AMPK Activation Inhibits Expression of Proinflammatory Mediators Through Downregulation of PI3K/p38 MAPK and NF-κB Signaling in Murine Macrophages

Abstract

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a central role in energy homeostasis and regulation of inflammatory responses. The present study is aimed to investigate the anti-inflammatory effects of ENERGI-F704, a nucleobase analogue isolated from bamboo leaves, on expression of proinflammatory mediators in murine macrophage RAW264.7 in response to lipopolysaccharide (LPS). ENERGI-F704 enhanced phosphorylation of AMPK(T172) but insignificantly affected the viability of RAW264.7 cells. Further investigation showed that ENERGI-F704 decreased mRNA expression of interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS) induced by LPS, as well as suppressed the production of prostaglandin E2 (PGE₂) and nitric oxide (NO). Additionally, the inhibitory effects of ENERGI-F704 on the LPS-induced proinflammatory mediators were diminished by pretreatment of AMPK inhibitor Compound C. ENERGI-F704 also inhibited LPS-triggered activation of nuclear factor kappa B (NF-κB), phosphatidylinositol 3-kinase (PI3K), and p38 mitogen-activated protein kinase (p38), whereas extracellular signal-regulated kinase (Erk)1/2 and c-Jun N-terminal kinase (JNK) were insignificantly influenced. Our findings indicate that ENERGI-F704 may exert anti-inflammatory activity on RAW264.7 cells in response to LPS through the activation of AMPK and suppression of PI3K/P38/NF-κB signaling and the consequent decreased expression of proinflammatory mediators, suggesting that ENERGI-F704 is beneficial to the amelioration of inflammatory disorders.

Introduction

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an energy sensor that regulates energy homeostasis and metabolic stress (Long and Zierath, 2006). AMPK is activated under conditions of glucose deprivation, heat shock, oxidative stress, and ischemia (Corton et al., 1994). Once activated, AMPK suppresses key enzymes involved in adenosine triphosphate (ATP)-consuming anabolic pathways and increases cellular ATP supply (Kemp et al., 1999). AMPK stimulates fatty acid oxidation by phosphorylating and inhibiting acetyl CoA carboxylase (ACC) (Hardie and Pan, 2002). In particular, a potential role for AMPK in the suppression of inflammatory responses is supported by evidence obtained using a common activator of AMPK, 5-aminoimidazole-4-carboxamide ribose (AICAR). AICAR reduces the synthesis of inducible nitric oxide synthase (iNOS) by adipocytes, macrophages, and glial cells (Giri et al., 2004).

Inflammation originates from the attempt at self-protection against harmful stimuli through the activation of serial immune responses. Among the immune systems participating in host defense, macrophages are the primary cells triggered by lipopolysaccharide (LPS). LPS binds to toll-like receptor (TLR)-4 and induces secretion of a variety of proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α (Raabe et al., 1998; Hsu and Wen, 2002). In addition, LPS also enhances expression of cyclooxygenase-2 (COX2) and iNOS, which produce potent inflammatory mediator prostaglandins and nitric oxide (NO) (Erwig and Rees, 1999).

Activation of the nuclear factor kappa B (NF-κB) plays a crucial role in inflammatory diseases (Tak and Firestein, 2001; Imanifooladi et al., 2010). NF-κB is a heterodimer composed of p50 and p65, which functions as a transcriptional activator to induce gene expression of proinflammatory mediators. In its inactive state, NF-κB is a cytoplasmic heterodimer that consists of three subunits: p50, p65, and inhibitor of κB (IκB). Upon stimuli such as LPS, IκBα is phosphorylated and then degraded through the ubiquitin–proteasome pathway, exposing nuclear localization signals on the p50-p65 heterodimer (Guha and Mackman, 2001).

In the present study, we investigated anti-inflammatory effects of an AMPK activator ENERGI-F704 identified from Bambusa oldhamii Munro on murine macrophage RAW264.7 in response to LPS. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. mRNA expression and protein production of proinflammatory mediators was determined by reverse transcription-polymerase chain reaction (RT-PCR), real-time (RT) quantitative PCR (qPCR), and quantitative sandwich enzyme-linked immunosorbent assay (ELISA). Kinase and NF-κB signaling cascades were investigated by immunoblotting analysis.

Materials and Methods

Chemicals and reagents

Aprotinin, leupeptin, LPS, MTT, Igepal CA-630, phenylmethylsulfonyl fluoride (PMSF), NaF, NaCl, Na₂HPO₄, Tris-HCl, Triton X-100, Tween-20, and Compound C (Dorsomorphin) were purchased from Sigma-Aldrich (St. Louis, MO). ENERGI-F704 was provided by Energenesis Biomedical Co. Ltd. (New Taipei City, Taiwan).

