Journal of Ethnopharmacology 173 (2015) 91–99

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Insight into the molecular mechanism of a herbal injection by integrating network pharmacology and in vitro Yi-min Ma, Xin-zhuang Zhang, Zhen-zhen Su, Na Li, Liang Cao, Gang Ding, Zhen-zhong Wang, Wei Xiao n State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Kanion Pharmaceutical Corporation, NO. 58, Haichang Road Lianyungang, People's Republic of China

art ic l e i nf o

a b s t r a c t

Article history: Received 20 March 2015 Received in revised form 19 June 2015 Accepted 16 July 2015 Available online 17 July 2015

Chinese medical herbs could treat complex diseases through the synergistic effect of multi-components, multi-targets and multi-channels. However, it was difficult to systematically investigate the pharmacological mechanisms of action due to the complex chemical composition and the lack of an effective research approach. Fortunately, network pharmacology as an integrated approach was proposed to systematically investigate and explain the underlying molecular mechanisms of Chinese medical herbs. Reduning injection (RDN) is one of the herbal injections for treatment of upper respiratory tract infections (URTIs). Previous studies revealed the molecular mechanism of RDN on URTIs through network pharmacology. In this work, the mechanism of RDN was verified by enzyme linked immunosorbent assay (ELISA), Western Blot, immunofluorescence assay and electrophoretic mobility shift assay (EMSA) in lipopolysaccharide (LPS)-induced RAW264.7 cells and enzyme assay. RDN dose-dependently suppressed the production of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-6 (IL-6) and interleukin-1β (IL1β), and reduced the protein expression of inducible NO synthetase (iNOS) and cyclooxygenase-2 (COX2), which could be related to its suppression on the phosphorylations of mitogen-activated protein (MAP) kinases, including extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase(JNK) and p38, as well as the activation and translocation of nuclear factor-κB (NF-κB). In addition, the activity of RDN on PGE2 was also partly attributed to the inhibition of COX-2 enzyme. Therefore, it can be concluded that RDN inhibited the production of inflammatory mediators and the macrophage activation to treat URTIs via down-regulating the activation of MAPK and NF-κB signaling pathways, which might pave a way to illustrate the molecular mechanism of herbs. & 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Herbs Reduning injection Upper respiratory tract infections Anti-inflammatory effect Mechanism of action

1. Introduction As an important complementary and alternative medical system, Chinese medical herbs have been widely used in clinic for thousands of years in China (Zhang et al., 2013a). However, along with the development of modern medicine, the scientific basis and action mechanism of herbs have been questioned by medical practitioners recently (Yang et al., 2013). Fortunately, network Abbreviations:: RDN, reduning injection; LPS, lipopolysaccharide; URTIs, upper respiratory tract infections; PG, prostaglandin; NO, nitric oxide; COX, cyclooxygenase; iNOS, inducible NO synthase; IL, interleukin; TNF-α, tumor necrosis factor-α; TLR, Toll-like receptor; MAPK, mitogen activated protein kinase; ERK, extracellular signal-related kinase; JNK, c-Jun N-terminal kinase; NF-κB, nuclear factor-κB; ELISA, enzyme linked immunosorbent assay; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; BSA, bovine serum albumin; EMSA, electrophoretic mobility shift assay; RSV, respiratory syncytial virus RSV n Corresponding author. E-mail address: [email protected] (W. Xiao). http://dx.doi.org/10.1016/j.jep.2015.07.016 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

pharmacology integrating systems biology and polypharmacology is expected to open a way to identify active ingredients and to reveal the action mechanisms of herbs at system-level, which is helpful to illustrating the scientific basis of herbs (Li et al., 2014a; Lv et al., 2014; Zhang et al., 2014). Reduning Injection (RDN) is a traditional Chinese medicine preparation, which was developed from an herbal formula that consisted of Lonicera Japonica Thunb. (Jinyinhua), Gardenia Jasminoides Ellis (Zhizi) and Artemisia Annua L. (Qinghao). It has been widely used to treat various diseases in clinic such as upper respiratory tract infections (URTIs) (Liu, 2014), fever and inflammation caused by viral infection (influenza viruses, respiratory syncytial virus, EV71, Dengue) (Li et al., 2014b, 2013; Zhang et al., 2013b). In previous work (Zhang et al., 2014), network pharmacology was employed to identify potential compounds and to uncover the mechanism of RDN on URTIs. The results revealed that the candidate active compounds of RDN could not only regulate mitogen

