Mixtures of recombinant growth factors inhibit the production of pro-inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 cells by inactivating the ERK and NF-κB pathways

  • Authors:
    • Yonghee Lee
    • Dohyun Lee
    • Kyotan Koo
    • Jay Lee
    • Yi Seop Song
    • Ho Sang Yoon
    • Yoo Mi Choi
    • Beom Joon Kim
  • View Affiliations

  • Published online on: May 28, 2014     https://doi.org/10.3892/ijmm.2014.1790
  • Pages: 624-631
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Abstract

Growth factors are important for regulating a variety of cellular processes and typically act as signaling molecules between cells. In the present study, we examined the mechanisms underlying the inhibitory effects of mixtures of recombinant growth factors (MRGFs) on nitric oxide (NO) and pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. We also examined whether these effects are mediated through the mitogen‑activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signal transduction pathways. NO production was assessed by measuring nitrite acucmulation using the Greiss reaction. Cytokine concentrations were measured using respective ELISA kits for each cytokine. Our results revealed that the MRGFs significantly attenuated the LPS-induced production of pro-inflammatory cytokines and NO in a dose-dependent manner. To elucidate the mechanisms underlying the inhibitory effects of MRGFs, we examined the effects of the LPS-induced phosphorylation of MAPKs and the activation of the NF-κB signaling pathway on the stabilization of NF-κB nuclear translocation and inhibitory factor-κB (IκB) degradation. Western blot analysis was performed to determine the total and phosphorylated levels of ERK, as well as the nuclear translocation of NF-κB, and IκB phosphorylation and degradation. Our results demonstrated that treatment with MRGFs resulted in a reduction in the phosphorylation of the ERK and NF-κB signaling pathways, whereas the phosphorylation of JNK and p38 was not affected. Taken together, our results suggest that MRGFs inhibit the production of pro-inflammatory cytokines and NO by downregulating inducible NO synthase gene expression and blocking the phosphorylation of the ERK and NF-κB signaling pathways. These findings may provide direct evidence of the potential application of MRGFs in the prevention and treatment of inflammatory diseases.

Introductions

Various growth factors, also known as cytokines or steroid hormones, regulate a variety of cellular processes, as well as the development of tumors, inflammation and wound healing. These growth factors differ from well-known polypeptide hormones, such as insulin and adrenocorticotropic hormone, not only in the response elicited, but also in the mode of delivery from the secreting to the responding cell. Growth factors also have different cell-type specificities and different functions (14). For example, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and keratinocyte growth factor (KGF) are thought to play a role in wound healing and the regulation of class II major histocompatibility complex, macrophages and lymphocytes. During this process, inflammation initiates healing. The regulation of inflammation is so important that homeostatic mechanisms have evolved to control this process (1,59). Insulin-like growth factor (IGF)-1 displays pleiotropic properties, including the ability to promote cellular proliferation and differentiation, as well as processes involved in metabolism/hypertrophy, such as nutrient transport, energy storage, gene transcription and protein synthesis (10). Superoxide dismutase (SOD), one of the most important antioxidants, is an enzyme that catalyzes the dismutation of superoxide (O2) into oxygen and hydrogen peroxide and serves as a key antioxidant in cells (11). The aforementioned growth factors, including EGF, bFGF, KGF, IGF-1 and SOD, act by binding to their respective receptor tyrosine kinases, followed by the downstream signaling and activation of the protein kinase C, AKT and ERK signaling pathways. In particular, mitogen-activated protein kinase (MAPK) signaling, including ERK, plays a critical role in innate immune responses (8,11,12).

MAPKs are serine/threonine-specific protein kinases (1316). They are important upstream factors that lead to the activation of nuclear factor-κB (NF-κB) (6,17). They are mainly composed of three subfamily members: ERK, JNK and p38. The MAPK signaling pathway regulates a wide variety of cellular events, including complex cellular programs, such as differentiation, proliferation, apoptosis and processes involved in immune response (18). The phosphorylation of MAPKs modulates the expression of a variety of genes involved in immune and inflammatory responses, including inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2).

