Antiinflammatory Activities of Crebanine by Inhibition of NF-κB and AP-1 Activation through Suppressing MAPKs and Akt Signaling in LPS-Induced RAW264.7 Macrophages

Abstract

Crebanine, an aporphine alkaloid, displays various biological activities such as anticancer and antimicrobial activities. In this study, we further investigated the suppressive effect of crebanine on lipopolysaccharide (LPS)-induced expression of proinflammatory mediators and the molecular mechanisms underlying these activities in RAW264.7 macrophages. Crebanine inhibited the production of proinflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor-alpha in LPS-induced RAW264.7 cells. Moreover, crebanine suppressed LPS-induced inducible nitric oxide (iNO) and prostaglandin E₂ and reduced the expression of iNO synthase and cyclooxygenase-2 in RAW264.7 cells. Crebanine suppressed LPS-induced phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), including extracellular signaling-regulated kinase 1/2, c-Jun NH₂-terminal kinase, and p38 MAPK signaling. In addition, the specific inhibitor of MAPKs and Akt reduced the expression of IL-6 and NO production in LPS-induced macrophages. Furthermore, crebanine inhibited LPS-induced nuclear factor kappa B (NF-κB) activation by reducing the phosphorylation of p65 at Ser536 but not the p65 translocation to the nucleus and inhibitory factor kappa B alpha degradation. Crebanine also suppressed phosphorylation and nucleus translocation of activator protein-1 (AP-1). These observations suggest that the antiinflammatory properties of crebanine may stem from the inhibition of proinflammatory mediators via suppression of the NF-κB, AP-1, MAPKs, and Akt signaling pathways.

Inflammation is an essential biological reaction in the body in response to infection and tissue injury which may be caused by a variety of physical, chemical, or biological stimuli.¹⁾ Even though inflammation is a helpful physiological process, chronic inflammation is a significant cause of many inflammatory disorders including cancer, cardiovascular diseases, diabetes, autoimmune disorders, and arthritis.^2–4) Macrophages are a main component of immunological system and play an important role in the inflammatory responses. The macrophages are activated by various inflammatory stimuli including, microbial lipopolysaccharide (LPS) and cytokines. During the progression of an inflammatory process, macrophages excessively produce the inflammatory mediators such as nitric oxide (NO), prostaglandin E₂ (PGE₂), and pro-inflammatory cytokines including interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-α).^5,6) The subsequent generation of NO and PGE₂ are produced by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) respectively.⁷⁾ The expression of iNOS and COX-2 can be considered markedly up-regulated inflammatory disorders.⁸⁾

Activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) are the transcription factors that play an important role in regulating inflammatory responses by increasing the expression of pro-inflammatory genes.⁹⁾ LPS initiated the inflammatory cascades in macrophages via Toll-like receptors 4 (TLR4). Upon stimulation of TLR4, the phosphorylation of Akt, c-Jun NH₂-terminal kinases (JNK), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (MAPKs) signaling pathway are activated.¹⁰⁾ These signaling pathways in turn mediate multiple downstream events leading to the activation of AP-1 and NF-κB, which coordinate the induction of a range of inflammatory proteins.¹¹⁾ Therefore, treatment aimed at inhibiting MAPKs, Akt, NF-κB and AP-1 may have potential therapeutic advantage for inflammatory diseases.^12,13)

Crebanine is an aporphine alkaloid that has been isolated from the tube of Stephania venosa. Crebanine exhibits a variety of biological properties including, anti-microbial activities, improvement of neurodegenerative diseases, as well as anti-invasion and anti-proliferation activities in cancer cells.^14–16) Moreover, our previous report demonstrated that crebanine reduced TNF-α induced lung cancer cells proliferation and invasion via suppressing NF-κB activity.¹⁷⁾ However, neither the anti-inflammatory activity nor the mechanism by which crebanine act on macrophages has been reported.

