© Birkhäuser Verlag, Basel, 2008 Inflamm. res. 57 (2008) 189–198 1023-3830/08/040189-10 DOI 10.1007/s00011-007-7190-3

Inflammation Research

A genome-wide microarray analysis reveals anti-inflammatory target genes of paeonol in macrophages H. Huang1, E. J. Chang1, Y. Lee1, J. S. Kim2, S. S. Kang2 and H. H. Kim1 1

Department of Cell and Developmental Biology, BK21 Program, and DRI, Seoul National University, School of Dentistry, 28 Yeongon-Dong, Chongno-Gu, Seoul 110-749, Korea, e-mail: [email protected] 2 Natural Products Research Institute and College of Pharmacy, Seoul National University, Seoul, Korea Received 3 October 2007; returned for revision 28 October 2007; received from final revision 22 November 2007; accepted by A. Falus 9 December 2007 Published Online First 26 March 2008

Abstract. Objective: Paeony root has long been used for its anti-inflammatory effects. In this study, the effects of albiflorin, paeoniflorin, and paeonol, compounds from paeony root, on gene expression profiles were examined in macrophages challenged with the inflammation inducer lipopolysaccharide (LPS). Methods: The RAW264.7 macrophages were treated with LPS in the presence or absence of albiflorin, paeoniflorin, or paeonol. Global mRNA expression levels were detected by using an oligonucleotide microarray platform covering the mouse whole genome. Results: Treatment with LPS caused expression level changes in 1,270 genes by 2 folds or more. Paeonol attenuated the induction level of 355 LPS-responsive genes. Classification of the genes targeted by paeonol according to the Panther group analysis revealed 20 biological processes, 24 molecular functions, and 22 signaling pathways. The Panther signaling pathways highly affected by paeonol included the ‘inflammation mediated by chemokine and cytokine signaling’, ‘interleukin signaling’, and ‘Toll receptor signaling’. Conclusion: Our results demonstrate that paeonol has extensive inhibitory effects on the regulation of inflammationassociated gene expression by LPS in macrophages. In addition, the predominant effect of paeonol among the tested compounds suggests that paeonol may be a major ingredient for the anti-inflammatory effect of paeony root. Key words: Gene profiling – Paeonol – Paeoniflorin – Albiflorin – Macrophage – Lipopolysaccharide – RAW264.7 – Microarray

Abbreviations: CCL, chemokine (C-C motif) ligand; CXCL, chemokine (C-X-C motif) ligand; GAPDH, glyceraldehyde3-phosphate dehydrogenase; IL, interleukin; IVT, in vitro

Correspondence to: H. H. Kim

transcription; LPS, lipopolysaccharide; MCP, monocyte chemotactic protein; PCR, polymerase chain reaction; Ptgs, prostaglandin-endoperoxide synthase; RT, reverse transcription; TLR, Toll-like receptor; TNF, tumor necrosis factor Introduction The inflammation process is crucial to defense against bacterial infection. Key events in the inflammatory process include expression of inflammatory cytokines, chemokines, and other mediators [1]. The recognition of invading pathogens by host cells starts with the binding of specific cellular receptors to pathogen molecules with distinct patterns [2]. The major pattern-recognition receptors identified to date consist of the Toll-like receptors (TLR). Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, is one of the most extensively studied molecular patterns of microorganisms [3]. Only small quantities of LPS or its bioactive center, lipid A induces a rapid and potent stimulation of pro-inflmmatory cytokine production by monocytes, macrophages, and neutrophils. LPS is recognized by TLR4 in association with CD14 and MD-2 [4, 5]. TLR4 signaling is mediated via the recruitment of adaptor proteins MyD88, TIRAP, TRAM, and TRIF, which leads to the activation of intracellular signaling pathways involving IRAK, TRAF6, IKKs, and MAPKs. These signaling pathways ultimately stimulates the transcription factors NFκB, IRF, and AP-1 needed for induction of inflammatory cytokines, including TNFα, interleukin-1β (IL-1β), and IL-6 [6, 7]. Paeony root (Paeoniae radix) has been used as a traditional medicine for more than a thousand years in Asian countries to alleviate inflammation, amenorrhea, epistaxis, abdominal pain, and other symptoms [8]. Recently, the paeony root extract was reported to suppress monocyte chemotactic proteins (MCP)-1 and MCP-3 [9], which may be an attribute of the anti-inflammatory property of paeony root. Many compounds have been isolated from the paeony root, including albiflorin, paeoniflorin, paeonol, oxypaeoniflorin,