In addition, NF-κB nuclear translocation, as well as inhibitory factor-κB (IκB) phosphorylation and degradation are important inflammatory factors. The expression of pro-inflammatory cytokines is mainly regulated by the NF-κB pathway (19). In unstimulated cells, NF-κB resides in the cytoplasm as an inactive NF-κB-IκB complex (20).

Although there are several studies focusing on the growth factors and pathways associated with inflammation (8,12,21), the mechanisms underlying the inflammatory response, particularly the response to a mixture of the five aforementioned growth factors, have not been investigated to date.

In this study, the anti-inflammatory effects of mixtures of recombinant growth factors (MRGFs) on the generation of several chemokines, cytokines and enzymes involved in the inflammatory process, such as inducible iNOS, COX-2, interleukin (IL)-1β, IL-6, IL-10, IL-12p40, granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and NO in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, were investigated. We also investigated whether MRGFs affect the LPS-stimulated ERK and NF-κB signaling pathways.

Materials and methods

Materials

Mixed in the same ratio, recombinant human EGF, recombinant human bFGF, recombinant human KGF, recombinant human IGF-1 and recombinant human SOD were provided by Nutrex Technology Co., Ltd. (Seoul, Korea). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and LPS were purchased from Sigma Chemical Co. (St. Louis, MO, USA); Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FBS), trypsin EDTA, phosphate-buffered saline (PBS) and penicillin/streptomycin were purchased from WelGENE Co. (Daegu, Korea). Antibodies specific to COX-2, and GAPDH were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); iNOS was purchased from Pharmingen BD Biosciences (San Diego CA, USA). Antibodies against phosphorylated (p-)ERK, ERK, p-JNK, JNK, p-p38, p38, NF-κB, p-IκB, IκB and lamin B1 were purchased from Cell Signaling Technology (Berverly, MA, USA). PD98059 and IKK inhibitor VII were purchased from Sigma Chemical Co. and Calbiochem (San Diego, USA), respectively. The secondary antibodies, anti-goat IgG, anti-mouse IgG and anti-rabbit IgG were purchased from Vector Laboratories (Burlingame, CA, USA). The iNOS, COX-2 and GAPDH oligonucleotide primers were obtained from Bioneer, Inc. (Seoul, Korea).

Culture of RAW 264.7 cells

The RAW 264.7 murine macrophage cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2 in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 10 μg/ml streptomycin.

Cell viability

Cell viability was assessed by MTT assay that was performed using a slightly modified version of the method described in the study by Twentyman and Luscombe (22). Seeded RAW 264.7 cells in a 12-well plate were treated with LPS and MRGFs after 24 h of incubation. The supernatant was removed and MTT solution was added to each well followed by further incubation for 4 h. Subsequently, 1.5 ml of dimethyl sulfoxide (DMSO) were added to each well to solubilize any deposited formazon. Following incubation for 10 min at room temperature, the optical density (OD) was determined at 540 nm on an ELISA plate reader (Thermomax, Molecular Devices, Sunnyvale, CA, USA).

NO assay

NO production was assessed by measuring nitrite accumulation. After the RAW 264.7 cells were seeded in a 12-well plate, LPS (100 ng/ml) and MRGFs at the indicated concentrations (0.01, 0.1, 1 and 10 μg/ml) were added to the culture medium, followed by incubation for 24 h. Cells treated with LPS only were used as controls. The concentration of nitrite in the spent culture medium was determined using the Griess reaction. Subsequently, 100 μl of supernatant from each well were transferred to a 96-well plate and mixed with 100 μl of Griess reagent (Thermo Fisher Scientific, Wilmington, DE, USA) in a separate 96-well plate. Following incubation for 10 min at room temperature, OD was determined at 540 nm on an ELISA plate reader.

