Aromadendrin Inhibits Lipopolysaccharide-Induced Nuclear Translocation of NF-κB and Phosphorylation of JNK in RAW 264.7 Macrophage Cells

Aromadendrin, a flavonol, has been reported to possess a variety of pharmacological activities such as anti-inflammatory, antioxidant, and anti-diabetic properties. However, the underlying mechanism by which aromadendrin exerts its biological activity has not been extensively demonstrated. The objective of this study is to elucidate the anti-inflammatory mechanism of aromadedrin in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Aromadendrin significantly suppressed LPS-induced excessive production of pro-inflammatory mediators such as nitric oxide (NO) and PGE2. In accordance, aromadendrin attenuated LPSinduced overexpression iNOS and COX-2. In addition, aromadendrin significantly suppressed LPS-induced degradation of IκB, which sequesters NF-κB in cytoplasm, consequently inhibiting the nuclear translocation of pro-inflammatory transcription factor NF- κB. To elucidate the underlying signaling mechanism of anti-inflammatory activity of aromadendrin, MAPK signaling pathway was examined. Aromadendrin significantly attenuated LPS-induced activation of JNK, but not ERK and p38, in a concentration-dependent manner. Taken together, the present study clearly demonstrates that aromadendrin exhibits anti-inflammatory activity through the suppression of nuclear translocation of NF-κB and phosphorylation of JNK in LPS-stimulated RAW 264.7 macrophage cells.


INTRODUCTION
Macrophages play essential roles in host defense against bacterial infection (Rehman et al., 2012). Macrophages orchestrate the innate immunity response to infection by expressing various infl ammatory cytokines such as TNF-α, IL-1, NO, and PGE 2 . The most common cause of macrophage activation is an exposure to LPS, the principal component of the outer membrane Gram-negative bacteria (Rietschel and Brade, 1992). However, aberrantly over-activated macrophages have also been attributed to detrimental responses in infl ammatory diseases including sepsis, septic shock, or systemic infl ammatory response syndrome (Rackow and Astiz, 1991;Shapiro et al., 2010). Therefore, suppression of aberrant activation of macrophage may have valuable therapeutic potential for the treatment of infl ammatory diseases such as sepsis.
Aromadendrin is a fl avonol, which is a type of fl avonoids.
Recently, it has been reported that aromadendrin possesses anti-diabetic activity by stimulating glucose uptake and ameliorating insulin resistance (Zhang et al., 2011). In addition, it has been also reported that aromadendrin derivatives exhibit various biological activities such as anti-proliferative (Duh et al., 2012) and osteogenic actions (Swarnkar et al., 2011). However, the underlying mechanism by which aromadendrin exerts its pharmacological activity, especially anti-infl ammatory, has not been clearly demonstrated. Aromadendrin has been reported to be present in various medicinal herbs including Chionanthus retusus (Kwak et al., 2009), Cudrania tricuspidata
In the present study, to provide a novel pharmacological agent that could suppress aberrantly activated macrophages, the anti-infl ammatory activity of aromadendrin and its underlying mechanism were elucidated in LPS-stimulated RAW 264.7 macrophage cells.

Reagents and cell culture
Bacterial lipopolysaccharide (LPS) from Escherichia coli serotype 055:B5 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Aromadendrin was isolated and identifi ed from Hemiptelea davidii (Fig. 1) (Chang et al., 2004). Aromadendrin was dissolved in dimethyl sulfoxide (DMSO) and added to the cell culture at the desired concentrations. The macrophage RAW 264.7 cell line was maintained in Dulbecco's modifi ed Eagle's medium (DMEM; Gibco BRL, Grand Island, NY, USA) containing 5% heat-inactivated fetal bovine serum and penicillin-streptomycin (Gibco BRL) at 37 o C, 5% CO 2 . In all experiments, cells were incubated in the presence of the indicated concentration of Aromadendrin before the addition of LPS (200 ng/ml).

Cell viability
Cell viability was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RAW 264.7 macrophage cells were seeded at 5×10 5 cells per well and incubated with aromadendrin at various concentrations for 24 hr at 37 o C. After incubation, MTT (0.5 mg/ml in PBS) was added to each well, and the cells were incubated for 3 hr at 37 o C and 5% CO 2 . The resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO). Absorbance was determined at 540 nm. The results were expressed as a percentage of surviving cells over control cells.

Nitrite quantifi cation assay
The production of NO was estimated by measuring the amount of nitrite, a stable metabolite of NO, using the Griess reagent as described . After aromadendrinpretreated RAW 264.7 macrophage cells were stimulated with LPS in 12-well plates for 24 hr, 100 μl of the cell supernatant was mixed with an equal volume of Griess reagent. Light absorbance was read at 540 nm. The results were expressed as a percentage of released NO from LPS-stimulated RAW 264.7 cells. To prepare a standard curve, sodium nitrite was used to prepare a standard curve.

