nuclear factor-kappa B in macrophages.

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by GEORGEWASHUNIVBF on 01/09/15 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

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Oxymatrine inhibits lipopolysaccharide-induced inflammation by down-regulating toll-like receptor 4/nuclear factor-kappa B in macrophages Zhang, Yu 1*, Yan, Ruhong 2*, Hu, Yae 3# 1 Department of Pediatrics, the First People’s Hospital of Nantong, Nantong 226001, Jiangsu Province, China 2 Department of Clinical Laboratory, Department of Medical Equipment, The Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China 3 Department of Pathophysiology, the Medical School of Nantong University, Nantong 226001, Jiangsu Province, China * These authors contributed equally to this work. # Corresponding author. Yae Hu, Department of pathophysiology, the Medical school of Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province, R. P. China. Tel: (+86 513) 85051733, E-mail: [email protected].

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Abstract: Oxymatrine (OMT) is the quinolizidine alkaloid extracted from Chinese herb Sophora flavescens Ait that has many pharmacological effects and is used for the treatment of some inflammatory diseases. In this study, RAW264.7 cells and THP-1 differentiated macrophages were pretreated with various concentrations of OMT 2 h prior to Lipopolysaccharide (LPS) (1 µg/ml) treatment for different times. We detected the anti-inflammatory effect of OMT in LPS-stimulated macrophages and tried to reveal its molecular mechanism. It was shown that OMT pretreatment significantly inhibited the LPS-induced secretion of nitric oxide (NO), interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in supernatant, attenuated the mRNA levels of

inducible nitric oxide synthase (iNOS), IL-1β, TNF-α and toll-like receptor 4 (TLR4),

increased TLR4, phosphorylation of inhibitor of kappa B-alpha (p-IBα) in cytosol, and decreased the nuclear level of nuclear factor-κB (NF-κB) p65 in macrophages. In conclusion, OMT exerts anti-inflammatory property in LPS-stimulated macrophages by down-regulating TLR4/NF-κB pathway. Keywords: Chinese herb; anti-inflammation; cytokines; RAW264.7; THP-1

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1. Introduction Oxymatrine (OMT), a traditional Chinese medicine, is the major quinolizidine alkaloid extracted from the root of Sophora flavescens Ait. The chemical structure of OMT is shown in Fig. 1a. Previous studies have reported that OMT had many pharmacological effects, such as anti-inflammation, anti-apoptosis, antitumor, anti-virus, anti-fibrosis and anti-arrhythmia (Deng et al. 2009;Ho et al. 2009;Runtao et al. 2011;Wang et al. 2011;Zheng et al. 2005). OMT is also proven to protect ischemia and reperfusion-induced liver, heart, intestinal and brain injury (Hong‐ li et al. 2008;Jiang et al. 2005;Liu et al. 2009;Zhao et al. 2008). Recently, more and more studies have pay attention to its effect against inflammatory diseases, such as colitis, acute pancreatitis in rats, and LPS-induced mastitis in mice (Yang et al. 2014;Zhang et al. 2012). However, the molecular mechanism of the anti-inflammatory actions of OMT is yet undefined. Macrophages, originated from circulating blood monocytes, locate throughout the body tissues and participate in inflammatory responses. They host antimicrobial defense and restoration of tissue homeostasis by ingesting dead tissue and fighting against invading pathogens (Murray and Wynn 2011). But in some cases, excessive activation of macrophages can even deteriorate the inflammatory processes. Therefore, controlling macrophage activities is regarded to be a promising strategy for anti-inflammatory therapies. RAW264.7 cells has been developed as a primary experimental macrophage model for the study of macrophage signaling pathways and the research of macrophage inflammations (Pascual et al. 2005). After differentiation into macrophages, THP-1 cells were previously shown to be a widely used cell line to study function and regulation of macrophages in vitro (Daigneault et al. 2010;Tsuchiya et al. 1980). Inflammation is a tissue reaction to irritation, injury, or infection, especially caused by various bacterial infections. The activation of macrophages is a common characteristic for the early stages of pathogens infection. Lipopolysaccharide (LPS), a major outer membrane component of Gram-negative bacteria, plays an important role in various inflammatory responses (Khan et al. 1998), and has been served as an important active component for pathogen-induced macrophage inflammation studies (Smith et al. 2001). LPS activates the expression of toll-like receptor 4 (TLR4) and promotes the secretion of pro-inflammatory factors and cytokines via binding to the CD14/TLR4/myeloid differentiation protein-2 (MD-2) receptor complex and