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benzoylpaeoniflorin, paeonin, and palbinone [10, 11]. Among them, both paeoniflorin and paeonol have been verified to have anti-inflammatory and anti-allergic activity [12, 13]. In addition, paeoniflorin was reported to induce the immune tolerance of mesenteric lymph node lymphocytes [14], neuroprotection in cerebral ischemia [15], apoptosis of T cells [8], and the attenuation of cognitive deficit [16]. Paeonol has been shown to have diverse effects on different cell types in various experimental conditions. The reported effects of paeonol include anti-oxidant [17], anti-diabetic [18], and anxiolytic-like [19] activities as well as apoptosis-inducing effects [20]. Anti-inflammatory effects of paeonol were demonstrated in a study with carrageenan-evoked thermal hyperalgesia model animals [13]. In the study, a decrease in the production of pro-inflammatory molecules, such as IL1β, TNFα, NO, and PGE2 and an increase in the production of the anti-inflammatory cytokine IL-10 were suggested to be involved in the anti-inflammatory effect of paeonol [13]. Similarly, paeonol attenuated trinitobezene sulfonic acid (TNBS)-induced colitis potentially by suppressing iNOS upregulation [21]. However, only few investigations have been conducted on albiflorin and other compounds and no clear biological functions for those components of the paeony root have been indicated to date. Overall, studies on the molecular explanation for pharmacological effects of the peony root ingredients are fairly scanty. With the human genome project, global gene expression profiling has become applied to a wide range of experimental systems for understanding the genetic regulation of cellular processes and disease pathogenesis. However, this genomic approach has just begun to be employed in studies on natural product medicine. In this study, we analyzed effects of paeony root compounds on the gene expression profiles in an inflammatory cell model using an oligonucleotide DNA microarray. The LPS-challenged RAW264.7 macrophage cells were treated with paeoniflorin, paeonol, and albiflorin. Paeonol was found to most extensively affect the expression profile of LPS-target genes, which includes many interleukines and chemokines.

Materials and methods Materials

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in a six-well plate and incubated for 24 h before being treated with the control vehicle (0.1 % DMSO), LPS (1 µg/ml), or LPS plus paeony root compound (1~100 µg/ml) for the indicated time.

Cytotoxicity assay RAW264.7 cells were seeded at a density of 104 cells per well in 96well-plates. In the next day, cells were treated with albiflorin, paeoniflorin, and paeonol at concentration of 0, 0.1, l, 10, or 100 µg/ml. Cells were culture for 24 h at 37 °C in an atmosphere of 95 % air and 5 % CO2. Viability of cells was measured by using the CCK-8 assay kit following the manufacturer’s instruction (Dojindo Laboratories).

Preparation of RNA Cells were washed three times with ice-cold PBS. 1 ml of Trizol reagent was added to each well of 6-well plates. The extract was transferred to a microfuge tube and centrifuged for 12,000 g for 15 min. The supernatant was collected, mixed with 500 µl isopropanol, incubated for 10 min at room temperature followed by centrifugation for 8 min. The supernatant was removed and the pellet washed in 1 ml of 75 % ethanol. The precipitated RNA was dissolved in 25 µl nuclease-free water. The concentration of RNA in the final solution was determined by spectrophotometry. The quality of RNA was examined by gel electrophoresis and RNA of good quality only was further processed.

Reverse transcription (RT)-polymerase chain reaction (PCR) One µg of total RNA obtained as above was heated at 65 °C for 10 min followed by chilling on ice. The denatured RNA was annealed with 0.5 µg of poly dT in a 20 µl volume containing 1 mM dNTP, 25 mM TrisHCl, 50 mM KCl, 2 mM DTT, and 5 mM MgCl2. One unit of RNase inhibitor and 20 units of reverse transcriptase were added. The mixture was incubated for 50 min at 42 °C and 15 min at 70 °C for reverse transcription reaction. One µl of the generated cDNA was used for PCR with the thermal cycle of denaturing at 94 °C, annealing at 58~60 °C, and extension at 72 °C for 30 s each. The number of amplification cycles was determined to be in a linear range of amplification, i. e. 22 cycles for GAPDH and 25~30 cycles for the others. The PCR primer sequences used are as follows: TNF-α, 5-ACACCGTCAGCCGATTTGC-3 (forward) and 5-CCCTGAGCCATAATCCCCTT-3 (reverse); IL-1α, 5-ATAACCTGCTGGTGTGTGAC-3 (forward) and 5-TGCAGACTCAAACTCCACTT-3 (reverse); IL-6, 5-TTGTGCAATGGCAATTCT-3 (forward) and 5-AGAGCATTGGAAATTGGG-3 (reverse); GAPDH, 5ACCACAGTCCATGCCATCAC-3 (forward) and 5-TCCACCACCCTGTTGCTGTA-3 (reverse).

