A polysaccharide isolated from the fruits of Physalis alkekengi L. induces RAW264.7 macrophages activation via TLR2 and TLR4-mediated MAPK and NF-κB signaling pathways

Journal Pre-proof A polysaccharide isolated from the fruits of Physalis alkekengi L. induces RAW264.7 macrophages activation via TLR2 and TLR4-mediated MAPK and NF-κB signaling pathways

Fan Yang, Xiaozhou Li, Ye Yang, Selina Mawunyo Ayivi-Tosuh, Feihe Wang, Hong Li, Guiyun Wang PII:

S0141-8130(19)33051-X

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.08.174

Reference:

BIOMAC 13151

To appear in:

International Journal of Biological Macromolecules

Received date:

29 April 2019

Revised date:

15 August 2019

Accepted date:

20 August 2019

Please cite this article as: F. Yang, X. Li, Y. Yang, et al., A polysaccharide isolated from the fruits of Physalis alkekengi L. induces RAW264.7 macrophages activation via TLR2 and TLR4-mediated MAPK and NF-κB signaling pathways, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.08.174

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© 2019 Published by Elsevier.

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A polysaccharide isolated from the fruits of Physalis alkekengi L. induces RAW264.7 macrophages activation via TLR2 and TLR4-mediated MAPK and NF-κB signaling pathways

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Fan Yang, Xiaozhou Li, Ye Yang, Selina Mawunyo Ayivi-Tosuh, Feihe Wang, Hong Li, Guiyun Wang*

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School of Life Science, Northeast Normal University, 5268 Renmin Street,

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Changchun, Jilin Province, 130024, People’s Republic of China

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*Corresponding author. Tel: + 86-431-85099590 E-mail address: [email protected]

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Journal Pre-proof Abstract

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Polysaccharides from Physalis alkekengi L. have been proven to possess many biological activities. In our previous study, a homogeneous polysaccharide (PPSB) was extracted and purified from the fruits of Physalis alkekengi L., and the structure characterization was analyzed. The present study aimed to investigate the effects of PPSB on RAW264.7 macrophage cells activation and the underlying molecular mechanism. PPSB could activate RAW264.7 cells by not only enhancing the pinocytic and phagocytic activity, but also promoting the production of NO, ROS, TNF-α, and IL-6 in RAW264.7 cells. Meanwhile, PPSB up-regulated the expression of major histocompatibility complex (MHC-I/II) and costimulatory molecules such as CD40, CD80 and CD86. Mechanism studies showed that PPSB induced the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) pathways. Moreover, the production of NO, TNF-α and IL-6 induced by PPSB in RAW264.7 cells were suppressed by specific MAPKs and NF-κB inhibitors. Further experiments with blocking antibodies demonstrated that the releases of NO, TNF-α and IL-6 and the activation of MAPKs and NF-κB induced by PPSB were decreased after TLR2 and TLR4 were blocked. Our date illustrated that PPSB was capable of activating the RAW264.7 cells via MAPKs and NF-κB signaling mediated by TLR2 and TLR4.

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Key words: Physalis alkekengi L.; Polysaccharide; RAW264.7 macrophages

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Journal Pre-proof 1. Introduction

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Polysaccharides are a kind of bioactive macromolecules which are widely distributed in animals, plants and microorganisms. In recent years, numerous studies have shown that polysaccharides isolated from plants exhibit a wide range of biological activities such as immunological regulation, anti-diabetics, anti-hypertension and anti-oxidative function [1]. Among these, more and more attentions are paid to the immunity-modifying property of plant polysaccharides [2-4], such as immune cells activation, complement system activation, cytokines production and antibodies promotion [5]. During the past decades, many studies indicated that polysaccharides are promising candidates for macrophages activation [1]. Macrophages are important innate immune cells and almost distributed in all major organs of the body. They are the first line of defense against invading pathogens and tumor cells [6]. Macrophages can not only engulf and kill microbes, but also recruit other immune cells to the infection site and promote inflammatory response through releasing cytokines, chemokines and inflammatory factors [7]. Moreover, macrophages also initiate the adaptive immune response by antigens presenting and cytokines secretion [8]. So, macrophages have been considered as the key target cells of some immunomodulatory The pathogen-associated molecular patterns (PAMP), which are commonly conserved in microorganisms but not present in the host itself, can be recognized by immune system through a small number of germline-encoded pattern recognition receptors (PRRs) [9]. There are multiple PRRs including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), and complement receptor type 3 (CR3) on macrophages [10, 11]. The combination of PRRs with microbial components triggers the activation of intracellular signaling cascades, such as mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB, leading to the transcription of genes which involved in inflammatory responses [12-14]. In this progress, macrophages are activated, characterized by the enhancement of phagocytosis, the promotion of cytotoxic molecules production such as nitric oxide (NO) and reactive oxygen species (ROS), as well as the secretion of various cytokines (IL-6, TNF-α, IL-12 and so on). In addition, the expression of surface molecules like major histocompatibility complex (MHC) and costimulatory molecules are up-regulated [15]. Physalis alkekengi L.(P. alkekengi) is a kind of medicinal plants widely distributed in Changbai Mountain of Jilin Province, China. It has been used as herbal medicine with a long history. P. alkekengi. polysaccharide, the main compound extracted from P. alkekengi., has been demonstrated to have immunological enhancement, hypoglycemic, anti-inflammatory and antioxidant activities. In our previous work, a novel water-soluble homogeneous polysaccharide (PPSB), which molecular weight was 27kDa, was extracted and purified from the fruits of P. alkekengi. The structure characterization of PPSB was analyzed by GC-MS, partial acid hydrolysis, periodate oxidation and Smith degradation. It was revealed that PPSB was composed of Ara, Gal, Glc, GalA with a molar ratio of 2.6:3.6:2:1. The backbone of PPSB was mainly composed of (1→5)-linked Ara and (1→6)-linked Gal. Meanwhile, it was also found 3