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downstream activation of nuclear factor-kappa B (NF-κB) signaling pathway in macrophages (Fu et al. 2013;Murata et al. 2013). Recently, LPS stimulated RAW264.7 cells and human THP-1 derived macrophages’ inflammatory model have been widely used to screen effective anti-inflammation drugs and to investigate their mechanism (Yuan et al. 2012;Zhang et al. 2014). In the present study, we evaluated the anti-inflammatory effects of OMT on the expression of pro-inflammatory factors and cytokines including nitric oxide (NO), interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in LPS-stimulated RAW 264.7 and THP-1 derived macrophages. Furthermore, we investigated whether OMT participated in modulating TLR4/NF-κB signaling pathways when presenting anti-inflammatory effects.

2. Materials and methods 2.1 Compounds OMT was purchased from Baoji F. S. Biological Development Company Limited (Shanxi, China). OMT was first dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Shanghai, China) to 1 M and then diluted in serum free culture medium to get a stock solution of 10 mM.

2.2 Cell Culture and Treatment The RAW 264.7 mouse macrophage cell line and the human monocytic cell line THP-1 was purchased from China Cell Line Bank (Shanghai, China) and cultured in DMEM and RPMI 1640 culture medium (GIBCO, USA) respectively supplemented with 10 % heat inactivated fetal bovine serum (GIBCO), 100 U/ml penicillin and 100 µg/ml streptomycin at 37 °C in a humidified incubator with 5 % CO2. Cells were passaged once every 48 h. For differentiation to macrophages, THP-1 monocytes (1×106 cells/well) were plated in 6-well plates in 1 ml completed medium and stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, Shanghai, China) for 72 h as previously described (Auwerx 1991). PMA-differentiated THP-1 macrophages were selected by keeping only the adherent cells to the plate. The culture medium containing non-adherent cells was discarded and a new medium without PMA was added (Kurosaka et al. 1998) . Cells were incubated with or without various concentrations of OMT that was added 2 h prior to lipopolysaccharide (LPS, from Escherichia coli 0111:B4, Sigma-Aldrich; 1 µg/mL) treatment. 4


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2.3 Cell Counting Kit-8 (CCK8) Assay Cell viability was determined by CCK8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacture's protocol. In brief, RAW 264.7 cells and THP-1 derived macrophages were plated in 96-well plates (4×104) cells. 24 h later, various concentrations of OMT (20, 50, 100, 200 µM) were added to cells for another 24 h. Then the supernatant was replaced by 100 µl of DMEM medium containing 10 µl of CCK8 and the cells were incubated for 1 h at 37 °C. The absorbance was measured at 450 nm using an ELX-800 microplate assay reader (BIO-TEK, Winooski, VT, USA). Cell viability was calculated by (absorbance

450nm

in treatment group/absorbance

450nm

in

control group) × 100%.

2.4 NO Content Assay The nitrite in the culture medium representing NO production was detected based on the Griess reaction. Various concentrations of OMT (20, 50, 100 µM) were added to RAW 264.7 cells and THP-1 derived macrophages, 2 h prior to stimulation of LPS (1 µg/ml) for 24 h in serum free DMEM. Culture supernatants were mixed with equal volume of Griess reagent [equal volumes of 1

%

(w/v)

sulfanilamide

in

5

%

(v/v)

phosphoric

acid

and

0.1

%

(w/v)

naphtylethylenediamine-HCl] and then incubated at room temperature for 10 min. The absorbance was read at 550 nm on a microplate reader and NO concentration was calculated with reference to standard curve of sodium nitrite.