Cell lysate preparation and western blot analysis

Treated whole cell extracts were lysed in RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 0.1% sodium dodecyl sulfate (SDS) (Sigma Chemical Co.) and a protease inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN, USA) for the preparation of cellular extracts. Cytoplasmic and nuclear proteins were extracted using buffer A [HEPES 10 mmol/l, pH 7.9, KCl 10 mmol/l, 2 mM EDTA, phenylmethylsulfonyl fluoride (PMSF) 1 mmol/l, 1 mM EGTA, dithiothreitol (DTT) 1 mmol/l, aprotinin 1 mg/l and protease inhibitor cocktail tablet 5 mg/ml] and buffer B (HEPES 20 mmol/l, pH 7.9, NaCl 420 mmol/l, edetic acid 0.1 mmol/l, egatazic acid 0.1 mmol/l, PMSF 1 mmol/l, DTT 1 mmol/l, aprotinin 1 mg/l and protease inhibitor cocktail tablet 1 mg/ml), as previously described (23). The protein concentration of the extracts was estimated using Bradford reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with bovine serum albumin as the standard, as previously described (24).

For western blot analysis, cell lysates containing 20 μg of proteins were resolved by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were washed with Tris-buffered saline (10 mM Tris, 150 mM NaCl) containing 0.05% Tween-20 (TBST) and blocked in TBST containing 5% non-fat dried milk. The membranes were further incubated with respective specific antibodies. The membranes were continuously incubated with appropriate secondary antibodies coupled to horseradish peroxidase and developed using enhanced chemiluminescence (ECL) western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions following treatment and quantified using an ND-1000 spectrophotometer (Thermo Fisher Scientific) with a ratio of absorbance at 260 nm. cDNA was synthesized with 2 μg of denatured total RNA in a final volume of 20 μl of buffer containing MgCl2, KCl, dNTPs and oligo(dT) reverse transcriptase by incubation at 42°C for 60 min. The cDNA obtained was amplified with the following primers: iNOS forward, 5′-CTA CCT ACC TGG GGA ACA CCT GGG-3′ and reverse, 5′-GGA GGA GCT GAT GGA GTA GTA GCG G-3′; COX-2 forward, 5′-CTG TAT CCC GCC CTG CTG GTG-3′ and reverse, 5′-ACT TGC GTT GAT GGT GGC TGT CTT-3′; and GAPDH forward, 5′-GCC AAA AGG GTC ATC ATC TC-3′ and reverse, 5′-GGT CCT CAG TGT AGC CCA AG-3′. Application was performed using PCR Master Mix (Takara Bio Inc., Shiga, Japan) in a total volume 20 μl. PCR cycling conditions consisted of denaturation at 94°C for 30 sec, annealing at 60°C for 1 min and extension at 72°C for 30 sec. The products were electrophoresed for 30 min at 100 V on a 1% agarose gel. Gels were visualized using the Molecular Imager® Gel Doc™ XR imaging system (Bio-Rad Laboratories, Inc.).

Real-time PCR

Total RNA and the cDNAs were generated as described above. Real-time PCR was performed with a C1000™ Thermal Cycler (Bio-Rad Laboratories, Inc.) using SYBR-Green (Takara Bio Inc.). Reactive mixtures were incubated for 40 cycles at 95°C for 15 sec, 58°C for 45 sec and 72°C for 20 sec. Gene expression was normalized to those of the housekeeping gene, GAPDH.

ELISA for the detection of cytokine production

After the RAW 264.7 cells were seeded in a 24-well plate, LPS and MRGFs were added to each well and followed by incubation for 24 h. The cytokine concentrations in the culture medium were measured using the ELISA kit for each cytokine (eBioscience, San Diego, CA, USA).

Statistical analysis

Statistical analyses were performed using SPSS version 18.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are expressed as the means ± standard deviation, as previously described (25). Data were analyzed using one-way ANOVA followed by a Duncan’s test for multiple comparison; a two-tailed value of P<0.05 was considered to indicate a statistically significant difference.

Results

Effect of MRGFs on cell viability and NO production

After examining the effects of each of the five growth factors (EGF, bFGF, KGF, IGF-I and SOD) on NO production and cell viability, we examined the effects of MRGFs on the production of NO. As none of the five growth factors decreased cell viability or NO production up to a concentration of 100 ng/ml in the LPS-stimulated RAW 264.7 cells (data not shown), we prepared MRGFs by mixing 100 ng/ml of each of the five growth factors in the same ratio. As the MRGFs did not decrease the viability of the RAW 264.7 cells up to a concentration of 10 μg/ml (data not shown), this concentration was used in the subsequent experiments. The RAW 264.7 cells were incubated with LPS (100 ng/ml) and MRGFs at concentrations of 0.01, 0.1, 1 and 10 μg/ml for 24 h, and the NO production was measured. The levels of NO production following treatment of the RAW264.7 cells with the MRGFs are shown in Fig. 1. Following treatment with 10 μg/ml of MRGFs, NO production significantly decreased in the RAW 264.7 cells compared to the LPS-stimulated control (P<0.05). These results demonstrate that MRGFs inhibit the production of NO by suppressing iNOS activity.