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activated protein kinase (MAPK) pathways via interacting MEK1, c-jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38 proteins, but also inhibit some key enzymes such as cyclooxygenase (COX), inducible NO synthetase (iNOS). Moreover, the process played an important role in the production of chemokines, inflammatory cytokines (interleukin-1β (IL-1β), interleukin-6 (IL-6), prostaglandin E2 (PGE2), nitric oxide (NO)) when the host was attacked by respiratory virus. Previous animal research indicated that RDN had antipyretic effect on fever in rabbit and inhibited inflammation by reducing the levels of IL-6, IL-1 and PGE2 in rat (Wang et al., 2013). According to these hints on the molecular mechanism of RDN, the molecular mechanism of RDN was clarified by ELISA, Western Blot, EMSA, immunofluorescence assay and Enzyme assay. The results would provide further reasonable practice of RDN in clinical use, and it might be a strategy to investigate the mechanism of action of herbs.

2. Materials and methods

2.3. Cell culture and sample preparation The murine macrophage RAW264.7 cell lines were cultured in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in humidified atmosphere containing 5% CO2, and 95% air. The stock solution (200 mg/mL) of RDN was dissolved in DMSO. In order to quantify and administer accurately, RDN powder was prepared by lyophilize action for all the cell assays and was dissolved in DMSO before administration. 2.4. Cell viability assay RAW264.7 cells were seeded in a 96-well plate at a density of 1
104 cells/well overnight. After treated with various concentrations of RDN (25, 50, 100, and 200 mg/mL) for 24 h, the cells were added with 20 μL MTT (5 mg/mL) each well and incubated for another 4 h. The supernatant was then removed and 150 ml DMSO was added to each well. The optical absorbance at 490 nm was measured by a microplate reader (Molecular Devices, Menlo Park, USA).

2.1. Chemicals and reagents RDN was supplied by Kanion Pharmaceutical Co. Ltd. (Lianyungang, China). Geniposidic Acid, Neochlorogenic Acid, Chlorogenic Acid, Cryptochlorogenic Acid, Caffeic Acid, Geniposide, Secoxyloganin, Isochlorogenic Acid B, Isochlorogenic Acid A, Isochlorogenic Acid C (Z98%) were purchased from National Institute for Food and Drug Control (Nanjing, China). HPLC grade methanol was purchased from Merck (Darmstadt, Germany), and deionized water was purified using the Milli-Q system (Millipore, Bedford, MA, USA). The murine macrophage RAW264.7 cell lines were purchased from Chinese Academy of Medical Sciences (Beijing, China). Mouse tumor necrosis factor-α (TNF-α), IL-1β and IL-6 enzyme linked immunosorbent assay (ELISA) kits were from eBioscience (Vienna, Austria). PGE2 ELISA kits were from Enzo Life Science (Farmingdale, NY, USA). Antibodies against iNOS, COX-2, p65, ERK1/2, JNK, p38, p-p65, p-ERK1/2, p-p38 and p-JNK were obtained from Cell Signaling Technology (Beverly, MA, USA). Horse radish peroxidase-conjugated secondary antibodies, DyLight 488conjugated secondary antibody and primary antibody against βactin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dulbecco's modified Eagle's medium (DMEM) was produced by Kaiji Biotechnology (Nanjing, China) and fetal bovine serum (FBS) was produced by Sijiqing (Hangzhou, China). Lipopolysaccharide (LPS, Escherichia coli O55:B5), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), cell culture grade DMSO and 4′,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich (St. Louis, MO, USA). 2.2. LC-MS analysis of the compounds of RDN The compounds of RDN were identified by using an Agilent Series 1290/6538 ultra-high performance liquid chromatograph/ QTOFMS (Agilent Technologies, Palo Alto, CA). The chromatographic separation of RDN was achieved by an Agilent Eclipse Plus C18 RRHD (1.8 μm, 2.1
100 mm2). The mobile phase was deionized water (0.1% formic acid, v/v) and methanol with a flow rate of 0.5 mL/min. The elution program was listed as follows: 0– 10 min, 12–30% methanol, 10–14 min, 30% methanol, 14–20 min, 30–49% methanol, 20–25 min, 49–100% methanol and 25–30 min, 100% methanol. Agilent 6538 Q-TOF mass spectrometer with electrospray ionization interface was used in LC-MS method. MS conditions were as follows: negative ion mode, drying gas N2, 8 L/ min, temperature 350 °C, pressure of nebulizer 40 psig and capillary voltage 3500 V, scan range 100–3000 u.