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2.1. Materials and reagents

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2. Materials and methods

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that there were three side chains, which were terminated with Gal or Glc, attached to O-3 of (1→6)-linked Gal, and GalA was primarily distributed in side chains. [16]. On basis of determining the structure characterization, we further discovered that PPSB, as an adjuvant, could enhance the specific antibodies titers (IgG, IgG1, and IgG2b) and the production of IL-2 and IL-4 in sera of mice immunized with DNA vaccine (pD-HSP90C) against systemic candidiasis. Importantly, PPSB improved the survival rate as well as decreased the colony forming unit in kidneys of mice which were challenged with living Candida albicans cells after immunization with pD-HSP90C containing PPSB [17]. However, it is not clear about how PPSB effects on the macrophages which play vital important role in innate immune and adaptive immune response. In this study, we investigated the effects of PPSB on the activation of RAW264.7 macrophage cells, including cell proliferation, pinocytosis and phagocytosis activities, NO, ROS and cytokine production as well as the expressions of MHC and costimulatory molecules. The more importantly, we focused on the underlying signaling pathways and the potential receptors involved in the activation of RAW264.7 cell mediated by PPSB.

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Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Anti-mouse CD282 (TLR2) mAb, anti-mouse CD284 (TLR4) mAb, anti-mouse CR3, Fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD40, CD80, CD86 and MHC-I and MHC-II mAb were purchased from eBioscience (San Diego, CA, USA). Rabbit monoclonal antibodies for Erk1/2, phospho-Erk1/2, p38, phospho-p38, SAPK/JNK, phosphor-SAPK/JNK, NF-κB p65, phosphor-NF-κB p65, phospho-IκBα, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit monoclonal antibodies for IκBα and anti-mannose receptor were purchased from Abcam (Cambridge, MA, USA). Rabbit monoclonal antibodies for Dectin-1 was purchased from R&D Systems, Inc (Minneapolis, MN, USA). NO assay kit, DAPI, MAPK and NF-κB inhibitor (SP600125, U0126, SB203580 and BAY11-7082) were purchased from Beyotime Biotechnology (Shanghai, China). Mouse TNF-α, IL-6 ELISA kits were purchased from UBIO Biotechnology (Beijing, China). Tissue or Cell Total Protein Extraction Kit was purchased from Sangon Biotech (Shanghai, China). BCA protein quantitation kit was purchased from BOSTER Biological Technology (Wuhan, China). ToxinSensor™ Chromogenic LAL Endotoxin assay kit was purchased from GenScript (New Jersey, USA). FITC-Dextran, 3-(4,5)-dimethyl-2-thiazolyl-2,5-diphenyltetrazolium bromide (MTT), Lipopolysaccharide (LPS; from Escherichia coli 055:B5), polymyxin B (PMB), neutral red, penicillin, streptomycin, dimethylsulfoxide (DMSO), 4

Journal Pre-proof 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and all other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Preparation of PPSB

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The methods of extraction and purification of PPSB were reported previously (Tong, Liang & Wang, 2008). In brief, crude polysaccharide was extracted from fresh fruits of Physalis alkekengi L. with distilled water at 100 ℃, and the supernatant was precipitated by ethanol precipitation. Afterwards, the crude polysaccharide was frozen-thawed repeatly and grading-alcoholic precipitation to obtain polysaccharide fraction (PPSA). The proteins in PPSA were removed by a combination of proteinase and Sevage method. After that, PPSA was purified by Sepharose CL-6B, and the main fraction (PPSB) was collected, dialyzing and freeze-drying. PPSB was filtered by 0.22 μm filters before the following experiments.

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2.3. Cell line and cell culture

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2.4. Cell proliferation assay

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RAW264.7 cells, a murine macrophages cell line, were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). Cells were cultured in DEME high glucose medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 ℃ in a humidified incubator with atmosphere of 5% CO2.

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A 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to investigate the proliferation of RAW264.7 cells. In brief, RAW264.7 cells were seeded in 96-well plates at a density of 1 × 105 cells/mL and treated with different concentrations of PPSB (40, 80, 160 μg/mL) or LPS (1 μg/mL) for 24 h. The medium was then removed and 100 μL/well of the MTT solution (1 mg/mL) was added. After 4 h incubation, the medium was discarded, and 100μL of DMSO was added to each well to solubilize the Formosan salt. Finally, the absorbance was recorded at 570 nm on the microplate reader. Cell proliferation was expressed as the percentage of the control which was set to 100%. 2.5. Determination of NO production RAW264.7 cells (2 × 105 cells/mL) were seeded in 12-well plates and incubated with different concentrations of PPSB (40, 80, 160 μg/mL) or LPS (1 μg/mL) for 24 h. After incubation, cell supernatants were collected for the measurement of NO using NO assay kit following the manufacturer’s instructions. Microplate reader was used to detect the OD value.

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Journal Pre-proof 2.6. Determination of TNF-α and IL-6 production RAW264.7 cells (2 × 105 cells/mL) were seeded in 12-well plates and incubated with different concentrations of PPSB (40, 80, 160 μg/mL) or LPS (1 μg/mL) for 24 h. In addition, RAW264.7 cells were treated with PPSB (160 μg/mL) or LPS (1 μg/mL) for 6, 12, 24 and 48 h for dynamic detection. After incubation, cell supernatants were collected for the measurement of TNF-α and IL-6 using TNF-α, IL-6 ELISA kits following the manufacturer’s instructions. Microplate reader was used to detect the OD value. 2.7. Determination of ROS generation

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Intracellular ROS level was determined by detecting the changes of fluorescence intensity resulting from the oxidation of the fluorescent probe DCFH-DA. In brief, RAW264.7 cells (2 × 105 cells/mL) were seeded in 12-well plates and incubated with different concentrations of PPSB (40, 80, 160 μg/mL) or LPS (1 μg/mL) for 24 h. Next, the cells were incubated with DCFH-DA (10 μM) for 30 min at 37 ℃ in dark. Later, cells were washed with PBS, and the mean fluorescence intensity (MFI) of RAW264.7 cells were measured by flow cytometry.

