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Journal of Ethnopharmacology
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j e t h p h a r mShikonin derivatives inhibited LPS-induced NOS in RAW 264.7 cells via
downregulation of MAPK/NF-
B signaling
Yu Wen Cheng
a, Ching Yi Chang
a, Kou Lung Lin
b, Chien Ming Hu
c,
Cheng Hui Lin
a, Jaw Jou Kang
b,∗aSchool of Pharmacy, Taipei Medical University, Taipei, Taiwan, ROC
bInstitute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC cEmergency Department, Taipei Medical University Hospital, Taipei, Taiwan, ROC
a r t i c l e i n f o
Article history: Received 17 June 2008
Received in revised form 1 September 2008 Accepted 3 September 2008
Available online 12 September 2008 Keywords: Shikonin Lipopolysaccharide Nitric oxide iNOS MAPKs NF-B
a b s t r a c t
Aim of the study: Shikonin/alkannin (SA) derivatives, analogs of naphthoquinone pigments, are the major components of root extracts of the Chinese medicinal herb (Lithospermum erythrorhizon; LE) and widely distributed in several folk medicines.
In the present study, the effect and the underline molecular mechanism of shikonin derivatives isolated from root extracts of Lithospermum euchroma on lipopolysaccharide (LPS)-induced inflammatory response were investigated.
Materials and methods: Effects of five SA derivatives, including SA, acetylshikonin, ,-dimethylacrylshikonin, 5,8-dihydroxy-1.4-naphthoquinone, and 1,4-naphthoquinone on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in mouse macrophage RAW264.7 cells were examined.
Results: Data suggested that SA derivatives inhibited LPS-induced NO and PGE2production, and iNOS
protein expression. RT-PCR analysis showed that SA derivatives diminished LPS-induced iNOS mRNA expression. Moreover, the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in LPS-stimulated RAW 264.7 cells was concentration-dependently suppressed by SA derivatives. SA inhibited NF-B activation by prevention of the degradation of inhibitory factor-B and p65 level in nuclear fractions induced by LPS.
Conclusions: Taken together, these results suggest that the anti-inflammatory properties of SA derivatives might result from inhibition of iNOS protein expression through the downregulation of NF-B activation via suppression of phosphorylation of ERK, in LPS-stimulated RAW 264.7 cells.
© 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Shikonin/alkannin (SA) derivatives, analogs of naphthoquinone pigments, the major components of root extracts of the Chinese medicinal herb (Lithospermum erythrorhizon; LE), are ingredients of several folk medicines. Treatment indications claimed for LE roots include burns, anal ulcers, hemorrhoids, infected crusts, bed-sores, external wounds, and oozing dermatitis (Papageorgiou et al., 1999). Shikonin was first isolated as its acetate form from the roots of LE by the Japanese chemistsMajima and Kuroda (1922),
∗ Corresponding author at: Institute of Toxicology, College of Medicine, National Taiwan University, No. 1, Jen Ai-Road, Section 1, Taipei 100, Taiwan,
ROC. Tel.: +886 2 23123456x88603; fax: +886 2 27374622. E-mail addresses:[email protected](Y.W. Cheng),
[email protected](J.J. Kang).
after several decades, such compounds have also been shown to exhibit significant antitumor, antibacterial, anti-HIV-1 (Ueba et al., 1993; Chen et al., 2003), anti-DNA topoisomerase, antiangiogenesis (Hisa et al., 1998), and anti-inflammatory activities (Papageorgiou et al., 1999). Studies suggest that the anti-inflammatory effects of shikonin derivatives may be attributable to several mechanisms of action, e.g., suppression of mast cell degranulation and pro-tection of the vasculature from mediator challenge (Wang et al., 1995), inhibition of phosphatidylinositol signaling in neutrophils (Wang and Kuo, 1997), selective blocking of chemokine bind-ing to CC chemokine receptor-1 (Chen et al., 2001), antioxidant and free radical-scavenging activities in vivo by interference with free radical processes (Kourounakis et al., 2002), and inhibition of phorbol 12-myristate 13-acetate-induced cyclooxygenase (COX)-2 expression (Subbaramaiah et al., 2001).Staniforth et al. (2004) reported that shikonin inhibits the transcriptional activity of the human tumor necrosis factor (TNF)-␣ promoter through inter-0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
ference with the basal transcription machinery, suggesting that shikonin may have clinical potential as an anti-inflammatory thera-peutic (Staniforth et al., 2004). Although a broad range of biological and pharmacological activities of shikonin have been reported, the exact mechanism of the inflammatory action of SA derivatives has not been fully elucidated.