2.5. Measurement of NO RAW264.7 cells (2
105 cells/well) were plated into 96-well plates overnight. The cells were then treated with various concentrations of RDN (50, 100, and 200 mg/mL) for 24 h at the presence or absence of 1 μg/mL LPS. In the supernatants, NO level was detected by Griess reaction (Luo et al., 2010). In brief, 100 μL of Griess reagent (1% sulfanilamide and 0.1% naphthylenediamine in 2.5% phosphoric acid) was mixed with an equal volume of the supernatant. The optical absorbance was measured at 540 nm after incubation in dark for 10 min. 2.6. Measurement of cytokine levels RAW264.7 cells were seeded in a 24-well plate at a density of 5
104 cells /well for PGE2 and TNF-α, as well as 1
105 cells/well and 2.5
105 cells/well for IL-6 and IL-1β, respectively. After incubated for 24 h, RAW264.7 cells were pretreated by RDN (50, 100, and 200 mg/mL) for 1 h, and then stimulated with LPS (1 μg/mL) for 24 h. The levels of PGE2, TNF-α, IL-6 and IL-1β in the supernatants were quantified using ELISA kits according to the manufacturer’s instruction. 2.7. Western blot analysis RAW264.7 cells were seeded in 6-well plates. The cells were pretreated with RDN at various concentrations of 50, 100 and 200 μg/mL for 3 h and stimulated with LPS (1 μg/mL) for 20 min. Then cells were homogenized in lysis buffer for 10 min. After determined by bicinchoninic acid (BCA) assay (Sigma Aldrich, St. Louis, MO, USA), the prepared protein extracts were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked at room temperature for 1 h with 5% non-fat dry milk in PBS buffer, and then washed five times in PBS containing 0.1% Tween 20 (PBST) for 5 min. The blots were sequentially incubated with specific primary antibodies (iNOS, COX-2, p65, ERK1/ 2, JNK, p38, p-p65, p-ERK1/2, p-P38, p-JNK and β-actin) in PBST containing 3% bovine serum albumin (BSA) overnight at 4 °C. The blots were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The bands were visualized using film exposure with enhanced chemiluminescence detection reagents (Pierce, Rockford, IL, USA).

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2.8. Nuclear factor-κB (NF-κB) nuclear translocation assay The nuclear translocation of NF-κB p-p65 was observed by an immunofluorescence assay (Zhang et al., 2012). Briefly, RAW264.7 cells were cultured in 96-well plates (1
104 cells/well) and pretreated with RDN (50, 100 and 200 μg/mL) for 3 h prior to incubation with LPS for 20 min. After fixed with a 4% paraformaldehyde for 20 min at 4 °C, permeabilized with 0.3% Triton X-100 for 20 min, and blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, the cells were incubated with primary anti-p-p65 antibody for 2 h at room temperature, followed by DyLight 488-conjugated secondary antibody. The cells were washed with PBS, and then were incubated in DAPI solution (10 μg/mL) for 10 min in the dark (Chen et al., 2014b; Jin et al., 2010). The live cell images were visualized by using a confocal laser scanning microscope (BIO-RAD, Hemel Hempstead, UK).