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2.8. Determination of endotoxin contamination in PPSB

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RAW264.7 cells (2 × 105 cells/mL) were seeded in 12-well plates and incubated with PPSB (160 μg/mL) or LPS (1 μg/mL) in the presence or absence of PMB (50 μg/mL) for 24 h. After treatments, cell supernatants were collected for the detection of NO production using NO assay kit following the manufacturer’s instructions. The content of endotoxin in PPSB was detected using Toxin sensor™ Chromogenic LAL Endotoxin Assay kit according to the manufacturer’s protocol. 2.9. Determination of pinocytosis and phagocytic activity in RAW264.7 The pinocytosis activity of RAW264.7 cells was measured by uptake of neutral red. Briefly, RAW264.7 cells (2 × 105 cells/mL) were seeded in 96-well plates and incubated with different concentrations of PPSB (40, 80, 160 μg/mL) or LPS (1 μg/mL) for 24h. Then, the medium was removed, and 100 μL of 0.1% neutral red was added into each well and incubated for 1h at 37 ℃. After being washed with PBS for three times, each of the wells was loaded with 100 μL of cell lysis solution (glacial acetic acid: ethanol = 1: 1). The cell culture plate was statically placed for 2h at room temperature. The absorbance at 540 nm was measured using a microplate reader. The phagocytic activity of RAW264.7 was measured by assaying the uptake of FITC-dextran using Flow cytometry and confocal laser-scanning microscopy respectively. For Flow cytometry analysis, RAW264.7 cells (2 × 105 cells/mL) were seeded in 12-well plates and incubated with different concentrations of PPSB (40, 80, 160 μg/mL) or LPS (1 μg/mL) for 24 h. After incubation, cells were collected and 6

Journal Pre-proof cultured with the medium containing FITC-dextran (1 mg/mL) for 1 h at 37 ℃. In the end, the cells were washed three times with PBS, and the MFI was measured by flow cytometry. For confocal laser-scanning microscopy observation, RAW264.7 cells (2 × 104 cells/mL) were spread on glass slides and exposed to different concentrations of PPSB (40, 80, 160 μg/mL) or LPS (1 μg/mL) for 24 h, then incubated with FITC-dextran (1 mg/mL) for 1h at 37 ℃. Next, cells were washed by PBS and fixed with 4% paraformaldehyde solution at room temperature for 15 mins. Nucleus was stained with DAPI for 10 mins at room temperature. Finally, the above cells were observed using a laser-scanning confocal microscope to determination the uptake of FITC-dextran.

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2.10. Determination of MHC and costimulatory molecule expression on RAW264.7 cells

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The detection of MHC and costimulatory molecule expression on RAW264.7 cells was performed by flow cytometry. The RAW264.7 cells were incubated at a density of 5 × 105 cells/mL with different concentrations (40, 80, 160 μg/mL) of PPSB, or 1μg/mL LPS at 37 ℃. After 24 h incubation, RAW264.7 cells were collected and washed with PBS containing 2% FBS, then incubated with anti-mouse FITC-CD40, FITC-CD80, FITC-CD86, FITC-MHC-I and FITC-MHC-II at 4 ℃ for 30 mins. After that, the cells were collected, washed and resuspended in cold PBS. The MFI was measured by flow cytometry.

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2.11. Determination of MAPKs and NF-κB pathway activation in RAW264.7 cells

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The determination of the activation of MAPKs and NF-κB pathway were performed by western blot. Firstly, for dynamic detection of the MAPKs and NF-κB pathway activation, RAW264.7 cells, at a density of 5 × 105 cells/mL, were incubated with 160 μg/mL of PPSB for different times (0.5, 1, 2, 4, 8, 12, 24 h) at 37 ℃. Next, in order to determine the effects of concentration of PPSB on MAPKs and NF-κB pathway activation, the RAW264.7 cells (5 × 105 cells/mL) were incubated with different concentrations (20, 40, 80, 160 μg/mL) of PPSB, or 1μg/mL LPS (as positive control) at 37 ℃ for 0.5 h. After various treatments mentioned above, the total proteins of cells were extracted using the protein extraction kit following the manufacturer’s instructions. The phosphorylation of MAPKs (p38, ERK1/2, JNK), NF-κB and IκB-α and the degradation of IκB-α were analyzed by western blot followed the method mentioned at 2.13. 2.12. Inhibition of MAPKs and NF-κB using specific inhibitors To investigate the roles of MAPKs and NF-κB signaling pathway in the activation of RAW264.7 cells induced by PPSB, RAW264.7 cells (2 × 105 cells/mL) were seeded in 12-well-plate and pretreated with p38 MAPK inhibitor SB203580 (25 μM), ERK inhibitor PD98059 (25 μM), JNK inhibitor SP600125 (25 μM), and NF-κB inhibitor 7

Journal Pre-proof BAY 11-7028 (10 μM) for 2 h at 37 ℃. After removing the cell supernatants, new medium alone or with PPSB (160 μg/mL) were added to the plates for 0.5 h or 24 h respectively to detect the signaling pathways activation and the production of NO and cytokines in RAW264.7 cells. In detail, cells were harvested and the total proteins were extracted by total protein extraction kit following the manufacturer’s instructions to detect the molecules (mentioned above) in the signaling pathways by western blot (shown at 2.14) after PPSB incubation for 0.5 h. The cell supernatants were obtained after 24 h incubation with PPSB to measure NO, TNF-α and IL-6 using NO assay kit and ELISA kit. 2.13. Determination of PPSB receptors on the surface of RAW264.7

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For identification of the role of cytomembrane receptors in RAW264.7 activation mediated by PPSB, an antibodies inhibition experiment was applied. RAW264.7 cells (2 × 105 cells/mL) were seeded in 12-well plates and pretreated with anti-TLR2 (10 μg/mL), anti-TLR4 (10 μg/mL), anti-MR (20 μg/mL), anti-CR3 (2.5 μg/mL), anti-Dectin-1 (5 μg/mL) for 2 h. Subsequently, the medium was renewed and RAW264.7 cells were incubated with PPSB (160 μg/mL) for 0.5 h or 24 h respectively to detect the signaling pathways activation and the production of NO and cytokines (TNF-α, IL-6) in RAW264.7 cells. The total protein of RAW264.7 cells incubated with PPSB for 0.5 h was extracted for western blot assay to detect the MAPKs and NF-κB pathways activation. The cell supernatants were obtained after 24 h incubation with PPSB to measure NO, TNF-α and IL-6 using NO assay kit and ELISA kit.