Cell culture and treatment

Murine macrophage cell line, RAW264.7, purchased from ATCC was cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Gaithersburg, MD) supplemented with 0.1% sodium bicarbonate, 2 mM glutamine, penicillin G (100 U/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum and maintained at 37°C in a humidified incubator containing 5% CO₂. Cells were plated at a density of 3×10⁵ cells in 60-mm Petri dish. After incubation with ENERGI-F704 at indicated concentrations for 3 h, the RAW264.7 cells were then treated with 0.1 μg/mL LPS for 6 h (immunoblotting, RT-PCR, and RT-qPCR analysis) or 24 h (cell viability assay, protein secretion, and NO production). For AMPK inhibition, pretreatment with 5 μM AMPK inhibitor Compound C for 1 h was performed before incubation with ENERGI-F704.

Cell viability assay

Cell viability was determined by mitochondrial-dependent reduction of MTT to formazan. Ten microliters of MTT solution (5 mg/mL in DMEM) was added to the culture medium and incubated for 4 h at 37°C. After removal of medium, 2-propanol was added into Petri dish and then solubilize formazan was quantitated by optical density at 570 nm obtained from a microplate reader (Benchmark; Bio-Rad Laboratories, Hercules, CA). The optical density of formazan generated by untreated cells was regarded as 100%.

RNA extraction, RT-PCR, and RT-qPCR

After treatments, total RNA was isolated from lysed cells using the RNeasy kit according to the manufacturer's instructions (Qiagen, Valencia, CA). The purified RNA was used as a template to generate the first-strand cDNA synthesis using RevertAid™ First Strand cDNA Synthesis Kit (Fermentas Life Sciences, St. Leon-Rot, Germany). The primer sequences used for RT-PCR and qPCR were listed in Table 1. qPCR was performed using the ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA). For mRNA quantitation, FastStart Universal SYBR Green Master kit (Roche Applied Science, Mannheim, Germany) was used for Taqman PCR. The threshold cycle numbers were calculated using the ΔΔCT relative value method and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Three independent experiments were performed for RT-PCR and RT-qPCR analysis. The correct size of the PCR products was checked by agarose gel electrophoresis.

Table 1.

Primer Sequences Used for Reverse Transcription-Polymerase Chain Reaction and Reverse Transcription Real-Time Quantitative Polymerase Chain Reaction

Gene name		RT-PCR	RT-qPCR
IL-6	F	5′-GTACTCCAGAAGACCAGAGG-3′	5′-CACGGCCTTCCCTACTTCAC-3′
	R	5′-TGCTGGTGACAACCACGGCC-3′	5′-TGCAAGTGCATCATCGTTGT-3′
IL-8	F	5′-AGATATTGCACGGGAGAA-3′	5′-CTCTTGGCAGCCTTCCTGATTT-3′
	R	5′-GAAATAAAGGAGAAACCA-3′	5′-CGCAGTGTGGTCCACTCTCAAT-3′
TNF-α	F	5′-ATGAGCACAGAAAGCATGATC-3′	5′-GTGGAACTGGCAGAAGAGGC-3′
	R	5′-TACAGGCTTGTCACTGGAATT-3′	5′-AGACAGAAGAGCGTGGTGGC-3′
COX2	F	5′-TGGAGGCGAAGTGGGTTT TA-3′	5′-GGGTGTGAAGGGAAATAAGG-3′
	R	5′-GAGTGGGAGGCACTTGCA TT-3′	5′-TCTCCACCAATGACCTGAT-3′
iNOS	F	5′-TCTTGGAGCGAGTTGTGG AT-3′	5′-AAGTCAAATCCTACCAAAGTGA-3′
	R	5′-GGGTCGTAATGTCCAGGA AGT-3′	5′-CCATAATACTGGTTGATGAACT-3′
GAPDH	F	5′-GATGGCATGGACTGTGGTCA-3′	5′-ACCACAGTCCATGCCATCAC-3′
	R	5′-GCAATGCCTCCTGCACCACC-3′	5′-TCCACCACCCTGTTGCTGTA-3′

COX2, cyclooxygenase-2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL-6, interleukin 6; IL-8, interleukin 8; iNOS, inducible nitric oxide synthase; RT-qPCR, reverse transcription real-time quantitative polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; TNF-α, tumor necrosis factor-α.

Quantitation of IL-6, IL-8, TNF-α, and PGE₂ using ELISA

For quantitative analysis of IL-6, IL-8, TNF-α, and prostaglandin E2 (PGE₂) production, cells were seeded in six-well plates at an initial density of 5×10⁵ cells/mL. After indicated treatments, the resulting culture supernatants were collected and the concentrations of IL-6, IL-8, TNF-α, and PGE₂ were determined using Mouse Quantikine^® ELISA Kits (IL-6, IL-8, TNF-α) and PGE₂ Parameter Assay Kit (R&D Systems, Abingdon, United Kingdom) according to the manufacturer's instructions.

NO assay

After treatments, the culture supernatants were gently mixed with the equal volume of modified Griess reagent (Sigma-Aldrich) and then absorbance at 540 nm was measured by the microplate reader (Benchmark; Bio-Rad Laboratories). Quantitation of NO was performed by using sodium nitrite (NaNO₂) as a standard (Green et al., 1982).

