Shikonins, Phytocompounds from Lithospermum erythrorhizon, Inhibit the Transcriptional Activation of Human Tumor Necrosis Factor α Promoter in Vivo
2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês
10.1074/jbc.m309185200
ISSN1083-351X
AutoresVanisree Staniforth, Sheng‐Yang Wang, Lie‐Fen Shyur, Ning‐Sun Yang,
Tópico(s)Morinda citrifolia extract uses
ResumoTumor necrosis factor α (TNF-α) contributes to the pathogenesis of both acute and chronic inflammatory diseases and has been a target for the development of new anti-inflammatory drugs. Shikonins, the naphthoquinone pigments present in the root tissues of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), have been reported to exert anti-inflammatory effects both in vitro and in vivo. In this study, we evaluated the effects of shikonin and its derivatives on the transcriptional activation of human TNF-α promoter in a gene gun-transfected mouse skin system by using a luciferase reporter gene assay. The crude plant extract of L. erythrorhizon as well as derived individual compounds shikonin, isobutyryl shikonin, acetyl shikonin, dimethylacryl shikonin and isovaleryl shikonin showed significant dose-dependent inhibition of TNF-α promoter activation. Among the tested compounds, shikonin and isobutyryl shikonin exhibited the highest inhibition of TNF-α promoter activation and also showed significant suppression of transgenic human TNF-α mRNA expression and protein production. We demonstrated that shikonin-inhibitory response was retained in the core TNF-α promoter region containing the TATA box and a 48-bp downstream sequence relative to the transcription start site. Further our results indicated that shikonin suppressed the basal transcription and activator-regulated transcription of TNF-α by inhibiting the binding of transcription factor IID protein complex (TATA box-binding protein) to TATA box. These in vivo results suggest that shikonins inhibit the transcriptional activation of the human TNF-α promoter through interference with the basal transcription machinery. Thus, shikonins may have clinical potential as anti-inflammatory therapeutics. Tumor necrosis factor α (TNF-α) contributes to the pathogenesis of both acute and chronic inflammatory diseases and has been a target for the development of new anti-inflammatory drugs. Shikonins, the naphthoquinone pigments present in the root tissues of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), have been reported to exert anti-inflammatory effects both in vitro and in vivo. In this study, we evaluated the effects of shikonin and its derivatives on the transcriptional activation of human TNF-α promoter in a gene gun-transfected mouse skin system by using a luciferase reporter gene assay. The crude plant extract of L. erythrorhizon as well as derived individual compounds shikonin, isobutyryl shikonin, acetyl shikonin, dimethylacryl shikonin and isovaleryl shikonin showed significant dose-dependent inhibition of TNF-α promoter activation. Among the tested compounds, shikonin and isobutyryl shikonin exhibited the highest inhibition of TNF-α promoter activation and also showed significant suppression of transgenic human TNF-α mRNA expression and protein production. We demonstrated that shikonin-inhibitory response was retained in the core TNF-α promoter region containing the TATA box and a 48-bp downstream sequence relative to the transcription start site. Further our results indicated that shikonin suppressed the basal transcription and activator-regulated transcription of TNF-α by inhibiting the binding of transcription factor IID protein complex (TATA box-binding protein) to TATA box. These in vivo results suggest that shikonins inhibit the transcriptional activation of the human TNF-α promoter through interference with the basal transcription machinery. Thus, shikonins may have clinical potential as anti-inflammatory therapeutics. Inflammation represents a cascade of physiological and immunological reactions as the first cellular response to noxious environmental stimuli in an effort to localize toxic materials or pathogens or to prevent tissue injury. One of the most important proinflammatory cytokines, tumor necrosis factor α (TNF-α) 1The abbreviations used are: TNF-αtumor