3β-Angeloyloxy-8β,10β-dihydroxyeremophila-7(11)-en-12,8α-lactone Inhibits Lipopolysaccharide-Induced Nitric Oxide Production in RAW264.7 Cells

Abstract

Farfugium japonicum (L.) KITAM, named “Lian-Peng-Cao” in China, has been traditionally used in Chinese folk medicine to treat sore throat, cold and cough due to its anti-inflammatory properties. In this study, the anti-inflammatory action of 3β-angeloyloxy-8β,10β-dihydroxyeremophila-7(11)-en-12,8α-lactone (FJ1) isolated from Farfugium japonicum and its molecular mechanism in RAW264.7 cells were investigated. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that FJ1 with or without 3 µg/mL lipopolysaccharide (LPS) had no significant cytotoxicity in RAW264.7 cells. The production of nitric oxide (NO) was identified with a Griess reagent kit. The mRNA expression of inducible nitric oxide synthase (iNOS) and cytokines, including tumor necrosis factor α (TNF-α) and cyclooxygenase-2 (COX-2), was measured by real-time polymerase chain reaction (PCR). Reactive oxygen species (ROS) production was detected by flow cytometry analysis. Western blot was used to examine the protein expression of nuclear factor-kappa B (NF-κB)/p65, inhibitor of kappa B (IκB)-α, phosphorylated IκB-α (p-IκB-α), mitogen-activated protein kinase (MAPK) molecules, iNOS, and TNF-α. We discovered that FJ1 possesses anti-inflammatory effects that inhibit the release of LPS-stimulated pro-inflammatory cytokines, including NO and ROS. The molecular mechanism of FJ1-mediated anti-inflammation is associated with decreasing phosphorylation of MAPK molecules, including extracellular signal-related kinase 1/2 (ERK1/2), p38 MAPK, and c-Jun NH2-terminal kinase (JNK), FJ1 also reverses IκB degradation and attenuates the mRNA and protein expression of NF-κB-related downstream inducible enzymes and cytokines, such as iNOS, TNF-α in RAW264.7 cells. The results suggest that FJ1 has anti-inflammatory properties, which indicates that F. japonicum can be utilized to treat inflammatory diseases. The potential mechanism is associated with the NF-κB and MAPK activation pathways in LPS-stimulated macrophages.

Macrophages are present in almost all body tissues. Macrophages as important effectors cells of the immune system provide defense against microbial invaders and malignancies owing to their phagocytic, cytotoxic, and intracellular killing capacities. Macrophages play important roles in innate and adaptive immune response to pathogens, including phagocytosis, antigen presentation, and cytokine secretion.¹⁾ Macrophages release a number of factors, such as nitric oxide (NO), prostaglandin, and cytokines, during immune response.^2–5) Overproduction of inflammatory mediators released by activated macrophages is associated with many inflammatory diseases.^6,7)

Natural products provide a great chemical structural diversity. Farfugium japonicum, known as da-wu-feng-cao in China, is mainly distributed in southeast and south China, Japan, and Korea. F. japonicum is utilized as folk medicine in China to treat sore throat, cold, cough and pain owing to its anti-inflammatory properties.⁸⁾ Besides, modern pharmacological studies have shown that the total extract of F. japonicum and its compounds have many pharmacological properties, including anti-tumor, anti-inflammatory, and analgesic properties.⁹⁾ Moreover, the total extract of F. japonicum exhibited significant inhibition effect on lipopolysaccharide (LPS)-stimulated NO production in RAW264.7 cells in our present study (Fig. 1B). In our continuing program to search for anti-inflammatory agents from this plant, we chose four higher yields compounds (FJ1, FJ3, FJ5, FJ12) to test inhibition potency on NO production. The striking activity and the highest yields of FJ1 suggested that FJ1 may pay an important role in the anti-inflammatory diseases of F. japonicum. In the present study, FJ1 was employed further to investigate the effects and molecular mechanisms on LPS-stimulated pro-inflammatory properties in RAW264.7 cells.