The inflammatory process involves activation of monocytes and/or macrophages; among these, macrophage activation is cru-cial for the progression of multiple inflammatory diseases via the release of inflammatory mediators, such as cytokines, nitric oxide (NO), and prostaglandins (PGs) (Liang et al., 1999). Physiologically, over-induction of NO leads to various harmful responses including tissue injury, septic shock (Petros et al., 1991; Xie et al., 1992), apop-tosis, and necrosis (Nagai et al., 2003). In some cases, the induction of NO by stimulating macrophages leads to organ destruction in some inflammatory (Heiss et al., 2001) and autoimmune diseases (Kleemann et al., 1993). Thus, the inhibition of NO production by blocking iNOS expression may be a useful strategy for treating var-ious inflammatory diseases.
In addition to NO, macrophages also release prostaglandins, which are COX products. COX is an enzyme that converts arachi-donic acid to PGs, and is found as two isoforms (COX-1 and -2). COX-1 is constitutively expressed while COX-2 is barely detectable under normal physiological conditions, and can be induced by proinflammatory cytokines and mediators by the same stimulus as iNOS (Chi et al., 2003; Aktan, 2004). NF-B is one of the most ubiquitous transcription factors, regulates the expressions of genes involved in cellular proliferation, inflammatory responses, and cell adhesion, and is activated in response to various extracellular stim-uli, including interferon␥ (INF)-␥, LPS, and oxidative stress. NF-B sites have been identified in the promoter region of the iNOS gene (Xie et al., 1993; Eberhardt et al., 1996) and COX-2 gene (Appleby et al., 1994).
LPS is a major inflammatory molecule that triggers the pro-duction of proinflammatory toxins, cytokines such as iNOS, COX-2, TNF-␣, and IL-1 in various cell types (MacMicking et al., 1997). In the present study we demonstrate that SA derivatives (Fig. 1) inhib-ited NO generation in LPS-induced RAW-264.7 macrophages, as well as levels of iNOS messenger (m)RNA and protein. The inhibition of iNOS protein expression appeared to be due to downregulation of NF-B activation by inhibition of ERK phosphorylation.
2. Materials and methods
2.1. Chemicals
Fetal bovine serum (FBS) was from Gibco BRL (Grand Island, NY), Dulbecco’s modified Eagle’s medium (DMEM), and medium sup-plement for cell culture were obtained from Invitrogen (Carlsbad, CA). LPS was obtained from Sigma (St. Louis, MO). 5,8-Dihydroxy-1,4-naphthoquinone and 5,8-Dihydroxy-1,4-naphthoquinone were purchased from ACROS (New Jersery, USA). The specific antibody for iNOS was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The NF-B oligonucleotide for the electrophoretic mobility shift assay (EMSA) was synthesized by MDBio (Taipei, Taiwan). Reagents for the gel shift assay system (Promega, Madison, WI) and radioactive [␥-32P]-ATP were purchased from Amersham International
(Buck-inghamshire, UK). All other chemicals were of the highest quality available.
2.2. Plant materials
Commercially available dried roots of Lithospermum euchroma were purchased from a local Chinese medicine drug store, and its authenticity was confirmed by Prof. Hui-Wen Cheng of Taipei
Fig. 1. Chemical structures of shikonin derivatives (SA, AC, and DM) and naphtho-quinone pigments (DH and NAP).
Medical University. A voucher specimen was kept and stored at the Institute of Pharmacy, Taipei Medical University, Taipei, Tai-wan.
2.3. Isolation of shikonin/alkannin and their derivatives
Shikonnin/alkannin (SA) was isolated following a procedure previously described byCheng et al. (1995). Briefly, dried roots of Lithospermum euchroma were extracted twice with n-hexane. The n-hexane extract was evaporated, and then the crude extract was dissolved in NaOH. After filtration, the solution was acidified with H2SO4to pH 6 and re-extracted with n-hexane. The n-hexane
layer was concentrated and fractionated through an open column of silica gel 60 (15 mm× 15 mm). Alternatively, acetylshikonin (AC) and,-demethylacrylshikonin (DM) were isolated from the crude n-hexane extract of Lithospermum euchroma using an open col-umn of silica gel 60 (15 mm× 15 mm). The chemical structures of SA, AC, DM, 5,8-dihydroxy-1.4-naphthoquinone (DH), and 1,4-naphthoquinone (NAP) (as shown inFig. 1) were determined by chemical and physical means and by comparison with data from the literature. A red crystal of shikonin/alkannin with an enantiomeric ratio of 17:83 was used in this study.