2.5 Enzyme-Linked Immunosorbent Assay (ELISA) To determine the effect of OMT on pro-inflammatory cytokines production from LPS-stimulated RAW264.7 cells, cells were plated on 24-well plates (4×105) and were pretreated with OMT (20, 50, 100 µM) 2 h prior to LPS (1 µg/ml) treatment for 24 h in serum free DMEM. Then supernatants were collected and IL-1β and TNF-α concentration were assayed using ELISA kits from R&D Systems (Minneapolis, MN, USA). Samples were analyzed according to the manufacturer’s recommendations. The absorbance value at 450 nm was measured using a microplate reader within 30 minutes.

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2.6 Quantitative Reverse Transcription-PCR (qRT-PCR) Analysis To observe the effect of OMT on gene expression in LPS-treated RAW 264.7 cells and THP-1 derived macrophages, cells were plated on six-well plates (1×106) and were pre-incubated with OMT (20, 50, 100 µM) 2 h before LPS (1 µg/ml) treatment for 6 h in serum free DMEM. Total RNA was extracted using TRIzol (Promega, USA) according to the manufacturer's explaination. The RNA was reverse transcribed into cDNA using a reverse transcriptase protocol (Promega, USA). cDNA was amplified on a StepOneTM real-time PCR detection system (ABI, Foster City, CA, USA) using SYBR® Green Supermix (Fermentas, Glen Burnie, MD, USA). β-actin was amplified in parallel with the target genes and used as a normalized control. The primers used for qRT-PCR in RAW 264.7 cells are as follows: iNOS (NM_010927.3): 5’- GGA GCG AGT TGT GGA TTG T-3’ and 5’- AGT GAT GTC CAG GAA GTA GGT G -3’; TLR4 (NM_019178): 5'- GGC TTC TAA CCT CAA CGA CC-3' and 5'-ACT GGG CCT TAG CCT CCT-3'; IL-1β (NM_0315122): 5'-GAT GAT GAC GAC CTG CTA GTG T-3' and 5'-CGT TGC TTG TCT CTC CTT GTA-3'; TNF-α (NM_0126753): 5'-CAC CAC GCT CTT CTG TCT ACT G-3' and 5'-GCT TGG TGG TTT GCT ACG AC-3'; β-actin (NM_031144): 5'-CA GGT CAT CAC TAT CGG CAA T-3' and 5'-AGG TCT TTA CGG ATG TCA ACG-3'. The primers used for qRT-PCR in THP-1 derived macrophages are as follows: iNOS (NM_000625.4): 5’- CCT CGG CTC CAG CAT GTA CCCTCG G-3’ and 5’- CGG AAG GCG TCCTCC TGC CCA CTG A -3’; TLR4 (NM_003266.3 ): 5'- ATC ATT GGT GTG TCG GTC CT-3' and 5'-AGC TCA TTC CTT ACC CAG TCC-3'; IL-1β (NM_000576): 5'-CTC GCC AGT GAA ATG ATG GCT-3' and 5'-GTC GGA GAT TCG TAGCTG GAT-3'; TNF-α (NM_000549): 5'-CTC TTC TCC TTC CTG ATC GTG -3' and 5'- GGT TCG AGA AGA TGA TCT GAC TG -3'; β-actin (NM_001101.3): 5'- AGG CCA ACC GCG AGA AGA TGA CC -3' and 5'- GAA GTC CAG GGC GAC GTA GCA C -3'. The amplifying conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95 °C for 15 s, 54 °C for 30 s, and 72 °C for 30 s. The levels of target gene were determined using the relative threshold cycle (CT) method. △CT＝(CT value of target gene – CT value of internal gene control), △△CT＝(CT value of target gene – CT value of internal gene control) sample A – (CT value of target gene – CT value of internal gene control) sample B. Fold change relative quantification (RQ) ＝ 2-∆∆CT.