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2.14. Western blot analysis

After various treatments, the cells were collected, and the total proteins were extracted using Tissue or Cell Total Protein Extraction Kit. The protein concentration was measured using BCA protein quantitation kit. After boiling at 100 ℃ for 5 mins, 20 μg of proteins from different groups were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane at voltage of 100V for 1 h. Afterwards, PVDF membranes were blocked by TBST containing 5% skim milk for 1h at room temperature and then incubated with different primary antibodies (1: 1000) over night at 4 ℃. Next, the PVDF membranes were incubated with goat anti-rabbit IgG-HPR (1: 5000) for 2 h at room temperature. The primary antibodies include Rabbit monoclonal antibodies for Erk1/2, phospho-Erk1/2, p38, phospho-p38, SAPK/JNK, phosphor-SAPK/JNK, NF-κB, phosphor-NF-κB, IκB-α, phospho-IκB-α, and β-actin. The signal was visualized using the enhanced chemiluminescence (ECL) western blot detection reagent. 2.15. Statistical analysis SPSS 17.0 software was used for statistical analysis. Data were expressed as mean ± 8

Journal Pre-proof SD from at least three independent experiments. Student’s t-text was used in analyzing all data for examining their statistical significance. P value of less than 0.05 was considered statistically significant.

3. Results and discussions 3.1. PPSB can promote the activation of macrophage 3.1.1. PPSB increases the proliferation of RAW264.7 cells

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To investigate the effect of PPSB on RAW264.7 cell proliferation, cells were treated with different concentrations of PPSB or LPS for 24 h. After incubation, MTT assay was used to detect the proliferation of cells. As shown in Fig. 1A, PPSB (80 and 160 μg/mL) significantly increased the proliferation rates of RAW264.7 cells (p ＜ 0.05) when compared with control after 24 h treatment. LPS, served as a positive control, significantly promoted the cell proliferation too (p ＜ 0.01).

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3.1.2. PPSB induces NO and ROS production of RAW264.7cells

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NO and ROS are important pro-inflammatory mediators in immune system. Activated macrophages promptly excite the expression of genes responsible for the synthesis of ROS and NO which play important roles in the regulation of the inflammatory response. NO is involved in the killing of microorganisms and tumor cells [18, 19]. ROS acts both a signaling molecule and secondary messengers in inflammation [20]. Firstly, we determined the NO production of RAW264 cells following PPSB or LPS treatment for 24 h using NO assay kit. The results demonstrated that PPSB (40, 80, and 160 μg/mL) promoted NO production in a dose-dependent manner, compared with control (p ＜ 0.01). (shown in Fig. 1B). To detect whether there was endotoxin contamination in PPSB to effect on the activation of RAW264.7 cells, NO production was evaluated in RAW264.7 cells treated with PPSB (160 μg/mL) or LPS (1 μg/mL) alone or with PMB (50 μg/mL) for 24 h. It is well-known that PMB inhibits the biological activities of LPS by binding the lipid A moiety [21]. As shown in Fig. 1C, no apparently differences in the generation of NO in RAW264.7 cells was found between group PPSB alone and PPSB with PMB (p ＞ 0.05). However, LPS-induced NO production were significantly inhibited by the presence of PMB (p ＜ 0.01) indicating that the activation of RAW264.7 cells activation was evoked by PPSB, not duo to endotoxin contamination. In addition, the concentration of endotoxin in PPSB was tested by Toxin sensor™ Chromogenic LAL Endotoxin Assay kit, and the result showed that there was no detectable endotoxin (< 0.01 EU/mL) in PPSB (1 mg/mL). To evaluate the production of ROS, RAW264.7 cells were incubated with different concentrations of PPSB and LPS for 24 h. After treatment, the changes of fluorescence intensity in cells was detected by FACS analysis. Our data (Fig. 1D and 9

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Fig. 1E) showed that the MFI of RAW264.7 cells was increased after PPSB (40, 80, and 160 μg/mL) treatment when compared with control (p ＜ 0.05). These results indicated that PPSB promoted the production of ROS in RAW264.7 cells. As positive control, LPS (1 μg/mL) significantly stimulated NO and ROS production as expected (p ＜ 0.01). These results illuminated that PPSB promoted the production of NO and ROS in RAW264.7 cells.

Fig.1 3.1.3. PPSB promotes the production of TNF-α and IL-6 in RAW264.7cells Activated macrophages can meditate immune response through secreting the inflammatory cytokines such as TNF-α and IL-6. TNF-α has a proinflammatory effect through an increased production of a variety of inflammatory factors. Meanwhile, TNF-α can enhance the functional responses of macrophages and monocytes through autocrine manner [22]. IL-6 is a kind of pivotal cytokines which can induce adaptive immune response by promoting the differentiation of the T cells and B cells [23, 24]. In present work, the expression levels of TNF-α and IL-6 from RAW264.7 cells， 10

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which treated with different concentrations of PPSB or LPS for 24 h or treated with PPSB (160 μg/mL) for 6, 12, 24 and 48 h, were evaluated by ELISA. As expected, PPSB (40, 80, 160 μg/mL) significantly promoted TNF-α and IL-6 production in contrast with control (p ＜ 0.01) in a dose-dependent manner (Fig. 2A and Fig. 2C). The secretion of TNF-α and IL-6 was also substantially increased (p ＜ 0.05) following PPSB (160 μg/mL) stimulation from 6-48 h compared with control (Fig. 2B and Fig. 2D). Similarly, LPS (1 μg/mL) could provoke TNF-α and IL-6 production of RAW264.7 cells. It was obvious that PPSB could promote the production of TNF-α and IL-6 in RAW264.7 cells.

