Pilloin, A Flavonoid Isolated from Aquilaria sinensis, Exhibits Anti-Inflammatory Activity In Vitro and In Vivo

Flavonoids, widely present in medicinal plants and fruits, are known to exhibit multiple pharmacological activities. In this study, we isolated a flavonoid compound, pilloin, from Aquilaria sinensis and investigated its anti-inflammatory activity in bacterial lipopolysaccharide-induced RAW 264.7 macrophages and septic mice. Pilloin inhibited NF-κB activation and reduced the phosphorylation of IκB in LPS-stimulated macrophages. Moreover, pilloin significantly suppressed the production of pro-inflammatory molecules, such as TNF-α, IL-6, COX-2 and iNOS, in LPS-treated RAW 264.7 macrophages. Additionally, pilloin suppressed LPS-induced morphological alterations, phagocytic activity and ROS elevation in RAW 264.7 macrophages. The mitogen-activated protein kinase-mediated signalling pathways (including JNK, ERK, p38) were also inhibited by pilloin. Furthermore, pilloin reduced serum levels of TNF-α (from 123.3 ± 7 to 46.6 ± 5.4 ng/mL) and IL-6 levels (from 1.4 ± 0.1 to 0.7 ± 0.1 ng/mL) in multiple organs of LPS-induced septic mice (liver: from 71.8 ± 3.2 to 36.7 ± 4.3; lung: from 118.6 ± 10.6 to 75.8 ± 11.9; spleen: from 185.9 ± 23.4 to 109.6 ± 18.4; kidney: from 160.3 ± 11.8 to 75 ± 10.8 pg/mL). In summary, our results demonstrate the anti-inflammatory potential of pilloin and reveal its underlying molecular mechanism of action.


Introduction
Inflammation, which is triggered by infection and injury, is an important process in the host defence system and homeostasis [1]. Upon pathogen recognition, macrophages are stimulated to produce pro-inflammatory molecules, such as tumor necrosis factor-alpha (TNF-α), nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and reactive oxygen species (ROS) to fight pathogens [2][3][4]. However, persistent inflammation is highly associated with a variety of diseases,

Isolation and Identification of Pilloin from A. sinensis
The EtOAc-soluble fractions (MeOH extract) from stem barks of A. sinensis were purified using a silica gel column and preparative thin layer chromatography (TLC), which yielded approximately 28.5 mg of pilloin. Its molecular weight was determined on the basis of the positive ESI-MS at m/z 315 [M + H] (Supplementary Figure S1) and the structure was identified by the 1 H-NMR data (Supplementary Figure S2). The structure was also confirmed by comparison of the UV and IR data of the isolated pilloin with those from the literature ( Figure 1A) [21,22].

Pilloin Suppresses NF-κB Activity in LPS-Induced RAW 264.7 Macrophages
LPS-activated macrophages serve as an in vitro system to study inflammation [12]. Our laboratory previously established an LPS-responsive macrophage cell clone, RAW 264.7/Luc-P1, in which the activity of NF-κB correlates with the expression of the reporter gene (luciferase) upon LPS treatment [23]. We applied this cell line to evaluate the effects of pilloin on LPS-stimulated NF-κB activity in RAW 264.7 macrophages. As shown in Figure 1B, pilloin inhibited NF-κB activation in a concentration-dependent manner. Consistent with this observation, the phosphorylation of IκB (a negative regulator of NF-κB) was also reduced by pilloin in LPS-stimulated macrophages in a concentration-dependent manner ( Figure 1C). Pilloin did not cause cytotoxicity at 3 or 10 µM, and only caused slight toxicity at 30 µM (85% viability), indicating pilloin has considerably low cytotoxicity ( Figure 1D).

Pilloin Suppresses the Production of Pro-Inflammatory Molecules Induced by LPS
Under LPS stimulation, NF-κB activation in macrophages results in the production of pro-inflammatory molecules, such as TNF-α, IL-6 and NO [7,10]. We next determined whether pilloin affects the production of pro-inflammatory molecules in LPS-induced macrophages. As shown in Figure 2, pilloin reduced TNF-α and IL-6 production at 30 M, as measured by ELISA. Pilloin also decreased NO production and iNOS expression relative to the vehicle group ( Figure  3A,B). The expression of COX-2, a downstream pro-inflammatory enzyme of NF-κB, was also suppressed by pilloin at 30 μM ( Figure 3C).