Fig. 1. (A) Chemical Structures of FJ1, FJ3, FJ5, and FJ12; (B) NO Production with Total Extract of F. japonicum in RAW264.7 Cells; (C) Effects of FJ1, FJ3, FJ5, and FJ12 on LPS-Stimulated NO Production in RAW264.7 Cells; (D) Cytotoxicity of FJ1 in RAW264.7 Cells

(A, B) The cells were incubated with LPS (3 µg/mL) only and with LPS plus the indicated concentrations of total extract for 24 h. The medium was analyzed for NO generation. The data are expressed as mean±S.D. (C) The cells were incubated with LPS (3 µg/mL) only and with LPS plus the indicated concentrations of FJ1 for 24 h. The medium was analyzed for NO generation. The data are expressed as mean ±S.D. * p<0.05 and ** p<0.01 compared with the LPS only group. (D) The cells were treated with the indicated concentrations of FJ1with the absence and presence of LPS for 24 h. Cell viability was evaluated through an MTT assay. The data represent at least three independent experiments and are expressed as mean±S.D. (% Cell viability).

MATERIALS AND METHODS

Isolation and Purification

Tested compounds (FJ1, FJ3, FJ5, FJ12) have been prepared as described in our group previous papers.^8,10) In brief, the air-dried rhizome of F. japonicum were extracted three times (seven days each time) with petroleum ether (bp 60–90°C)–Et₂O–CH₃OH at room temperature. The extract was concentrated under reduced pressure. After that, the residue was subjected to silica gel CC eluting with a gradient of n-hexane–acetone to give a few fractions which were subsequently isolated by silica gel CC eluted with suitable gradient n-hexane–acetone elution. Then, compound FJ1 was further purified by repeated silica gel CC eluted with n-hexane–acetone as eluent, FJ3 and FJ5 were purified by silica gel CC with n-hexane–EtOAc as eluent, and FJ12 was purified by low pressure C-18 CC with H₂O–CH₃OH elution. The yields of FJ1, FJ3, FJ5, FJ12 were 1.05%, 0.25%, 0.45%, 0.17%, respectively.

Chemicals and Reagents

FJ1 (Fig. 1A) isolated from F. japonicum, with a purity exceeding 98%, was produced by the Nature Products Department of our institute. FJ1 was dissolved in dimethyl sulfoxide (DMSO) as a 10 mM stock solution and diluted according to experimental requirements. Lipopolysaccharide (LPS) and 3-(4,5-dimethylthiazol)-2,5-diphenylterazolium bromide (MTT) were purchased from Sigma Co., U.S.A. All other chemicals utilized in the experiments were commercial products and of reagent grade.

Cell Culture

The murine macrophage cell line RAW264.7, obtained from the Shanghai Institute for Biological Science (SIBS), was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Invitrogen, U.S.A.) at 37°C in a humidified atmosphere containing 5% CO₂. All culture media were supplemented with 10% fetal bovine serum (TBD, Tianjin, China), 100 units/mL streptomycin and 100 units/mL penicillin. The cells in the exponential phase of growth were utilized in the experiments.

MTT Assay

The cytotoxicity of FJ1 was measured through an MTT assay. Briefly, Cells (3×10³ to 4×10³ per well) were seeded in 96-well culture plates. After incubating for 24 h, the cells were treated with FJ1 at diverse concentrations (5, 10, 15 µM) for another 24 h. Then, 20 µL of MTT solution (5 mg/mL) was added to each well for 4 h. The medium was removed and the formazan precipitates were solubilized in 150 µL of DMSO. Optical density of each well was measured by a microtiter reader (Bio-Rad 680) at 570 nm. Wells with DMSO but untreated with FJ1 were used for control cell viability and represented 100% cell survival, and wells without cells for blanking the spectrophotometer.   

Nitrite Assay with a Griess Reagent

The nitrite accumulation in the supernatant was assessed with a Griess reagent kit (Beyotime Institute of Biotechnology, China). According to the manufacturer’s instructions, each 50 µL of culture supernatant was mixed with 50 µL of Griess reagent I and 50 µL of Griess reagent II at room temperature. Absorbance was measured with a microplate reader at 540 nm. A series of known concentrations of sodium nitrite was considered the standard.

Measurement of Intracellular Reactive Oxygen Species (ROS)

Generation ROS levels were quantified by measuring the oxidative conversion of cell permeable 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Molecular Probes, Eugene, OR, U.S.A.) into fluorescent dichlorofluorescein (DCF). ROS kit (Beyotime Institute of Biotechnology, China) with FACS-SCAN apparatus (Becton Dickinson, U.S.A.) was utilized. The RAW264.7 cells grown in 6-well plates were treated with FJ1 (5, 10, 15 µM) at 37°C for 24 h. The culture medium was removed and the cells were washed three times with DMEM after incubation. The cells were incubated for 20 min at 37°C in DMEM containing 5 µM of DCFH-DA to label intracellular ROS. The cells were then immediately analyzed by flow cytometry. The difference between the values of the experimental and control samples represented the decrease in intracellular ROS levels.