Immunoblotting analysis

Cells (3×10⁶ cells) were harvested, washed twice with ice-cold PBS, and incubated with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% v/v Igepal CA-630, 1 mM PMSF, 1 mM NaF, and 10 μg/mL aprotinin and leupeptin). The cell lysates were centrifuged at 14,000 g for 10 min at 4°C to remove debris, and then, the resulting supernatants were collected. Crude protein concentrations were quantitated using BCA™ protein assay kit (Pierce, Rockford, IL) and bovine serum albumin as standard. Proteins were electrophoresed on 12.5% SDS–polyacrylamide gel and transferred onto a nitrocellulose membrane (Millipore, Bedford, MA). The blotted membrane was blocked with 5% w/v skimmed milk in phosphate-buffered saline for 1 h and then incubated with 1/1000 dilution of the primary antibodies for 2 h. The primary antibodies included antibody against phosphorylated-threonine172 AMPK [p-AMPK(T172)], AMPKα1, phosphorylated phosphatidylinositol 3-kinase (p-PI3K), total PI3K (t-PI3K), phosphorylated extracellular signal-regulated kinase 1/2 (p-Erk1/2), total Erk1/2 (t-Erk1/2), phosphorylated c-Jun N-terminal kinase (p-JNK), total JNK (t-JNK), phosphorylated p38 mitogen-activated protein kinase (p-p38), total p38 (t-p38; Cell Signaling Technologies, Danvers, MA) and GAPDH (Abcam, Cambridge, MA). Bound antibodies were detected using 1/3000 dilution of peroxidase-conjugated secondary antibodies (Sigma-Aldrich), and signals were developed using ECL chemiluminescence reagent (Millipore) as the substrate system. Quantitative analysis was performed using chemiluminescent densitometry.

Subcellular fractionation

Cells were washed with normal saline and incubated with a lysis buffer (10 mM HEPES, pH7.6; containing 15 mM KCl, 2 mM MgCl₂, 0.1 mM EDTA, 1 mM dithiothreitol, 0.05% v/v Igepal CA-630 and 1 mM PMSF, 1 mM Na₃VO₄, 50 mM NaF, 10 μg/mL leupeptin, and 10 μg/mL aprotinin) for 10 min. After centrifugation at 2500 g for 10 min at 4°C, the supernatant was transferred into a new Eppendorf, further centrifuged at 20,000 g for 15 min at 4°C, and then the resulting supernatant was collected as cytosolic fraction. The pellets containing nuclei were washed with normal saline, incubated with a nuclear buffer (25 mM HEPES, pH7.6, 0.1% v/v Igepal CA-630, 1 M KCl, 0.1 mM EDTA, 1 mM PMSF, 1 mM Na₃VO₄, 2 mM NaF, 10 μg/mL leupeptin, and 10 μg/mL aprotinin), and then centrifuged at 10,000 g for 15 min at 4°C. The resulting supernatants were collected as nuclear fraction.

Statistical analysis

Data were presented as means±SD of the three to five independent experiments. Statistical comparisons are made by a one-way analysis of variance, followed by the Duncan multiple comparison test. Differences are considered to be significant when the p-values are <0.05.

Results

Effects of ENERGI-F704 on AMPK phosphorylation and viability of murine macrophage RAW264.7 cells

To examine whether ENERGI-F704 induced AMPK activation in RAW264.7 cells without or with exposure to LPS (0.1 μg/mL), the level of AMPK with phosphorylation at threonine172 was determined by immunoblotting. The results showed that both ENERGI-F704 alone or ENERGI-F704 combing with LPS dose dependently enhanced the phosphorylation of AMPK [p-AMPK(T172)] in RAW264.7 cells compared to the control (Fig. 1A). In parallel, LPS alone slightly increased the level of p-AMPK(T172).

FIG. 1.

ENERGI-F704 induced AMPK phosphorylation but insignificantly affected the viability of RAW264.7 cells. (A) Cells were incubated with ENERGI-F704 alone (5–40 μM) or ENERGI-F704 combining LPS (10–40 μM) for 24 h and then lysed for immunodetection of AMPKα1 and p-AMPK(T172). (B) Cells were incubated with LPS alone, LPS combining with ENERGI-F704 (10–160 μM), or ENERGI-F704 alone (160 μM) for 24 h, and then, the cells were subjected to the cell viability assay. *p<0.05 compared to negative control. AMP, adenosine monophosphate; AMPK, AMP-activated protein kinase; LPS, lipopolysaccharide.

Cytotoxic effects of ENERGI-F704 on viability of RAW264.7 cells were next demonstrated. Viability of RAW264.7 cells cultured with LPS alone (0.1 μg/mL) or LPS combining with ENERGI-F704 at a serial concentration (10, 20, 40, 80, and 160 μM) for 24 h was determined using the MTT assay. As shown in Figure 1B, our findings revealed that LPS alone and LPS combining with ENERGI-F704 (10, 20, 40, and 80 μM) insignificantly affected the viability of RAW264.7 cells (p>0.05 compared to control). However, high concentration of ENERGI-F704 (160 μM) alone or combing with LPS reduced the cell viability to 78.9%±2.4% and 76.3%±4.1% of control, respectively (p<0.05 compared to control). Accordingly, ENERGI-F704 at concentrations of 20 and 40 μM were used for the subsequent investigation of anti-inflammatory activity.