necrosis factor αCMVcytomegalovirusGAPDHglyceraldehyde-3-phosphate dehydrogenaseLELithospermum erythrorhizonBPBidens pilosaSHshikoninASacetyl shikoninIBSisobutyryl shikoninDMASdimethylacryl shikoninIVSisovaleryl shikoninTFtranscription factorTBPTATA box-binding proteinHPLChigh pressure liquid chromatographyRTreverse transcriptionELISAenzyme-linked immunosorbent assayBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPBSphosphate-buffered salineErkextracellular signal-regulated kinaseEMSAelectrophoretic mobility shift assay.1The abbreviations used are: TNF-αtumor necrosis factor αCMVcytomegalovirusGAPDHglyceraldehyde-3-phosphate dehydrogenaseLELithospermum erythrorhizonBPBidens pilosaSHshikoninASacetyl shikoninIBSisobutyryl shikoninDMASdimethylacryl shikoninIVSisovaleryl shikoninTFtranscription factorTBPTATA box-binding proteinHPLChigh pressure liquid chromatographyRTreverse transcriptionELISAenzyme-linked immunosorbent assayBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPBSphosphate-buffered salineErkextracellular signal-regulated kinaseEMSAelectrophoretic mobility shift assay. has been shown to play a pivotal role in immune and inflammatory responses (1Vassalli P. Annu. Rev. Immunol. 1992; 10: 411-452Crossref PubMed Scopus (1804) Google Scholar, 2Beutler B. J. Investig. Med. 1995; 43: 227-235PubMed Google Scholar). Inappropriate or overexpression of TNF-α is a hallmark of a number of inflammatory and autoimmune diseases, including rheumatoid arthritis, inflammatory bowel disease, psoriasis, asthma, multiple sclerosis, diabetes, and AIDS (3Feldmann M. Brennan F.M. Elliott M. Katsikis P. Maini R.N. Circ. Shock. 1994; 43: 179-184PubMed Google Scholar, 4Murch S.H. Braegger C.P. Walker-Smith J.A. MacDonald T.T. Gut. 1993; 34: 1705-1709Crossref PubMed Scopus (496) Google Scholar, 5Ackermann L. Harvima I.T. Arch. Dermatol. Res. 1998; 290: 353-359Crossref PubMed Scopus (135) Google Scholar, 6Shah A. Church M.K. Holgate S.T. Clin. Exp. Allergy. 1995; 25: 1038-1044Crossref PubMed Scopus (114) Google Scholar, 7Rieckmann P. Albrecht M. Kitze B. Weber T. Tumani H. Broocks A. Luer W. Helwig A. Poser S. Ann. 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Kamm M.A. Lancet. 1997; 349: 521-524Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). tumor necrosis factor α cytomegalovirus glyceraldehyde-3-phosphate dehydrogenase Lithospermum erythrorhizon Bidens pilosa shikonin acetyl shikonin isobutyryl shikonin dimethylacryl shikonin isovaleryl shikonin transcription factor TATA box-binding protein high pressure liquid chromatography reverse transcription enzyme-linked immunosorbent assay 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol phosphate-buffered saline extracellular signal-regulated kinase electrophoretic mobility shift assay. tumor necrosis factor α cytomegalovirus glyceraldehyde-3-phosphate dehydrogenase Lithospermum erythrorhizon Bidens pilosa shikonin acetyl shikonin isobutyryl shikonin dimethylacryl shikonin isovaleryl shikonin transcription factor TATA box-binding protein high pressure liquid chromatography reverse transcription enzyme-linked immunosorbent assay 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol phosphate-buffered saline extracellular signal-regulated kinase electrophoretic mobility shift assay. Naphthoquinone compounds present in root extracts of a traditional Chinese medicinal herb, Lithospermum erythrorhizon Sieb. et Zucc. (LE), have been reported to confer many medicinal properties such as antibacterial, wound healing, anti-inflammatory, antithrombotic, and antitumor effects (12Papageorgiou V.P. Assimopoulou A.N. Couladouros E.A. Hepworth David Nicolaou K.C. Angew. Chem. Int. Ed. Engl. 1999; 38: 270-300Crossref PubMed Scopus (531) Google Scholar). Among these, shikonin, one of the active components of the roots of LE, has been shown to inhibit the capillary permeability induced by an intradermal injection of histamine and edema caused by a thermal injury to the skin of rats (13Tanaka S. Tajima M. Tsukada M. Tabata M. J. Nat. Prod. 1986; 49: 466-469Crossref PubMed Scopus (175) Google Scholar). Subcutaneous administration of shikonin has been reported to inhibit ear edema induced by croton oil in mice and paw swelling induced by yeast in rats (14Wang W.J. Bai J.Y. Liu D.P. Xue L.M. Zhu X.Y. Yaoxue Xuebao. 