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RDN (62.5, 125, 250, 500, 1000, 2000 and 4000 ng/mL) or solvent 2.5 μL, assay buffer 35 μL, heme 2.5 μL, and COX-2 2.5 μL were mixed and incubated for 120 min. Chemiluminescent substrate 10 μL and AA 5 μL were added to the reaction at room temperature, and immediately read in a luminoerter microplate reader (Molecular Devices, Menlo Park, USA) for 5 s. The peroxidative activity of COX-2 was determined by a specific chemiluminescent substrate. The inhibitory potency (IC50) of RDN on the COX-2 was calculated by GraphPad Prism 5.0. 2.11. Statistical analysis All results were expressed as mean 7 S.E.M. Statistical differences were analyzed by one-way analysis of variance (ANOVA) and Student's t test. Differences were considered to be significant when the values were of pr0.05.

2.9. Electrophoretic motility shift assay (EMSA) 3. Results RAW264.7 cells were cultured in 100 mm dishes (2
106 cells/ dish) and grown in complete DMEM until confluent. The cells were pretreated with RDN (50, 100 and 200 μg/mL) for 3 h prior to incubation with LPS for 20 min. After being washed once with cold PBS, the cells were scraped off and centrifuged to collect the pellets. Then the nuclear extracts were prepared according to the methods described by Wang et al. (2007). The NF-κB DNA binding activity assay was performed as previously described (Duvoix et al., 2004). Briefly, oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′, IDTDNA Technologies; Coralville, IA) was synthesized as a probe which was labeled with biotin for the gel retardation assay. 10 μg nuclear extract protein and biotinylated DNA were incubated for 20 min in 10 μL buffer (0.1 M Tris, pH 7.5, 0.5 M KCl, 10 mM dithiothreitol (DTT)). The reaction mixture was separated by a 5% nondenaturing polyacrylamide gel, and then transferred to nylon membranes. The biotinylated DNA was detected by using a LightShift chemiluminescent EMSA kit (Thermo scientific, Rockford, IL, USA). 2.10. COX-2 enzyme activity assay The COX-2 inhibitory assay was carried out by using a COX Activity Kit (Enzo Life Science, Farmingdale, NY, USA)) according to the instructions. The ovine COX-2 and arachidonic acid (AA) not contained in the kit were supplied by Caymen Chemicals. Briefly,

3.1. Identification of the components in RDN The peaks were identified by comparing their retention time and accurate molecular weight to standard substances. The LC-MS analysis of RDN showed that most of these constituents were classified into two structural groups, polyphenol compounds and iridoids (Fig. 1). 3.2. Effect of RDN on the viability of RAW264.7 cells The effect of RDN on the viability of RAW264.7 cells was determined by MTT assay. RDN did not display cytotoxicity at the indicated concentrations (Table 1). Thus, the effects of RDN were observed with concentration at 200 mg/mL during the experiment. The cells were treated with the indicated concentrations of RDN. Cell viability was measured by MTT assay. The results were expressed as mean 7 SD from three independent experiments. 3.3. Inhibition of RDN on NO, PGE2 and inflammatory cytokines production To assess the anti-inflammatory effects of RDN, the ability to inhibit LPS-induced inflammatory response in RAW264.7 cells was investigated. The DMSO vehicle without LPS was considered as the

Fig. 1. LC-MS analysis of the compounds from RDN. Total ion chromatograms of RDN are displayed. (1) Geniposidic Acid, (2) Neochlorogenic Acid, (3) Chlorogenic Acid, (4) Cryptochlorogenic Acid, (5) Caffeic Acid, (6) Geniposide, (7) Secoxyloganin, (8) Isochlorogenic Acid B, (9) Isochlorogenic Acid A, (10) Isochlorogenic Acid C.