ENERGI-F704 inhibited both mRNA expression and secreted protein level of IL-6, IL-8, and TNF-α in RAW264.7 cells in response to LPS

Effects of ENERGI-F704 on mRNA expression and protein production of proinflammatory cytokines in RAW2647 cells exposed to LPS were next investigated by using RT-PCR, RT-qPCR, and sandwich ELISA. RT-PCR analysis revealed that LPS alone significantly enhanced mRNA expression of IL-6, IL-8, and TNF-α, and the increased mRNA expressions were suppressed by pretreatment of ENERGI-F704 in a dose-dependent manner (Fig. 2A). Involvement of AMPK activation in ENERGI-F704-lowered mRNA expression of the proinflammatory mediators is accessed by using Compound C, a cell-permeable pyrazolopyrimidine compound that inhibits the AMPK kinase activity (IC₅₀=234.6 nM) and is commonly used in combination with AMPK activators, such as Metformin for studying AMPK-dependent cellular events in vitro (Zhou et al., 2001). Our results revealed that the suppressive effects of ENERGI-F704 on the mRNA expression were diminished by cotreatment with the AMPK inhibitor Compound C (Fig. 2A). Subsequent quantitative analysis using RT-qPCR showed the similar influences as RT-PCR. LPS alone significantly increased mRNA levels of IL-6, IL-8, and TNF-α up to 13.5±2.5-fold, 22.6±0.7-fold, and 8.1±0.6-fold of control (p<0.01), respectively. Compared to LPS alone, pretreatment of 40 μM ENERGI-F704 reduced LPS-induced mRNA levels of IL-6, IL-8, and TNF-α to 3.8±0.5-fold, 7.5±1.7-fold, and 3.4±0.2-fold of control, respectively (p<0.05) (Fig. 2B). Besides, the ENERGI-F704-suppressed mRNA levels of IL-6, IL-8, and TNF-α was restored to 10.5±0.6-fold, 17.5±1.4-fold, and 6.9±0.2-fold of negative control, respectively, by cotreatment with Compound C (p<0.05 compare to LPS combining with 40 μM ENERGI-F704) (Fig. 2B).

FIG. 2.

ENERGI-F704 suppressed mRNA expression and protein production of proinflammatory cytokines, IL-6, IL-8, and TNF-α, in LPS-stimulated RAW264.7 cells. Cells were preincubated with ENERGI-F704 (20 or 40 μM) for 3 h, treated with 0.1 μg/mL LPS for 24 h, and then, the treated cells and resulting supernatants were individually collected for analysis of mRNA expression (A, B) and protein production (C) by using RT-PCR, RT-qPCR, and quantitative sandwich ELISA, respectively. Quantitative data were expressed as mean±SD of the three independent experiments. ^a p<0.05 compared to negative control. ^b p<0.05 compared to LPS alone. ^c p<0.05 compared to LPS combining with 40 μM ENERGI-F704. IL, interleukin; RT-qPCR, reverse transcription real-time quantitative polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; TNF-α, tumor necrosis factor-α.

In addition to mRNA expression, protein production of the three proinflammatory mediators was also determined. As shown in Figure 2C, LPS alone increased protein production of IL-6, IL-8, and TNF-α to 121.3±11.2, 168.3±13.2, and 507.2±27.3 pg/mL, respectively (p<0.05 compared to control). Treatment with 40 μM ENERGI-F704 combining with LPS lowered protein production of IL-6, IL-8, and TNF-α to 55.6±8.8, 68.5±10.6, and 352.2±20.5 pg/mL, respectively (p<0.05 compared to LPS alone). Similar to the mRNA expression, the ENERGI-F704-suppressed protein production of IL-6, IL-8, and TNF-α was elevated to 82.7±7.2, 111.2±8.2, and 386.9±17.2 pg/mL, respectively, by cotreatment with Compound C (p<0.05 compared to LPS combining with 40 μM ENERGI-F704, Fig. 2C).

ENERGI-F704 inhibited mRNA expression of COX2 and iNOS in RAW264.7 cells and production of PGE₂ and NO in response to LPS

Both COX2 and iNOS are known to play important roles in the development and progression of inflammation; therefore, the effects of ENERGI-F704 on mRNA expression of COX2 and iNOS in LPS-stimulated RAW264.7 cells were analyzed. RT-PCR analysis showed that LPS alone significantly elevated mRNA expression of COX2 and iNOS, and the increase of these mRNA expressions was inhibited by pretreatment of ENERGI-F704 in a dose-dependent manner (Fig. 3A). In addition, the inhibitory effects of ENERGI-F704 on COX2 and iNOS were diminished by cotreatment with Compound C. Further quantitative analysis using RT-qPCR revealed that LPS alone significantly increased mRNA levels of COX2 and iNOS to 33.8±2.2-fold and 7.1±0.2-fold of negative control, respectively (p<0.05) (Fig. 3B). Comparing to LPS alone, pretreatment of ENERGI-704 (40 μM) inhibited LPS-induced mRNA levels of COX2 and iNOS to 8.8±0.9-fold and 2.9±0.2-fold of negative control, respectively (p<0.05) (Fig. 3B). Moreover, the ENERGI-F704-suppressed mRNA levels of IL-6, IL-8, and TNF-α was restored to 26.8±1.5-fold and 4.6±0.3-fold of negative control, respectively, by cotreatment with Compound C (p<0.05 compared to ENERGI-F704 combining with LPS) (Fig. 3B). These findings showed that ENERGI-F704 significantly suppressed mRNA expression of COX2 and iNOS in response to LPS, and AMPK activation was involved in the suppression of COX2 and iNOS mRNA expression.