2.4. Cell culture
The mouse macrophage-like cell line, RAW 264.7, was purchased from American Type Culture Collection (ATCC TIB71, Manassas, VA), RAW 264.7 macrophages were grown in DMEM supplemented with 10% heat-inactivated FBS and 1% antibiotics under 5% CO2at
den-sity of 1× 106cells/60-mm plate. After pre-incubation for 24 h, the
test compounds were added 30 min before co-treatment with LPS (1g/ml) for another 18 h. Test compounds were dissolved in DMSO on the day of the experiment and diluted with serum-free DMEM at appropriate concentrations. The final concentration of DMSO was adjusted to 0.1% (v/v). Control groups also received the same amount of DMSO. Before stimulation with LPS, the medium was refreshed with 3 ml DMEM and treated with shikonin derivatives or naphthoquinone pigments and then stimulated with 1g/ml LPS for the indicated times in the figure legends.
2.5. NO determination
For determining the NO concentration, the stable conver-sion product of NO, nitrite (NO2−), was measured using the
Griess reagent (1.0% sulfanilic acid and 0.1% N-(1)-naphthyl-ethylenediamine-dihydrochloride) incubated at room temperature for 5 min. NaNO2was used to generate a standard curve, and NO
production was determined by measuring the optical density at 550 nm with a microplate reader (Spectra Max, Molecular Devices, Sunnyvale, CA). In order to determine the direct inhibitory effects of the test compounds of the iNOS enzyme on cell levels, RAW 264.7 cells were cultured in 96-well plates using DMEM with-out phenol red and were pretreated with different concentrations of SA derivatives before being incubated for 30 min. Cellular NO production was induced by adding 1g/ml LPS and incubating for 18 h. After incubation, 100l of conditioned media contain-ing NO2−(the primary stable oxidation product of NO) was mixed
with the same volume of Griess reagent and incubated for 18 h. LPS alone was used as the positive control from the medium, and the NO concentration was measured by the optical density at 550 nm on an enzyme-linked immunosorbent assay (ELISA) plate reader (Molecular Devices). Nitrite production was determined by com-paring the optical density with the standard curve obtained with NaNO2.
2.6. PGE2determination
RAW264.7 cells (1× 105cells/well) were cultured in 96-well
plates using DMEM without phenol red and pretreated with dif-ferent concentrations of SA derivatives before being incubated for 30 min. Cellular PGE2production was induced by adding 1g/ml
LPS and incubating for 18 h. LPS alone was taken as the positive con-trol. After incubated, culture supernatants were collected, and the PGE2 concentration was measured by ELISA using a monoclonal
antibody, as specified by the manufacturer. The PGE2 content in
the medium was assayed according to the protocol provided by the manufacturer (PGE2EIA kit, Cayman, Chemicals, Ann Arbor, MI).
2.7. Lactate dehydrogenase (LDH) assay
The LDH released into the culture medium was used as an index of cell death. Toxicity was quantitated by determining the percent leakage of the cytosolic enzyme, LDH, from cells into the medium. RAW 264.7 cells (1× 105cells/ml) were cultured in
96-well plates using DMEM without phenol red, in an incubator at 37◦C for 24 h. After being pretreated with different concentrations of SA derivatives and incubated for 30 min, cellular LDH production was induced by adding 1g/ml LPS, and incubated for 18 h. Briefly, the substrate reaction buffer (0.5 mM lactic acid, 0.1N NaOH, and 0.1 M glycine buffer) was added to the medium. Dinitrophenylhydrazine (0.02%) was added as a chromogenic agent, and the absorbance values at 460 nm were read in a spectrophotometer (Beckman DU 640).