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Table 1 Effect of RDN on the viability on RAW264.7 cells. Concentration (μg/mL)

Cell viability

– 12.5 25.0 50.0 100.0 200.0

1007 0.00 99.737 2.08 99.60 7 2.31 98.60 7 1.69 98.727 2.29 98.85 7 2.95

control group for all the experiments. As showed in Fig. 2, RDN dramatically reduced PGE2 production in LPS-induced RAW264.7 cells, and the highest inhibitory activity was up to 92% (Fig. 2B). LPS-induced IL-1β and IL-6 levels were significantly inhibited by RDN in a dose-dependent manner (Fig. 2C and D) when compared to their corresponding controls. Likewise, RDN showed the highest inhibitory activity with reduction of LPS-induced NO levels by 38% (Fig. 2A). However, it has also been observed that RDN displayed a slight decline in TNF-α production (data not showed). 3.4. RDN inhibited LPS-induced expression of iNOS and COX-2 Among the above cytokines, NO and PGE2 production were reported to be dependent on iNOS and COX-2 enzyme, respectively

(Chen et al., 2014b; Zhao et al., 2014). Thus, the expression of the two proteins in LPS-induced RAW264.7 cells was examined by using Western Blot analysis. LPS (1 μg/mL) significantly increased the protein expression of iNOS and COX-2. However, RDN dramatically suppressed the up-regulation of iNOS and COX-2 protein in a dose-dependent manner (Fig. 3). It suggested that the suppressive activities of RDN on NO and PGE2 were related to the inhibition of the two protein expressions. 3.5. Effects of RDN on LPS-induced phosphorylations of MAPKs in RAW264.7 cells The above results showed that RDN significantly inhibited LPSinduced inflammatory cytokines (IL-1β, IL-6) and inflammatory mediators (NO, PGE2), as well as the expression of two enzymes. It has been widely reported that activation of NF-κB and MAPK (such as ERK1/2, JNK, and p38) played a critical role in the signaling pathways that induced inflammatory cytokines and iNOS, COX-2 protein expression in RAW264.7 cells treated with LPS (Shao et al., 2013; Sun et al., 2012). Thus, the effects of RDN on the phosphorylation of MAPKs were firstly observed by Western Blot assay. Activations of MAPKs were detected after 20 min exposure to LPS in RAW264.7 cells. When the cells were pretreated with RDN for 3 h, ERK1/2, JNK and p38 phosphorylations were significantly

Fig. 2. Effects of RDN on the production of inflammatory mediators in LPS-induced RAW264.7 cells. The cells were treated with the LPS (1 μg/mL) alone or LPS plus various concentrations of RDN (50, 100, 200 μg/mL) for 24 h. The production of NO in the medium was assayed using Griess reagent and the amounts of secreted PGE2, IL-6 and IL-1β in the culture supernatant were quantified by ELISA. Celecoxib and Luteolin were positive controls. Data were expressed as means 7 SD from three independent experiments. ##p o 0.01, compared with control. np o0.05, nnpo 0.01, compared with model.

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Fig. 3. Effects of RDN on COX-2 and iNOS protein expression in LPS-stimulated RAW264.7 cells. The cells were treated with the indicated concentrations of RDN at the presence or absence of LPS (1 μg/mL) for 24 h. The protein levels of COX-2 and iNOS were determined by Western Blot with specific antibodies. The gray values of protein expression were quantified by Image Tool 3.0. The figure showed was the representative of three independent experiments.

inhibited in a concentration-dependent manner (Fig. 4). These results indicated that RDN suppressed the phosphorylations of MAPKs, and that the anti-inflammatory activity of RDN was likely to the blockade of MAPKs pathway. 3.6. Effect of RDN on phosphorylation of p65 in LPS-induced RAW 264.7 cells Besides MAPKs pathway, the activity of RDN on NF-κB was also investigated in LPS-stimulated RAW264.7 cells, which was also a key regulator in the production of inflammatory cytokines and inflammatory mediators. When p65 was phosphorylated at specific residues, it could translate into the nucleus to bind the promoter regions of many cytokines genes (Hanada and Yoshimura, 2002; Jung et al., 2010). As showed in Fig. 5, treatment with LPS significantly increased the phosphorylation of p65 in RAW264.7 cells. However, in the presence of RDN, the phosphorylation of p65 was significantly inhibited in a dose-dependent manner. 3.7. Effect of RDN on translocation of NF-κB in LPS-induced RAW 264.7 cells To further investigate the effects of RDN on the regulation of the cytokines, immunofluorescence assay and EMSA were employed to observe the inhibition of RDN on the translocation of NF-