Pilloin Suppresses the Production of Pro-Inflammatory Molecules Induced by LPS
Under LPS stimulation, NF-κB activation in macrophages results in the production of pro-inflammatory molecules, such as TNF-α, IL-6 and NO [7,10]. We next determined whether pilloin affects the production of pro-inflammatory molecules in LPS-induced macrophages. As shown in Figure 2, pilloin reduced TNF-α and IL-6 production at 30 µM, as measured by ELISA. Pilloin also decreased NO production and iNOS expression relative to the vehicle group ( Figure 3A,B). The expression of COX-2, a downstream pro-inflammatory enzyme of NF-κB, was also suppressed by pilloin at 30 µM ( Figure 3C).

Pilloin Suppresses the Production of Pro-Inflammatory Molecules Induced by LPS
Under LPS stimulation, NF-κB activation in macrophages results in the production of pro-inflammatory molecules, such as TNF-α, IL-6 and NO [7,10]. We next determined whether pilloin affects the production of pro-inflammatory molecules in LPS-induced macrophages. As shown in Figure 2, pilloin reduced TNF-α and IL-6 production at 30 M, as measured by ELISA. Pilloin also decreased NO production and iNOS expression relative to the vehicle group ( Figure  3A,B). The expression of COX-2, a downstream pro-inflammatory enzyme of NF-κB, was also suppressed by pilloin at 30 μM ( Figure 3C).

Pilloin Suppresses LPS-Induced Morphological Alterations, Phagocytic Activity and ROS Elevation in RAW 264.7 Macrophages
Activated macrophages typically display a distinct morphology and enhanced phagocytic activity [23,24]. As shown in Figure 4A, vehicle-treated RAW 264.7 cells were round and refractive, while LPS-treated cells were polygonal and more adherent. Notably, pilloin treatment attenuated LPS-induced morphological changes. Furthermore, the phagocytic activity of RAW 264.7 was reduced by pilloin ( Figure 4B). LPS-activated macrophages exhibit elevated ROS. As shown in Figure 4C, ROS levels in LPS-treated macrophages were diminished by pilloin in a concentration-dependent manner. Together, the above results (Figures 1-4) suggest that pilloin exhibits anti-inflammatory activity via attenuating LPS-induced macrophage activation.

Pilloin Inhibits Mitogen-Activated Protein Kinase (MAPK)-Mediated Signalling Pathways
MAPKs are also involved in LPS-induced pro-inflammatory responses [25]. Therefore, we measured the effect of pilloin on the activation status of MAPKs. Expression of both the total and active forms of MAPK family proteins, JNK, ERK and p38, in pilloin-pretreated cells were detected by Western blot analysis. As shown in Figure 5, pilloin at 30 μM suppressed the activation of all MAPK signalling pathways in LPS-activated macrophages.

Pilloin Suppresses LPS-Induced Morphological Alterations, Phagocytic Activity and ROS Elevation in RAW 264.7 Macrophages
Activated macrophages typically display a distinct morphology and enhanced phagocytic activity [23,24]. As shown in Figure 4A, vehicle-treated RAW 264.7 cells were round and refractive, while LPS-treated cells were polygonal and more adherent. Notably, pilloin treatment attenuated LPS-induced morphological changes. Furthermore, the phagocytic activity of RAW 264.7 was reduced by pilloin ( Figure 4B). LPS-activated macrophages exhibit elevated ROS. As shown in Figure 4C, ROS levels in LPS-treated macrophages were diminished by pilloin in a concentration-dependent manner. Together, the above results (Figures 1-4) suggest that pilloin exhibits anti-inflammatory activity via attenuating LPS-induced macrophage activation.

Pilloin Inhibits Mitogen-Activated Protein Kinase (MAPK)-Mediated Signalling Pathways
MAPKs are also involved in LPS-induced pro-inflammatory responses [25]. Therefore, we measured the effect of pilloin on the activation status of MAPKs. Expression of both the total and active forms of MAPK family proteins, JNK, ERK and p38, in pilloin-pretreated cells were detected by Western blot analysis. As shown in Figure 5, pilloin at 30 µM suppressed the activation of all MAPK signalling pathways in LPS-activated macrophages.