Lipopolysaccharide (LPS) was purchased from Sigma (St. Louis). αMEM, penicillin G, streptomycin, and fetal bovine serum (FBS) were from Gibco BRL (Grand Island). Trizol reagent and Superscript reverse transcription kit were from Invitrogen (Carlsbad). Mouse genome microarray chip (MGABI-002), chemiluminescent RT-IVT labeling kit v.2.0, and chemiluminescence detection kit were obtained from Applied Biosystems (Foster City). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Tabaru, Japan). Albiflorin, paeoniflorin, and paeonol were isolated from the roots of P. lactiflora and identified according to the previously described method [22].

RT-in vitro transcription (IVT) labeling

Cell culture

Hybridization and detection

RAW264.7 cells were grown in α-MEM medium supplemented with 100 U/ml penicillin G, 100 µg/ml streptomycin, and 10 % heat-inactivated FBS in a humidified incubator with an atmosphere of 95 % air and 5 % CO2 at 37 °C. The cells were seeded at a density of 5 × 105 cells/well

The microarray chip was pre-hybridized for 1 h at 55 °C. DIG-labeled cRNA was fragmented for 30 min at 60 °C following the manufacturer’s protocol. The fragmented cRNA was added to prehybridized chip and the chip was incubated for 16 h at 55 °C with constant rocking at

Digoxigenin (DIG)-labeled cRNA was generated from the total RNA prepared as above by using the chemiluminescent RT-IVT labeling kit as per the manufacturer’s instruction. 2 µg of total RNA was reverse transcribed for 2 h at 42 °C. The second strand DNA was synthesized for 2 h at 16 °C. The double stranded cDNA was purified and mixed with DIG-UTP, IVT enzyme mix, and IVT buffer mix. IVT reaction was performed for 9 h at 37 °C. DIG-labeled cRNA was purified and quantitated by spectrophotometry.

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100 rpm. The microarray chip was washed and incubated with antidigoxigenin-alkaline phosphatase for 20 min at 22 °C. After washing, chemiluminescence substrate was added and the chip was immediately scanned with Applied Biosystems 1700 Analyzer.

Data processing and analysis The data were filtered for values with more than 3 of signal to noise ratio and 0 Flags using the software in the scanner system. The filtered data were processed by Lowess normalization method. Genes showing more than 2-fold increase or decrease in LPS or LPS plus paeony root compound samples compared to the control vehicle-treated samples were collected. The whole process of experiment, i. e. cell treatment, RNA processing, microarray hybridization, was repeated three times and genes showing changes in all three experiments were only considered. The fold change presented is the mean of the three replicates. The genes with more than 4-fold change were processed for categorizing by Panther classification (http://www.pantherdb.org/). The gene expression data were deposited to Gene Expression Omnibus (GEO) databank (http:// www.ncbi.nlm.nih. gov/projects/geo/, accession number GSE9632).