FIG. 3.

ENERGI-704 inhibited mRNA expression of COX2 and iNOS in LPS-stimulated RAW264.7 cells and reduced the production of PGE₂ and NO. Cells were preincubated with ENERGI-F704 (20 or 40 μM) for 3 h, treated with 0.1 μg/mL LPS for 24 h, and then lysed for mRNA extraction. mRNA expression level was analyzed by RT-PCR (A) and quantitated by RT-qPCR (B), and the cultured medium was collected for quantitation of PGE₂ (C) and NO production (D) as described in the Materials and Methods section. Quantitative data were expressed as mean±SD of the three independent experiments. ^a p<0.05 compared to negative control. ^b p<0.05 compared to LPS alone. ^c p<0.05 compared to LPS combining with 40 μM ENERGI-F704. COX2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; NO, nitric oxide; PGE₂, prostaglandin E2.

In parallel to the analysis of COX2 and iNOS mRNA expression, the effects of ENERGI-F704 on production of PGE₂ and NO in response to LPS were also demonstrated. As shown in Figure 3C and D, LPS alone increased PGE₂ and NO production to 228.3±16.2 pg/mL and 27.5±1.5 mM, respectively (p<0.05 compared to negative control). Comparing to LPS alone, pretreatment of ENERGI-F704 (40 μM) inhibited LPS-induced production of PGE₂ and NO to 154.5±2.1 pg/mL and 15.5±1.1 mM, respectively (p<0.05) (Fig. 3C, D). In addition, the ENERGI-F704-suppressed production of PGE₂ and NO was restored to 191.6±8.2 pg/mL and 20.7±0.8 mM, respectively, by cotreatment of Compound C (p<0.05 compared to LPS combining with 40 μM ENERGI-F704) (Fig. 3C, D).

ENERGI-F704 suppressed phosphorylation of PI3K, p38 MAPK, and IκBα and reduced nuclear NF-κB in LPS-stimulated RAW264.7 cells

Activation of PI3K and MAPKs and the consequent NF-κB signaling is reported to involve in LPS-induced production of proinflammatory cytokines (Deng et al., 2013; Huo et al., 2013). Therefore, the effects of ENERGI-F704 on phosphorylation of PI3K, Erk1/2, JNK, p38, and IκBα and nuclear fraction of NF-κB in LPS-stimulated RAW264.7 cells were investigated. As shown in Figure 4A, phosphorylation of PI3K, Erk1/2, JNK, and p38 was significantly enhanced in RAW264.7 cells in response to LPS (p<0.05 compared to negative control). Pretreatment with ENERGI-F704 inhibited LPS-induced phosphorylation of PI3K and p38 in a dose-dependent manner (p<0.05 compared to LPS alone) but insignificantly affected phosphorylation of Erk1/2 and JNK (Fig. 4A, lower panel). In addition, cotreatment with Compound C restored the ENERGI-F704-inhibited phosphorylation of PI3K and p38 and slightly affected the phosphorylation of Erk1/2 and JNK (Fig. 4A, lower panel).

FIG. 4.

ENERGI-F704 suppressed the phosphorylation of PI3K and p38 and reduced nuclear level of NF-κB in LPS-stimulated RAW264.7 cells. Cells were preincubated with ENERGI-704 (20 or 40 μM) for 3 h, treated with 0.1 μg/mL LPS for 24 h, and then lysed for crude protein extraction and subcellular fractionation. Target proteins were detected using immunoblotting. (A) Phosphorylation of PI3K, Erk1/2, JNK, and p38. (B) Phosphorylation of IκBα and level of nuclear NF-κB(p65). Quantitation of immunosignals was performed by densitometric analysis using GAPDH and Histone H1 as cytosolic and nuclear internal control, respectively. Quantitative data were expressed as mean±SD of the three independent experiments. ^a p<0.05 compared to negative control. ^b p<0.05 compared to LPS alone. ^c p<0.05 compared to LPS combining with 40 μM ENERGI-F704. Erk1/2, extracellular signal-regulated kinase 1/2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IκBα, inhibitor of κBα; JNK, c-Jun N-terminal kinase; NF-κB, nuclear factor kappa B.