Inhibitory effects of MRGFs on iNOS and COX-2 protein and mRNA expression in LPS-stimulated RAW 264.7 cells

We investigated the effects of MRGFs on LPS-stimulated iNOS and COX-2 protein expression by western blot analysis and mRNA expression by RT-PCR and real-time PCR. After the cells were co-treated with LPS (100 ng/ml) and MRGFs at a concentration of 0.01, 0.1, 1 and 10 μg/ml for 24 and 6 h, we harvested the protein and mRNA samples. MRGFs at a concentration of 10 μg/ml significantly decreased iNOS and COX-2 protein and mRNA expression in the LPS-stimulated cells (Fig. 2). Therefore, the MRGFs showed a significant inhibitory effect on the production of pro-inflammatory mediators, such as iNOS and COX-2 in the LPS-stimulated RAW 264.7 cells.

Inhibitory effects of MRGFs on MAPK phosphorylation in LPS-stimulated RAW 264.7 cells

We examined the effects of MRGFs on LPS-stimulated MAPK phosphorylation in the RAW 264.7 cells. After the cells were stimulated with LPS, western blot analysis was performed to determine the total and phosphorylated levels of ERK. Compared with the control, the levels of phosphorylated ERK significantly increased after the cells were stimulated with LPS for 2 h (Fig. 3A). The cells were then co-treated with LPS (100 ng/ml) and MRGFs at a concentration of 0.01, 0.1, 1 and 10 μg/ml for 2 h. At the concentration of 10 μg/ml, the MRGFs inhibited the phosphorylation of ERK, but not that of JNK and p38 in the LPS-stimulated RAW 264.7 cells (Fig. 3B). To confirm the causal link between the inhibition of the phosphorylation of MAPKs and MRGFs, the RAW 264.7 cells were pre-treated with ERK inhibitor (PD98059, 30 μM) for 1 h. Following treatment with LPS (100 ng/ml) and MRGFs (10 μg/ml) for 2 h, we confirmed that the production of LPS-stimulated pro-inflammatory mediators, such as iNOS and COX-2, as well as ERK phosphorylation were inhibited by 30 μM/ml of PD98059 and 10 μg/ml of MRGFs (Fig. 3C).

Inhibitory effects of MRGFs on NF-κB nuclear translocation, and IκB phosphorylation and degradation in LPS-stimulated RAW 264.7 cells

The cells were co-treated with LPS (100 ng/ml) and MRGFs at a concentration of 0.01, 0.1, 1 and 10 μg/ml for 4 h. The effects of MRGFs on NF-κB nuclear translocation, and IκB phosphorylation and degradation in the cytoplasm and nucleus were then assessed by western blot analysis. Fig. 4A shows that the LPS-stimulated IκB phosphorylation and degradation was significantly inhibited by MRGFs (10 μg/ml) in the cytoplasm of RAW 264.7 cells. Moreover, the amount of NF-κB in the nucleus was markedly increased upon exposure to LPS alone, but MRGFs (10 μg/ml) inhibited the LPS-stimulated nuclear translocation of NF-κB. To confirm whether the MRGFs suppressed the activation of NF-κB, the cells were pre-treated with the upstream inhibitor of NF-κB, IKK inhibitor VII, for 2 h and exposed to LPS (100 ng/ml) and MRGFs for 4 h. We confirmed that the activation of the LPS-stimulated NF-κB pathway was inhibited by both 1 μM/ml of IKK inhibitor VII and 10 μg/ml of the MRGFs (Fig. 4B).