Fig. 4. Effect of RDN on LPS-induced activation of MAPKs in RAW264.7 cells. Cells were preincubated with different concentrations of RDN (50, 100, 200 μg/mL) for 3 h, then stimulated with LPS (1 μg/mL) for 20 min. Total protein extracts were prepared and subjected to 10% SDS-PAGE. The protein levels of ERK1/2, JNK, p38, p-ERK1/2, p-JNK and p-p38 were detected by Western Blot with specific antibodies. p-ERK1/2, p-JNK and p-p38: phosphorylation of ERK1/2, JNK, p38; T-ERK1/2, T-JNK and T-p38: total ERK1/2,JNK and p38. The gray values of protein expression were quantified by Image Tool 3.0. The figure showed was the representative of three independent experiments.

κB and NF-κB-DNA binding. The inhibition of LPS-induced translocation of NF-κB by RDN was detected by immunofluorescence assay in RAW264.7 cells. In unstimulated cells, p-p65 was almost absent. Treatment by LPS led to p-p65 significantly translocation to the nucleus. The level of p-p65 in the nucleus was dramatically reduced by pretreatment with RDN at the concentration of 200 μg/mL (Fig. 6A). The inhibition of LPS-induced NF-κB-DNA binding activity was detected by EMSA. The NF-κB-DNA complex was significantly increased after stimulation with LPS for 20 min, while a concentration-dependent reduction of NF-κB-DNA complexes was observed in cells pretreated with RDN (Fig. 6B).

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Fig. 7. Effects of RDN on COX-2 activity. Results were expressed as means 7 SD of three independent experiments.

3.8. RDN inhibited LPS-induced COX-2 activity in RAW264.7 cells In addition, we investigated the effect of RDN on COX-2 enzyme as shown in Fig.7. RDN had good activity against COX-2, with an apparent IC50 ¼328.2 ng/mL. These findings suggested that RDN not only was active at the level of COX-2 protein synthesis but also blocked the activity of COX-2 enzyme.

4. Discussion

Fig. 5. Effect of RDN on LPS-induced activation of NF-κB in RAW264.7 cells. Cells were preincubated with different concentrations of RDN (50, 100, 200 μg/mL) for 3 h, then stimulated with LPS (1 μg/mL) for 20 min. Total protein extracts were prepared and subjected to 10% SDS-PAGE. The protein levels of p-p65, total p65 and β-actin were detected by Western Blot with specific antibodies. p-p65: phosphorylations of p65; T-p65: total p65. The gray values of protein expression were quantified by Image Tool 3.0. The figure showed was the representative of three independent experiments.

Chinese medical herbs are commonly used to treat complex diseases in clinic (Gao et al., 2013). However, it is difficult to systematically investigate the molecular mechanism of action of herbs due to its multi-component and multi-target properties. Likely, RDN, a widely used traditional Chinese injection, encountered the issue. Recently, network pharmacology is introduced to illustrate the molecular mechanism of herbs (Hu et al., 2014; Xu et al., 2014; Yang et al., 2013). In our previous study, the molecular mechanism of RDN was predicted to directly target the key proteins in the respiratory viral life cycle and indirectly regulate cell signaling pathways through combining with multi-target