2.8. Reverse-transcription (RT)-PCR
Total cellular RNA from treated cells was isolated using an NE-PERTM kit (Pierce, Rockford, IL) according to the manu-facturer’s instructions. From each sample, 3g of RNA was reverse-transcribed for 1 h at 37◦C in a reaction mixture con-taining 5 U RNase inhibitor (Invitrogen), 0.5 mM dNTP (Boeringer Mannheim, Indianapolis, IN), 2 mM random hexamer (Promega, Madison, WI), 1× RT buffer, and 5 U reverse transcriptase. A PCR analysis was then performed on aliquots of the complemen-tary cDNA preparations to detect iNOS-actin gene expression using a PTC-150 Minicycler MJ (MJ Research, Watertown, MA). The reaction was carried out in a volume of 25 ml contain-ing 1 unit of Taq DNA polymerase, 0.2 mM dNTP, 10× reaction buffer, and 0.2mol of the 5- and 3-primers. After initial
denaturation for 5 min at 95◦C, 30 amplification cycles were performed including 1 min of denaturation at 95◦C, 1 min of annealing at 56 (for iNOS) or 53◦C (for -actin), and 2 min of extension at 72◦C. The PCR primers used in this study were as follows. Two specific probes: iNOS (5 -TGG-GAA-TGG-AGA-CTG-TCC-CAG/3-GGG-ATC-TGA-ATG-TGA-TGT-TTG) and -actin (5-AAG-GTG- TGA-TGG-TGG-GAA-TG/3 -ATG-GCT-ACG-TAC-ATG-GCT-GG) were used, and these were then run on 2% agarose gels for the cDNA fragment determination. The condition of reverse transcription was based on protocols provided by the manufac-turer. Analysis of the resulting PCR products on 1% agarose gels showed single-band amplification products with the expected sizes.
2.9. Western blotting
Cellular proteins were extracted from control and SA derivative-treated RAW264.7 cells. Cells were collected by centrifugation and washed twice with phosphate-buffered saline (PBS). The washed cell pellets were resuspended in extraction lysis buffer (50 mM HEPES (pH 7.0), 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothre-itol, 5 mM NaFl, and 0.5 mM sodium orthovanadate) containing 5g/ml each of leupeptin and aprotinin and then incubated at 4◦C for 20 min. Cell debris was removed by microcentrifugation, followed by quick freezing of the supernatants. The concentration of cell lysates was determined using a Bio-Rad Protein Assay Kit (Bio-Red, Richmond, CA) according to the manufacturer’s instruc-tions. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS–PAGE) was performed on 10% polyacrylamide gels and then transferred onto PVDF membranes by the PantherTMSemidry
Elec-troblotter (Owl Scientific, Portsmouth, NH). The immunoblot was incubated overnight with blocking solution (5% skim milk) at 4◦C, and then incubated with primary antibodies that recognize iNOS (BD transduction),-actin (Sigma), COX-2, IB-␣, p65, MAPKs (ERK, JNK, p38, pERK, pJNK, and pp38), and Histone-1 (Santa Cruz). After washing with TBST, horseradish peroxidase (HRP)-conjugated sec-ondary antibodies (Amersham, Piscataway, NJ) (1:3000 dilution in TBST) were applied, and the blots developed by an enhanced chemi-luminescence (ECL) detection system (Pierce Biotechnology). 2.10. Electrophoretic mobility shift assay (EMSA)
RAW264.7 macrophage cells were plated in 100-mm dishes (1× 106cells/ml). Cells were preincubated with each of the
shikonin derivatives for 30 min before the addition of 1g/ml LPS for 18 h. Cells were then washed twice, scraped off into cold PBS, and centrifuged. Pellets were resuspended in hypo-tonic extraction buffer containing 10 mM HEPES, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.2 mM PMSF,
4g/ml leupeptin, and 20 g/ml aprotinin, and incubated on ice for 10 min. Then, cells were lysed by the addition of 0.5% Nonidet P-40 and vigorous vortexing for 10 s. The nuclei were pelleted by centrifugation at 12,000× g for 10 min at 4◦C and
resuspended in high-salt buffer (20 mM HEPES (pH 7.9), 25% glycerol, 400 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT,
1 mM NaF, and 1 mM sodium orthovanadate). Five micrograms of nuclear extract, poly(dI-dC), and biotin-labeled double-strain NF-B oligonucleotide (5-AGTTGAGGGGACTTTCCCAGGC-3) were
end-labeled with [␥-32P]dATP (the underlining indicates a B con-sensus sequence or a binding site for the NF-B/cRel homodimeric or heterodimeric complex). Binding reactions were performed according to the manufacturer’s instructions: mixing with the binding buffer (Lightshift Chemiluminescent EMSA kit; Pierce Chemical) to a final volume of 20M, and then incubation at room temperature for 30 min. An unlabeled double-stranded NF-B oligonucleotide (5-AGTTGAGGcGACTTTCCCAGGC-3), and an AP-1 oligonucleotide (5-CGCTTGATGAGTCAGCCGGAA-3) were also used to confirm specific binding. The P65 antibody was used to induce a supershift, indicating the binding affinity of NF-B. The nuclear protein–DNA complex was separated by 5% Tris/boric acid/EDTA-polyacrylamide gel at 100 V in 0.5× TBE buffer. The gels were vacuum-dried for 1 h at 80◦C and exposed to X-ray film at −70◦C for 24 h.