Cell Fractionation

The cells were separated into two fractions consisting of a nucleus and cytosol with a nuclear and cytoplasmic protein extraction kit (Beyotime Inst., Haimen, China). The cells (2×10⁷) were washed with ice-cold phosphate buffered saline (PBS) and suspended in 200 µL of Reagent A containing 1 mM of phenylmethyl sulfonylfluoride (PMSF). The cells were incubated in ice for 15 min after vortexing for 5 s. Then, 10 µL of Reagent B was added. The above operation was repeated. The nuclei were separated from the cytosolic extracts by centrifugation (16000×g, 5 min at 4°C). The supernatant was the cytosol fraction. The nuclear pellets were resuspended in a nuclear protein reagent for 30 min at 4°C under agitation. The supernatants containing the nucleus proteins were collected for Western blot analysis after centrifugation (16000×g, 10 min at 4°C).

Western Blot Analysis

The total cell lysates, cytosolic and nucleus proteins were analyzed through Western blot. The samples were subjected to 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was washed in distilled water and then blocked with 5% nonfat milk in TBS-T (10 mM Tris–HCl at pH 7.8, 150 mM NaCl, and 0.05% [v/v] Tween-20 buffer, pH 7.8) for at least 1 h at room temperature. After a short washing in TBS-T buffer, the membrane was incubated overnight in a solution of monoclonal antibodies specific for extracellular signal-regulated kinase (ERK) 1/2, c-Jun N terminal kinase (JNK), p38, p-ERK1/2, p-JNK, p-p38, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB/p65), inhibitor of nuclear factor-kappa B (IκB)-α, phosphorylated IκB-α (p-IκB-α), nitric oxide synthase (iNOS) and TNF-α (All 1 : 1000, Santa Cruz Biotechnology) at 4°C. Then the membrane was incubated with secondary horseradish peroxidase (HRP) goat anti-mouse immunoglobulin G (IgG) or anti-rabbit IgG (1 : 1000; Santa Cruz Biotechnology). Proteins on the membrane were detected with an enhanced chemiluminescence detection system (ECL^®, Amersham Biosciences). Protein expression was quantified by densitometry with the Image J and Java software.

RNA Extraction and Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted with an RNA easy kit (Bioecon Biotec, China) according to the manufacturer’s instructions. The purity of the total RNA was evaluated by OD_260/280 of the RNA samples (>1.8). cDNA was synthesized through reverse transcription with an M-MLV reverse transcriptase and Oligo (dT) primer. The expression of iNOS, TNF-α, cyclooxygenase-2 (COX-2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes was detected through real-time PCR assay. PCR amplification was performed thrice in an 8-tube strip format (Axygen, CA, U.S.A.). Each reaction contained 1×SYBR Green PCR Master mix, 1 µL of forward primer and reverse primer and 1 µL of template cDNA in a final volume of 20 µL. A Mastercycler ep realplex apparatus (Eppendorf, Germany) was utilized. Primers were based on the iNOS gene (forward: 5′-CGC TAC AAC ATC CTG GAG GAA GT-3′ and reverse: 5′-GTC TTT CCA GAG CGA GGC CAG-3′), TNF-α (forward 5′-CAT CTT CTC AAA ATT CGA GTG ACA A-3′ and reverse 5′-TGG GAG TAG ACA AGG TAC AAC CC-3′), and COX-2 (forward: 5′-TGA GTA CCG CAA ACG CTT CTC-3′ and reverse: 5′-TGG ACG AGG TTT TTC CAC CAG-3′). The evaluation of the GAPDH expression utilized as a control for the RNA amount was conducted with a primer (forward: 5′-GAC GGC CGC ATC TTC TTG T-3′ and reverse: 5′-CAC ACC GAC CTT CAC CAT TTT-3′).^11,12) Amplification was performed for 45 cycles of sequential denaturation (95°C for 2 min, annealing (60°C for 15 s), and extension (72°C for 20 s). Data acquisition and analysis of the real-time PCR assay were performed with a Mastercycler ep realplex apparatus (Eppendorf, Germany). Each fluorescent reporter signal was measured against the internal reference dye signal to normalize non-PCR-related fluorescence fluctuations in the wells. All samples were collected in triplicate independent experiments. All the primers were synthesized by Sangon Co., Ltd. (Shanghai, China).