Pilloin Attenuates LPS-Induced Cytokine Expression In Vivo
Sepsis caused by the complicated interactions between pathogens and the host immune system results in a cytokine storm [26]. Previous studies have demonstrated that LPS stimulates the production of pro-inflammatory cytokines, such as IL-6 and TNF-α [26,27]. Therefore, we evaluated the effects of pilloin on TNF-α and IL-6 levels in an LPS-induced sepsis model. As shown in Figure 6, mice treated with pilloin (10 mg/kg) exhibited decreased serum TNF-α and IL-6 levels compared with vehicle-treated LPS mice. Moreover, pilloin reduced IL-6 production in liver, lung, spleen and kidney (Figure 7). The serum levels of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and creatinine (CRE) are close to those of control groups, indicating pilloin does not affect renal and liver function (Supplementary Figure S3).

Pilloin Attenuates LPS-Induced Cytokine Expression In Vivo
Sepsis caused by the complicated interactions between pathogens and the host immune system results in a cytokine storm [26]. Previous studies have demonstrated that LPS stimulates the production of pro-inflammatory cytokines, such as IL-6 and TNF- [26,27]. Therefore, we evaluated the effects of pilloin on TNF-α and IL-6 levels in an LPS-induced sepsis model. As shown in Figure 6, mice treated with pilloin (10 mg/kg) exhibited decreased serum TNF-α and IL-6 levels compared with vehicle-treated LPS mice. Moreover, pilloin reduced IL-6 production in liver, lung, spleen and kidney (Figure 7). The serum levels of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and creatinine (CRE) are close to those of control groups, indicating pilloin does not affect renal and liver function (Supplementary Figure S3).   Figure 6A, and data were collected from mice exposed to various treatments for 12 h

Pilloin Attenuates LPS-Induced Cytokine Expression In Vivo
Sepsis caused by the complicated interactions between pathogens and the host immune system results in a cytokine storm [26]. Previous studies have demonstrated that LPS stimulates the production of pro-inflammatory cytokines, such as IL-6 and TNF- [26,27]. Therefore, we evaluated the effects of pilloin on TNF-α and IL-6 levels in an LPS-induced sepsis model. As shown in Figure 6, mice treated with pilloin (10 mg/kg) exhibited decreased serum TNF-α and IL-6 levels compared with vehicle-treated LPS mice. Moreover, pilloin reduced IL-6 production in liver, lung, spleen and kidney (Figure 7). The serum levels of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and creatinine (CRE) are close to those of control groups, indicating pilloin does not affect renal and liver function (Supplementary Figure S3).   Figure 6A, and data were collected from mice exposed to various treatments for 12 h  Figure 6A, and data were collected from mice exposed to various treatments for 12 h after LPS challenge. The production of IL-6 was determined by ELISA. The values were shown as mean ± SEM. * indicates statistically significant (p < 0.05).