In parallel to activation of PI3K and MAPKs, the effects of ENERGI-F704 on NF-κB signaling were also investigated. As shown in Figure 4B, LPS alone significantly increased phosphorylation of IκBα (p-IκBα) and nuclear fraction of NF-κB [(nu)NF-κB(p65)]. Pretreatment of ENERGI-704 reduced both the LPS-induced p-IκBα and (nu)NF-κB(p65). In addition, cotreatment of Compound C restored the ENERGI-F704-reduced p-IκBα and (nu)NF-κB(p65) (Fig. 4B, lower panel, p<0.05 compared to ENERGI-F704 combining with LPS). Taken together, these results showed that ENERGI-F704 suppressed the activation of PI3K, p38, and NF-κB signaling.

Discussion

Inflammatory response induced by tissue injury or infection is highly associated with energy-intensive process. In the presence of LPS, macrophages can immediately switch from a resting state to a highly active state, displaying a great increase in the production of host defense factors, enhanced phagocytosis, and antigen presentation. During the process, ATP generation undergoes a switch from predominant oxidative phosphorylation to glycolysis (Krawczyk et al., 2010). Therefore, it is proposed that alteration of energy metabolism may modulate the inflammatory signaling, interpreting the dual effects of AMPK in macrophage-oriented inflammation (O'Neill and Hardie, 2013). Recent studies have shown that AMPK activator, such as AICAR or resveratrol, exhibits anti-inflammatory activities through AMPK-mediated B-cell lymphoma-6 protein (Bcl-6), poly[ADP-ribose] polymerase 1 (PARP-1), and NF-κB signaling (Yi et al., 2011; Gongol et al., 2013). Similarly, our results showed that an AMPK activator ENERGI-F704 isolated from bamboo activated AMPK by inducing phosphorylation of threonine 172 in RAW264.7 cells, and the ENERGI-F704-induced AMPK activation was involved in suppressing mRNA expression and protein production of IL-6, IL-8, TNF-α, COX2, and iNOS in response to LPS. Moreover, ENERGI-F704 also lowered PGE₂ and NO production by RAW264.7 cells exposed to LPS. These results indicate that ENERGI-F704 possesses inhibitory activity on inflammatory responses triggered by murine macrophage with the stimulation of LPS.

In addition to macrophage, neutrophil infiltrating into injury sites is also known as a remarkable inflammatory event in skin wounding (Li et al., 2005). Similarly, our preliminary results also revealed that the external use of ENERGI-F704 exerted anti-inflammatory effects on surgical incision wounding of mice, including reduced neutrophil infiltration (Supplementary Fig. S1 and Table S1; Supplementary Data are available online at www.liebertpub.com/dna) and IL-1β production, as well as improved the dermal healing and reepithelialization (data not shown), suggesting that ENERGI-F704 may also alleviate dermal inflammation in vivo in response to incision injury. However, further investigation is still needed for exploration of in vivo anti-inflammatory activity of ENERGI-F704.

Macrophage is one of the major effector cells during inflammatory process (Murakami et al., 2005). The main function of macrophage is to modulate immune responses through the production of various cytokines, reactive oxygen and nitrogen species, growth factors, and chemokines in response to pathogens or potent immunogens, such as bacterial LPS (Fujiwara and Kobayashi, 2005). Although the bioactive factors produced by macrophages have valuable outcomes in inflammation, these factors were also exhibited unfavorable and damaging effects (Heumann and Roger, 2002). Therefore, the regulation of these products provides a potential target to restrain inflammatory diseases. IL-6 has various biological properties in a number of chronic endothelial dysfunctions, such as modulation of hematopoiesis, proliferation and differentiation of lymphocytes, and induction of acute-phase reactions (Desai et al., 2002). IL-8 is known as a key intermediate regulator in acute inflammatory responses (Noda et al., 2008). TNF-α was a pivotal mediator produced by activated macrophages and was shown to affect various biological processes, including the regulation and the production of other cytokines (Hume et al., 2001; Aggarwal et al., 2002). Our findings revealed that ENERGI-F704 significantly inhibited mRNA expression of IL-6, IL-8, and TNF-α in LPS-stimulated RAW264.7 cells, suggesting that ENERGI-F704 may attenuate the LPS-induced activation of RAW264.7 cells and the consequent inflammatory mediators produced by the murine macrophage.

COX2 and iNOS are important enzymes that catalyze the synthesis of inflammatory mediators in promotion of inflammation. COX is the common target protein for analgesic and anti-inflammatory therapies that have been used for hundreds of years. The COX enzyme consists of at least two isoforms: COX1 and COX2. In mammals, the expression of COX1 is constitutive in normal tissues, such as production of prostaglandin precursors for thromboxane in platelets and the regulation of blood flow. In contrast to COX1, COX2 protein is only slightly expressed in most resting tissues. However, upon inflammatory stimulation, the expression levels of COX2 protein and mRNA are increased (Lee et al., 1992). NOSs are a family of enzymes that catalyze the production of NO from L-arginine. Three isoforms of NOS have been characterized, including neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). nNOS and eNOS mainly exists in neuronal tissues and vascular endothelial cells, respectively, whereas iNOS can be detected in a variety of cell types, including macrophages, microglial cells, astrocytes, and keratinocytes (Andrew and Mayer, 1999). In addition, iNOS is overexpressed in the cells with infectious and proinflammatory stimuli. Our results showed that ENERGI-F704 dose dependently suppressed mRNA expression of COX2 and iNOS, as well as PGE2 and NO production by RAW264.7 cells in response to LPS, indicating that ENERGI-F704 may alleviate adverse reactions attributing to excess production of PGE₂ and NO.