Statistical Analysis

All experiments were performed at least thrice. Statistical analysis was performed with ANOVA (SPSS 17.0, Chicago, U.S.A.) followed by Tukey’s t-test. A p-values <0.05 were considered to be statistically significant.

RESULTS

Effect of FJ1 on LPS-Stimulated NO Production and Cell Viability in RAW264.7 Cells

We detected levels of NO in LPS-stimulated RAW264.7 cells to identify the anti-inflammation effect of total extract and compounds isolated from F. japonicum. The cells were treated with various concentration of total extract and tested compounds in the presence or absence of LPS (3 µg/mL) through Griess assay. The results showed that the total extract of F. japonicum inhibited LPS-stimulated NO production from 0.1 mg/mL to 100 mg/mL (peak value at 20 mg/mL with the inhibition rate was 77.14%) in RAW264.7 cells (Fig. 1B). The total extract of F. japonicum has no significant cytotoxicity in the same dose. And then we tested the further NO production inhibition effect of the four higher yield compounds on RAW264.7 cells. The results showed that the pretreatment of RAW264.7 cells with compounds (FJ1, FJ3, FJ5, FJ12) significantly inhibited LPS-stimulated NO production (Fig. 1C). Among these compounds, FJ1 exhibited the highest potency. FJ1 inhibited LPS-stimulated NO production in a concentration-dependent manner. The inhibition rates were 28.61%, 43.42%, and 71.80% at the concentration of 5 µM, 10 µM, and 15 µM, respectively (Fig. 1C).

We performed an MTT colorimetric assay to determine the cytotoxic effect of FJ1 and to assess the effect of FJ1 on cell viability of RAW264.7 cells. Figures 1C and D indicate that the treatment of FJ1 (0–15 µM) with LPS at 3 µg/mL resulted in slight toxicity but exhibited a more significant NO production compared with the vehicle control. Figure 1D showed that FJ1 with and without LPS pretreatment did not display significant cytotoxicity up to 15 µM in RAW264.7 cells (the cell viability with LPS was 96.37%, 89.78%, 82.55%, 83.68% and without LPS was 100%, 96.07%,92.19%, 94.44%, respectively). This finding suggests that the anti-inflammatory effect of FJ1 is not related to cytotoxicity.

Effect of FJ1 on LPS-Induced ROS Production

Considering that ROS is important for LPS-induced inflammation,¹³⁾ we investigated whether FJ1 inhibits LPS-induced intracellular accumulation of ROS. The results of the flow cytometric assay indicate that a greater ROS accumulation was occurred in the LPS-treated RAW264.7 cells compared to the cells that had not been treated with FJ1. The increase in LPS-stimulated ROS levels was significantly attenuated by FJ1 in a dose-dependent manner (Fig. 2).

Fig. 2. Effects of FJ1 on LPS-Induced ROS Production in RAW264.7 Cells

The cells were treated with the indicated concentrations of FJ1 (5, 10, 15 µM) for 4 h before LPS (3 µg/mL) treatment for 24 h. Flow cytometry was performed to detect ROS production. ROS accumulation was quantified by measuring DCF-derived fluorescence. The fluorescence intensity of cells without FJ1 and LPS treatment was set at 100% (FJ1 0 µM, LPS 0 µM). (A) Representative peak observed during flow cytometric analysis of ROS accumulation in RAW264.7 cells. (B) Column bar graphs show the fluorescence intensities of ROS accumulation in RAW264.7 cells. Each column represents the mean±S.D. * p<0.05 and ** p<0.01 compared with the LPS only group.

FJ1 Inhibits LPS-Induced Activation of Mitogen-Activated Protein Kinase (MAPK) and NF-κB

Many signaling pathways, such as MAPK and NF-κB pathways, are involved in the regulation of microglial activity and contribute to the production of inflammatory mediators.^14–16) The phosphorylation of three MAPK molecules, ERK1/2, p38, and JNK, were examined in this study by Western blot analysis. LPS can stimulate the phosphorylation of ERK1/2, p38, and JNK in RAW264.7 cells. FJ1 suppressed the expression of p-ERK, p-JNK, and p-p38 in a concentration dependent manner in LPS-stimulated macrophages. These results indicate that signal transduction by the MAPK molecules was blocked by FJ1 in LPS-activated macrophages (Fig. 3).