Fig.2

3.1.4. PPSB enhances the pinocytic and phagocytic activity of RAW264.7 cells As one of the most important professional phagocyte, macrophages play scavenger role in clearance of foreign matters and pathogens [25]. The pinocytic and phagocytic activity were increased after macrophages activation [26, 27]. The effect of PPSB on the pinocytic activity of RAW264.7 cells was determined by the uptake of neutral red. Meanwhile, the uptake of FITC-dextran was measured by FACS and laser scanning confocal to detect the effect of PPSB on the phagocytic activity in RAW264.7 cells. For pinocytic and phagocytic activities determination, RAW264.7 cells were incubated with different concentrations of PPSB or LPS for 24 h. After treatment, neutral red uptake assay was used to evaluate the pinocytic activity of RAW264.7 cells. Meanwhile, the phagocytic activity of RAW264.7 cells was measured by assaying the uptake of FITC-dextran with FACS analysis. As shown in Fig. 3A, PPSB (40, 80, and 160 μg/mL) significantly increased the absorbance at 540 nm in lysis solution of RAW264.7 cells when compared to control (p ＜ 0.05), which was in a 11

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dose-dependent manner. The results suggested that PPSB enhanced the pinocytic activity of RAW264.7 cells. FACS analysis (Fig. 3B and Fig. 3C) illuminated that the MFI in RAW 264.7 cells was obviously increased (p ＜ 0.01) after treatment of PPSB. Moreover, laser scanning confocal image (Fig. 3D) indicated clearly that the intensity of green fluorescence (FITC-dextran) in cells treated with PPSB were higher than the cells in control group. These results showed that the phagocytic activity of RAW264.7 cells was also enhanced by PPSB. As expected, LPS (1 μg/mL), as positive control, promoted the pinocytic and phagocytic activity of RAW264.7 cells (p ＜ 0.01). Therefore, it could be confirmed that PPSB increased the pinocytic and phagocytic activity in RAW264.7 cells.

Fig. 3

3.1.5. PPSB up-regulates the expression of MHC and costimulatory molecules on RAW264.7 cells Macrophage is one of important antigen-presenting cells which are responsible for antigen processing and presenting to antigen-specific T cells. Activated macrophages 12

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can augment the expression of MHC-I and MHC-II and costimulatory molecules such as CD40, CD80 and CD86, which promote continuous stimulatory interaction with T cells to induce the adaptive immunity [8, 28]. The expressions of CD40, CD80, CD86, MHC-I and MHC-II on RAW264.7 cells treated with different concentrations of PPSB or LPS were measured by flow cytometry. As shown in Fig. 4, when compared to control, PPSB (40, 80, and 160 μg/mL) and LPS (1 μg/mL) (as positive control) significantly increased the MFI of CD40, CD80, CD86, MHC-I and MHC-II in RAW264.7 cells (p ＜ 0.05). It was clear that PPSB could promoted the expression of MHC-I/II and costimulatory molecules (CD40, CD80 and CD86) on RAW264.7 cells.

Fig.4 3.2. PPSB activates RAW264.7 cells by MAPK and NF-κB signaling pathways MAPKs are protein Ser/Thr kinases which play roles in converting extracellular 13

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stimuli into extensive cellular responses. Conventional MAPKs mainly consist of the extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun amino (N)-terminal kinases (JNK), and p38 isoforms. MAPKs can be activated by many factors like inflammatory cytokines, environmental stresses, and numerous mediators of inflammations such as LPS. MAPKs play a fatal role in immune and inflammatory response such as promoting cell proliferation and regulating cytokine expression through modulating transcription factors. The functions regulated by the MAPKs are generally mediated by phosphorylation [12, 29, 30]. NF-κB regulates many physiological processes, including innate- and adaptive-immune response, cell death and inflammation. In resting cells, NF-κB is retained in the cytoplasm by specific inhibitor IκB-α. When stimulated by pro-inflammatory cytokines or PAMPs through different receptors such as tumor necrosis factor receptor (TNFR) and TLRs, IκB kinase (IKK) complex was activated, causing the phosphorylation and degradation of IκB-α. Afterwards, NF-κB is released and translocates to the nucleus, phosphorylated by nuclear kinases and binding to DNA, which lead to the transcriptions of proinflammatory related gene [31-33]. In order to gain insight into the mechanism responsible for the activation of macrophages induced by PPSB, we detected the effect of PPSB on MAPKs (p38, ERK1/2, JNK) and NF-κB activation, and determined the role of MAPKs and NF-κB in the activation of RAW264.7 cells evoked by PPSB using MAPKs and NF-κB specific inhibitors. 3.2.1. PPSB induces the activation of MAPKs and NF-κB pathways in RAW264.7 cells

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The effect of PPSB on the activation of MAPKs and NF-κB were detected by western blot. Firstly, we detected the dynamic activation of MAPKs and NF-κB induced by PPSB through incubating RAW264.7 cells with PPSB (160 μg/mL) for different times (0.5, 1, 2, 4, 8, 12, 24 h). Next, we investigated the effects of different concentrations of PPSB (20, 40, 80, 160 μg/mL) on the activation of MAPKs and NF-κB for 0.5 h stimulation. It was clear (shown in Fig. 5A) that the phosphorylation of ERK1/2, JNK, and p38 evoked by PPSB occurred at 0.5 h and sustained for 12 hours at least. It was also found (Fig. 5B) that PPSB (20, 40, 80, and 160 μg/mL) caused the phosphorylation of ERK1/2, JNK, and p38 in a dose-dependent manner for 0.5h stimulation. Accordingly, the phosphorylation and the degradation of IκB-α, as well as the phosphorylation of NF-κB occurred (Fig. 5C) when RAW264.7 cells were treated with PPSB for 0.5 h. Meanwhile, PPSB at different concentrations (20, 40, 80, and 160 μg/mL) induced the phosphorylation and the degradation of IκB-α and NF-κB phosphorylation in a dose-dependent manner after 0.5 h stimulation (Fig. 5D). As a positive control, LPS (1 μg/mL) enhanced the degradation of IκB-α and increased the phosphorylation of MAPKs and NF-κB as expected. The above data demonstrated that PPSB could activate MAPKs and NF-κB signaling pathways in RAW264.7 cells.