Discussion
In this study, the anti-inflammatory potential of pilloin was demonstrated in LPS-activated macrophages and in LPS-induced septic mouse model. Pilloin inhibited NF-κB and MAPK signalling pathways in LPS-activated macrophages (Figures 1 and 5). Pro-inflammatory cytokines (e.g., TNF-α and IL-6), as well as enzymes (e.g., iNOS and COX-2) were also downregulated by pillion (Figures 2  and 3). In addition, the phenotypes and functions of activated macrophages (i.e., ROS production and phagocytic activity) were also suppressed by pillion (Figure 4). Furthermore, pilloin attenuated the LPS-stimulated production of cytokines (i.e., TNF-α and IL-6) in serum and in tissues in vivo (Figures 6 and 7). Although previous studies have described that pilloin reduced NO production in mouse peritoneal macrophages and decreased ROS generation in rabbit neutrophils [28,29], our study fully explored the anti-inflammatory activity of pilloin both in vitro and in vivo. Our study is also the first to describe the in vivo efficacy and the molecular mechanism of pilloin-mediated anti-inflammatory activity.
According to previous studies, stimulation of TLR4 by LPS in macrophages triggers myeloid differentiation factor 88-dependent (MyD88) and the MyD88-independent signalling pathways [30][31][32][33]. In MyD88-dependent signalling pathway, activation of tumor necrosis factor-receptor-associated factor 6 (TRAF6) results in the activation of nuclear factor-κB (NF-κB) through phosphorylation of IκB via IκB kinase (IKK), as well as the activation of MAPK (ERK, p38 and JNK) which subsequently leads to activation of AP-1 transcription factors. As a consequence, activation of NF-κB and AP-1 induces the expression of downstream pro-inflammatory molecules such as iNOS, COX-2, TNF-α, IL-6 [30][31][32][33]. Our data showed that pilloin inhibited IκB phosphorylation and suppressed the transcriptional activity of NF-κB in LPS-treated macrophages (Figure 1). In addition, the known downstream targets of NF-κB pathways, such as TNF-α, IL-6, iNOS and COX-2 were all suppressed upon pilloin treatment (Figures 2  and 3), suggesting the NF-κB pathway as an essential pathway underlying the anti-inflammatory effects of pilloin. On the other hand, the activities of MAPKs as well as its downstream molecules (iNOS, TNF-α and ROS) were correspondingly suppressed by pilloin (Figures 2-5), indicating the possibility that MAPK pathway is another target of pilloin. Since both NF-κB and MAPKs are downstream of LPS-mediated TLR4 signalling pathway, it is possible that pilloin acts on an upstream regulator of LPS-mediated signalling pathway, which leads to simultaneous inhibition of both NF-κB and MAPK pathways. Alternatively, there might be a sequential relationship between the inhibition of MAPK and NF-κB based on current publications [34][35][36]. Certainly, the direct molecular targets of pilloin and the detailed molecular mechanism of pillion-mediated anti-inflammatory effects merit further investigation.
According to previous studies, stimulation of TLR4 by LPS in macrophages triggers myeloid differentiation factor 88-dependent (MyD88) and the MyD88-independent signalling pathways [30][31][32][33]. In MyD88-dependent signalling pathway, activation of tumor necrosis factor-receptor-associated factor 6 (TRAF6) results in the activation of nuclear factor-κB (NF-κB) through phosphorylation of IκB via IκB kinase (IKK), as well as the activation of MAPK (ERK, p38 and JNK) which subsequently leads to activation of AP-1 transcription factors. As a consequence, activation of NF-κB and AP-1 induces the expression of downstream pro-inflammatory molecules such as iNOS, COX-2, TNF-α, IL-6 [30][31][32][33]. Our data showed that pilloin inhibited IκB phosphorylation and suppressed the transcriptional activity of NF-κB in LPS-treated macrophages (Figure 1). In addition, the known downstream targets of NF-κB pathways, such as TNF-α, IL-6, iNOS and COX-2 were all suppressed upon pilloin treatment (Figures 2  and 3), suggesting the NF-κB pathway as an essential pathway underlying the anti-inflammatory effects of pilloin. On the other hand, the activities of MAPKs as well as its downstream molecules (iNOS, TNF-α and ROS) were correspondingly suppressed by pilloin (Figures 2-5), indicating the possibility that MAPK pathway is another target of pilloin. Since both NF-κB and MAPKs are downstream of LPS-mediated TLR4 signalling pathway, it is possible that pilloin acts on an upstream regulator of LPS-mediated signalling pathway, which leads to simultaneous inhibition of both NF-κB and MAPK pathways. Alternatively, there might be a sequential relationship between the inhibition of MAPK and NF-κB based on current publications [34][35][36]. Certainly, the direct molecular targets of pilloin and the detailed molecular mechanism of pillion-mediated anti-inflammatory effects merit further investigation.
Sepsis is characterized by a cytokine storm [37]. It has been reported that the plasma levels of TNF-α are increased in sepsis patients and in animal models [26]. Furthermore, previous studies have demonstrated that IL-6 is a crucial cytokine in the pathophysiology of severe sepsis and that increased levels of IL-6 are related with the highest risk of death in sepsis patients [17]. Our data, for the first time, demonstrated that pilloin reduces IL-6 and TNF-α levels in an LPS-induced sepsis model (Figures 6  and 7). Furthermore, the values of serum hepatic and renal biomarkers in pilloin-treated mice are within normal ranges, indicating pilloin does not cause toxicity (Supplementary Figure S3). Together, our data support the anti-sepsis potential of pilloin.
Flavonoids exert diverse biological activities such as anti-inflammation and anti-oxidation. The crucial structural features underlying the anti-inflammatory activities of flavonoids are the unsaturation in the C ring, the carbonyl group at C-4 and the number and position of the hydroxyl groups. Flavonoids containing hydroxyl groups at C-5 and C-7 in the aromatic A ring or at C-3 and C-4 in the B aromatic ring positions exhibit higher inflammatory activities [38]. Pilloin contains two hydroxyl groups at C-3 and C-5, consistent with its anti-inflammatory potential observed herein. Currently, there is no pharmacokinetic study on pilloin. It has been reported that methylation of the free hydroxyl groups of flavonoids increases metabolic stability and augments intestinal absorption and oral bioavailability [39]. Since pilloin contains two methoxy groups at C4 and C7, we speculate that this compound may display fair bioavailability, but this aspect should be further investigated.
Many anti-inflammatory drugs, such as corticosteroids and nonsteroidal anti-inflammatory drugs, have been clinically used to attenuate inflammatory and autoimmune diseases. However, they all cause serious side effects [13]. Therefore, finding other anti-inflammatory compounds is important. Our study demonstrated that pilloin is a potential anti-inflammatory compound both in vitro and in vivo. Previous studies have shown that pilloin exhibited anti-diabetic potency by inhibiting the formation of advanced glycation end products (AGEs) and exerted cytotoxicity on transformed lymphoblasts [28,40]. Therefore, pilloin displays multiple biological functions, and can be further developed as a nutraceutical or pharmaceutical agent.