1994; 29: 161-165Google Scholar). A derivative of shikonin, MDS-004, has strongly inhibited ear edema in a delayed type hypersensitivity model induced by oxazolone and dinitrofluorobenzene (15Seto Y. Motoyoshi S. Nakamura H. Imuta J. Ishitoku T. Isayama S. Yakugaku Zasshi. 1992; 112: 259-271Crossref PubMed Scopus (22) Google Scholar). Recent studies suggest that the anti-inflammatory effects of shikonin derivatives may be attributable to several mechanisms of action, e.g. inhibition of leukotriene B4 biosynthesis (14Wang W.J. Bai J.Y. Liu D.P. Xue L.M. Zhu X.Y. Yaoxue Xuebao. 1994; 29: 161-165Google Scholar), suppression of mast cell degranulation and protection of the vasculature (16Wang J.P. Raung S.L. Chang L.C. Kuo S.C. Eur. J. Pharmacol. 1995; 272: 87-95Crossref PubMed Scopus (21) Google Scholar), inhibition of neutrophil respiratory burst by attenuation of protein tyrosine phosphorylation and failure of NADPH oxidase complex formation (17Wang J.P. Tsao L.T. Raung S.L. Hsu M.F. Kuo S.C. Br. J. Pharmacol. 1997; 121: 409-416Crossref PubMed Scopus (14) Google Scholar), impairment of phosphatidylinositol signaling (18Wang J.P. Kuo S.C. Biochem. Pharmacol. 1997; 53: 1173-1177Crossref PubMed Scopus (20) Google Scholar), blockade of chemokine ligands binding to CC chemokine receptor 1 (19Chen X. Oppenheim J. Howard O.M. Int. Immunopharmacol. 2001; 1: 229-236Crossref PubMed Scopus (60) Google Scholar), and inhibition of phorbol 12-myristate 13-acetate-induced COX-2 expression (20Subbaramaiah K. Bulic P. Lin Y. Dannenberg A.J. Pasco D.S. J. Biomol. Screen. 2001; 6: 101-110PubMed Google Scholar). Although the efficacy of shikonin and its derivatives has been demonstrated in vitro and in vivo, their precise mode of action and the molecular basis for their anti-inflammatory actions in vivo warrants further investigation. Skin is an immune-competent organ that serves as a first line of defense to various assaults, such as exogenous stress, environmental antigens, or pathogens. The skin immune system has been defined as a cutaneous complex of interacting immune response-related cells (21Bos J.D. Clin. Exp. Immunol. 1997; 107: 3-5PubMed Google Scholar). In skin, TNF-α is a prominent cytokine that seems to be important in allergic and irritant contact dermatitis and in other inflammatory conditions (22LaDuca J.R. Gaspari A.A. Dermatol. Clin. 2001; 19: 617-635Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Modulating TNF-α expression in skin may provide therapeutic benefits for a variety of skin disorders. A simple and direct in vivo transfection method, particle-mediated gene transfer by gene gun, has been used for in vivo characterization of mammalian promoters in skin and liver tissues of rats and mice (23Cheng L. Ziegelhoffer P.R. Yang N.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4455-4459Crossref PubMed Scopus (272) Google Scholar). In this study, we have used a gene gun-transfected mouse skin system to evaluate the effects of a crude extract and individual compounds, shikonin and its derivatives, isolated from the roots of L. erythrorhizon on the transcriptional activity of a transgenic human TNF-α promoter. The current study on the inhibition of TNF-α promoter activity by shikonins has provided us with an additional insight into the molecular mechanism underlying the anti-inflammatory properties of these phytocompounds. This study also demonstrated an in vivo quantitative molecular screening system for the evaluation of anti-inflammatory agents. Mice—Female BALB/c mice (National Laboratory Animal Breeding and Research Center, Taipei, Taiwan, Republic of China) were maintained under pathogen-free conditions on standard laboratory chow and water in the animal facilities of the Institute of Biological Chemistry, Academia Sinica. All mice used in our experiments were 7-8 weeks old. Chemicals—Hydrocortisone and betamethasone were purchased from Sigma. Chemical solutions were prepared immediately prior to the use in 70% polyethylene glycol 400 and 30% ethanol solvent. Croton oil was purchased from Fluka Chemie GmbH (Buchs, Switzerland) and diluted in acetone prior to use. Plasmid Constructs—The pGL2 luciferase reporter vector