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2.7 Western Blotting Analysis The RAW 264.7 cells and THP-1 derived macrophages were plated on six-well plates (1×106) and were pretreated with OMT (20, 50, 100 µM) 2 h before LPS (1 µg/ml) stimulation for 6 h in serum free DMEM. Cells were washed twice with ice-cold PBS and lysed in buffer (50 mM Tris, pH 7.4, 150 mM sodium chloride, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate, supplemented with protease inhibitor cocktail) on ice for 30 min. For nuclear extraction, cells were lysed with NE-PERTM (Pierce, Rockford, IL, USA). Cell proteins were quantified using a BCA-100 Protein Quantitative Analysis Kit (Sigma, USA) and same quantity of proteins were separated on 10 % gels, and then transferred to polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Each membrane was blocked with 5 % milk in TBS-T (0.1 M Tris-HCl, pH 7.4, 0.9 % NaCl, 0.1 % Tween-20) at room temperature for 1 h, and then incubated with rabbit anti-TLR4 polyclonal antibody (1:200, Santa Cruz, CA, USA), mouse anti-β-actin monoclonal antibody (1:2000, Santa Cruz), mouse anti-Histone H1 monoclonal antibody (1:500, Millipore, Billerica, MA, USA), rabbit anti-NF-κB p65 polyclonal antibody (1:1000, Kangchen BIO-TECH,

shanghai,

China),

mouse

anti-IκBα

monoclonal

antibody

and

mouse

anti-phospho-IκBα monoclonal antibody (1:1000, Kangchen BIO-TECH) at 4°C overnight. The membrane was washed 3 times for 10 min each using TBS-T. Afterwards, it was incubated in the horseradish peroxidase-conjugated goat anti-mouse IgG or the horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1000, Santa Cruz) at room temperature for 2 h. Enhanced chemiluminescence were used for detection. Immunoreactive bands were scanned and quantified by Scion Image software (Scion, Maryland, USA), and the amount was normalized with histone or β-actin values in the same lane.

2.8 Flow cytometry Cell surface expression of TLR4 was assessed by flow cytometric assay. Briefly, RAW 264.7 and THP-1 derived macrophages were pretreated with OMT (20, 50, 100 µM) 2 h before LPS (1 µg/ml) stimulation for 6 h. Cells were collected and washed three times with PBS followed by incubation with purified rabbit anti-TLR4 antibody (1:500, Santa Cruz) for 1 h. Subsequently, cells were washed three times with PBS and incubated with phycoerythrin-labeled goat anti-rabbit IgG mAb (R&D, Minneapolis, MN, USA) for 30min. Then, cells were washed three times with 7


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PBS and immediately analyzed on a FACS Calibur flow cytometer [Becton Dickinson (BD), Franklin Lakes, NJ, USA], and the data were analyzed by BD Cell-Quest software. Nonspecific fluorescence

was determined by using isotype IgG as control.

2.9 Statistical analysis All results are expressed as Mean ± Standard deviation. Statistical analysis was performed using Stata 8.0 software (computer resource center, USA). The data were analyzed using one-way analysis of variance (ANOVA) followed by Scheffé test. A value of P < 0.05 was considered statistically significant.

3. Results 3.1 Effects of OMT on cell viability The potential cytotoxicity of OMT was examed by CCK8 assay, and the results showed that cell viability was not affected by OMT treatment at the concentrations used (20, 50, and 100 µM) while OMT at 200 µM decreased cell viability both in RAW 264.7 and THP-1 derived macrophages (Fig. 1b and 1c). So we used OMT (20, 50, and 100 µM) to observe the anti-inflammatory effect and its mechanism.

3.2 OMT inhibits LPS-induced NO production and iNOS mRNA expression To determine the anti-inflammatory effect of OMT, we detected its effect on LPS-induced NO releasing by measuring nitrite content accumulated in the culture medium by Griess reaction. Compared with control group, LPS (1 µg/ml) stimulation significantly increased NO production in RAW264.7 cells and THP-1 derived macrophages, pretreated with OMT (20, 50, 100 µM) decreased NO concentration (Fig. 2a and 2b). iNOS is a upstream regulator for NO production. To detect the effect of OMT on LPS-induced iNOS mRNA expression, qRT-PCR was performed. We found that LPS significantly increased iNOS mRNA expression in RAW 264.7 cells and THP-1 derived macrophages, while OMT pretreatment decreased iNOS mRNA expression (Fig. 2c and 2d).