2.11. Statistical analysis
Results are expressed as the mean± S.D. of at least three experiments performed using different in vitro cell preparations. Statistically significant values were compared using Student’s t-test followed by Dunn’s test. Statistical significance was set at p < 0.05.
3. Results
3.1. Effects of SA derivatives on LPS-induced NO and PGE2
production by LPS-treated RAW 264.7 cells
To assess the effect of SA derivatives on LPS-induced NO produc-tion in RAW 264.7 macrophages, cells were treated with various concentrations of SA derivatives for 30 min, and NO and PGE2
were induced by co-treatment with LPS (1g/ml) for another 18 h. Neither LPS nor samples were added to the control group. Cell cul-ture medium was harvested before treatment, and NO levels were quantified using the Griess reaction. All SA derivatives, including SA, DH, AC, and DM (0.3–3g/ml), and NAP (3–10 g/ml) showed dose-dependent inhibition of LPS-induced NO production (Fig. 2A). To determine whether SA derivatives inhibited PGE2 production,
cells were treated with various concentrations of SA derivative for 30 min, and PGE2was induced by co-treatment with LPS (1g/ml)
for another 18 h. As shown inFig. 2B, only higher concentration of SA derivatives, including 3g/ml SA, DH, AC, and DM, and 10 g/ml NAP, significantly inhibited the production of PGE2. Among these,
SA was found to have the most-potent effect on inhibition of PGE2
accumulation. The cytotoxic effects of these five compounds were also evaluated with the LDH assay, and only higher concentrations of 3g/ml SA, DH, AC, and DM, and 10 g/ml NAP showed an effect on cell viability (data not shown). Thus, the inhibitory effects of SA derivatives on NO production at 0.3–1g/ml were not attributable to any cytotoxic effect.
3.2. Effects of SA derivatives on iNOS and COX-2 protein expressions by LPS-treated RAW 264.7 cells
Since SA derivatives were found to inhibit the production of NO and PGE2induced by LPS-treated RAW 264.7 cells, we further
Fig. 2. Shikonin derivatives and naphthoquinone pigments dose-dependently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) but partially inhibited prostaglandin E2(PGE2) production in RAW 264.7 macrophages. Cells were treated
with 0.3, 1, or 3M of SAs or NAP at 3 or 10 M at 37◦C for 30 min before the
addi-tion of 1g/ml LPS for 18 h. At the end of incubaaddi-tion, 100 and 500 l of medium were collected for the NO (A) and PGE2(B) assays, respectively. C, control; L, LPS
(1g/ml) treated. Data were obtained from three independent experiments and are expressed as the mean± S.D. *Statistically significant difference compared to 18-h positive control at the p < 0.05 level using Student’s t-test.
investigated their effects on LPS-induced iNOS and COX-2 pro-tein expression levels by Western blotting. The immunoblot assay showed that iNOS and COX-2 proteins were undetectable in resting RAW264.7 macrophages and were highly induced in the presence of LPS. SA derivatives significantly inhibited this iNOS production in a dose-dependent manner (Fig. 3B). A similar pattern was observed when the effect of SA was examined on LPS-induced COX-2 expres-sion levels but not on DH, AC, DM, or NAP. However, none of these treatments affected the expression of the housekeeping gene, -actin (Fig. 3A).
3.3. Effects of SA derivatives on iNOS mRNA expression by LPS-treated RAW 264.7 cells
Since iNOS expression decreased at the protein level, we investigated whether the decrease in NO production was due to downregulation of iNOS gene expression at the mRNA level using RT-PCR. In unstimulated RAW264.7 cells, the iNOS mRNA level was almost undetectable. However, in response to LPS, the expression of iNOS was markedly augmented, and SA derivatives significantly inhibited iNOS mRNA induction in a concentration-dependent manner (Fig. 4A and B).
Fig. 3. Shikonin derivatives and naphthoquinone pigments dose-dependently inhibited lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) protein expression in RAW 264.7 macrophages. (A) Cells were treated with 0.3, 1, or 3M of SAs or 3 or 10 M NAP at 37◦C for 30 min before the addition of 1g/ml
LPS for 18 h. Equal amounts of total proteins (60g/lane) were subjected to 10% SDS–PAGE, and expressions of iNOS, COX-2, and-actin protein were detected by Western blotting using specific anti-iNOS, COX-2, and anti--actin antibodies. (B) Quantified data. Data were obtained from three independent experiments and are expressed as the mean± S.D. *Statistically significant difference compared to the 18-h positive control at the p < 0.05 level using Student’s t-test.