containing the human TNF-α promoter (a generous gift from Dr. Robert L. Danner, National Institutes of Health, Bethesda, MD) was used as a template to generate a -1049 to +48 bp TNF-α promoter region flanked by MluI and BglII restriction sites using PCR and cloned into the pGL3-Basic vector (Promega, Madison, WI). The resultant plasmid was designated as pTNFP-Luc. Plasmid pIL2P-Luc was constructed by isolating a -1437 to +50 bp region of human interleukin-2 promoter flanked by MluI and BglII restriction sites from human genomic DNA through PCR and cloning into the pGL3-Basic vector. The promoterless pGL3-Basic vector was used as a negative control plasmid for luciferase assays. The plasmid pCMV-Luc containing a CMV immediate early gene enhancer/promoter has been described elsewhere (24Thompson T.A. Gould M.N. Burkholder J.K. Yang N.S. In Vitro Cell Dev. Biol. 1993; 29: 165-170Crossref Scopus (54) Google Scholar). Clone PE4 (ATCC) was used as a template to isolate the 720-bp coding region of human TNF-α gene flanked by NcoI and XbaI restriction sites and subcloned into pTNFP-Luc by replacing the luciferase gene and designated as pTNFP-TNFC. All plasmid constructs were verified by DNA sequencing. A series of 5′-deletions of the human TNF-α promoter were created by PCR using pTNFP-Luc (-1049/+48 bp) as template with specific 5′ primers of defined lengths relative to the transcription start site (-924/+48, -745/+48, -495/+48, -285/+48, -124/+48, and -29/+48 bp) flanked by an MluI site and a common 3′ primer flanked by a BglII site. The PCR products were cloned into pGL3-Basic vector and verified by sequencing. All plasmids used in transient transfection assays were isolated using an endotoxin-free megaplasmid purification kit (Qiagen GmbH). In Vivo Particle-mediated Gene Transfer—The Helios gene gun system (Bio-Rad) was used to transfect the plasmid constructs into mouse skin. Plasmid DNA was precipitated onto 2-μm gold particles in the presence of spermidine and CaCl2 and coated onto the inner surface of Tefzel tubing (25Kuo C.F. Wang J.H. Yang N.S. Gene Therapy Protocols. 2nd Ed. Humana Press Inc., Totowa, NJ2002: 137-147Google Scholar). The tubing was cut into 0.5-inch-length cartridges, resulting in the delivery of 0.5 mg of gold and 1.25 μg of plasmid DNA/bombardment. Female BALB/c mice were shaved on a restricted area of the abdomen and disinfected with 70% ethanol. No visual damage to epidermal skin tissue was observed. For each treatment, the target skin area was bombarded twice to deliver 2.5 μg of plasmid DNA coated onto 1 mg of gold particles with a 380 p.s.i. helium gas pressure. The bombarded area was marked with an ink stamp containing a circular sign of 2-cm diameter (3.14 cm2). Isolation and Administration of Herbal Compounds—The roots of the LE plants were air-dried, ground into powder, and extracted by n-hexane at room temperature. The solvent was removed under vacuum, and the resultant material was used as crude extract. Individual compounds were separated and purified by semipreparative HPLC (Waters HPLC system equipped with a Waters 600 controller, Waters Delta 600 pump, and 2487 Dual λ absorbance detector). A 5-μm C18 column (250 × 10 mm, Merck) was used with two solvent systems, acetonitrile-water (80:20, v/v) (A) and methanol (B). Elution was performed as follows: 0-8 min, A:B = 95:5 (isocratic); 8-15 min, 95-80% A to B (linear gradient); 15-40 min, 80-50% B (linear gradient) with a flow rate of 3.5 ml/min with the detection wavelength set at 254 nm. Molecular weights of isolated compounds were as follows: shikonin (SH), 288.30; acetyl shikonin (AS), 330.34; isobutyryl shikonin (IBS), 358.39; β,β-dimethylacryl shikonin (DMAS), 370.40; and isovaleryl shikonin (IVS), 372.41, as determined by mass spectrometry. Molecular structures were assigned based on various spectroscopic techniques including electron ionization mass spectrometry, Fourier transform infrared spectroscopy, and NMR analysis (Fig. 1). Bidens pilosa L. var. radiata Schult. Bip. (BP), a folk herb reputed for anti-inflammatory activities (26Chih H.W. Lin C.C. Tang K.S. Am. J. Chin. Med. 1995; 23: 273-278Crossref PubMed Google Scholar), was tested in parallel in this study. The dried aerial parts of BP were