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3.2.2. The inhibition of MAPKs and NF-κB suppresses the activation of RAW264.7 induced by PPSB To further clarify whether the MAPKs and NF-κB signaling were involved in the activation of RAW264.7 cells mediated by PPSB, we further traced the effect of inhibitors of MAPKs (ERK, JNK, p38) and NF-κB on the phosphorylation of ERK1/2, JNK, p38 and degradation of IκB-α by western blot analysis when RAW264.7 cells were pretreated with inhibitor for 2 h before PPSB (160 μg/mL) stimulation; subsequently, NO, TNF-α as well as IL-6 from supernatants of RAW264.7 cells were detected to confirm the role of MAPKs and NF-κB signaling in this process. As expected, the application of inhibitors of MAPKs significantly reduced the phosphorylation of ERK1/2, JNK, p38 induced by PPSB in RAW264.7 cells in contrast with PPSB stimulation alone respectively (Fig. 6A). Noteworthy, as shown in Fig. 6B, inhibitors of p38, ERK1/2 and JNK, significantly suppressed the secretion of NO, TNF-α as well as IL-6 evoked by PPSB in RAW264.7 cells respectively, when they were compared with PPSB treatment alone (p ＜ 0.05). Similarly, data from inhibitor of NF-κB revealed that pretreatment of NF-κB inhibitor reduced the degradation of IκB-α induced by PPSB in RAW264.7 cells (Fig. 7A). Meanwhile, it was obviously shown that (Fig. 7B) the amount of NO, TNF-α and 15

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IL-6 production induced by PPSB was inhibited significantly by inhibitor of NF-κB when compared with only PPSB stimulation (p ＜ 0.01). Thus, it was further confirmed that MAPKs and NF-κB signaling pathways were involved in PPSB-induced activation of RAW264.7 cells.

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3.3. TLR2 and TLR4 were involved in the activation of RAW264.7 induced by PPSB

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Macrophages activation by polysaccharide is thought to be mediated primarily through the recognition of polysaccharide receptors. These receptors are also known as pattern recognition molecules which can recognize PAMPs [1]. Studies have showed that polysaccharides can be recognized by TLR2, TLR4, Complement receptor 3 (CR3), Mannose receptor (MR), and Dectin-1 on macrophages. For examples, Zhou et al found that TLR4 was involved in the productions of TNF-α and IL-6 in macrophages caused by Astragalus polysaccharides [34]. Hong’s work showed that the polysaccharides isolated from fruiting body of Cordyceps militaris mediated macrophages activation through dectin-1 and TLR2 [35]. In addition, Han et al reported that polysaccharides extracted from the radix of Platycodon grandiflorum could enhance the NO production of macrophages via CR3 [36]. Li et al demonstrated that Ganoderma atrum polysaccharides can elevate the expression of MR and mediate the IL-1β and TNF-α production through MR in macrophages [37]. To detect the receptors which interacted with PPSB on macrophages, RAW264.7 cells were pretreated with function-blocking antibodies of Dectin-1, MR, CR3, TLR2 and TLR4 for 2 h respectively, and then the cells were incubated with PPSB (160 μg/mL). The results showed that (Fig. 8) the production of NO, TNF-α and IL-6 from the treatment of anti-TLR2 antibody group were nearly 42.8%, 41.2% and 53.5% lower compared with PPSB treatment alone group respectively. Meanwhile, it was also demonstrated that the production of NO, TNF-α and IL-6 were remarkably reduced by 43.8%, 53.8% and 53.9% after treatment of anit-TLR4 antibody compared with only PPSB stimulation group. However, when we employed antibodies of Dectin-1, MR and CR3 to treat RAW264.7 cells, no decrease was observed in NO, 17

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TNF-α and IL-6 production compared with PPSB group (p ＞ 0.05). We further blocked TLR2 and TLR4 simultaneously with anti-TLR2 and anti-TLR4 before PPSB incubation. The data from Fig. 9A showed that the levels of NO, TNF-α and IL-6 in the groups treated with anti-TLR2 and anti-TLR4 simultaneously were lower than the levels of the groups treated with only anti-TLR2 or anti-TLR4 (p ＜ 0.05). However, the production of NO, TNF-α and IL-6 was still higher than the levels of control group (p ＜ 0.05). The above results showed TLR2 and TLR4 could be the receptors of PPSB, but they may not the only receptors.

Fig.8

To further detect the role of TLR2 and TLR4 in RAW264.7 cell activation caused by PPSB, we focused on the effect of PPSB on MAPKs and NF-κB activation when RAW264.7 cells were pretreated with antibodies of TLR2 or TLR4 respectively or both anti-TLR2 and anti-TLR4 before PPSB stimulation. Western blot analysis showed that after blocking with anti-TLR2 and TLR4 before employing PPSB, the phosphorylation level of p38, ERK1/2 and JNK were obviously lower than the group of PPSB stimulation alone respectively (Fig. 9B). Besides, the phosphorylation and degration of IκB-α and the phosphorylation of NF-κB were also suppressed by anti-TLR2 or anti-TLR4 antibodies in contrast with the group PPSB stimulation alone (Fig. 9C). Moreover, it was also found that (shown in Fig. 9B and Fig. 9C) being treated with anti-TLR2 and anti-TLR4 simultaneously could further attenuate not only the phosphorylation of p38, ERK1/2 and JNK, but also the phosphorylation of NF-κB 18

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and IκB-α and the degration of IκB-α compared with anti-TLR2 or anti-TLR4 separately used in RAW264.7 cells；However, they could not completely abolish the activation of MAPKs and NF-κB when compared with control group. On basis of the results above, it can be suggested that TLR2 and TLR4 were involved in the intracellular signaling cascades activation triggered by PPSB in RAW264.7 cells.