Inhibitory effects of MRGFs on inflammatory cytokine production in LPS-stimulated RAW 264.7 cells

Inflammatory cytokines, such as IL-1β, IL-6 and TNF-α, were released as a result of the activation of the NF-κB pathway in the LPS-stimulated RAW 264.7 cells. We have already confirmed that MRGFs inhibit the activation of the NF-κB pathway in the above-mentioned results. The effects of the MRGFs on the release of cytokines were assessed by ELISA. Table I shows the inhibitory effects of the MRGFs on the production of inflammatory cytokines (IL-1β, IL-6, IL-10, IL-12p40, GM-CSF, MCP-I and TNF-α) in the LPS-stimulated RAW 264.7 cells. Specifically, MRGFs, at a concentration of 10 μg/ml, decreased the production of IL-10, IL-12p40, GM-CSF, MCP-I and TNF-α in the LPS-stimulated RAW 264.7 cells (Fig. 5). These results indicate that MRGFs can modulate the synthesis of several cytokines involved in the inflammatory process.

Table I

Effect of MRGFs on pro-inflammatory cytokine production in LPS-stimulated RAW 264.7 cells.

Table I

Effect of MRGFs on pro-inflammatory cytokine production in LPS-stimulated RAW 264.7 cells.

Pro-inflammatorymediator (pg/ml)ControlLPS 100 ngConcentration (μg/ml) of MRGFs (administered with LPS)

0.010.1110
IL-1β7.18±0.7271.88±3.2969.76±1.3667.43±3.1872.76±3.8562.23±12.81
IL-68.27±5.171064.17±103.811055.74±51.341008.82±91.78983.42±57.75951.45±87.95
IL-1046.32±5.24402.60±24.32381.04±24.64393.50±20.91309.34±7.04a 284.58±11.80a
IL-12p40102.65±7.20882.71±36.55932.01±132.61954.55±52.16765.64±125.92 514.27±76.87a
GM-CSF295.91±27.692007.73±100.711858.30±96.601836.32±128.421500.67±461.97 959.34±288.08a
MCP-11539.87±377.353013.51±273.403224.93±238.522732.03±361.922433.15±321.58 2089.91±344.59a
TNF-α216.79±142.111981.54±98.781900.50±51.691854.94±81.89 1634.96±22.18a 1128.78±46.50a

{ label (or @symbol) needed for fn[@id='tfn1-ijmm-34-02-0624'] } The concentration of cytokines in the culture medium was measured after 24 h of incubation. Data are the means ± SD (n=3).

a P<0.05 vs. LPS-stimulated cytokine level.

{ label (or @symbol) needed for fn[@id='tfn3-ijmm-34-02-0624'] } LPS, lipopolysaccharide; IL, interleukin; TNF-α, tumor necrosis factor-α.

Discussion

The major finding of this study is that MRGFs play an important role in the LPS-stimulated inflammatory response. Previous studies (14) have demonstrated that various growth factors regulate a variety of cellular processes, such as cell growth, differentiation and proliferation, by binding to specific high-affinity cell membrane receptors. A number of studies have found a correlation between growth factors and wound healing, cancer and DNA synthesis in various cells (13,16). Nonetheless, little information is available regarding the molecular mechanisms underlying the anti-inflammatory effects of various combinations of growth factors.

The activation of macrophages plays an important role in the initiation and propagation of inflammatory responses by inflammatory mediators (26). Therefore, LPS-stimulated macrophage activation increased the production of cytokines, such as IL-1β, TNF-α, GM-CSF and NO, which is modulated by the upregulation of iNOS (27). iNOS and COX-2 are often present together, share a number of similarities and play fundamental roles in similar pathophysiological conditions, such as inflammation and cancer (28,29).

The MAPK and NF-κB signaling pathways are important in the regulation of inflammatory mediators. Members of the MAPK family, including ERK, JNK and p38, are frequently involved in LPS-stimulated inflammation and play a critical role in the regulation of cell growth and differentiation, and in the control of cellular responses to cytokines and stresses.

The transcription factor, NF-κB, controls a number of inflammatory mediators, such as iNOS, COX-2 and cytokines, and is important for immunity and inflammation (30). Previous studies have reported that the LPS-induced stimulation of NF-κB signaling activity leading to the activation of MAPK is a major mechanism underlying NO production by iNOS (31). When RAW 264.7 cells are stimulated by LPS, IκB is phosphorylated and separated from NF-κB, resulting in the translocation of NF-κB to the nucleus (32).