Fig. 6. Effect of RDN on translocation of NF-κB in LPS-induced RAW 264.7 cells. (A) In the immunofluorescence assay for NF-κB p-p65 nuclear translocation, cells were pretreated with RDN for 3 h, and then LPS (1 μg/mL) was treated for 20 min. Images, from left to right, represented the nucleus, the p-p65 protein and merged images with nucleus and p-p65 protein. Nuclei were stained by DAPI (blue); p-p65 was detected by DyLight 488-labeled immunostaining (green). (B) RAW264.7 cells were pretreated with RDN at different concentrations of 50, 100 and 200 μg/mL for 3 h, and then exposed to LPS (1 μg/mL) for another 20 min. Nuclear extracts were prepared and then assayed by EMSA on a 5% polyacrylamide gel with a biotin-labeled oligonucleotide containing the NF-κB consensus sequence. Binding competition assays were performed with a 100-fold excess of unlabeled NF-κB oligonucleotide as the competitor (cold NF-κB). The figure showed was the representative of three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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ingredients by network pharmacology (Zhang et al., 2014). Based on the results, a series of macrophage cell-based assays were applied to validate the predicted mechanism of RDN on URTIs. Inflammation is one of the first host defense systems to infection, while the symptoms of URTLs are mainly attributed to the inflammatory response of the body to viral infection (Zhang et al., 2014). The process also involves various cytokines including proinflammatory mediator (PGE2, IL-1β, IL-6, TNF-α, NO) and chemokines, as well as some key enzymes (iNOS and COX-2), which was regulate by a series of signaling cascades such as MAPKs and NF-κB (Akira, 2003; Cheung et al., 2013; Iontcheva et al., 2004). Meanwhile, the treatment of RDN on URTIs was mainly attributed to the anti-inflammatory effects. Therefore, the molecular mechanism of RDN on the inflammatory reaction of URTLs was explored. RAW264.7 stimulated by LPS is a classic model of inflammation. After activated by LPS, RAW264.7 cells could release pro-inflammatory cytokines/mediators (Luo et al., 2010). Of these, IL-1β, IL-6, PGE2 are regarded as endogenous inflammatory mediators, which is associated with fever. TNF-α plays a key role in inflammation due to reactions by activating T cells and macrophages through upregulating other pro-inflammatory cytokines and endothelial adhesion molecules, which enhance the recruitment of leukocytes to the site of inflammation (Andreakos et al., 2002; Chen et al., 2014a). Overproduction of NO triggers a number of cell functions including production of numerous pro-inflammatory mediators, signal transduction and apoptosis of local cells contributing to the tissue damage (Chen et al., 2014a). In this work, RDN dramatically reduced the production of NO, PGE2, IL-6 and IL1β at a dose-dependent manner in LPS-induced macrophages, and the results revealed that RDN possessed the anti-inflammatory effect through inhibiting the production of inflammatory mediators. This was also consistent with previous animal experiments (Wang et al., 2013). Network analysis (Zhang et al., 2014) predicted that RDN could regulate MAPK pathway (ERK1/2, p38 and JNK), and further influence the expression of chemokines and cytokines (Chen et al., 2014a). Likely, MAPKs and NF-κB are well known to two pivotal pathways in the expression of inflammatory mediators (Choi et al., 2012; Corriveau and Danner, 1993). In addition, It has been reported that the expression of iNOS and COX-2, as two key enzyme to produce NO and PGE2, was regulated by MAPKs and/or NF-κB, when RAW264.7 cells were stimulated by LPS (Choi et al., 2011). In this work, we investigated the effect of RDN on the expression of iNOS. RDN decreased the iNOS protein expression in a dose-dependent manner. This indicated that the inhibition of RDN on NO production was partly due to the inhibition of iNOS protein expression. We also discovered that RDN suppressed the COX-2 protein expression as well as the activity of COX-2 in a dose-dependent manner. Our findings indicated that the suppression of RDN on PGE2 was directly toward the protein expression and the activity of COX-2. MAPK pathways are activated to induce migration and accumulation of leukocytes, as well as production of cytokines and proinflammatory mediators (Hsu et al., 2013). Thus, we examined the effect of RDN on the LPS-induced phosphorylation of MAPKs in RAW264.7 cells. The results revealed that treatment of RDN significantly suppressed the phosphorylation of ERK1/2, JNK and p38, which were responsible for the inhibition of inflammatory mediator production. The results suggested that suppression of phosphorylation of MAPKs might be involved in the treatment of RDN on URTIs. NF-κB plays an crucial role in inflammation through its ability to induce the transcription of pro-inflammatory genes (Fan et al., 2013). Many natural and synthetic agents, which were reported to inhibit NF-κB activation, target different steps in the NF-κB signal