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4. Conclusion

In this study, we illuminated the effects of PPSB on the activation of RAW264.7 macrophages and the underlying mechanisms. Firstly, it was found that PPSB not only promoted the cell proliferation, NO, ROS, TNF-α, and IL-6 productions and pinocytosis and phagocytosis activities in RAW264.7 cells, but also up-regulated the expressions of MHC (I/II) and costimulators (CD40/80/86) on RAW264.7 cells, indicating that PPSB contributed to the activation of RAW264.7 cells. Based on the PPSB prominent stimulation on the activation of RAW264.7 cells mentioned above, we further focused on the underlying molecular mechanism including the effect of PPSB on the signaling pathways (MAPKs and NF-κB) and the involvement of receptors during this process. Signaling pathways studies showed that PPSB could activate MAPKs signaling through evoking the phosphorylation of MAPKs (p38, ERK1/2, and JNK). In addition, PPSB induced the phosphorylation and degradation of IκB-α as well as the phosphorylation of NF-κB, indicating that PPSB 19

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could activate NF-κB signaling pathway. More importantly, the PPSB-induced productions of NO, TNF-α, and IL-6 were suppressed when MAPKs and NF-κB were inhibited by specific inhibitors in RAW264.7 cells. These results showed that MAPKs and NF-κB were involved in the activation of RAW264.7 cells by PPSB. In order to investigate the potential receptors of PPSB, antibodies for Dectin-1, MR, CR3, TLR2 and TLR4 were applied to block the receptors respectively before PPSB stimulation, and the productions of NO, TNF-α, and IL-6 were measured. The results showed that the NO, TNF-α, and IL-6 production induced by PPSB were attenuated after TLR2 or TLR4 were blocked. Furthermore, the MAPKs as well as NF-κB activation caused by PPSB were also inhibited by anti-TLR2 or anti-TLR4 antibodies. However, the employing of anti-TLR2 and anti-TLR4 simultaneously did not completely suppress the productions of NO, TNF-α and IL-6, nor did they fully inhibit the activation of MAPKs and NF-κB of RAW264.7. It could be suggested from the results above that TLR2 and TLR4 were involved in RAW264.7 activation evoked by PPSB, but TLR2 and TLR4 may not the only receptors for PPSB. We will elucidate its potential mechanism in future work. Taken together, PPSB are capable of inducing RAW264.7 macrophages activation through MAPKs and NF-κB signaling pathways via TLR2 and TLR4, which help us to have a better understanding of the mechanisms of PPSB in immunomodulatory activity.

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References

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This work was supported by the Natural Science Foundation of China (Grant No. 30970639, 31570342). In this study, the funders had no role in design of experiments, data collection and analysis, or manuscript writing.

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Figure Captions

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Fig. 1. Effects of PPSB on cell proliferation, NO and ROS production of RAW264.7 cells. RAW264.7 cells were treated with PPSB (40, 80, 160 μg/mL) for 24h. The proliferation of RAW264.7 cells were detected by the MTT assay (A). The releases of NO were determined by using NO assay kit (B). Endotoxin contamination test was proceeded through determining the effects of PMB on PPSB or LPS induced NO production in RAW264.7 cells (C). The intracellular ROS was detected by flow cytometry. The MFI of RAW264.7 cells in the histogram represents the amount of ROS produced from RAW264.7 cells (D). The figure shown is representative of three independent experiments. The gray solid lines in the figure represent control groups, and the black hollow lines represent PPSB or LPS stimulation group (E). Each experiment was performed in triplicate, and values were represented as means ± SD. Significant difference was analyzed with a student’s t test. Asterisk symbols indicate significant differences when compared to control group, *p ＜ 0.05 and **p ＜ 0.01. Hash symbols indicate significant difference between the tested groups, ##p ＜ 0.01. ns, not significant difference between the tested groups.

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Fig. 2. Effects of PPSB on cytokines production of RAW264.7 cells. RAW264.7 cells were treated with PPSB (40, 80, 160 μg/mL) for 24h or treated with PPSB (160 μg/mL) for 6 h, 12 h, 24 h and 48 h. The production of TNF-α (A, B) and IL-6 (C, D) were measured by ELISA kit. Each experiment was performed in triplicate, and values were represented as means ± SD. Significant difference was analyzed with a student’s t test. Asterisk symbols indicate significant differences when compared to control group. *p ＜ 0.05 and **p ＜ 0.01. Fig. 3. Effects of PPSB on pinocytosis and phagocytosis activity of RAW264.7 cells. The pinocytosis activities of RAW264.7 cells were determined by the uptake of neutral red assay (A). The phagocytosis activity was analyzed via detecting the internalization of FITC-dextran by flow cytometry and laser scanning confocal microscope. MFI in the histogram represents the phagocytosis activity of RAW264.7 cells (B). The figure shown is representative of three independent experiments of flow cytometry. The gray solid lines in the figure represent control groups, and the black hollow lines represent PPSB or LPS stimulation group (C). Laser scanning confocal image of PPSB induced phagocytosis. Blue: nucleus; Green: FITC-Dextran (D). Each experiment was performed in triplicate, and values were represented as means ± SD. Significant difference was analyzed with a student’s t test. Asterisk symbols indicate significant differences when compared to control group. *p ＜ 0.05 and **p ＜ 0.01. Fig. 4. Effects of PPSB on the expression of MHC molecules and costimulatory molecules on the surface of RAW264.7 cells. RAW264.7 cells were treated with PPSB (40, 80, 160 μg/mL) for 24h. The expression of CD40, CD80, CD86, MHC-I and MHC-II were analyzed by flow cytometry. The 25

Journal Pre-proof figure shown is representative of three independent experiments of flow cytometry. The gray solid lines in the figure represent control groups, and the black hollow lines represent PPSB or LPS stimulation group (A). MFI in the histogram represents the amount of expression from CD40, CD80, CD86, MHC-I and MHC-II (B). Each experiment was performed in triplicate, and values were represented as means ± SD. Significant difference was analyzed with a student’s t test. Asterisk symbols indicate significant differences when compared to control group. *p ＜ 0.05 and **p ＜ 0.01.