In this study, we determined the effect of crebanine on LPS-induced production of pro-inflammatory mediators including NO, PGE₂, IL-6 and TNF-α. The potential mechanism of crebanine on the expression of COX-2 and iNOS were also examined. Moreover, we evaluated the molecular inflammatory mechanism through MAPKs, Akt signaling pathway and the activation of NF-κB and AP-1 transcription factors.

MATERIALS AND METHODS

Materials

Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin, and trypsin–ethylenediaminetetraacetic acid (EDTA) were purchased from GIBCO-BRL (Grand Island, NY, U.S.A.). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, U.S.A.). Mouse IL-6 and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were purchased from Biolegend, Inc. (San Diego, CA, U.S.A.). PGE₂ Immunoassay ELISA kit was purchased from Abcam (Cambridge, MA, U.S.A.). Antibodies specific to β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Antibodies specific to iNOS, COX-2, phospho-p38, p38, phospho-ERK1/2, ERK1/2, phospho-JNK, JNK, phospho-NF-κB p65, NF-κB p65, phospho-c-Jun, c-Jun, phospho-Akt, Akt, LY294002 and PD98059 were purchased from Cell Signaling Technology Inc. (Beverly, MA, U.S.A.). SB202190 and SP600125 were purchased from InvivoGen (San Diego, CA, U.S.A.). Griess reagent was purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Crebanine was isolated and purified from ethyl acetate fraction of the tube of Stephania vernosa as our previously described.^15,18) It was obtained as light yellow crystals, melting point at 117.0–117.8°C. The structure of crebanine was elucidated by extensive spectroscopic analysis (UV, IR, MS, ¹H-NMR, ¹³C-NMR) and the molecular formula was identified as crebanine, C₂₀H₂₁NO₄.

Cell Culture

RAW264.7 cells line were cultured in DMEM supplemented with 10% FBS, 100 units/mL of penicillin and 100 µg/mL of streptomycin. The cell cultures were maintained in a humidified incubator with an atmosphere of 5% CO₂ at 37°C. For the crebanine treatment, crebanine was dissolved in dimethyl sulfoxide (DMSO) and diluted with culture medium leaving the final concentration of DMSO was less than 0.1% (v/v).

Measurement of Cell Viability

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.¹⁹⁾ Briefly, RAW264.7 cells were plated at 7.5×10³ cells/well into 96-well plates in DMEM with 10% FBS. The cells were treated with various concentrations of crebanine (7.4–58.9 µM) and incubated for 24 h. Fifteen microliters of MTT solution (5 mg/mL) was added at the end of the treatment and further incubated for 4 h. After the incubation, the formazan crystals in each well were dissolved in 200 µL of DMSO for 15 min, and the optical density (OD) at 570 nm with a reference wavelength of 630 nm was measured using a Microplate Reader. Cell viability in control medium, absent of any treatment was represented as 100%.

Measurement of Pro-inflammatory Cytokines (TNF-α and IL-6) Production

RAW264.7 cells were plated at 6.0×10⁵ cells/well into 12-well plates in DMEM with 10% FBS. The cells were pretreated with crebanine (7.4–29.5 µM) for 2 h and then the cells were stimulated with or without LPS (1 µg/mL) for 24 h. The supernatants were collected and the level of IL-6 and TNF-α were determined with ELISA kits (BioLegend, Inc.) as following the manufacturer’s protocol.²⁰⁾

Measurement of NO and PGE₂ Concentrations

The nitrite concentration was determined using Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine dihydrochloride and 2.5% phosphoric acid) as an indicator of NO production.²¹⁾ Briefly, RAW264.7 cells were plated at 3.5×10⁵ cells/well into 12-well plates in DMEM with 10% FBS. The cells were pretreated with crebanine (7.4–29.5 µM) for 2 h and then the cells were stimulated with or without LPS (1 µg/mL) for 24 h. After incubation, 150 µL of the culture supernatants was mixed with 150 µL of Griess reagent. The absorbance of the mixtures was measured at 540 nm with a microplate reader. The nitrite concentrations in the supernatants were calculated from a standard curve of nitrite solution. The production of PGE₂ from activated macrophage was determined using enzymes immune assay kit (Abcam, Cambridge, MA, U.S.A.). The procedure was performed in accordance with the manufacturer’s instructions.²²⁾ The concentration of PGE₂ was calculated with standard curve of PGE₂.