LPS is known to activate several signaling kinases, including PI3K, Erk1/2, MAP kinase kinase (MEK) (Geppert et al., 1994), JNK (Hambleton et al., 1996), P38, and other MAP kinase family members (Han et al., 1994), and the consequent NF-κB signaling contributing to expression of proinflammatory mediators. Our findings show that ENERGI-F704 inhibits the activation of PI3K and P38 but not Erk1/2 and JNK, as well as reduced phosphorylation of IkBα and nuclear level of NF-κB, suggesting that the inhibitory effects of ENERGI-F704 on expression of proinflammatory cytokines, COX2, and iNOS may attribute to the modulation of PI3K and P38 and the consequent NF-κB signaling. In conclusion, these findings indicate that ENERGI-F704 possesses potent anti-inflammatory activity on murine macrophage RAW264.7 cells with stimulation of LPS, attributing to the inhibition of PI3K and P38 activation and NF-κB signaling and the consequent suppressed expression of proinflammatory cytokines, COX2, and iNOS.

Footnotes

Acknowledgments

We thank the support of real-time quantitative PCR machine by the Instrument of Center, Chung Shan Medical University, which is partially supported by the Ministry of Education and the Chung Shan Medical University.

Disclosure Statement

The authors have declared that no competing financial interests exist.

References

Aggarwal

B.B.

, Shishodia

, Ashikawa

, and Bharti

A.C.

(2002). The role of TNF and its family members in inflammation and cancer: lessons from gene deletion. Curr Drug Targets Inflamm Allergy, 1, 327–341.

Andrew

P.J.

, and Mayer

(1999). Enzymatic function of nitric oxide synthases. Cardiovasc Res, 43, 521–531.

Corton

J.M.

, Gillespie

J.G.

, and Hardie

D.G.

(1994). Role of the AMP-activated protein kinase in the cellular stress response. Curr Biol, 4, 315–324.

Deng

, Maitra

, Morris

, and Li

(2013). Molecular mechanism responsible for the priming of macrophage activation. J Biol Chem, 288, 3897–3906.

Desai

T.R.

, Leeper

N.J.

, Hynes

K.L.

, and Gewertz

B.L.

(2002). Interleukin-6 causes endothelial barrier dysfunction via the protein kinase C pathway. J Surg Res, 104, 118–123.

Erwig

L.P.

, and Rees

A.J.

(1999). Macrophage activation and programming and its role for macrophage function in glomerular inflammation. Kidney Blood Press Res, 22, 21–25.

Fujiwara

, and Kobayashi

(2005). Macrophages in inflammation. Curr Drug Targets Inflamm Allergy, 4, 281–286.

Geppert

T.D.

, Whitehurst

C.E.

, Thompson

, and Beutler

(1994). Lipopolysaccharide signals activation of tumor necrosis factor biosynthesis through the ras/raf-1/MEK/MAPK pathway. Mol Med, 1, 93–103.

Giri

, Nath

, Smith

, Viollet

, Singh

A.K.

, and Singh

(2004). 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside inhibits proinflammatory response in glial cells: a possible role of AMP-activated protein kinase. J Neurosci, 24, 479–487.

10.

Gongol

, Marin

, Peng

I.C.

, Woo

, Martin

, King

, et al. (2013). AMPKalpha2 exerts its anti-inflammatory effects through PARP-1 and Bcl-6. Proc Natl Acad Sci U S A, 110, 3161–3166.

11.

Green

L.C.

, Wagner

D.A.

, Glogowski

, Skipper

P.L.

, Wishnok

J.S.

, and Tannenbaum

S.R.

(1982). Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem, 126, 131–138.

12.

Guha

, and Mackman

(2001). LPS induction of gene expression in human monocytes. Cell Signal, 13, 85–94.

13.

Hambleton

, Weinstein

S.L.

, Lem

, and DeFranco

A.L.

(1996). Activation of c-Jun N-terminal kinase in bacterial lipopolysaccharide-stimulated macrophages. Proc Natl Acad Sci U S A, 93, 2774–2778.

14.

Han

, Lee

J.D.

, Bibbs

, and Ulevitch

R.J.

(1994). A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science, 265, 808–811.

15.

Hardie

D.G.

, and Pan

D.A.

(2002). Regulation of fatty acid synthesis and oxidation by the AMP-activated protein kinase. Biochem Soc Trans, 30, 1064–1070.

16.

Heumann

, and Roger

(2002). Initial responses to endotoxins and Gram-negative bacteria. Clin Chim Acta, 323, 59–72.

17.

Hsu

H.Y.

, and Wen

M.H.

(2002). Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of interleukin-1 gene expression. J Biol Chem, 277, 22131–22139.