Fig. 3. Western Blot Analysis of the MAPK Expression in Cells Treated with FJ1 at Various Concentrations for 24 h

ERK1/2(A), p38(B), and JNK(C), and their phosphorylation protein expressions were analyzed by Western blot after the RAW264.7 cells were treated with FJ1 for 24 h. The corresponding histograms quantified through densitometry are presented on the right side. * p<0.05 and ** p<0.01 compared with the LPS only group.

NF-κB is also a well-known critical transcription factor in the induction of a wide variety of genes involved in the regulation of immune and inflammatory responses. We determined NF-κB/p65, IκB-α, and phosphorylation of IκB-α by Western blot analysis to investigate the effect of FJ1 on the stimulation of NF-κB activity in RAW264.7 cells.¹⁵⁾ Figure 4 shows that the FJ1-treated RAW264.7 cells reduced the expression of NF-κB/p65 in the nucleus and augmented the phosphorylation of IκB-α stimulated by LPS in the cytosol in a dose-dependent manner.

Fig. 4. Effect of FJ1 on the LPS-Induced Nucleus Translocation of NF-κB /p65 and Degradation and Phosphorylation of IκB-α

The cells were pretreated with FJ1 for 2 h and then stimulated with LPS (3 µg/mL) for 1 h. (A) The decreased expression of NF-κB/p65 in the nucleus fraction was detected through Western blotting and its corresponding histogram quantified through densitometry. (B) The increased expression of IκB-α in the cytosolic extracts was determined through Western blot and its corresponding histogram-quantified through densitometry. Actin protein was utilized as an internal control, and experiments with similar results were repeated at least three times. * p<0.05 and ** p<0.01 compared with the LPS only group.

Effect of FJ1 on Inflammation-Related Genes and Proteins in RAW264.7 Cells

The above data demonstrated the inflammation-associated activities and NF-κB pathway activities of FJ1 induced in RAW264.7 cells. Further experiments show that FJ1 has effects on NF-κB-related downstream inflammation genes. The expressions of iNOS, TNF-α, and COX-2 genes in RAW264.7 cells were evaluated by quantitative real-time PCR after incubating FJ1 with and without pretreatment with LPS for 24 h. Besides, the expressions of iNOS and TNF-α proteins were checked using a Western blot analysis. Figures 5A, B, and C indicated that FJ1 significantly reduced the LPS-induced the upregulation of iNOS, COX-2, and TNF-α mRNA levels, consisting with the expression of iNOS and TNF-α proteins (Figs. 5D, E), which further confirmed the distinct effects of FJ1 on the modulation of LPS-induced inflammation.

Fig. 5. Effect of FJ1 on Inflammation-Related Genes and Proteins in LPS-Stimulated RAW264.7 Cells

The RAW264.7 cells were pre-incubated with different concentrations of FJ1 for 2 h and then treated with LPS (3 µg/mL) for another 24 h. The mRNA expressions of iNOS (A), COX-2 (B), and TNF-α (C) were analyzed through real-time PCR (RT-PCR). The protein expressions of iNOS and TNF-α (D) were checked by Western blot. The corresponding histograms quantified through densitometry are presented on the right side (E). Data are expressed as means±S.D. with three independent experiments. ** p<0.01 compared with the LPS only group.

DISCUSSION

Inflammation is a complex biological process. Macrophages play a critical role in the initiation, maintenance and resolution as well as the overproduction of pro-inflammatory cytokines and inflammatory mediators such as NO, ROS, COX-2, and TNF-α.¹⁷⁾ NO, the broadest screening inflammatory mediator, is the product of inducible iNOS.^18,19) NO production triggers a rapid but transient release of ROS and activates the inflammatory pathways through redox reactions.^20–22) Many reports have demonstrated LPS-stimulated inflammatory responses in mouse macrophage RAW264.7 cells. Inflammatory response stimulated by LPS is a suitable method to identify anti-inflammatory activity compounds in vitro.^11,23–25) LPS stimulates macrophages and activates the expression of genes responsible for the synthesis of inflammatory mediators, such as reactive oxygen and nitrogen species. These free radicals are important mediators to provoke or sustain inflammatory processes, thereby protecting hosts from harmful stimuli. However, high levels of these free radicals can damage cells and tissues resulting in inflammation.^26,27)