Enzyme-Linked Immunosorbent Assay (ELISA)
RAW 264.7 cells (2 × 10 5 cells in 24-well plates) were treated with pilloin, vehicle (0.1% DMSO) or andrographolide (positive control) for 1 h, followed by LPS (10 ng/mL) for 24 h. For measurements of serum and tissue cytokines, blood samples and supernatant of the homogenized tissues were collected from treated mice as described previously [13]. The levels of TNF-α and IL-6 in the medium of cultured RAW 264.7 macrophages, in the serum and in tissue extracts were measured by ELISA (eBioscience, San Diego, CA, USA). A450 nm and A550 nm (reference absorbance) were determined on a Model 680 Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA).

Phagocytosis Assay
Phagocytosis activity was measured using Vybrant Phagocytosis Assay Kit (Molecular Probes, Eugene, OR, USA). In brief, RAW 264.7 cells (2 × 10 5 cells in 24-well plates) were treated with pilloin, fisetin (positive control) or vehicle (0.1% DMSO) for 24 h and incubated with 50 µg/mL of bioparticles (fluorescein-labelled Escherichia coli)) for 30 min. The supernatant was removed and then the cells were treated with Trypan Blue to quench the remaining extracellular bioparticles, washed with PBS, trypsinised and analysed under excitation/emission wavelengths of 480 nm/520 nm using Infinite ® 200 PRO (Tecan Group Ltd.). The relative phagocytic activity of the treated cells is determined according to the manufacturer's protocol.

LPS-Induced Inflammatory Animal Model
Male C57BL/6 mice (10-12 weeks old) were purchased from the Animal Center of National Yang-Ming University (Taipei, Taiwan) and maintained in a specific pathogen-free area of this center. The endotoxin model was used to induce inflammation responses as described previously [44,45]. In brief, mice were divided into four groups for the following treatments: vehicle group (0.6% DMSO in 0.1% carboxymethyl cellulose (CMC)), pilloin group (10 mg/kg pilloin in vehicle + LPS 20 mg/kg), LPS group (vehicle + LPS 20 mg/kg) and pyrrolidine dithiocarbamate (PDTC) group (positive control; 50 mg/kg PDTC in vehicle + LPS 20 mg/kg). The vehicle or drug was intraperitoneally (i.p.) delivered into mice 1 h before LPS injection. After 12-h treatment, mice were sacrificed and the serum as well as tissues was collected for ELISA analysis. Serum glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and creatinine (CRE) were measured using the FUJI DRI-CHEM 4000i (Fujifilm Corp., Tokyo, Japan). The animal protocol was reviewed and approved by the Animal Care and Use Committee of National Yang Ming University (No. 1050902).

Statistical Analyses
Results are expressed as the mean ± SD from at least three independent experiments. The in vivo data are presented as the mean ± SEM. Comparisons between groups were performed using ANOVA followed by post hoc Dunnett's test. A p value of <0.05 was considered statistically significant.
Supplementary Materials: The following are available online. Figure S1: ESI-MS spectrum of pillion, Figure S2