Real-time PCR Quantitative PCR was performed with the Applied Biosystems 7300 instrument (Applied Biosystems) in triplicates using the standard curve method. Each PCR reaction contains 1 µl of RT product and 0.2 µM of each primer in the SYBR GREEN PCR master mix (Applied Biosystem). The thermal cycling condition was one time 5 min incubation at 95 °C followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The PCR primer sequences used are as follows: IL-1α, 5-ACAGGAACCTTCCTCACCCT-3 (forward) and 5-GTATCATATGTCGGGGTGGC-3 (reverse); CCL2, 5-TCACCAAGCTCAAGAGAGAG-3 (forward) and 5-CATTCAAAGGTGCTGAAGA-3 (reverse); CCL3, 5-TGGAGCTGACACCCCGACTG-3 (forward) and 5-ATGACACCTGGCTGGGAGCA-3 (reverse); CXCL2, 5- ACAGAAGTCATAGCCACTCTC-3 (forward) and 5-CCTTGCCTTTGTTCAGTATC-3 (reverse); Ptgs2, 5-GCTGTACAAGCAGTGGCAAA-3 (forward) and 5-TTCTGCAGCCATTTCCTTCT-3 (reverse); Tnc, 5-TGTGTGCTTCGAAGGCTATG-3 (forward) and 5GCAGACACACTCGTTCTCCA-3 (reverse); HPRT, 5-CCTAAGATGAGCGCAAGTTG-3 (forward) and 5-CCACAGGGACTAGAACACCT-3 (reverse). Quantitation of the amount of target in unknown samples is accomplished by measuring the fractional cycle number (Ct) using the standard curve. All quantitations were normalized to a housekeeping gene HPRT. The relative quantitation value for each target gene compared to the calibrator for that target is expressed as 2–(Ct-Cc) (Ct and Cc are the mean threshold cycle differences after normalizing to HPRT). The relative expression levels of samples are presented by semi-log plot.

Results Effect of albiflorin, paeoniflorin, and paeonol derived from paeony root on LPS-stimulated induction of cytokines To understand the anti-inflammatory effects of paeony root at the transcriptome level, we set out DNA microarray analyses. RAW264.7, a mouse macrophage cell line, was chosen to evaluate three single compounds isolated from paeony root, albiflorin, paeoniflorin, and paeonol. In order to determine the treatment concentrations of the compounds at a range with no apparent cytotoxicity, the cells were incubated with 1~100 µg/ml of each compound for 24 h and the cell viability was assessed. All three compounds were without elicit toxicity in RAW264.7 cells at the tested concentrations (Fig. 1A). Therefore, we used 100 µg/ml as the highest concentra-

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tion in following experiments. LPS plays a fundamental role in the pathogenesis of a number of inflammatory diseases by inducing a distinctive pattern of inflammatory cytokine release. These cytokines include TNFα, IL-1, and IL-6. We used LPS to produce inflammatory conditions in RAW264.7 cells. Treatment of the cells with 1 µg/ml increased in mRNA levels of TNF-α and IL-1α from 30 min after stimulation (Fig. 1B). The stimulated mRNA expression of TNF-α and IL-1α was sustained up to 24 h (Fig. 1B). LPS also stimulated IL-6 mRNA expression in RAW264.7 cells. The IL-6 level was prominently elevated during 3~12 h after stimulation, but it returned to near basal level at 24 h (Fig. 1B). As 6 h was the time point that showed maximal or near maximal induction of TNFα, IL-1α, and IL-6, we stimulated cells with LPS for 6 h in following experiments. We next examined the potential effects of albiflorin, paeoniflorin, and paeonol on the induction of TNFα, IL-1α, and IL-6 by LPS. RAW264.7 cells were treated with 1 µg/ml LPS for 6 h in the presence of 1~100 µg/ml of the paeony root compounds. The induction of TNFα was attenuated by paeoniflorin and paeonol while that of IL-1α was suppressed only by paeonol (Fig. 1C). LPS-induced expression of IL-6 was inhibited by all three compounds (Fig. 1C). Expression profiles of LPS-responsive genes in RAW264.7 cells treated with paeony root compounds The global gene expression changes by albiflorin, paeoniflorin, and paeonol were analyzed by using the Mouse Genome Survey Microarray system (Applied Biosystems). Based on data presented above (Fig. 1), RAW264.7 cells were treated with 1~100 µg/ml compound and 1 µg/ml LPS for 6 h. mRNA was prepared and processed for hybridization to the microarray chip. Hybridized signals were detected and normalized. Comparing the signals of LPS treated samples with those of the control vehicle-treated samples revealed that a total of 1,270 genes were significantly increased or decreased by ≥2-fold (Table 1). Among them 314 genes showed ≥4-fold change (Table 1). A clustering analysis of those 314 genes showed that much more number of genes up-regulated than the genes down-regulated by LPS (Fig. 2). The pattern of expression changes in response to albiflorin, paeoniflorin, and paeonol was not proportional to concentrations in many genes (Fig. 2). Among the 1,270 genes responding to LPS, we analyzed those genes for which levels were attenuated by more than 1.5-fold upon treatment with albiflorin, paeoniflorin, and paeonol at the maximal concentration. Albiflorin, paeoniflorin, and paeonol affected 19, 68, and 355 genes, respectively (Table 1). It is striking that about 28 % of the LPS-responsive genes were influenced by paeonol. This result suggests that paeonol is likely to be the most effective compound among the three paeony root components in attenuating the LPS-induced responses. Among those genes influenced by the compound, 11 were commonly affected by albiflorin and paeoniflorin, while 12 were changed by both albiflorin and paeonol (Fig. 3). More substantial number of genes (55 genes) was simultaneously targeted by both peoniflorin and paeonol (Fig. 3). Of note, there were 9 genes for which expression was changed by all three compounds (Fig. 3).