18.

Hume

D.A.

, Underhill

D.M.

, Sweet

M.J.

, Ozinsky

A.O.

, Liew

F.Y.

, and Aderem

(2001). Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state. BMC Immunol, 2, 11.

19.

Huo

, Gao

, Jiang

, Cui

, Duan

, Deng

, et al. (2013). Suppression of LPS-induced inflammatory responses by gossypol in RAW 264.7 cells and mouse models. Int Immunopharmacol, 15, 442–449.

20.

Imanifooladi

A.A.

, Yazdani

, and Nourani

M.R.

(2010). The role of nuclear factor-kappaB in inflammatory lung disease. Inflamm Allergy Drug Targets, 9, 197–205.

21.

Kemp

B.E.

, Mitchelhill

K.I.

, Stapleton

, Michell

B.J.

, Chen

Z.P.

, and Witters

L.A.

(1999). Dealing with energy demand: the AMP-activated protein kinase. Trends Biochem Sci, 24, 22–25.

22.

Krawczyk

C.M.

, Holowka

, Sun

, Blagih

, Amiel

, DeBerardinis

R.J.

, et al. (2010). Toll-like receptor-induced changes in glycolytic metabolism regulate dendritic cell activation. Blood, 115, 4742–4749.

23.

Lee

S.H.

, Soyoola

, Chanmugam

, Hart

, Sun

, Zhong

, et al. (1992). Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. J Biol Chem, 267, 25934–25938.

24.

, Dasgeb

, Phillips

, Li

, Chen

, Garner

, et al. (2005). Wound-healing perspectives. Dermatol Clin, 23, 181–192.

25.

Long

Y.C.

, and Zierath

J.R.

(2006). AMP-activated protein kinase signaling in metabolic regulation. J Clin Invest, 116, 1776–1783.

26.

Murakami

, Nishizawa

, Egawa

, Kawada

, Nishikawa

, Uenakai

, et al. (2005). New class of linoleic acid metabolites biosynthesized by corn and rice lipoxygenases: suppression of proinflammatory mediator expression via attenuation of MAPK- and Akt-, but not PPARgamma-, dependent pathways in stimulated macrophages. Biochem Pharmacol, 70, 1330–1342.

27.

Noda

, Kinoshita

, Sakurai

, Matsumoto

, Mugishima

, and Tanjoh

(2008). Hyperglycemia and lipopolysaccharide decrease depression effect of interleukin 8 production by hypothermia: an experimental study with endothelial cells. Intensive Care Med, 34, 109–115.

28.

O'Neill

L.A.

, and Hardie

D.G.

(2013). Metabolism of inflammation limited by AMPK and pseudo-starvation. Nature, 493, 346–355.

29.

Raabe

, Bukrinsky

, and Currie

R.A.

(1998). Relative contribution of transcription and translation to the induction of tumor necrosis factor-alpha by lipopolysaccharide. J Biol Chem, 273, 974–980.

30.

Tak

P.P.

, and Firestein

G.S.

(2001). NF-kappaB: a key role in inflammatory diseases. J Clin Invest, 107, 7–11.

31.

C.O.

, Jeon

B.T.

, Shin

H.J.

, Jeong

E.A.

, Chang

K.C.

, Lee

J.E.

, et al. (2011). Resveratrol activates AMPK and suppresses LPS-induced NF-kappaB-dependent COX-2 activation in RAW 264.7 macrophage cells. Anat Cell Biol, 44, 194–203.

32.

Zhou

, Myers

, Li

, Chen

, Shen

, Fenyk-Melody

, et al. (2001). Role of AMP-activated protein kinase in mechanism of metformin action. Clin Invest, 108, 1167–1174.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.34 MB

0.02 MB

AMPK Activation Inhibits Expression of Proinflammatory Mediators Through Downregulation of PI3K/p38 MAPK and NF-κB Signaling in Murine Macrophages

Abstract

Introduction

Materials and Methods

Chemicals and reagents

Cell culture and treatment

Cell viability assay

RNA extraction, RT-PCR, and RT-qPCR

Quantitation of IL-6, IL-8, TNF-α, and PGE2 using ELISA

NO assay

Immunoblotting analysis

Subcellular fractionation

Statistical analysis

Results

Effects of ENERGI-F704 on AMPK phosphorylation and viability of murine macrophage RAW264.7 cells

ENERGI-F704 inhibited both mRNA expression and secreted protein level of IL-6, IL-8, and TNF-α in RAW264.7 cells in response to LPS

ENERGI-F704 inhibited mRNA expression of COX2 and iNOS in RAW264.7 cells and production of PGE2 and NO in response to LPS

ENERGI-F704 suppressed phosphorylation of PI3K, p38 MAPK, and IκBα and reduced nuclear NF-κB in LPS-stimulated RAW264.7 cells

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

Supplementary Material

Quantitation of IL-6, IL-8, TNF-α, and PGE₂ using ELISA

ENERGI-F704 inhibited mRNA expression of COX2 and iNOS in RAW264.7 cells and production of PGE₂ and NO in response to LPS