3.4. Effect of SA on LPS-induced NF-B activation
To further investigate the mechanism of the SA derivative-mediated inhibition of iNOS transcription, we focused on NF-B, which is activated in cells challenged by LPS and other
inflamma-Fig. 4. Shikonin derivatives and naphthoquinone pigments dose-dependently inhibited lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) mRNA expression in RAW 264.7 macrophages. (A) Cells were treated with 0.3, 1, or 3M SAs of 3 or 10 M NAP at 37◦C for 30 min before the addition of 1g/ml
LPS for 18 h. Reverse transcription was performed on total RNA (3g) as described in Section2. Subsequently, PCR was conducted on cDNA using specific primers to iNOS and-actin and then run on 2% agarose gels to examine the cDNA fragments. C, control; L, LPS (1g/ml) treated. (B) Quantified data of SA on LPS-induced iNOS mRNA expression. Data were obtained from three independent experiments and are expressed as the mean± S.D. *Statistically significant difference compared to the 18-h positive control at the p < 0.05 level using Student’s t-test.
tory stimuli, and is involved in the transcriptional activation of the responsive genes of iNOS and COX-2 (Surh et al., 2001; Lappas et al., 2002). Among these five chemicals, SA is a well-known chemi-cal for anti-inflammation and was the only one which could inhibit COX-2 protein expression in our study. To elucidate the mechanism, we next determined whether SA inhibited the DNA-binding activ-ity of NF-B in LPS-stimulated RAW 264.7 macrophages. As shown inFig. 5A, treatment with LPS caused a significant increase in the DNA binding activity of NF-B. In the presence of SA (0.3–1 g/ml), however, LPS-induced NF-B–DNA binding was markedly sup-pressed in a dose-dependent manner (Fig. 5A and B). In addition, co-treatment with an unlabeled NF-B oligonucleotide caused a significant decrease in the DNA-binding activity of NF-B in the absence of SA, and when co-treated with the AP-1 oligonucleotide, the inhibition was markedly reversed. The addition of the p65 anti-body caused a significant supershift of the NF-B-DNA complex, further confirming the binding affinity of p65 and NF-B (Fig. 5A). Because p65 is the major component of NF-B activated by LPS in macrophages, the translocation of p65 into the nucleus was deter-mined using immunocytochemistry. Western blot analysis showed that the LPS-induced p65 level in nuclear fractions was reduced by SA (Fig. 5C).
3.5. Effect of SA on LPS-induced IB degradation
The nuclear translocation and DNA binding of the NF-B tran-scription factor are preceded by degradation of the inhibitory factor-B␣ (I-B␣) (Rice and Ernst, 1993). The cytoplasmic levels of I-B␣ were examined by a Western blot analysis at the indicated concentrations of SA derivatives to determine whether inhibition of NF-B-DNA-binding by SA is related to I-B␣ degradation. I-B␣ protein expression was barely detected at 15 min after LPS treat-ment. However, SA caused a significant recovery of the level of I-B␣ protein (Fig. 6). A similar pattern was observed when the effects on the recovery of LPS-treated I-B␣ degradation by DH, AC, DM, and NAP.
3.6. Effects of SA derivatives on MAPK phosphorylation of LPS-treated RAW 264.7 cells
We investigated the molecular mechanism of NF-B inhibi-tion by its inhibitory effect on the activainhibi-tions of MAPKs (ERK1/2, p38, and JNK). Maximum phosphorylation of p38, ERK1/2, and JNK expression levels are known to occur 30 min after LPS (1g/ml) treatment in RAW 264.7 macrophages. However, the SA derivatives, SA, DH, AC, and DM (3g/ml), and NAP (10 g/ml) significantly sup-pressed the LPS-induced activations of ERK 1/2 but not p38 MAPK or SAPK/JNK (Fig. 7A). The concentration-dependent inhibition of ERK phosphorylation is shown inFig. 7B.