Isolation of nuclear extract
The isolation of the nuclear fraction was performed according to the protocol of NF-κB activation assay kits (FIVE photon).

Statistical analysis
All values shown in the fi gures are expressed as the mean ± SD obtained from at least three independent experiments.
Statistical signifi cance was analyzed by two-tailed Student's t-test. Data with values of p<0.05 were considered as statistically signifi cant. Single (* and # ) and double (** and ## ) marks represent statistical signifi cance in p<0.05 and p<0.01, respectively.

Aromadendrin inhibits NO and PGE 2 production in LPSstimulated RAW 264.7 macrophage cells
Given the previous reports that pro-infl ammatory mediators such as NO and PGE 2 play key roles in the progression of infl ammation (Ock et al., 2009;Lee et al., 2012), the inhibitory ef- fects of aromadendrin on NO and PGE 2 release in LPS-stimulated RAW 264.7 macrophage cells. Cells were incubated with aromadendrin (10, 50, 100, or 200 μM) for 1 hr prior to LPS treatment (200 ng/ml) LPS. LPS markedly increased NO and PGE 2 production in RAW 264.7 cells. However, aromadendrin signifi cantly inhibited NO and PGE 2 release in LPS-stimulated RAW 264.7 cells in concentration dependent manners (Fig. 2  A and B). Furthermore, to examine the anti-infl ammatory magnitude of aromadedrin, inhibitory potency of aromadendrin on LPS-induced NO production was compared to prototype fl avo-noids, such as wogonin and quercetin ( Fig. 2A). Although aromadendrin signifi cantly attenuated LPS-induced NO production in a concentration-dependent manner, the inhibitory effect of aromadendrin was less potent than those of wogonin and quercetin. In addition, aromadendrin showed no signifi cant cytotoxicity in concentration ranges used in the study (Fig. 2C).

Aromadendrin inhibits LPS-induced mRNA and protein expressions of iNOS and COX-2
As aromadendrin inhibited LPS-induced NO and PGE 2 production in RAW 264.7 cells (Fig. 2), whether the suppression of NO and PGE 2 were attributable to downregulation of iNOS and COX-2 expression was examined in the presence or absence of aromadendrin. LPS stimulation of the RAW  www.biomolther.org 264.7 cells strongly upregulated iNOS and COX-2 protein levels. Pretreatment of aromadendrin resulted in a signifi cant decrease in LPS-induced iNOS and COX-2 overexpression levels in a concentration-dependent manner (Fig. 3A). Consistent with the decrease of protein levels, LPS-induced up-regulation of iNOS and COX-2 mRNA expression was signifi cantly attenuated with aromadendrin (Fig. 3B), indicating that attenuated release of NO and PGE 2 is due to decreased expression of their responsible genes by aromadendrin.

Aromadendrin inhibits IκB degradation and NF-κB translocation in in LPS-stimulated RAW 264.7 cells
Activation of the transcription factor NF-κB has been reported to result in the increased transcription of pro-infl ammatory mediators including iNOS and COX-2 (Baldwin, 1996;Moynagh, 2005). IκB has been demonstrated to inhibit nuclear translocation of NF-κB by retaining it in cytoplasm Rehman et al., 2012). It has been also reported that LPS causes activation of IκB kinases, which phosphorylate IκB and then lead to proteasomal degradation of IκB (Baeuerle and Baichwal, 1997). Therefore, in the present study, whether aromadendrin could suppress LPS-induced IκB degradation was examined. LPS treatment resulted in obvious degradation of IκB. However, aromadendrin signifi cantly attenuated LPS-induced degradation of IκB in a concentration-dependent manner (Fig. 4A). Furthermore, whether aromadendrin could prevent nuclear translocation of the p65 subunit of NF-κB was examined. LPS treatment caused a signifi cantly increased nuclear translocation of NF-κB. However, aromadendrin resulted in a signifi cant reduction of LPS-induced nuclear translocation of NF-κB in a concentration-dependent manner (Fig. 4B).

Aromadendrin inhibits the phosphorylation of JNK in LPS-stimulated RAW 264.7 cells
It has been previously reported that MAP kinase signaling pathway is implicated in the LPS-induced infl ammatory responses (Guha and Mackman, 2001;Rushworth et al., 2005). In the present study, MAP kinase pathway was examined to understand the underlying signaling mechanism by which aromadendrin exhibits its anti-infl ammatory activity. The effect of aromadendrin on LPS-stimulated phosphorylation of JNK, ERK, and p38 kinase in RAW 264.7 macrophage cells was determined. Cells were pretreated with aromadendrin at indicated concentrations (10, 50, 100, and 200 μM), and then were treated with LPS (200 ng/ml) for 30 min. Aromadendrin signifi cantly suppressed the LPS-induced phosphorylation of JNK in a concentration-dependent manner in RAW 264.7 cells  ing pathway in RAW 264.7 macrophage cells. RAW 264.7 cells were stimulated with 200 ng/ml LPS in the absence or presence of aromadendrin. Western blot analysis was then performed to evaluate the activation of MAP kinase signaling pathway. LPSinduced phosphorylation of JNK was signifi cantly attenuated with aromadendrin treatment, but phosphorylation of ERK and p38 was not decreased with aromadendrin treatment, suggesting that JNK signaling might play a key role in the LPS-induced overactivation of RAW 264.7 cells. β-Actin was used as an internal control. Images are representative of three independent experiments that shows reproducible results. ARD stands for aromadendrin. http://dx.doi.org/10.4062/biomolther.2013.023 (Fig. 5). However, no signifi cant attenuation of ERK and p38 phosphorylation was observed with aromadendrin. The data strongly suggest that JNK signaling plays a key role in LPSinduced infl ammatory response in RAW 264.7 cells.