3.3 OMT inhibits LPS-induced pro-inflammatory cytokines expression 8


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To analyze the potential anti-inflammaory effects of OMT, we also determined whether OMT affected the expression of pro-inflammatory cytokines in LPS-stimulated cells. The production of IL-1β and TNF-α in the medium and their gene expression were detected by ELISA and qRT-PCR respectively. LPS stimulation activated RAW 264.7 cells and THP-1 derived macrophages, IL-1β and TNF-α were significantly enhanced, while pretreatment with OMT (20, 50, 100 µM) 2 h before LPS treatment suppressed TNF-α and IL-1β expression in LPS-stimulated RAW 264.7 cells both in protein (Fig. 3a and 3b) and mRNA levels (Fig. 3c and 3d).

3.4 OMT inhibits LPS-induced NF-κB activation NF-κB is an important signaling molecule in the pathophysiologic process of inflammation. To determine whether OMT mediates the inhibition of the inflammatory response through NF-κB pathway, cytosol levels of phospho-inhibitor of kappa B α (p-IκBα), cytosol IκBα and nuclear levels of NF-κB p65 subunit were analyzed by western blot analysis. The results showed that LPS stimulation for 6 h significantly decreased cytosol IκBα, enhanced cytosol p-IκBα and nuclear NF-κB p65. However, pretreatment with OMT obviously enhanced cytosol IκBα, decreased cytosol p-IκBα and nuclear NF-κB p65. Results indicate that OMT inhibited NF-κB activation by reducing phosphorylation of IκBα (Fig. 4).

3.5 OMT inhibits LPS-induced TLR4 mRNA and protein expression To determine whether TLR4 signaling is involved in anti-inflammatory effect of OMT, TLR4 mRNA and protein in LPS-stimulated RAW 264.7 cells were evaluated by qRT-PCR, western blot assay and flow cytometric assay. Results showed that LPS exposure for 6 h, TLR4 mRNA and protein level were enhanced. OMT significantly decreased LPS-induced TLR4 expression both in mRNA and protein level (Fig. 5).

4. Discussion The increasing interest in naturally derived herbs and plant extracts has promoted research into illuminating mechanism of effect attributed to these compounds. OMT, a traditional Chinese herbal product has shown promising beneficial effects. Although several studies have investigated the anti-inflammatory activity of OMT, the molecular mechanism of OMT inhibiting LPS-induced 9


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macrophages inflammation remains unclear. In this study, we observed the anti-inflammatory effects and discussed the potential molecular mechanism of OMT on LPS-stimulated RAW 264.7 cells and THP-1 derived macrophages. CCK8 assay is based on the dehydrogenase activity detection in viable cells. Although CCK8 results do not necessarily indicate the cell viability if protein synthesis is intact, it is still a good method for cell viability. In our study, CCK8 assay showed that OMT have no cytotoxic effect in concentration of 0 to 100 µM when treating RAW 264.7 cells and THP-1 derived macrophages, suggesting that OMT showed inhibitory effect not because of declining cells viability. As innate immune cells, macrophages initiate inflammation and immune response. When stimulated by LPS, macrophages are activated and release various pro-inflammatory factors and cytokines, whose excessive release may result in extensive tissue damage and pathological changes (Rossol et al. 2011). NO is an important regulatory molecule in human body that is produced from L-arginine by the family of NOS enzymes (Lee et al. 1992). The production of NO by iNOS can be triggered by multiple inflammatory stimuli, resulting in generation of reactive intermediates of NO and promoting pro-inflammatory responses (Kundu and Surh 2008). In addition, pro-inflammatory cytokines (e.g., IL-1β and TNF-α) also play an important role in inflammatory diseases (Miao et al. 2007). In our experiments, we used LPS as the typical inflammatory stimulator because of its ability to initiate the production of pro-inflammatory mediators (Abreu and Arditi 2004). To confirm the potential anti-inflammatory effects of OMT in vitro, effects of OMT on the production of NO, IL-1β and TNF-α were evaluated. We found that in LPS-stimulated RAW264.7 cells, OMT pretreatment significantly inhibited overexpression of iNOS and thus decreased the secretion of NO in supernatant. Also this study indicated that OMT could prevent and reduce inflammatory responses by attenuating IL-1β and TNF-α mRNA expression as well as their production. LPS is an effective activator of NF-κB. NF-κB is well known to play a crucial role in controlling most inflammatory responses in macrophages and thus becomes target for developing novel treatments for inflammatory disease (Hanada and Yoshimura 2002). Under normal condition, NF-κB presents in the cytoplasm as either a homodimer or a heterodimer and binds to the inhibitory kappa B (IκB) protein to form an inactive complex IκB-NF-κB. When challenged with LPS, the IκBα is phosphorylated, which facilitates the degradation of the IκB proteins and the 10