Preparation of Whole Cell Lysates, Cytoplasmic and Nuclear Fraction

The whole cell lysates were used to determine the expression level of COX-2, iNOS and the activation of MAPKs and Akt signaling proteins in activated RAW264.7 cells. Briefly, the cells were pretreated with crebanine (7.4–44.2 µM) and stimulated with or without LPS (1 µg/mL). The treated cells were collected with ice-cold phosphate buffered saline (PBS) and extracted by lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL leupeptin, 10 µg/mL aprotinin) on ice for 15 min. The insoluble matter was removed by centrifugation at 12000×g for 15 min at 4°C, and the supernatant fraction (whole cell lysate) was collected and protein concentration was determined with the Bradford protein assay.

The cytoplasm and nuclear fraction were used to determine the level of AP-1 and NF-κB proteins. The treated cells were collected and washed twice with ice-cold PBS. The cell pellet was suspended in hypotonic buffer (10 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.9, 10 mM KCL, 0.1 mM EDTA, 0.1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT), 0.5 mM PMSF, 1 mM NaF and 1 mM Na₃VO₄). The cells were allowed to swell on ice for 20 min, after which 15 µL of 10% of Nonidet P-40 was added. The tubes were agitated on a vortex for 15 s and centrifuged at 12000×g for 1 min. The supernatant was collected and represent the cytoplasmic fraction. The nuclear pellets were gently washed with ice-cold PBS and resuspended in extraction buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EGTA, 1 mM DTT, 1 mM NaF and 1 mM Na₃VO₄) on ice for 15 min and centrifuged at 12000×g for 10 min. The supernatant was collected which represents the nuclear fraction.²³⁾

Western Blot Analysis

To determine the level of protein in the whole cell lysate, cytoplasmic and nuclear fractions, the protein samples were separated by 8% or 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by electro blotting. Then, the membrane was immediately blocked with 5% (w/v) non-fat milk in PBS containing 0.5% (v/v) Tween-20 (PBST buffer) at room temperature for 1 h, to reduce non-specific binding. After blocking, the membrane was washed and probed with a specific primary antibody at 4°C overnight. The membrane was washed and probed with horseradish peroxidase-conjugated secondary antibody in 5% (w/v) non-fat dry milk in PBST buffer at room temperature for 2 h. The membrane was washed and the antibody labeling was visualized with a Supersignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, U.S.A.) according to the manufacturer’s instructions and exposed to X-ray film (GE Healthcare Ltd., U.K.).

Statistical Analysis

All experiments were performed in triplicate. Quantifications are defined as the mean±standard deviation (S.D.) of three independent experiments and expressed as percentage of the control, which was considered to be 100%. Statistical significances of difference throughout this study were calculated by one-way ANOVA, offered by Dunnett’s test (SPSS 17.0 software). A difference between the experimental groups was considered statistically significant whenever the p value are <0.01 and <0.05.

RESULTS

Crebanine Inhibits IL-6 and TNF-α Production in LPS-Induced RAW264.7 Cells

We first examined the cytotoxicity of crebanine in macrophages using an MTT assay. The cells were treated with various concentrations of crebanine (7.4–58.9 µM) for 24 h. As shown in Fig. 1A, no cytotoxicity was observed (% cells survival >80%) when the cells were treated with crebanine concentration lower than 44.2 µM. To investigate whether crebanine could regulate inflammatory cytokine, IL-6 and TNF-α production, the level of the cytokines was measured in LPS-stimulated macrophages after crebanine treatment. As shown in Figs. 1B, C, stimulation of macrophages with LPS increases the level of IL-6 and TNF-α. Whereas, pretreatment of the cells with crebanine inhibited the level of IL-6 and TNF-α in LPS-stimulated cells in a concentration-dependent manner.