Cytokines can be used as markers of inflammation (33). The cytokines, TNF-α and IL-1β, are closely related to each other and share many biological activities, i.e., pyrogenicity, activation of T lymphocytes, stimulation of fibroblast proliferation and neutrophil activation (34). IL-10 is a pleiotropic cytokine that modulates the adaptive immune-related cell function. It possesses immune stimulatory properties, including the ability to activate T cells, B cells, natural killer (NK) cells and mast cells (35,36). In addition, previous studies have demonstrated that IL-6, MCP-1, GM-CSF and IL-12p40 are related inflammatory mediators (35). In particular, IL-12p40 has been reported to markedly upregulate the expression of TNF-α and induce the expression of iNOS in a dose-dependent manner (37).

In the present study, we treated LPS-stimulated RAW 264.7 cells with various concentrations of MRGFs. The aim was to elucidate the pharmacological, biological and inhibitory effects of MRGFs, which include EGF, bFGF, KGF, IGF-I and SOD, on the production of inflammatory mediators in macrophages. Our data clearly indicated that MRGFs suppressed the production of NO, iNOS and COX-2 in the LPS-stimulated RAW 264.7 cells. In addition, inhibiting the phosphorylation of ERK and NF-κB decreased the production of these inflammatory mediators. Furthermore, our data demonstrated that treatment with MRGFs decreased the production of inflammatory cytokines, such as IL-10, IL-12p40, GM-CSF, MCP-I and TNF-α, in LPS-stimulated RAW 264.7 cells.

In conclusion, MRGFs have the potential to decrease the production of inflammatory mediators, such as iNOS, COX-2, IL-10, IL-12p40, GM-CSF, MCP-I and TNF-α, by inhibiting the phosphorylation of the ERK and the activation of the NF-κB signaling pathways. These findings suggest that MRGFs may prevent inflammatory diseases by suppressing MAPK- and NF-κB-mediated inflammation.

Acknowledgements

This study was financially supported by the Ministry of Knowledge Economy (MKE) and the Korea Institute for Advancement of Technology (KIAT) through the Inter-ER Cooperation Projects.

Abbreviations:

NO

nitric oxide

MRGFs

mixtures of recombinant growth factors

MAPK

mitogen-activated protein kinase

LPS

lipopolysaccharide

NF-κB

nuclear factor-κB

IκB

inhibitory factor-κB

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August-2014
Volume 34 Issue 2

Print ISSN: 1107-3756
Online ISSN:1791-244X

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Copy and paste a formatted citation
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Spandidos Publications style
Lee Y, Lee D, Koo K, Lee J, Song YS, Yoon HS, Choi YM and Kim BJ: Mixtures of recombinant growth factors inhibit the production of pro-inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 cells by inactivating the ERK and NF-κB pathways. Int J Mol Med 34: 624-631, 2014
APA
Lee, Y., Lee, D., Koo, K., Lee, J., Song, Y.S., Yoon, H.S. ... Kim, B.J. (2014). Mixtures of recombinant growth factors inhibit the production of pro-inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 cells by inactivating the ERK and NF-κB pathways. International Journal of Molecular Medicine, 34, 624-631. https://doi.org/10.3892/ijmm.2014.1790
MLA
Lee, Y., Lee, D., Koo, K., Lee, J., Song, Y. S., Yoon, H. S., Choi, Y. M., Kim, B. J."Mixtures of recombinant growth factors inhibit the production of pro-inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 cells by inactivating the ERK and NF-κB pathways". International Journal of Molecular Medicine 34.2 (2014): 624-631.
Chicago
Lee, Y., Lee, D., Koo, K., Lee, J., Song, Y. S., Yoon, H. S., Choi, Y. M., Kim, B. J."Mixtures of recombinant growth factors inhibit the production of pro-inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 cells by inactivating the ERK and NF-κB pathways". International Journal of Molecular Medicine 34, no. 2 (2014): 624-631. https://doi.org/10.3892/ijmm.2014.1790