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Gene group analysis of paeonol target genes Paeonol treated at 100 µg/ml led to an attenuation of the fold changes by LPS in 139 genes by ≥1.5-fold among the 314 genes that increased or decreased by ≥4-fold in response to LPS (Table 1). Those 139 paeonol target genes were classified for the biological process, molecular function, and signaling pathway by the Panther gene group analysis (http://www.pantherdb.org/). 20 biological process, 24 molecular function, and 22 signaling pathway categories were hit (Fig. 4) among the total 31, 29, and 129 groups, respectively. Among the biological process groups, the ‘immunity and defense’ (17.0 % of total hits), the ‘nucleotide and nucleic acid metabolism’ (14.3 %), the ‘protein metabolism and modification’ (8.9 %), and the ‘signal transduction’ (12.9 %) categories, besides the ‘unclassified’, contained a high number of hits (Fig. 4A). The genes belonging to the ‘immunity and defense’ group are listed in Table 2. In classification of the panther molecular function, the ‘nucleic acid

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binding’ (15.6 %), ‘signaling molecule’ (8.1 %), ‘transcription factor’ (6.4 %), and ‘receptor’ (6.9 %) categories had a high number of genes (Fig. 4B). Among the 22 panther pathways, the ‘inflammation mediated by chemokine and cytokine signaling’ (15.2 %), ‘interleukin signaling’ (10.9 %), ‘Toll receptor signaling’ (10.9 %), and ‘apoptosis signaling’ (10.9 %) pathways displayed high numbers (Fig. 4C). Genes in the ‘interferon-gamma signaling’ and ‘JAK/STAT signaling’ pathways were also influenced by paeonol (Fig. 4C). The expression changes of the genes in the ‘inflammation mediated by chemokine and cytokine signaling pathway’ are shown in Table 3. Confirmation of microarray results by real-time PCR To validate the results of microarray analysis, 6 genes associated with the immunity process and the inflammatory pathway in the Panther classification system were analyzed

Fig. 1. Reverse transcriptionPCR analyses of LPS target genes. (A) RAW264.7 cells were treated with the indicated concentrations of the compounds. The cells in the control group (0 µg/ml) were treated with vehicle (0.1 % DMSO). After 24 h incubation, cell viability was measured with CCK assay kit. (B) RAW264.7 cells were treated with 1 µg/ml LPS for 0.5~24 h. Total RNA was isolated and the mRNA expression levels of TNFα, IL-1α, and IL-6 were detected by RT-PCR. (C) RAW264.7 cells were treated with 1 µg/ ml LPS for 6 h in the presence of the indicated concentration of albiflorin, paeoniflorin, and paeonol. The mRNA expression levels of TNFα, IL-1α, and IL-6 were assessed by RT-PCR. Bar graphs in (B) and (C) show band intensity of PCR products. The values are mean ±S.D. * p < 0.01; ** p < 0.001. Af, albiflorin; Pf, paeoniflorin; Pn, paeonol.

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Table 1. The numbers of LPS-responsive genes and the number of genes for which LPS-induced change was attenuated by the paeony root compounds. Genes regulated by LPS

# of LPS-regulated genes attenuated by PR compounds by > 1.5-fold

Folds of change

# of genes

Albiflorin

Paeoniflorin

Paeonol

≤ –4 –4 < ~ ≤ –2 2≤~<4 4≤~<8 ≥8 Total

32 491 465 156 126 1270

0 10 8 1 0 19

5 23 13 11 16 68

9 102 114 58 72 355

Fig. 2. Clustering of LPS-responsive genes influenced by albiflorin, paeoniflorin, and paeonol. The genes for which expression changed by > 4-fold were subjected to a hierarchical clustering analysis. Green indicates a lower and red indicates a higher expression level than the control value (black) obtained from the vehicletreated cells. The genes for which expression was highly up-regulated by LPS and their LPS-stimulated up-regulation was attenuated by the paeony root compounds are clustered in the upper part. The genes that were decreased by LPS and for which decrease was suppressed in the presence of the paeony root compounds are clustered in the bottom part. Af, albiflorin; Pf, paeoniflorin; Pn, paeonol. Error bars represent S.D.