Fig. 1. Effect of Crebanine on Pro-inflammatory Cytokines Production in LPS-Induced RAW264.7 Cells

Cells were incubated with various concentrations of crebanine for 24 h and then cell viability was determined by MTT assay (A). Cells were pretreated with the indicated concentrations of crebanine 2 h before incubation with LPS (1 µg/mL). After incubation for 24 h, the levels of IL-6 (B) and TNF-α (C) that were present in the supernatants were measured using ELISA kits. The results represent as the mean±S.D. of three independent experiments. ** p<0.01 was considered statistically significant with LPS alone.

Effect of Crebanine on LPS-Induced NO Production and iNOS Expression in RAW264.7 Cells

In order to investigate the effect of crebanine on LPS-induced NO production in RAW264.7 cells, nitric oxide production was evaluated by measuring the level of nitrite released in the culture supernatants using the Griess reagent. As shown in Fig. 2A, a low level of NO was observed in unstimulated RAW264.7 cells. However, after LPS treatment, the level of NO production markedly increased. Crebanine inhibited the NO production in LPS-induced RAW264.7 cells in a concentration-dependent manner. Crebanine at 29.5 µM reduced the level of NO to 70% when compared with the control. Since NO is produced by iNOS, we next evaluated the effect of crebanine on the expression of iNOS expression in LPS-stimulated RAW264.7 cells. As the results showed in Fig. 2B, when the cell was induced by LPS, the expression of iNOS protein expression was markedly increased and crebanine inhibited the iNOS protein expression on LPS-induced RAW264.7 cells in a concentration-dependent manner.

Fig. 2. Effect of Crebanine on LPS-Induced Nitric Oxide Production and iNOS Expression in RAW264.7 Cells

RAW264.7 cells were pretreated with crebanine (7.4–29.5 µM) for 2 h and stimulated with LPS (1 µg/mL) for 24 h. Nitric oxide levels in the supernatant were detected using ELISA (A). iNOS protein levels were analyzed in whole cell lysates using Western blots (B), β-actin expression was used as an internal control. The results are expressed as the mean±S.D. of three different experiments. ** p<0.01 was considered statistically significant with LPS alone.

Crebanine Reduced PGE₂ Production and COX-2 Expression in LPS-Induced RAW264.7 Cells

COX-2 is related with the production of pro-inflammatory mediators, especially PGE₂. The inhibitory effects of crebanine on LPS-induced PGE₂ production in RAW264.7 cells were assessed. As shown in Fig. 3A, LPS markedly increased PGE₂ production in the culture supernatant by approximately 5 times when compared with untreated control cells, and crebanine inhibited PGE₂ production in a concentration-dependent manner. Furthermore, to determine whether the inhibitory activity of PGE₂ production by crebanine is associated with COX-2 expression, Western blot analysis was performed. As shown in Fig. 3B, LPS markedly induced COX-2 expression when compared to untreated control cells. Pretreatment of the cells with the indicated concentration of crebanine inhibited COX-2 expression in a concentration-dependent manner.

Fig. 3. Effect of Crebanine on LPS-Induced PGE₂ Production and COX-2 Expression in RAW264.7 Cells

RAW264.7 cells were pretreated with crebanine (7.4–29.5 µM) for 2 h and incubated with LPS (1 µg/mL) for 24 h. PGE₂ levels in the supernatant were detected using ELISA (A). COX-2 protein levels were analyzed in whole cell lysates using Western blots (B). The results are expressed as the mean±S.D. of three different experiments. * p<0.05 indicates significant differences from the LPS-treated group.