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translocation of free NF-κB from cytosol to the nucleus (Saccani et al. 2001). To illuminate whether the possible mechanism of OMT inhibiting the production of NO, IL-1β and TNF-α is mainly by suppressing the activity of NF-κB, we detect the expression of IκBα and p-IκBα in cytoplasm and NF-κB p65 in nuclear by Western blot. The results showed that OMT pretreatment significantly inhibited LPS-induced NF-κB activation by suppressing the phosphorylation and degradation of IκBα and thus increased NF-κB p65 in nuclear. Dong et al found that OMT had similar inhibition of NO cytokines and NF-κB nuclear translocation in BV2 microglia cells (Dong et al. 2013). However, Guzman et al reported that OMT decreased LPS-induced p65 nuclear translocation independent of IκBα degradation/phosphorylation in rat IEC and murine BMDC cells (Guzman et al. 2013). These differences may be due to different cell models. TLR4 expressing on various pro-inflammatory cells is the major receptor in LPS-induced inflammatory responses (Baker et al. 2011). Activation of TLR4 in immune cells by LPS initiates signaling cascades that result in the activation of NF-κB, which leads to the production of cytokines and other inflammatory mediators (Botos et al. 2011). To further verify whether the anti-inflammatory effect of OMT was manifested through TLR4 mediated signaling, we detected TLR4 mRNA expression by qPCR and detected protein level using Western blot analysis. Western blot technique demonstrates both intracellular and cell surface protein. Only when intracellular TLR4 trafficked to cell membrane, it is functional. So we measured cell surface expression of TLR4 by flow cytometry. The results showed that OMT inhibited the expression of TLR4 mRNA and cell surface protein up-regulated by LPS in RAW 264.7 cells and THP-1 derived macrophages, indicating that OMT inhibited the activation of NF-κB by suppressing TLR4 expression. Recently, a number of studies showed that other natural products also have similar effects on inflammation. Xu et al found that Punicalagin could inhibit LPS-induced inflammation via the suppression of TLR4-mediated MAPKs and NF-κB activation (Xu et al. 2014). Also, Young et al reported that oral administration of Curcumin in mice could suppress LPS-induced inflammation by inhibiting macrophage migration via NF-κB signaling (Young et al. 2014). Further studies are needed to determine the difference about anti-inflammatory effect and its mechanism between OMT and other natural products. In conclusion, the results of this study demonstrate that OMT decreases the production of 11


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pro-inflammatory factors and cytokines including NO, TNF-α and IL-1β in LPS-stimulated RAW 264.7 cells and THP-1 derived macrophages by suppressing the activation of TLR4/NF-κB signaling pathway. Therefore, these data suggest that OMT could be a promising therapeutic medicine in the treatment of various inflammation related diseases in macrophage. However, further studies are necessary to fully understand the overall intracellular process related to the anti-inflammatory mechanism of OMT.

Acknowledgements We would like to acknowledge Dr Ruhong Yan for checking the language carefully. This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions; the Nantong social enterprise innovation and demonstration of technology program (Grant No. S11953) and the National Natural Science Foundation of China (Grant No. 81301494).

Conflict of Interest. The authors declare that they do not have any conflict of interest.

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4912-4915. PMID: 16097071. Figure captions: Fig. 1. Chemical structure of OMT and effect of OMT on cell viability. (a) Chemical structure of OMT. (b) (c) Effect of OMT on the cell viability of RAW 264.7 cells and THP-1 derived macrophages was tested by CCK8 assay. OMT (20, 50, 100, 200 µM) were added to cells for 24 h. Cell viability was tested by CCK8 assay. Data represent the mean ± SEM of six independent experiments and differences between mean values were assessed by one-way ANOVA. *p＜0.05.