4. Discussion and conclusions
Shikonin is an enantiomer of alkannin, which has historical references for treatment of ulcers as early as the 4th century BC and is found in the roots of European dye-plant Alkana tinctoria (Hazra et al., 2004). In addition, shikonin is currently used in vari-ous medicinal and cosmetics and dyestuffs in Japan, while alkannin is mainly used for food coloring and cosmetics in Europe and the US (Papageorgiou et al., 1999). Although the efficacy of alkannin and shikonin derivatives has been demonstrated in vivo, the precise model of their anti-inflammatory actions remains undetermined. The purpose of this study was to elucidate the molecular mech-anism of the anti-inflammatory action of shikonin (SA) and its derivatives (AC and DM), and the naphthoquinone pigments (DH
Fig. 5. Shikonin dose-dependently inhibits lipopolysaccharide (LPS)-induced NF-B activation. Cells were seeded 1 × 106cells/ml 10 ml in 100-mm plates. After 24 h
of incubation, the medium was refreshed and treated with 0.3, 1, or 3M of SAs at 37◦C for 30 min before the addition of 1g/ml LPS for 18 h. (A) Nuclear extracts
(5g) were used for the electrophoretic mobility gel shift assay (EMSA). (B) Quanti-fied data. (C) Effects of shikonin derivatives on LPS-induced p65 translocation. Equal amounts of total proteins (30g/lane) were subjected to 10% SDS–PAGE, and expres-sions of p65 and histone-1 protein were detected by Western blotting using specific anti-p65 and anti-histone-1 antibodies. Histone-1 protein was used as an inter-nal control. C, control; L, LPS (1g/ml) treated; CE, cytosolic extracts; NE, nuclear extracts. Data were obtained from three independent experiments and are expressed as the mean± S.D. * Indicates a statistically significant difference compared to the 18-h positive control at the *p < 0.05 level using Student’s t-test.
and NAP) using the LPS-induced inflammatory responses in murine RAW 264.7 macrophage cells.
In murine RAW 264.7 macrophage cells, LPS induces iNOS tran-scription and translation, which is followed by NO production; therefore, suppression of the production of NO using drugs might be useful for treating inflammatory diseases and ectotoxin shock. Herein, we found that SA derivatives inhibited LPS-induced proin-flammatory molecules including NO and PGE2in dose-dependent
manners. To further investigate the mechanisms underlying these inhibitions by SA derivatives, expression levels of iNOS and COX-2 protein and the iNOS mRNA level were respectively examined by Western blotting and RT-PCR. Inhibition of iNOS gene expres-sion was evidenced by a reduction in its mRNA levels, and this occurred simultaneously and in concentration-dependent man-ners (Fig. 4), suggesting that the inhibition of NO release might be attributable to the suppression of iNOS mRNA transcription and thus protein expression. In addition, we studied another important mediator of inflammation, COX-2, which acts on arachidonic acid to release PGs that further orchestrate the process of inflammation (Willoughby et al., 2000). Among these five SA derivatives, all inhib-ited PGE2induction at the higher concentration (Fig. 2), however,
only SA showed an inhibitory effect on COX-2 protein expression (Fig. 3). Seto and co-workers studied the effects of shikonin and several derivatives on various aspects of wound healing, and their anti-inflammatory effects (Seto et al., 1992). Shikonin was shown to be more active than acetylshikonin and the other SA deriva-tives. The anti-inflammatory effects of extracts of Arnebia euchroma (alkanin esters), and,-dimethylalkannin in particular (Lin et al., 1980), have been studied and showed similar results (Seto et al., 1992). In our study, SA showed a more-potent effect than the other derivatives of inhibiting NO and PGE2induced by LPS-treated RAW
Fig. 6. Shikonin derivatives and naphthoquinone pigments block lipopolysaccha-ride (LPS)-induced NF-B activation via abrogation of IB-␣ degradation in RAW 264.7 macrophages. (A) Cells were treated with 0.3, 1, or 3M SAs or NAP at 3 or 10M at 37◦C for 30 min before the addition of 1g/ml LPS for 18 h. Equal amounts
of total proteins (30g/lane) were subjected to 10% SDS–PAGE, and the expressions of IB-␣ and -actin proteins were detected by Western blotting using specific anti-IB-␣ and anti--actin antibodies. -Actin protein was used as the internal control. C, control; L, LPS (1g/ml) treated. (B) Effects of SA derivatives on LPS-induced IB-␣ degradation in dose-dependent manners. Cells were treated with 1 g/ml of LPS only or LPS plus 0.3, 1, or 3M of shikonin for the indicated times. Equal amounts of total proteins (30g/lane) were subjected to 10% SDS–PAGE, and the expressions of IB-␣ and -actin proteins were detected by Western blotting using specific anti-IB-␣ and anti--actin antibodies. -Actin protein was used as the internal control. C, control, no treatment; L, LPS at 1g/ml.