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Table 2. List of LPS-responsive genes targeted by paeonol in the “immunity and defense” category.

Fig. 3. Schematic diagram of gene numbers affected by albiflorin, paeoniflorin, and paeonol. The genes of which expression increased or decreased by ≥2-fold by LPS treatment and the LPS-induced changes were affected by ≥1.5-fold in the presence of 100 µg/ ml of albiflorin, paeoniflorin, or paeonol were analyzed. Some of the genes were commonly affected by two or all three compounds of paeony root.

by real-time PCR: IL-1α, Tnc (tenascin C), chemokine (CC motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-X-C motif) ligand 2 (CXCL2), and prostaglandin-endoperoxide synthase 2 (Ptgs2). All of the genes exhibited a similar expression pattern in microarray and real-time PCR experiments (Fig. 5). Some of them showed a very high concordance between microarray and real-time PCR data. For example, the level of LPS-stimulated expression of IL-1α was reduced by paeonol by 60.5 % in the real-time PCR experiment, while the extent of reduction was 68.2 % in the microarray experiment. The effect of paeonol on CCL2 was 42.0 % in the real-time PCR and 39.6 % in the microarray experiments. Ptgs2 showed somewhat lower extent of paeonol’s effect in the real-time PCR (29.4 %) than the extent in the microarray (54.2 %) experiments. Discussion Paeony root has been reported to have anti-inflammatory, anti-allergic and immunoregulatory effects. Albiflorin, paeoniflorin, and paeonol are ingredients isolated from paeony root. Both paeoniflorin and paeonol have been demonstrated to have anti-inflammatory and immunoregulatory activities while the effect of albiflorin is unclear. We showed for the first time the gene expression profiles of compounds from paeony root by microarray analyses in a cell model system for inflammation.

Gene Name

GeneBank accession no.

Folds of LPS/CTL

Folds of (LPS + Pn)/CTL

Ccl2 Ccrl2 CD40 Cfb Cxcl11 Cxcl2 Fcgr1 Fcrl3 Gbp3 Gbp6 H2-T22 Hck Hsp70 Ifi16 Ifi204 Ifi205 Ifit1 Ifit2 Ifit3 Il13ra2 Il15 Il18 Il1a Il1rn Lgals8 Lgals9 Malt1 Oas3 Oasl2 Ptgs2 Rel Saa3 Slfn9 Tap1 Tap2 Tnf

NM_011333.1 NM_017466.3 NM_170704.1 NM_008198.1 NM_019494.1 NM_009140.1 NM_010186.2 NM_144559.1 NM_018734.2 NM_145545.2 NM_010399.2 NM_010407.3 NM_010479.2 NM_008329.1 AK076726.1 NM_172648.3 NM_008331.1 NM_008332.2 NM_010501 NM_008356.1 NM_008357.1 NM_008360.1 NM_010554.3 NM_031167.3 NM_018886.2 NM_010708.1 NM_172833.1 NM_145226.1 NM_011854.1 M64291.1 NM_009044.1 NM_011315.2 NM_181545.2 NM_013683.1 NM_011530.2 NM_013693.1

