

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and endotoxin.31 In this current study, we found that 5 to 10 µg mL-1 of Slp significantly

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inhibited ROS production (Figures 2B and 2C) and the secretion of TNF-α and IL-1β (Figures

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4A and 4B). A previous study showed that SlpA isolated from L. helvetius MIMLh5 exerted


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anti-inflammatory effects by reducing the expression of TNF-α and the activation of NF-κB in

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LPS-stimulated human macrophage U937 cells,13 which is consistent with those results of our

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study.

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MAPKs proteins (including ERK, JNK, and p38 protein kinases) are activated to act on

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their respective substrates to regulate the inflammatory response in LPS-stimulated

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macrophage cells.32 Laura et al.33 demonstrated that inhibition of the p38 pathway effectively

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reduced the iNOS and COX-2 protein expression, thus further inhibiting the inflammatory

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response. Therefore, the MAPK signaling pathway is crucial in the expression of

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inflammatory factors. In our study, 10 µg mL-1 Slp could exert its anti-inflammatory actions

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through the iNOS and COX-2 proteins down-regulation expression (Figures 3C and 5A) and

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inhibiting the phosphorylation of JNK and ERK proteins in the MAPK signaling pathway in

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LPS-induced macrophage cells (Figure 5A). This suggested that Slp could suppress the

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inflammatory response, which is related to the inhibition of the MAPK signaling pathway.

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Studies have indicated that activation of the MAPK signaling pathway by LPS could

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indirectly activate the downstream NF-κB pathway, initiate protein expression, and stimulate

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complex physiological responses.34

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NF-κB, as an important downstream pathway in LPS-mediated signal response, is

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closely related to tumor growth, inflammation, and apoptosis.35 When macrophage cells are

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stimulated, the inactive IκBα/p65 complex connected to IκBα in the cytoplasm is

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ubiquitinated and degraded, and active p65 is released into the nucleus to bind to

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pro-inflammatory genes, which results in inflammatory responses.36,37 Therefore, the IκBα

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protein expression and the p65 transcriptional active form are important markers of

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inflammatory reaction. In this current study, we found that 10 µg mL-1 of Slp exerted



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anti-inflammatory and protective effects by down-regulating the expression of p-IκBα protein,

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impeding the phosphorylation of IκBα (Figure 5B), and inhibiting the p65 nuclear

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translocation (Figure 6) in an LPS-stimulated inflammatory reaction.

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Taken together, the results demonstrate that Slp can inhibit the LPS-induced

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inflammatory response by suppressing the expression of proteins in MAPK and NF-κB

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signaling pathways to exert anti-inflammatory effects. Numerous studies have confirmed that

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the inflammatory response of LPS-stimulated cells is accomplished via a series of

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intracellular and extracellular protein interactions, including toll-like receptor 4 (TLR4) and

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other LPS-binding proteins.26, 32, 38 The TLR4 receptor is expressed on the surface of cells in

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the TLR family and recognizes lipids, proteins, and other outer membrane components.39, 40

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We can thus speculate that the anti-inflammatory effects of Slp may be connected with the

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TLR4 on the macrophage cell membrane. Specifically, Slp might recognize the TLR4 initially

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and then exert its anti-inflammatory effects via modulation of inflammatory mediators. The

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modulatory actions are ascribed to the inhibition of inflammatory cytokines production and

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relational protein expressions by blocking of NF-κB and MAPK signaling pathways.

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Increasing literatures have ascertained that the immunomodulatory capacity of proteins

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extracted from agricultural products relies on the hydrophobicity amino acid content and

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composition.41,

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asparagine, aspartic acid, glutamine, and tyrosine promote the immunomodulatory activities

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of food protein.42, 43 A previous study by our group, Meng 18 confirmed that the hydrophobic

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amino acid contents in Slps from L. acidophilus fb116 and L. acidophilus fb214 was 35.77%

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and 34.00%, respectively. Together with the current findings, it can be deduced that

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hydrophobic amino acids contribute to the anti-inflammatory actions of Slp.

42

Some researchers have reported that hydrophobic amino acids and


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In summary, we successfully evaluated the anti-inflammatory activities of Slp from L.

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acidophilus NCFM in LPS-induced murine macrophage cells. Slp was found to exhibit

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effective inhibition of ROS, NO, PGE2, and inflammatory cytokines production through

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attenuating the MAPK and NF-κB activation. Moreover, TLR4 may be related to the

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anti-inflammatory action of Slp in activated macrophages, and further study is required to

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determine whether TLR4 is involved in the signaling pathway. Overall, these consequences

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provide more valuable information on the molecular mechanisms by which Slp inhibits

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murine macrophage cells activation. Slp may thus have potential applications as a food

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additive or a new oral adjuvant for immunodeficiency diseases.