Fig. 7. Effect of shikonin derivatives and naphthoquinone pigments on the lipopolysaccharide (LPS)-induced phosphorylation of MAPKs in RAW 264.7 macrophages. (A) RAW 264.7 macrophages were treated with 0.3, 1, or 3M of SAs or 3 or 10M NAP at 37◦C for 30 min before the addition of 1g/ml LPS for 18 h. (B)
Concentration-dependent effect of SA derivatives on ERK phosphorylation. Whole-cell lysates were analyzed by Western blotting using various antibodies against activated MAPKs. Equal amounts of total proteins (60g/lane) were subjected to 10% SDS–PAGE, and the expressions of p-ERK, p-JNK, p-P38, ERK, JNK, P38 and -actin protein were detected by Western blotting using specific anti-p-ERK, p-JNK, p-p38, ERK, JNK, P38 and-actin antibodies. Data were obtained from three inde-pendent experiments and are expressed as the mean± S.D. *Statistically significant difference compared to the 18-h positive control at the p < 0.05 level using Student’s t-test. **p < 0.01, ***p < 0.001 vs. the positive control.
264.7 macrophages, as well as iNOS and COX-2 protein expressions, especially on mRNA induction of iNOS. In order to further investi-gate the mechanism involved in the anti-inflammatory effect of SA derivatives, we used SA to study the involvement of the NF-B signaling pathway. The promoter of the iNOS gene is known to contain two transcriptional regions: an enhancer and a basal promoter (Lowenstein et al., 1993). There are a number of binding sites for transcription factors includingB sites, which are located in both the enhancer and basal promoter (Xie et al., 1994), and NF-B, which is essential for LPS-mediated NO production. Therefore, we examined NF-B-DNA binding activity to confirm that the inhi-bition of the expression of iNOS and COX-2 are influenced by the NF-B signaling pathway. Our results indicated that DNA binding activity was inhibited in a concentration-dependent manner by SA (Fig. 5A and B) and these results corresponded with its inhibition of
the expressions of iNOS. However, when cytoplasmic levels of I-B␣ were examined by a Western blot analysis, we found that SA caused a significant recovery in the level of the I-B␣ protein (Fig. 6). A sim-ilar pattern was observed when the effects of DH, AC, DM, and NAP were examined on the recovery of LPS-treated I-B␣ degradation. Our results indicated that the inhibition of iNOS and COX-2 levels by SA was due to the inhibition of NF-B activation. Downregulation of the NF-B gene was further confirmed by inhibiting the respective whole-cell proteins p65 by SA. Furthermore, the protein level of p65 in the nucleus confirmed its downregulated expressions following treatment with SA (Fig. 5C).
It has been reported that activation of MAP kinase phos-phorylation transduces signals to activate the transcription of NF-B-mediated proinflammatory cytokines (Ren and Chung, 2007; Rajapakse et al., 2008). Ajizian et al. (1999) suggested that activation of ERK is thought to be involved in LPS-induced macrophage responses; in addition, JNK and p38 are activated by LPS stimulation and have been postulated to play important roles in controlling iNOS gene expression (Hambleton et al., 1996; Bhat et al., 1998). In our study, we showed that ERK, JNK, and p38 were phosphorylated with LPS stimulation. Furthermore, phospho-rylation of ERK was inhibited by SA derivatives at 30 min of LPS stimulation, whereas there was no effect of SA derivatives on pp38 or pJNK (Fig. 7). MAPKs are also likely targets for the development of novel anti-inflammatory drugs; however, signaling from MAPKs to transcription factors mediating iNOS and COX-2 expression is not fully understood.
The anti-inflammatory effect of shikonin was first reported from success in treating burns, leprous ulcers, and acute fissures (Michaelides et al., 1993), dating back to the 1600 s. More recently, shikonin was shown to inhibit the biosynthesis of leukotriene B4 and 5-hydroxyeicosatetranoic acid (Wang et al., 1994).Chiu and Yang (2007), demonstrated that shikonin downregulates the splicing process of TNF-␣ pre-mRNA through inactivation of the PKR pathway in LPS-stimulated THP-1 monocytes. Alto-gether, although several research groups have investigated the biological and pharmacological mechanisms of shikonin deriva-tives, their precise mode of action remains undetermined. Our findings are consistent with the fact that SA derivatives are potentially potent anti-inflammatory agents, and this is the first investigation showing that SA markedly inhibits LPS-induced NO production and iNOS expression by blocking NF-B activation and inhibits the phosphorylation of ERK in RAW 264.7 macrophages. The effect of SA on anti-inflammation not only comes from the iNOS/NO but also the COX-2/PGE2 pathway. Our findings
provide useful and additional mechanistic explanations for the anti-inflammatory effect of shikonin and highlight its pharmaceu-tical value.
Acknowledgement
This study was supported in part by a grant from the National Science Council, Taiwan, ROC.
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