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Fig. 5. PPSB activated the MAPKs and NF-κB pathways. RAW264.7 cells were treated with 160 μg/mL of PPSB for different time (0, 0.5, 1, 2, 4, 8, 12, 24h), and the phosphorylation level of MAPKs (p38, ERK1/2, JNK) were determined by western blot (A). RAW264.7 cells were treated with different concentrations (20, 40, 80, 160μg/mL) of PPSB for 0.5h, and the phosphorylation levels of MAPKs (p38, ERK1/2, JNK) were determined by western blot (B). RAW264.7 cells were treated with 160μg/mL of PPSB for different time (0, 0.5, 1, 2, 4, 8, 12, 24h), and the phosphorylation level of NF-κB and IκB-α, and the degradation of IκB were analyzed by western blot (C). RAW264.7 cells were treated with different concentrations (20, 40, 80, 160μg/mL) of PPSB for 0.5h, and the phosphorylation levels of NF-κB and IκB-α, and the degradation of IκB-α were analyzed by western blot (D). Each experiment was performed in triplicate.

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Fig. 6. MAPKs pathways were involved in RAW264.7 cells activation induced by PPSB. RAW264.7 cells were pretreated with 25 μM of PD98059, SP600125 or SB203580 for 2h and then exposed to 160 μg/mL of PPSB for 30mins. Total protein of cells was extracted, and the phosphorylation of ERK1/2, JNK, and p38 were analyzed by western blot (A). After being pretreated with PD98059, SP600125 or SB203580, cells were treated with 160 μg/mL of PPSB for 24h, and the release of NO, TNF-α, and IL-6 were assessed by NO assay kit and ELISA kit (B). Each experiment was performed in triplicate, and values were represented as means ± SD. Significant difference was analyzed with a student’s t test. Asterisk symbols indicate significant differences between the tested groups. *p ＜ 0.05 and **p ＜ 0.01. Fig. 7. NF-κB pathway was involved in RAW264.7 cells activation induced by PPSB. RAW264.7 cells were pretreated with 10 μM of BAY 11-7082 for 2h, and then exposed to 160 μg/mL of PPSB for 0.5h. Total protein of cells was extracted, and the degradation of IκB-α were analyzed by western blot (A). After being pretreated with BAY 11-7082, cells were treated with 160 μg/mL of PPSB for 24h, and the release of NO, TNF-α, and IL-6 were assessed by NO assay kit or ELISA kit (B). Each experiment was performed in triplicate, and values were represented as means ± SD. Significant difference was analyzed with a student’s t test. Asterisk symbols indicate significant differences between the tested groups. *p ＜ 0.05 and ** p ＜ 0.01.

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Journal Pre-proof Fig. 8. Anti-TLR2 and anti-TLR4 antibodies attenuated the production of NO, TNF-α and IL-6 of RAW264.7 cells induced by PPSB. RAW264.7 cells were pretreated with anti-TLR2 (10μg/mL), anti-TLR4 (10μg/mL), anti-MR (20μg/mL), anti-CR3 (2.5μg/mL), anti-Dectin-1 (5μg/mL) respectively for 2h, then exposed to 160 μg/mL of PPSB for 24h. The releases of NO, TNF-α, and IL-6 were assessed. Each experiment was performed in triplicate, and values were represented as means ± SD. Significant difference was analyzed with a student’s t test. Asterisk symbols indicate significant differences, compared to PPSB treatment group. *p ＜ 0.05 and **p ＜ 0.01.

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Fig. 9. TLR2 and TLR4 were involved in the activation of RAW264.7 cells induced by PPSB. RAW264.7 cells were pretreated with anti-TLR2 (10μg/mL), anti-TLR4 (10μg/mL) respectively or with both anti-TLR2 (10μg/mL) and anti-TLR4 (10μg/mL) for 2h, then exposed to 160 μg/mL of PPSB for 24h. The releases of NO, TNF-α, and IL-6 were assessed (A). RAW264.7 cells were pretreated with anti-TLR2 (10μg/mL), anti-TLR4 (10μg/mL) respectively or with both anti-TLR2 (10μg/mL) and anti-TLR4 (10μg/mL) simultaneously for 2h, then exposed to 160 μg/mL of PPSB for 0.5h. The total protein of cells was extracted. The phosphorylation levels of MAPKs (p38, ERK1/2, JNK) were analyzed by western blot (B). The degradation of IκB-α and phosphorylation level of NF-κB and IκB-α were analyzed by western blot (C). Each experiment was performed in triplicate, and values were represented as means ± SD. Significant difference was analyzed with a student’s t test. Asterisk symbols indicate significant differences between the tested group. *p ＜ 0.05 and **p ＜ 0.01.

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Highlights

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PPSB not only significantly enhanced the cell proliferation, pinocytic and phagocytic activity, but also promoted the production of NO, ROS, TNF-α, and IL-6 in RAW264.7 cells.

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PPSB up-regulated the expression of major histocompatibility complex (MHC-I/II) and costimulatory molecules such as CD40, CD80 and CD86 on RAW264.7 cells.

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PPSB activated the MAPKs and NF-κB signaling pathways in RAW264.7 cells

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TLR2 and TLR4 were involved in PPSB-induced activation of RAW264.7 cells

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