The Ability of Crebanine on the Activation of MAPKs and Akt Signaling Pathways

Since exposure of LPS in macrophage activates MAPKs and Akt signaling proteins, we tested the effect of crebanine on LPS-induced phosphorylation of Akt and several MAPKs, including ERK1/2, p38 and JNK in RAW264.7 cells by Western blot analysis. As shown in Fig. 4A, LPS significantly increased the phosphorylation of MAPKs signaling proteins, while pretreatment with crebanine significantly reduced the phosphorylation of ERK1/2, p38, and JNK in RAW264.7 cells. Moreover, crebanine treatment also inhibited the phosphorylation of Akt induced by LPS (Fig. 4B). To confirm the regulatory effects of MAPKs and Akt signaling pathways in LPS-induced inflammatory genes expression, we examined the effect of SB202190 (p38 inhibitor), SP600125 (JNK inhibitor), PD98059 (ERK1/2 inhibitor) and LY294002 (Akt inhibitor) on the level of IL-6 and NO production in LPS-induced RAW264.7 cells. As shown in Fig. 4C, MAPKs and Akt inhibitors significantly reduced the level of IL-6. On the other hand, SB202190, SP600125 and LY294002 significantly inhibited LPS-induced NO production, while PD98059 showed a minimal effect (Fig. 4D).

Fig. 4. Effects of Crebanine on LPS-Induced Activation of MAPKs and Akt Signaling Pathways in RAW264.7 Cells

RAW264.7 cells were pretreated with crebanine (7.4–44.2 µM) for 2 h and then incubated for 15 min with LPS (1 µg/mL). Phosphorylation and total protein expression of MAPKs (ERK1/2, p38, JNK) (A) and Akt (B) signaling proteins were analyzed in whole cell lysates using Western blot. To determine the roles of MAPKs and Akt signaling pathways in LPS-induced inflammatory gene expression, RAW264.7 cells were pretreated with MAPKs and Akt inhibitors for 2 h and induced with LPS (1 µg/mL) for 24 h. The level of IL-6 (C) and NO (D) in the supernatant were detected using ELISA. The results are expressed as the mean±S.D. of three different experiments. * p<0.05, ** p<0.01 was considered statistically significant with LPS alone.

Effect of Crebanine on NF-κB Activation in LPS-Induced RAW264.7 Cells

NF-κB is a transcription factor that regulates gene expression of inflammatory mediators. To investigate the regulation of crebanine on the transcriptional activity of NF-κB, we determined the nucleus translocation and phosphorylation of NF-κB. In resting cells, NF-κB is located in the cytoplasm as an inactive complex bound to its inhibitor, inhibitor of kappa B-α (IκB-α). Once induced with LPS, IκB-α is rapidly phosphorylated and degraded by IκB-α kinase, which results in translocation of NF-κB into the nucleus. As shown in Figs. 5A and B, treatment of RAW264.7 cells with LPS-induced IκB-α degradation and nucleus translocation of NF-κB p65 are displayed, respectively. However, pretreatment of the cells with crebanine did not have an effect on IκB-α degradation and the nuclear level of NF-κB. Several studies have shown that p65 phosphorylation on Ser536 residue mediated dimerization and the DNA binding activity of NF-κB. To investigate whether crebanine mediated NF-κB activation by regulating the phosphorylation of p65, the cytoplasmic fraction from LPS-induced RAW264.7 cells were prepared and analyzed by Western blot analysis. As shown in Fig. 5C, pretreatment of RAW264.7 cells with crebanine reduced LPS-mediated phosphorylation of p65 at Ser536 in a concentration-dependent manner. To examine the involvement of MAPKs and Akt signaling pathways in the phosphorylation of p65 at Ser536, we treated the cells with p38, JNK, ERK1/2 and Akt inhibitors and used Western blot analysis to determine the phosphorylation of p65. As shown in Fig. 5D, treatment of the cells with SB202190, SP600125, PD98059 and LY294002 significantly reduced LPS-inducement of the phosphorylation of p65 at Ser536.