22.5 ± 1.2 13.6 ± 0.6 142.2 ± 13.4 65.8 ± 7.5 57.0 ± 12.2 29.2 ± 9.1 63.8 ± 32.1 14.5 ± 8.2 185.3 ± 61.1 60.7 ± 10.8 200.2 ± 5.8 88.4 ± 17.9 9.5 ± 3.0 4.0 ± 1.5 6.2 ± 1.5 3.0 ± 0.5 110.5 ± 32.6 54.9 ± 8.4 83.5 ± 6.4 49.4 ± 17.8 9.0 ± 1.5 5.4 ± 1.3 4.8 ± 0.6 3.0 ± 0.2 17.9 ± 2.9 11.2 ± 2.6 34.2 ± 8.3 15.9 ± 4.4 171.5 ± 46.8 58.2 ± 23.2 10.1 ± 2.1 5.8 ± 1.1 258.1 ± 78.0 141.6 ± 69.7 167.4 ± 29.4 60.3 ± 36.7 493.1 ± 134.1 151.1 ± 90.7 8.4 ± 0.7 3.5 ± 0.8 25.6 ± 5.0 13.9 ± 0.8 5.7 ± 0.9 3.0 ± 0.4 23.9 ± 9.9 6.9 ± 0.3 18.3 ± 4.9 8.1 ± 2.3 4.6 ± 0.7 2.9 ± 0.4 9.9 ± 2.2 5.4 ± 1.1 6.4 ± 1.7 3.4 ± 0.6 4.2 ± 0.8 2.6 ± 0.8 69.6 ± 19.5 31.5 ± 8.9 34.9 ± 5.5 15.9 ± 1.3 4.3 ± 0.6 2.8 ± 0.2 92.8 ± 27.3 34.9 ± 6.7 9.5 ± 1.3 5.4 ± 1.9 10.2 ± 1.1 6.2 ± 2.1 5.0 ± 1.0 3.0 ± 0.5 24.6 ± 2.8 14.8 ± 1.4

Folds of LPS/CTL, ≥ 4-fold; Folds of LPS + Pn/Pn, > 1.5-fold; Pn, paeonol. Values, mean ± S.D.

LPS plays a crucial role in inflammatory responses by inducing expression of cytokine and other immune regulator genes in macrophages. In our study, the RAW264.7 macrophage cell line was stimulated with LPS to mimic the inflammatory conditions. Among total 33,012 gene probes, signals of 11467 gene probes were significantly above background when the ‘Flag’ threshold was set at 0 and the S/N (ratio of signal/SDEV) was set at more than 3. Expression of about 11 % (1270 gene probes) of the 11467 significant signals was changed ≥2-fold by treatment with LPS. 523 genes were down-regulated while 747 genes were up-regulated. Our data

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Table 3. List of LPS-responsive genes targeted by paeonol in the “inflammation mediated by chemokine and cyokine pathway” category. Gene Name

GeneBank accession no.

Folds of LPS/CTL

Ccl2 Cxcl2 Il18 Ptgs2 Rel Stat1 Tnc

NM_011333.1 NM_009140.1 NM_008360.1 M64291.1 NM_009044.1 NM_009283.2 NM_011607.1

22.5 ± 1.2 200.2 ± 5.8 5.7 ± 0.9 34.9 ± 5.5 4.3 ± 0.6 9.2 ± 1.4 107.6 ± 40.3

Folds of (LPS + Pn)/CTL 13.6 ± 0.6 88.4 ± 17.9 3.0 ± 0.4 15.9 ± 1.3 2.8 ± 0.2 5.8 ± 0.8 22.9 ± 6.6

Folds of LPS/CTL, ≥ 4-fold; Folds of LPS + Pn/Pn, > 1.5-fold; Pn, paeonol. Values, mean ± S.D.

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also revealed that paeonol caused changes in 355 genes by ≥1.5-fold among the 1,270 genes responded to LPS. The number of genes affected by paeonol covered 21.2 % of the genes down-regulated by LPS and 32.7 % of those up-regulated. In comparison, the numbers of genes of which expression was influenced by paeoniflorin and albiflorin by ≥1.5fold were only 68 and 19, respectively. Overall, our studies suggest that paeonol has the most extensive inhibitory effects among the three components of paeony root on the regulation of gene expression by LPS. Therefore, it may be inferable that paeonol is the major ingredient for the anti-inflammatory effect of paeony root. When the biological function of the genes that were up- or down-regulated ≥4-fold by LPS and of which LPS-induced changes were attenuated ≥1.5-fold by paeonol were ana-

Fig. 4. Classification of paeonol target genes in LPS-challenged macrophage cells. The genes of which mRNA levels decreased or increased by ≥4-fold by LPS treatment of RAW264.7 cells and the LPS-induced changes were affected by ≥1.5-fold in the presence of 100 µg/ml of paeonol were subjected to analyses. Analyses of the biological process (A), the molecular function (B), and the signaling pathway (C) of the Panther database were performed.