This study explores the anti-inflammatory components of the Chinese herb F. japonicum, which is used to treat various inflammatory diseases. We perform the drug screening process of compounds isolated from F. japonicum by LPS-stimulated RAW264.7 cells. We utilize a Griess reagent kit to screen the compounds. The compounds (FJ1, FJ3, FJ5, and FJ12) effectively inhibited the LPS-stimulated production of NO in RAW264.7 cells (Fig. 1C). Among these compounds, FJ1 exhibited the strongest anti-inflammatory ability with the highest NO inhibition rates of 28.61%, 43.42%, and 71.80% at concentrations of 5 µM, 10 µM, and 15 µM, respectively. Further results by flow cytometry analysis demonstrate the capacity of FJ1 to decrease intracellular ROS production.

MAPK and NF-κB are known to regulate the expression of inflammatory mediators. Primary inflammatory stimuli and cytokines come into work through the Toll receptors, IL-1 receptor (TIR) family, or the TNF receptor family. The activation of receptors triggers major intracellular signaling pathways: MAPK pathways and the pathway leading to the activation of the transcription factor NF-κB.¹⁶⁾ There are three major groups of distinctly regulated MAPK cascades, ERK1/2, JNK, and p38 MAPK, regulating gene expression in humans. NF-κB is a multi-protein complex that can activate a variety of genes involved in the early defense reactions of higher organisms.¹⁴⁾ NF-κB resides in the cytoplasm as an inactive complex with the inhibitor IκB in non-stimulated cells. Various stimulus, including LPS, cytokines, and viruses, activate NF-κB via several signal transduction pathways, leading to the phosphorylation of IκB. The phosphorylated IκB becomes rapidly ubiquitinated and degraded by the proteasome complex. Following IκB degradation, NF-κB translocates to the nucleus, binds to DNA control elements, and induces the synthesis of various types of mRNA.²⁸⁾ The transcription factors present in the cytoplasm or nucleus are phosphorylated and activated upon the activation of MAPK and NF-κB, leading to the expression of target genes and resulting in a biological response.

Therefore, the two major kinase-mediated signaling pathways activated by LPS stimulation, namely, MAPK and NF-κB, are examined to explore the possible underlying mechanism of FJ1. Western blot analysis indicates that LPS enhanced the phosphorylation of the three MAPK molecules: ERK1/2, p38, and JNK. Treatment of FJ1 significantly inhibited the LPS-stimulated phosphorylation of ERK1/2, p38, and JNK, which are responsible for triggering over-reactive inflammatory responses. Next, we perform Western blot analysis of sub-cellular fractions to detect the NF-κB signal. FJ1 significantly inhibited the LPS-induced phosphorylation and degradation of IκB-α as well as the translocation of activated NF-κB/p65 to the nucleus in RAW264.7 cells.

The activation of MAPK and NF-κB contributes to the expression of inflammatory genes in activated macrophages.^3,14,15,29) Reports exhibited that MAPK pathway positively regulates the stability of several inflammatory mediator mRNAs, including TNF-α and COX-2.^20,22) Several anti-oxidant compounds are also known to reduce inflammation by inhibiting the expression of cytokines consistent with NF-κB.³⁰⁾ The suppressive effects of these anti-oxidant compounds on the production of associated inflammatory mediators confirm that ROS enhances the expression of pro-inflammatory cytokine genes by activating NF-κB.³¹⁾ The upregulations of mRNA and protein levels involving LPS-induced TNF-α, iNOS, and COX-2, were strongly attenuated by FJ1 in RAW264.7 cells. FJ1 also inhibited the production of LPS-induced ROS and NO in the same cells. The potential mechanism is associated with MAPK and NF-κB activity in RAW264.7 cells.

In conclusion, the results of this study suggest that FJ1 possesses anti-inflammatory effects that decrease NO and ROS. We identified the anti-inflammatory mechanisms of FJ1 by modulating MAPK and NF-κB signaling pathways.