Fig. 5. Effect of Crebanine on NF-κB (p65) Activation in LPS-Induced RAW264.7 Cells

The cells were pretreated with crebanine (7.4–44.2 µM) for 2 h and then incubated with LPS (1 µg/mL) for 30 min. Cytoplasmic and nuclear extracts were prepared to analyze IκB-α degradation (A) and nucleus translocation of p65 (B), respectively. The level of these proteins were analyzed by Western bolt analysis. The levels of phosphorylation of p65 at Ser536 in cytoplasmic fraction were investigated by Western blot (C). To determine the regulatory effect of the MAPKs and Akt signaling pathways on the LPS-induced phosphorylation of p65, RAW264.7 cells were pretreated with MAPKs and Akt inhibitors for 2 h and induced with LPS (1 µg/mL) for 30 min, the phosphorylation of p65 at Ser536 was analyzed using Western blots (D). All experiments were repeated three times and the representative results are shown.

Crebanine Inhibited LPS-Induced AP-1 Activation in RAW264.7 Cells

Along with NF-κB, the transcription factor AP-1, is known to be involved in the transcription regulation process of the inflammatory responses. Therefore, we investigated the effect of crebanine on nucleus translocation and phosphorylation of AP-1 (c-Jun). As shown in Fig. 6A, treatment of RAW264.7 cells with LPS alone significantly increases the level of c-Jun in nucleus and pretreatment of the cells with crebanine significantly reduced the nucleus level of c-Jun in a concentration-dependent manner. Moreover, pretreatment of RAW264.7 cells with crebanine significantly decreased the phosphorylation of c-Jun (Fig. 6B).

Fig. 6. Effect of Crebanine on Nucleus Translocation and Phosphorylation of AP-1 in RAW264.7 Cells

RAW264.7 cells were pretreated with crebanine (7.4–44.2 µM) for 2 h and incubated with LPS (1 µg/mL) for 30 min. The nuclear extracts were prepared to analyze the nucleus translocation of c-Jun (A). The phosphorylation levels of c-Jun at Ser63 in the cytoplasmic extract were analyzed using Western blots (B). All experiments were repeated three times and the representative results are shown.

DISCUSSION

The overproduction of pro-inflammatory mediators from macrophages can be harmful and result in various inflammatory diseases such as cancer, atherosclerosis and arthritis.^2,4) Therefore, the agent that reduced the level of the inflammatory mediators and cytokines is regarded as an effective therapeutic strategy for relieving a chronic inflammatory disease. In this study, we reported for the first time that crebanine purified from Stephania venosa exhibited anti-inflammatory properties by reducing the level of cytokines, IL-6 and TNF-α from LPS-induced macrophages. In addition, we also demonstrated that crebanine exerts its anti-inflammatory effects by inhibiting LPS-induced iNOS and COX-2 expression and subsequently reduced the production of NO and PGE₂ in macrophages.

Previous studies report that alpha-7 nicotinic acetylcholine receptor (α7-nAChR) play a vital role in regulating cholinergic anti-inflammatory pathway.²⁴⁾ The expression of α7-nAChR has been found on various immune cells including macrophage. Activation of the α7-nAChR on macrophage exhibited anti-inflammatory via down-regulating the production of pro-inflammatory cytokines TNF-α and IL-6. It appears that the α7-nAChR is a promising target for developing novel anti-inflammation drugs. The specific agonists of α7-nAChR including nicotine have been reported to exhibit significant anti-inflammatory effect in vitro and in vivo model.²⁵⁾ On the other hand, treatment of an α7-nAChR antagonist such as α-bungarotoxin increased the severity of inflammation.²⁶⁾ However, there have not been reported evidence for anti-inflammation effect of crebanine on α7-nAChR. Recently, Rojsanga et al. reported that administration of crebanine improved memory impairments induced by scopolamine in mice.²⁷⁾ Their docking result indicated that crebanine specific bound to active site of α7-nAChR and functional assay showed that crebanine is an antagonist of α7-nAChR. From aforementioned reports the mode of action of crebanine binding on α7-nAChR remains controversial therefore other mechanism in addition to α7-nAChR may contribute to the observed in anti-inflammatory activities of crebanine.