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pathway including phosphorylation, ubiquitination, degradation, nuclear translocation and DNA binding (Zhang et al., 2012). p-p65 at serine 536 (Ser536) has been showed to be important in initiating transcription of NF-κB in macrophages, and the phosphorylation on Ser536 increased the p65 transcription activity (Chen et al., 2014a). In our study, the level of p-p65 (Ser536) significantly increased by the stimulation of LPS in 20 min. However, RDN could inhibit the level of LPS-induced p-p65 on serine 536 in RAW264.7 cells in a concentration-dependent manner. In parallel, the nuclear translocation and binding of NF-κB were investigated. We observed that the p-p65 protein was significantly translocated into the nucleus after 20 min of LPS treatment, and this translocation was suppressed by the preincubated RDN. The EMSA assay further confirmed that RDN inhibited p65 DNA binding activity. All these results suggested that RDN might inhibit the transcriptional initiation of NF-κB via inhibition of the phosphorylation of p65 on serine 536, the translocation of NF-κB to the nucleus and the binding ability of NF-κB to DNA. A network pharmacology approach was employed to uncover the molecular mechanism of Chinese medical herbs by Zhang et al., 2014. It was revealed that RDN could regulate MAPK pathways via interacting MEK1, JNK, ERK, p38 proteins as well as some key enzymes (COX and iNOS). In vitro experiments verified that RDN exactly inhibited the production of inflammatory mediators to treat URTIs via down-regulating the activation of MAPK and NFκB signaling pathways as well as the COX-2 and iNOS enzymes. Considering the successful experimentally validation of theoretically predictions on RDN, it could provide a strategy to uncover the mechanism of action of herbs which was helpful to the modernization and internationalization of Chinese medical herbs. Generally, herbal medicine includes hundreds of pharmacological compounds and interacts with several cellular targets to treat complex diseases. The underlying mechanism is normally not clear (Zhang et al., 2014). It was reported that polyphenol compounds and iridoids were the main components in RDN, including Chlorogenic Acid, Caffeic Acid, Isochlorogenic Acid B, Isochlorogenic Acid A, Isochlorogenic Acid C,Geniposide and so on (Wang et al., 2014; Zhang et al., 2014). Recent studies demonstrated that Chlorogenic Acid, Isochlorogenic Acid A, Isochlorogenic Acid B and Isochlorogenic Acid C could attenuate the release of PGE2 and IL-6 in macrophage (Li et al., 2013; Shan et al., 2009). Caffeic Acid suppressed LPS-stimulated pro-inflammatory response in RAW264.7 cells (Bufalo et al., 2013; Uwai et al., 2008). However, to date, no scientific study has been done on the combination effects of the compounds in RDN. Nevertheless, the interaction among the compounds is of great interest in present pharmacology research. Hence, it is valuable to study the interaction among compounds in RDN in the future. It may support the current quality control standard of RDN and reveal new light on the further mechanisms and the underlying material basis of RDN on URTIs.

5. Conclusion In conclusion, the current study investigated the molecular mechanism of RDN on URTIs based on the previous predictions of network pharmacology. Consistent with the previous predictions, our experiment results validated that RDN inhibited the production of inflammatory mediator and the activation of macrophages to treat URTIs via down-regulating the activation of MAPK and NFκB signaling pathways (Fig. 8). In view of the above findings, one question was raised about the relationship between MAPK and NFκB pathways. Some reports showed that the activation of NF-κB was dependent on MAPK. However, other reports showed negative regulation between MAPK and NF-κB (Ahmed et al., 2006; Jung

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Fig. 8. Putative RDN inhibition pathway in macrophage-mediated inflammatory signaling.

et al., 2005; Shao et al., 2013). We therefore wonder whether the inactivation of NF-κB by RDN is dependent on the inactivation of MAPKs. The relationship is still unclear and needs further investigation. Furthermore, the interactions of active compounds in RDN can be further applied to elucidate the further underlying material basis of RDN. Therefore, it can be concluded that integrating network pharmacology and in vitro offered an alternative way to illustrate the molecular mechanism and effective substances of herbs to impulse the development of Chinese medical herbs.

Acknowledgments This work was supported by the National Science and Technology Major Project ‘Key New DrugCreation and Manufacturing Program’ (Grant no. 2013ZX09402203). We are grateful to Yanjing Li, Zhenzhen Zhang, Weihua Wang and Guoxiang Wang for excellent technical assistance throughout the project.

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