Acknowledgment

This work was supported by Grant from the National Natural Science Foundation of China (No. 81273532).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

REFERENCES

• 1) Dunn DL, Barke RA, Ewald DC, Simmons RL. Macrophages and translymphatic absorption represent the first line of host defense of the peritoneal cavity. Arch. Surg., 122, 105–110 (1987).
• 2) Berenbaum F. Proinflammatory cytokines, prostaglandins, and the chondrocyte: mechanisms of intracellular activation. Joint Bone Spine, 67, 561–564 (2000).
• 3) Lawrence T, Gilroy DW, Colville-Nash PR, Willoughby DA. Possible new role for NF-kappaB in the resolution of inflammation. Nat. Med., 7, 1291–1297 (2001).
• 4) Lowenstein CJ, Hill SL, Lafond-Walker A, Wu J, Allen G, Landavere M, Rose NR, Herskowitz A. Nitric oxide inhibits viral replication in murine myocarditis. J. Clin. Invest., 97, 1837–1843 (1996).
• 5) Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature, 333, 664–666 (1988).
• 6) Chen TH, Kao YC, Chen BC, Chen CH, Chan P, Lee HM. Dipyridamole activation of mitogen-activated protein kinase phosphatase-1 mediates inhibition of lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 cells. Eur. J. Pharmacol., 541, 138–146 (2006).
• 7) Crespo A, Filla MB, Russell SW, Murphy WJ. Indirect induction of suppressor of cytokine signalling-1 in macrophages stimulated with bacterial lipopolysaccharide: partial role of autocrine/paracrine interferon-alpha/beta. Biochem. J., 349, 99–104 (2000).
• 8) Shen T, Yang X, Wang XR, Row KH. Eremophilane sesquiterpenoids from Farfugium japonicum. Bull. Korean Chem. Soc., 32, 350–352 (2011).
• 9) Kim JY, Oh TH, Kim BJ, Kim SS, Lee NH, Hyun CG. Chemical composition and anti-inflammatory effects of essential oil from Farfugium japonicum flower. J. Oleo Sci., 57, 623–628 (2008).
• 10) Zhao J-H, Shen T, Yang X, Zhao H, Li X, Xie W-D. Sesquiterpenoids from Farfugium japonicum and their inhibitory activity on NO production in RAW264.7 cells. Arch. Pharm. Res., 35, 1153–1158 (2012).
• 11) Liu MC, Lin TH, Wu TS, Yu FY, Lu CC, Liu BH. Aristolochic acid I suppressed iNOS gene expression and NF-kappaB activation in stimulated macrophage cells. Toxicol. Lett., 202, 93–99 (2011).
• 12) Wang QS, Xiang Y, Cui YL, Lin KM, Zhang XF. Dietary blue pigments derived from genipin, attenuate inflammation by inhibiting LPS-induced iNOS and COX-2 expression via the NF-kappaB inactivation. PLoS ONE, 7, e34122 (2012).
• 13) Morgan MJ, Liu ZG. Crosstalk of reactive oxygen species and NF-kappaB signaling. Cell Res., 21, 103–115 (2011).
• 14) Baima ET, Guzova JA, Mathialagan S, Nagiec EE, Hardy MM, Song LR, Bonar SL, Weinberg RA, Selness SR, Woodard SS, Chrencik J, Hood WF, Schindler JF, Kishore N, Mbalaviele G. Novel insights into the cellular mechanisms of the anti-inflammatory effects of NF-kappaB essential modulator binding domain peptides. J. Biol. Chem., 285, 13498–13506 (2010).
• 15) Jung WK, Choi I, Lee DY, Yea SS, Choi YH, Kim MM, Park SG, Seo SK, Lee SW, Lee CM, Park YM, Choi IW. Caffeic acid phenethyl ester protects mice from lethal endotoxin shock and inhibits lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW264.7 macrophages via the p38/ERK and NF-kappaB pathways. Int. J. Biochem. Cell Biol., 40, 2572–2582 (2008).
• 16) Kaminska B. MAPK signalling pathways as molecular targets for anti-inflammatory therapy—from molecular mechanisms to therapeutic benefits. BBA-Proteins Proteomics, 1754, 253–262 (2005).