MAPKs are highly conserved protein serine/threonine kinase and three major subfamilies including ERK1/2, p38 and JNK have been found in mammalian cells.²⁸⁾ MAPKs have been involved in pro-inflammatory signaling cascades and a good deal of evidence has demonstrated that the activation of ERK1/2, JNK and p38 is involved in up-regulation of IL-6, TNF-α, iNOS and COX-2 in LPS-activated macrophages.¹⁰⁾ Therefore, anti-inflammatory mechanism are closely related with inhibition of the phosphorylation of MAPKs in activated RAW264.7 cells. This study demonstrated that phosphorylation of ERK1/2, JNK and p38 by LPS was inhibited by crebanine. More recent studies have demonstrated that the phosphatidylinositol 3′-kinase/Akt (PI3-K/Akt) signaling pathway is involved in LPS-stimulated signal transduction and leads to an induction of pro-inflammatory genes expression.^13,29) Thus, the effect of crebanine on Akt pathway was investigated to further explore the signaling pathway required for regulated pro-inflammatory gene expression. We found that phosphorylation of Akt was significantly inhibited with crebanine in LPS-stimulated macrophages. Moreover, in this study we confirm that the inhibition of inflammatory mediator expression is related to the down-regulation of MAPKs and Akt pathway through the use of the inhibitors of MAPKs and Akt. Our result has shown that MAPKs and Akt inhibitors blocked LPS induced IL-6 and NO production. Consistent with previous reports, activation of ERK1/2, JNK, p38 and Akt signaling molecules are involved in LPS-induced expression of IL-6 and iNOS.^30,31) Collectively, these results suggest that crebanine suppresses the increased levels of inflammatory mediators by inhibiting activation of MAPKs and Akt in LPS-stimulated macrophages.

Transcription factors, such as NF-κB and AP-1, regulate the expression of inflammatory mediators including IL-6, TNF-α, COX-2 and iNOS in inflammatory processes.^32,33) A commonly known NF-κB consists of p50/p65 heterodimer, and NF-κB signaling is governed by the IκB kinase (IKK) complex and the downstream substrate IκB-α.³⁴⁾ Upon activation of TLR4 with LPS, myeloid differentiation primary response gene 88 (MyD88) recruits and confer a signal to interleukin-1 receptor-associated kinases (IRAKs). The released IRAKs can activate TNF receptor associated factor 6 (TRAF6) lead to the phosphorylation of transforming growth factor (TGF)-beta-associated kinase 1 (TAK1). TAK1, in turn, phosphorylates MAPK and the IKK complex.³⁵⁾ The activated IKK complex induces phosphorylation of IκB-α, leads to IκB-α degradation and enhanced NF-κB nucleus translocation. In the nucleus, NF-κB binds to cognate sequence in the promoter region of many inflammatory genes.³³⁾ Therefore, we examined the effect of crebanine on the degradation of IκB-α and the translocation of p65 into the nucleus. Despite many compounds such as curcumin³⁶⁾ and quercetin³⁷⁾ have been reported that they could inhibit LPS-induced NF-κB activity via reduced classical signaling pathway (IKK/IκB-α/p65 translocation). However, in this study crebanine did not affect IκB-α degradation and p65 translocation. Based on this finding indicate that crebanine did not effect on classical signaling cascade of LPS induced TLRs mediated IKK- IκB-α pathway.