DISCUSSION
The present study clearly demonstrated that aromadendrin suppresses infl ammatory responses through the suppression of nuclear translocation of NF-κB and phosphorylation of JNK in LPS-stimulated RAW 264.7 macrophage cells. Aromadendrin signifi cantly inhibited LPS-stimulated excessive production of NO and PGE 2 with the suppression of their responsible proteins, iNOS and COX-2, respectively. Aromadendrin also attenuated LPS-induced IκB degradation, nuclear NF-κB translocation, and JNK phosphorylation.
Although macrophages play essential roles in innate immunity for the host defense against infections (Rehman et al., 2012), macrophages also play a key role in sepsis by producing pro-infl ammatory mediators, expression of adhesion molecules and coagulation factors, phagocytosis, and cytoskeletal rearrangement (Sweet and Hume, 1996). It has been reported that aberrantly activated macrophages are implicated in the pathogenesis of many infection-associated disorders including sepsis and septic shock (Rackow and Astiz, 1991;Shapiro et al., 2010). Therefore, regulation of aberrant macrophage activation may have valuable therapeutic potential for the treatment of infl ammatory diseases such as sepsis. It has been reported that LPS activates gene transcription by binding to its membrane receptor in macrophages, TLR4, which induces signal transcription pathways leading to the phosphorylation of kinases such as IκB kinases, protein kinase C, PI3K, and MAPKs. Subsequently, these kinases activate various transcription factors such as NF-κB and AP-1 families (O'Connell et al., 1998;Guha and Mackman, 2001). In accordance, the present study showed that aromadendrin resulted in the signifi cant attenuation of LPS-induced degradation of IκB and nuclear NF-κB translocation. However, effects of aromadendrin on other pro-infl ammatory transcription factors have not been studied. Therefore, further studies are necessary to clearly understand the role of aromadendrin on various infl ammation-related transcription factors.
It has been reported that LPS is a potent activator of all three MAPK pathways in human monocytic cells (Guha and Mackman, 2001). Many studies have shown that LPS activates ERK1/2, JNK, and p38 in macrophages (Sweet and Hume, 1996) and that many of the downstream targets of MAPK pathways are transcription factors, which regulate various genes encoding infl ammatory mediators (Zhang et al., 2006;Lee et al., 2009;Zhang et al., 2011). In the present study, LPS treatment exhibited increased phosphorylation of all three MAPKs. However, aromadendrin selectively attenuated LPS-induced JNK activation in a concentration-dependent manner. No noticeable changes were observed in phosphorylation of ERK and p38 with aromadendrin. Selective suppression of JNK activation appeared to be suffi cient to exert anti-infl ammatory effects of aromadendrin in RAW 264.7 cells, suggesting that JNK pathway rather than ERK and p38 plays a key role in pro-infl ammatory responses in RAW 264.7 cells.
NF-κB is a major transcription factor for the expression of pro-infl ammatory mediators such as iNOS, IL-1β, and TNF-α (Siebenlist et al., 1994;Kuprash et al., 1995) and aberrant activation of NF-κB and its downstream genes have been implicated in various pathological conditions including sepsis and autoimmune diseases (Karin et al., 2001;Li and Verma, 2002). LPS has been reported to cause the nuclear translocation of NF-κB through IκB degradation (Moon et al., 2007;Zheng et al., 2008). Accordingly, the present study showed that aromadendrin signifi cantly attenuated LPS-induced IκB degradation and nuclear translocation of NF-κB in LPS-induced RAW 264.7 macrophages.
In conclusion, the present data demonstrate that aromadendrin possesses anti-infl ammatory activity such as suppression of NO and PGE 2 production, and inhibition of nuclear transclocation of NF-κB and phosphorylation of JNK in LPS-stimulated RAW 264.7 macrophage cells. The present study strongly suggests that aromadendrin might be a valuable therapeutic agent in the treatment of infl ammation-related pathologies including sepsis. However, further studies are necessary to clearly elucidate the exact mechanism by which aromadendrin inhibits LPS-induced activation of JNK signaling pathway and the possible crosstalk between NF-κB activation and JNK signaling pathway.