Fig. 2. Effect of OMT on LPS-induced NO production and iNOS mRNA expression. (a) (b) Effect of OMT on LPS-induced NO production in RAW 264.7 cells and THP-1 derived macrophages. Cells were pretreated with OMT (20, 50, 100 µM) 2 h prior to stimulation of LPS (1 µg/ml) for 24 h, and NO production in the supernatant was measured by Griess reaction. (c) (d)

Effect of OMT on LPS-induced iNOS mRNA expression in RAW 264.7 cells and THP-1 derived macrophages. Cells were pre-incubated with OMT (20, 50, 100 µM) 2 h before LPS (1 µg/ml) treatment for 6 h, and iNOS mRNA expression was tested by qRT-PCR. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ## p＜0.01 indicates significant differences compared with control group. *p＜0.05, **p＜0.01 indicate significant differences compared with LPS-treated group.

Fig. 3. Effect of OMT on LPS-induced pro-inflammatory cytokines secretion and their gene expression. (a) (b) Effect of OMT on the production of IL-1β and TNF-α in RAW 264.7 cells and THP-1 derived macrophages. Cells were pretreated with OMT (20, 50, 100 µM) 2 h prior to stimulation of LPS (1 µg/ml) for 24 h. Levels of IL-1β and TNF-α in the culture supernatants

were measured by ELISA. (c) (d)Effect of OMT on the mRNA expression of IL-1β and TNF-α in RAW 264.7 cells and THP-1 derived macrophages. Cells were pre-incubated with OMT (20, 50, 100 µM) 2 h before LPS (1 µg/ml) treatment for 6 h, and IL-1β and TNF-α mRNA expression was tested by qRT-PCR. Data represent the mean ± SEM of six or three independent experiments and differences between mean values were assessed by one-way ANOVA. ## p＜ 0.01 indicates significant differences compared with control group. *p＜0.05, **p＜0.01 indicate significant differences compared with LPS-treated group. 16


Page 17 of 23

Fig. 4. OMT inhibits LPS-induced NF-κB activation. Cells were pretreated with OMT (20, 50, 100 µM) 2 h prior to stimulation of LPS (1 µg/ml) for 6 h. (a) (b) Protein samples of in RAW

264.7 cells and THP-1 derived macrophages were analyzed by western blot analysis with specific antibodies. Histone and β-actin were used as an internal control. (c) (d) The mean densitometry values of neuclear NF-κB p65/Histone, cytosol IκB/β-actin and cytosol p-IκB/β-actin in RAW 264.7 cells and THP-1 derived macrophages were analyzed. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ## p＜0.01 indicates significant differences compared with control group. *p＜0.05, **p＜0.01 indicate significant differences compared with LPS-treated group.

Fig. 5. Effect of OMT on LPS-induced TLR4 mRNA and protein expression. (a) (b) Relative quantitation of TLR4 mRNA induced by LPS was detected by qRT-PCR in RAW 264.7 cells and THP-1 derived macrophages. Cells were pretreated with OMT (20, 50, 100 µM) 2 h prior to stimulation of LPS (1 µg/ml) for 6 h. (c) (d) TLR4 protein levels in each group were detected by western blot analysis in RAW 264.7 cells and THP-1 derived macrophages. β-actin was used as an internal control. Cells were pretreated with OMT (20, 50, 100 µM) 2 h prior to stimulation of LPS (1 µg/ml) for 6 h. (e) (f) The mean densitometry values of TLR4/β-actin in various groups were analyzed and expressed as bar charts to represent the relative expression of TLR4 protein. (g) (h) (i) (j) Expression of cell surface TLR4 protein was analyzed by flow cytometry. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ## p＜0.01 indicates significant differences compared with control group. *p＜0.05, **p＜0.01 indicate significant differences compared with LPS-treated group.