Although the phosphorylation of IκB-α and the subsequent nuclear translocation of NF-κB are the key steps for NF-κB activation, recent evidence has indicated that the phosphorylation of NF-κB subunit p65 modulates NF-κB transcription activity.³⁸⁾ Multiple lines of evidence have been indicated that the DNA binding and trans-activating capacity of NF-κB are upregulated by phosphorylation of p65 on Ser536 residue, which is independent of IκB-α degradation.³⁹⁾ Here we show that crebanine inhibited LPS-induced phosphorylation of p65 at Ser536. Several kinases including IKK, PI3-K/Akt, and glycogen synthase kinase 3 beta (GSK3β) have been identified as the regulators for p65 phosphorylation under certain inflammatory stimulus.⁴⁰⁾ Recent studies have shown that PI3-K/Akt signaling pathway can activate transactivation of NF-κB through phosphorylation of p65 on Ser536 but have no effect on IκB-α degradation.⁴¹⁾ Moreover, it has been reported that PI3-K/Akt is the downstream signaling component of the MyD88-dependent TLR4 signaling pathway. Therefore we investigated whether PI3-K/Akt signaling was involved in LPS-induced phosphorylation of p65. Our results have shown that specific inhibitor of Akt suppressed LPS-induced phosphorylation of p65 on Ser536. Although other stimulus such as TNF-α and oxidative stressed regulated phosphorylation of p65 via MAPKs signaling pathway.⁴²⁾ By using inhibitors of p38, JNK and ERK1/2, our result demonstrated for the first time that LPS-induced phosphorylation of p65 on Ser536 in RAWmacrophages via p38, JNK and ERK1/2 MAPK signaling cascade. Because the degradation of IκB-α and translocation of p65 were not changed by crebanine, it is possible that the phosphorylation of p65 may be a major mechanism by which crebanine inhibits NF-κB activity. Together, these results suggest that crebanine attenuated NF-κB activity via inhibited phosphorylation of p65 by modulation of Akt and MAPKs pathway.

AP-1 is another transcription factor known to be activated by the phosphorylation of MAPKs and Akt.⁴³⁾ The promoter of TNF-α, IL-6, iNOS and COX-2 genes contain the AP-1 binding site, indicating the involvement of AP-1 in the modulation of these genes.^32,44) A good deal of evidence has demonstrated that activation of JNK, p38 and ERK1/2 is involved in up-regulation of inflammatory mediators via the activation of AP-1 in LPS-stimulated immune cells.¹²⁾ The proximal signaling events in LPS-triggered activation of JNK, p38 and ERK1/2 are involved with MyD88, TRAF6 and TAK1. Moreover, involvement of the TLRs-associated PI3-K/Akt signaling pathway in the expression of inflammatory mediators in macrophages through the activation of AP-1 has also been demonstrated.⁴⁵⁾ Here, we observed the activation of AP-1 by investigating the translocation and phosphorylation of c-Jun, a major component of the AP-1 family, in LPS-induced macrophages. We showed that crebanine reduced LPS-induced c-Jun phosphorylation and translocation to the nucleus of RAW264.7 cells. Based on the results above, we suggested that suppression of LPS-induced NF-κB and AP-1 activation by crebanine resulted in the interruption of downstream signaling molecules that involved in TLR-induced MAPKs and PI3-K/Akt pathway as shown in Fig. 7. However, the exact molecular targeting of crebanine in signaling cascade of TLR-induced MAPKs and PI3K-Akt need to be further investigated.

Fig. 7. Proposed Mechanism of Crebanine Inhibition of LPS-Induced Inflammation in RAW264.7 Cells

The inhibitory effect of crebanine on expression of pro-inflammatory mediators in LPS-stimulated RAW264.7 cells may be mediated by blocking NF-κB and AP-1 activation through the upstream signaling molecules of the MAPKs and Akt pathway.

In conclusion, we provide evidence that crebanine exerts inhibitory effects on LPS-induced iNOS and COX-2 expression and the subsequent production of NO and PGE₂ in the murine macrophages. Moreover, crebanine inhibited the production of pro-inflammatory cytokines including, IL-6 and TNF-α. The effects of crebanine were found to be associated with an inactivation of NF-κB and AP-1 transcription factors via a blockade of MAPKs and Akt phosphorylation. Therefore, our results suggest that crebanine could be considered for use as a potential treatment for inflammatory disorders.

Acknowledgment

This work was supported by the Faculty of Medicine Research Fund, Faculty of Medicine, Chiang Mai University, Thailand.

Conflict of Interest

The authors declare no conflict of interest.

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