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ABBREVIATIONS USED

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Slp, surface layer protein;

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SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel;

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LPS, lipopolysaccharide;

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DMSO, dimethyl sulphoxide;

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DCFH-DA, 2,7-Dichlorodihydrofluorescein diacetate;

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DMEM, dulbecco’s modified eagle’s medium;

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ELISA, enzyme-linked immunosorbent;

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ROS, reactive oxygen species;

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TNF-α, tumor necrosis factor-α;

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IL-1β, interleukin-1β;

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NO, nitric oxide;

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PGE2, prostaglandin E2 ACS Paragon Plus Environment


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PBS, phosphate-buffered saline;

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COX-2, cyclooxygenase-2;

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MAPK, mitogen-activated protein kinase;

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ERK, extracellular signal-regulated kinase;

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JNK, jun-amino-terminal kinase;

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NF-κB, nuclear factor-kappa B;

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IκBα, inhibitory factor kappa B alpha;

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iNOS, nitric oxide synthase;

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FUNDING

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This study was supported by a grant from the National Natural Science Foundation of China

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(No. 31471696).

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Notes

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Authors declare they have no conflicts of interest.

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Supporting Information

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Slp were analyzed by SDS-PAGE and cation exchange chronmatography (Figure S1).

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This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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(1) Ng, E. W.; Yeung, M.; Tong, P. S. Effects of yogurt starter cultures on the survival of

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Figure captions

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Figure 1. Cell viability, and morphology analysis of Slp in LPS-stimulated RAW264.7

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cells. (A) Cell cytotoxicity. Cells were treated with the different concentration of Slp for 24 h,

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cell cytotoxicity was analyzed by MTT assay. (B) Effects of Slp on the cell viability in

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LPS-induced RAW264.7 cells. Cells were pretreated with varying concentration of Slp for 4 h,

479

and then stimulated by LPS (1 µg mL-1) for 20 h. (C) Effects of Slp on cellular morphology

480

changes in LPS-stimulated RAW264.7 cells. Scale bar = 2 µm. Values are expressed as the

481

mean ± SD (n = 3). #P < 0.05, # # P < 0.01, # # # P < 0.001: vs. control group (A, B); *P < 0.05,

482

** P < 0.01, *** P < 0.001: vs. LPS group (B).

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Figure 2. Slp inhibit LPS-stimulated ROS production in RAW 264.7 cells. (A)

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Fluorescence microscopy images of cells represented for ROS. Scale bar = 10 µm. (B, C) The

485

qualitative and quantitative analysis of intracellular ROS formation. Cells were pretreated for

486

4 h with various concentration of Slp, then exposed to LPS (1 µg mL-1) for 20 h. Values are

487

expressed as the mean ± SD (n = 3). #P < 0.05,

488

*P < 0.05, ** P < 0.01, *** P < 0.001: vs. LPS group.

489

Figure 3. Slp inhibit LPS-stimulated NO and PGE2, COX-2 and iNOS proteins

490

expression in RAW264.7 cells. (A) NO production. (B) PGE2 production. (C, D) Slp inhibit

491

LPS-induced COX-2 and iNOS proteins expressions. Cells were pretreated with various

492

concentration of Slp for 4 h before stimulation of LPS (1 µg mL-1) for 20 h. The protein

493

expression was analyzed by Western blot. GAPDH served as protein control. Values are

494

expressed as the mean ± SD (n = 3). #P < 0.05,

495

*P < 0.05, ** P < 0.01, *** P < 0.001: vs. LPS group.

496

Figure 4. Slp inhibit LPS-stimulated TNF-α and IL-1β formation in RAW264.7 cells. (A)

##

##

P < 0.01, # # # P < 0.001: vs. control group;

P < 0.01, # # # P < 0.001: vs. control group;



497

TNF-α production. (B) IL-1β production. Cells were pretreated with varying concentration of

498

Slp for 4 h before stimulation of LPS (1 µg mL-1) for 20 h. The concentration of TNF-αand

499

IL-1β in the culture medium was determined using ELISA. Values are expressed as mean ±

500

SD (n = 3). #P < 0.05,

501

*** P < 0.001: vs. LPS group.

502

Figure 5. Slp inhibit LPS-induced inflammatory effects through MAPK and NF-κB

503

pathways in RAW264.7 cells. Cells were pretreated for 4 h with various concentration of Slp

504

before stimulation of LPS (1 µg mL-1) for 20 h. The protein expression was analyzed by

505

Western blot. β-actin served as protein control. Values are presented as the mean ± SD (n = 3).

506

#

507

vs. LPS group.

508

Figure 6. Slp inhibit LPS-stimulated the NF-κB p65 nuclear translocation in RAW264.7

509

cells. Nuclear translocation of NF-κB p65 was assessed under a fluorescence microscope,

510

scale bar = 10 µm. RAW264.7 cells were pretreated with various concentration of Slp for 2 h

511

before stimulation of LPS (1 µg mL-1) for 1 h. Cells were incubated with NF-κB p65 primary

512

antibody and Cy3-conjugated secondary antibody, respectively. The red area (representative

513

of the contained p65 area) and blue area (representative of the DAPI conjugated nucle part)

514

images were overlaid to create a purple fluorescence in areas of co-localization.

##

P < 0.01, # # # P < 0.001: vs. control group; *P < 0.05, ** P < 0.01,

P < 0.05, # # P < 0.01, # # # P < 0.001: vs. control group; *P < 0.05, ** P < 0.01, *** P < 0.001:

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Figure 3.

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Figure 5.

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TOC Graphic

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