• 17) Jayasooriya RG, Kang CH, Seo MJ, Choi YH, Jeong YK, Kim GY. Exopolysaccharide of Laetiporus sulphureus var. miniatus downregulates LPS-induced production of NO, PGE(2), and TNF-alpha in BV2 microglia cells via suppression of the NF-kappaB pathway. Food Chem. Toxicol., 49, 2758–2764 (2011).
• 18) Hwang BY, Lee JH, Koo TH, Kim HS, Hong YS, Ro JS, Lee KS, Lee JJ. Kaurane diterpenes from Isodon japonicus inhibit nitric oxide and prostaglandin E2 production and NF-kappaB activation in LPS-stimulated macrophage RAW264.7 cells. Planta Med., 67, 406–410 (2001).
• 19) Park EK, Shin YW, Lee HU, Kim SS, Lee YC, Lee BY, Kim DH. Inhibitory effect of ginsenoside Rb1 and compound K on NO and prostaglandin E2 biosyntheses of RAW264.7 cells induced by lipopolysaccharide. Biol. Pharm. Bull., 28, 652–656 (2005).
• 20) Torres M, Forman HJ. Redox signaling and the MAP kinase pathways. (Reprinted from Thiol Metabolism and Redox Regulation of Cellular Functions). Biofactors, 17, 287–296 (2003).
• 21) Haddad JJ, Land SC. Redox/ROS regulation of lipopolysaccharide-induced mitogen-activated protein kinase (MAPK) activation and MAPK-mediated TNF-alpha biosynthesis. Br. J. Pharmacol., 135, 520–536 (2002).
• 22) Saldeen J, Welsh N. p38 MAPK inhibits JNK2 and mediates cytokine-activated iNOS induction and apoptosis independently of NF-κB translocation in insulin-producing cells. Eur. Cytokine Netw., 15, 47–52 (2004).
• 23) Islam S, Hassan F, Mu MM, Ito H, Koide N, Mori I, Yoshida T, Yokochi T. Piceatannol prevents lipopolysaccharide (LPS)-induced nitric oxide (NO) production and nuclear factor (NF)-kappaB activation by inhibiting IkappaB kinase (IKK). Microbiol. Immunol., 48, 729–736 (2004).
• 24) Kim HG, Yoon DH, Lee WH, Han SK, Shrestha B, Kim CH, Lim MH, Chang W, Lim S, Choi S, Song WO, Sung JM, Hwang KC, Kim TW. Phellinus linteus inhibits inflammatory mediators by suppressing redox-based NF-kappaB and MAPKs activation in lipopolysaccharide-induced RAW264.7 macrophage. J. Ethnopharmacol., 114, 307–315 (2007).
• 25) Wang QS, Cui YL, Wang YF, Chi W. Effects of compounds from bi-qi capsule on the expression of inflammatory mediators in lipopolysaccharide-stimulated RAW264.7 macrophages. J. Ethnopharmacol., 136, 480–487 (2011).
• 26) Billack B. Macrophage activation: role of toll-like receptors, nitric oxide, and nuclear factor kappa B. Am. J. Pharm. Educ., 70, 102 (2006).
• 27) Burk DR, Senechal-Willis P, Lopez LC, Hogue BG, Daskalova SM. Suppression of lipopolysaccharide-induced inflammatory responses in RAW264.7 murine macrophages by aqueous extract of Clinopodium vulgare L. (Lamiaceae). J. Ethnopharmacol., 126, 397–405 (2009).
• 28) Lee JI, Burckart GJ. Nuclear factor kappa B: important transcription factor and therapeutic target. J. Clin. Pharmacol., 38, 981–993 (1998).
• 29) Hehner SP, Heinrich M, Bork PM, Vogt M, Ratter F, Lehmann V, Schulze-Osthoff K, Droge W, Schmitz ML. Sesquiterpene lactones specifically inhibit activation of NF-kappaB by preventing the degradation of I kappaB-alpha and I kappaB-beta. J. Biol. Chem., 273, 1288–1297 (1998).
• 30) Sae-wong C, Tansakul P, Tewtrakul S. Anti-inflammatory mechanism of Kaempferia parviflora in murine macrophage cells (RAW264.7) and in experimental animals. J. Ethnopharmacol., 124, 576–580 (2009).
• 31) Asehnoune K, Strassheim D, Mitra S, Kim JY, Abraham E. Involvement of reactive oxygen species in Toll-like receptor 4-dependent activation of NF-kappaB. J. Immunol., 172, 2522–2529 (2004).