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lyzed, the immunity and defense group was the most highly affected with 17 % of total genes being affected. The LPSinduced up-regulation of TNFα and IL-1α has been well documented in macrophages [23, 24]. Consistent with previous reports, we found that the level of TNFα signal increased 24.5-fold in LPS-treated cells compared to the control vehicle-treated RAW264.7 cells. However, the increase was only 14.7-fold in cells treated with both LPS and paeonol. Likewise, IL-1α increased 21.7-fold in LPS-treated cells and 6.9-fold in cells treated with LPS plus paenol. In addition, some of CC and CXC chemokines have been reported to be targets of LPS in immune cells [16]. In our microarray study, the induction of CCL2, CXCL2, CXCL11, and CCRL2 by LPS was reduced by paeonol. Groups of interferon-activated genes (Ifi16, Ilfi204, and Ifi205) and interferon-inducible genes (Ifit1, Ilifit2, and Ifit3) were also changed by treatment with paeonol. Ifit1, Ilifit2, and Ifit3 have been implicated in interferon-mediated immunity. Ifi16, Ilfi204, and Ifi205 have been reported to be involved not only in interferon-mediated immunity but also in cell proliferation and differentiation [25]. Besides chemokines and Ifi/Ifit families, paeonol suppressed CD40 level to approximately half the level induced by LPS. CD40 is one of crucial co-stimulation receptors for T cell and macrophage activation [26–28]. Among the LPS-induced genes, the expression level of genes related to inflammation was also largely changed by

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paeonol. Those genes include ptgs2, IL-18, and Tnc. COX2, the gene product of ptgs2, is a key enzyme for the synthesis of pro-inflammatory prostaglandins, in particular PGE [29, 30]. IL-18 is an important pro-inflmmatory cytokine that is also induced by interferon-gamma [31]. IL-18 is elevated in synovial tissues of rheumatoid arthritis and promotes production of GM-CSF, nitric oxide, and TNFα in synovial culture [32]. In our study, STAT1 (signal transducer and activator of transcription) and Rel were up-regulated by LPS and the up-regulation was attenuated by paeonol. Rel family NFκB proteins are the critical transcription factors activated by many inflammatory cytokines as well as LPS. STAT1 is phosphorylated on tyrosine by Jak family kinases in response to interferons and subsequently dimerizes with another member of the STAT family prior to nuclear localization and stimulation of interferon target gene transcription [33]. LPS causes a rapid and potent release of type I interferons in macrophages [34]. LPS may coordinate an efficient interferon response in the cell by causing release of interferons on one hand and elevation of STAT1 level needed for interferon signaling on the other hand. Paeonol was reported to induce apoptosis in HepG2 cells [19]. We observed the significantly changed genes by paeonol among the LPS-induced genes included genes associated with apoptosis signaling. For example, the expression of heat shock protein 70 (HSP70) was up-regulated by LPS to 17.7fold of the control level, while the induction was 10.9-fold in Fig. 5. Real-time PCR analyses of paeonol target genes identified by the microarray analysis. RAW264.7 cells were treated with 1 µg/ml LPS or the control vehicle (0.1 % DMSO) in the presence or absence of 100 µg/ml paeonol for 6 h. Total RNA was prepared and real-time PCR analyses were performed for IL-1α, CXCL2, CCL2, CCL3, Tnc, and Ptgs2 RNA levels. Pn, paeonol.

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the presence of paeonol. HSP70 has been reported to protect hepatocytes from TNFα-induced apoptosis [35] and myeloid/lymphoid cells from heat shock- or ethanol-induced apoptosis [36]. Down-regulation of mRNA for Daxx (Fas death domain-associated protein) by paeonol was also discovered in our present study. Although Daxx has been implicated in apoptosis in many studies, whether it is pro- or anti-apoptotic is controversial [37, 38]. Its precise mechanisms of action for or against apoptosis also remain to be clearly understood. In summary, the expression levels of LPS-induced genes in macrophages were altered by treatment with paeonol, paeoniflorin, and albiflorin by different extents. From the comparison of expression changes of LPS-induced genes, paeonol was found to most widely influence the LPS response in gene expression. A major portion of the genes for which LPS-stimulated expression was attenuated by paeonol induced genes related to inflammation and immunity, implying that the anti-inflammatory effect of paeony root and paeonol may be through regulation of the transcription of these genes. Our study exemplifies an example that gene expression profiling using microarray analyses can be an effective approach to gain global insight into the mechanism of action of compounds isolated from natural products. Acknowledgements. This work was supported by the Korea Food & Drug Adminstration through the National Center for Standardization of Herbal Medicine. Electronic supplementary material The online version of this article (DOI: 10.1007/s00011-007-7190-3) contains supplementary material, which is available to authorized users.

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