17


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Fig. 1. Chemical structure of OMT and effect of OMT on cell viability. (a) Chemical structure of OMT. (b) (c) Effect of OMT on the cell viability of RAW 264.7 cells and THP-1 derived macrophages was tested by CCK8 assay. OMT (20, 50, 100, 200 µM) were added to cells for 24 h. Cell viability was tested by CCK8 assay. Data represent the mean ± SEM of six independent experiments and differences between mean values were assessed by one-way ANOVA. *p＜0.05. 209x297mm (300 x 300 DPI)


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Fig. 2. Effect of OMT on LPS-induced NO production and iNOS mRNA expression. (a) (b) Effect of OMT on LPS-induced NO production in RAW 264.7 cells and THP-1 derived macrophages. Cells were pretreated with OMT (20, 50, 100 µM) 2 h prior to stimulation of LPS (1 µg/ml) for 24 h, and NO production in the supernatant was measured by Griess reaction. (c) (d) Effect of OMT on LPS-induced iNOS mRNA expression in RAW 264.7 cells and THP-1 derived macrophages. Cells were pre-incubated with OMT (20, 50, 100 µM) 2 h before LPS (1 µg/ml) treatment for 6 h, and iNOS mRNA expression was tested by qRT-PCR. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ## p＜0.01 indicates significant differences compared with control group. *p ＜0.05, **p＜0.01 indicate significant differences compared with LPS-treated group. 122x78mm (300 x 300 DPI)


Page 20 of 23

Fig. 3. Effect of OMT on LPS-induced pro-inflammatory cytokines secretion and their gene expression. (a) (b) Effect of OMT on the production of IL-1β and TNF-α in RAW 264.7 cells and THP-1 derived macrophages. Cells were pretreated with OMT (20, 50, 100 µM) 2 h prior to stimulation of LPS (1 µg/ml) for 24 h. Levels of IL-1β and TNF-α in the culture supernatants were measured by ELISA. (c) (d)Effect of OMT on the mRNA expression of IL-1β and TNF-α in RAW 264.7 cells and THP-1 derived macrophages. Cells were preincubated with OMT (20, 50, 100 µM) 2 h before LPS (1 µg/ml) treatment for 6 h, and IL-1β and TNF-α mRNA expression was tested by qRT-PCR. Data represent the mean ± SEM of six or three independent experiments and differences between mean values were assessed by one-way ANOVA. ## p＜0.01 indicates significant differences compared with control group. *p＜0.05, **p＜0.01 indicate significant differences compared with LPS-treated group. 124x83mm (300 x 300 DPI)


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Fig. 4. OMT inhibits LPS-induced NF-κB activation. Cells were pretreated with OMT (20, 50, 100 µM) 2 h prior to stimulation of LPS (1 µg/ml) for 6 h. (a) (b) Protein samples of in RAW 264.7 cells and THP-1 derived macrophages were analyzed by western blot analysis with specific antibodies. Histone and β-actin were used as an internal control. (c) (d) The mean densitometry values of neuclear NF-κB p65/Histone, cytosol IκB/β-actin and cytosol p-IκB/β-actin in RAW 264.7 cells and THP-1 derived macrophages were analyzed. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ## p＜0.01 indicates significant differences compared with control group. *p＜0.05, **p＜0.01 indicate significant differences compared with LPS-treated group. 116x94mm (300 x 300 DPI)


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Fig. 5. Effect of OMT on LPS-induced TLR4 mRNA and protein expression. (a) (b) Relative quantitation of TLR4 mRNA induced by LPS was detected by qRT-PCR in RAW 264.7 cells and THP-1 derived macrophages. Cells were pretreated with OMT (20, 50, 100 µM) 2 h prior to stimulation of LPS (1 µg/ml) for 6 h. (c) (d) TLR4 protein levels in each group were detected by western blot analysis in RAW 264.7 cells and THP-1 derived macrophages. β-actin was used as an internal control. Cells were pretreated with OMT (20, 50, 100 µM) 2 h prior to stimulation of LPS (1 µg/ml) for 6 h. (e) (f) The mean densitometry values of TLR4/β-actin in various groups were analyzed and expressed as bar charts to represent the relative expression of TLR4 protein. (g) (h) (i) (j) Expression of cell surface TLR4 protein was analyzed by flow cytometry. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ## p＜0.01 indicates significant differences compared with control group. *p ＜0.05, **p＜0.01 indicate significant differences compared with LPS-treated group. 63x85mm (300 x 300 DPI)

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by GEORGEWASHUNIVBF on 01/09/15 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 23 of 23

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