ground into powder and extracted by 70% ethanol at room temperature. The solvent was removed under vacuum, and the resultant material was used as a crude extract. Crude extracts and pure compounds were dissolved in an organic solvent containing 70% polyethylene glycol (polyethylene glycol 400) and 30% absolute ethanol prior to administration. A final volume of 20 μl of test extract was pipetted onto the skin area immediately after DNA transfection, spread evenly, and allowed to completely air dry. Untreated transfected skin and transfected skin treated with solvent alone, crude extract of BP, or commercial drugs were used as positive or negative controls. Tissue Extraction and Luciferase Assay—Animals were sacrificed 16 h post-transfection, and transfected skins (2-cm-diameter circles), treated or untreated with test agents, were removed and frozen in liquid nitrogen. Skin samples were prepared in 500 μl of lysis buffer (1× phosphate-buffered saline (PBS), 0.1% Triton X-100, and protease inhibitors) by scissor mincing followed by sonication and centrifugation of cell debris. Samples were analyzed for luciferase activity (Promega) with a Lumat LB9507 luminometer (Berthold). Duplicate analyses of two aliquots from each test skin sample were performed, and the data were averaged. Promoter activities were measured as total relative light units/site/mouse. Induction of promoter activity was expressed as -fold increase over the control, and inhibition was expressed as percentage of the control. RT-PCR Analysis—The expression of endogenous mouse TNF-α and transgenic human TNF-α mRNAs in mouse skin tissues was analyzed by reverse transcription-polymerase chain reaction. Frozen mouse skin samples were homogenized in liquid nitrogen. The total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and resuspended in 25 μl of diethyl pyrocarbonate-treated water. RT-PCRs were carried out by using the AccessQuick RT-PCR system (Promega) according to the manufacturer's instructions. Briefly 1 μg of total RNA from each sample was added to the reaction mixture containing 1× AccessQuick master mixture (Tfl DNA polymerase, avian myeloblastosis virus/Tfl reaction buffer, 25 mm MgSO4, and 10 mm dNTP mixture), a 10 μm concentration of each of specific sense and antisense primers, 5 units of avian myeloblastosis virus reverse transcriptase, and nuclease-free water to obtain a final volume of 50 μl. Reactions were incubated at 48 °C for 60 min, and PCR amplification was carried out after denaturing at 95 °C for 2 min. The primers contained the following sequences: mouse TNF-α sense primer, 5′-ATGAGCACAGAAAGCATGATCCGCGACG-3′, and antisense primer, 5′-GACTCCAAAGTAGACCTGCCCGGACTC-3′; mouse GAPDH sense primer, 5′-CATCACTGCCACCCAGAAGACTGTGGA-3′, and antisense primer, 5′-TACTCCTTGGAGGCCATGTAGGCCATG-3′; human TNF-α sense primer, 5′-GAAAGCATGATCCGGGACGTGGA-3′, and antisense primer, 5′-GTTGGATGTTCGTCCTCCTCACA-3′. The PCR products were separated on 1.5% agarose gels (Ultrapure, Invitrogen) at 55 V for 75 min along with a molecular weight marker, GeneRuler 100-bp DNA Ladder (MBI Fermentas) and visualized by UV illumination after staining with 0.5 μg/ml ethidium bromide solution. Gels were photographed with type 55 positive/negative film (Polaroid Corp., Cambridge, MA). Images were scanned, and densitometry analysis of the captured image was performed using BIO-1D image analysis software. The signal intensities of the test genes in different samples were normalized to the respective mouse GAPDH signal intensity. ELISA for TNF-α—Endogenous mouse TNF-α and transgenic human TNF-α cytokine concentrations in mouse skin tissues were determined using OptEIA mouse TNF-α and OptEIA human TNF-α ELISA kits, respectively (Pharmingen). The capture and detection antibodies used were specific for mouse and human TNF-α cytokines. Briefly skin samples were prepared in 1 ml of lysis buffer as described for the luciferase assay. 96-well plates were coated with 100 μl/well anti-mouse or anti-human TNF-α capture antibody (1:500) and incubated overnight at 4 °C. Wells were washed three times with wash buffer (PBS with 0.05% Tween 20). Plates were blocked with assay diluent (PBS saline with 10% fetal bovine serum, pH 7.0) for 1 h and then washed. 100 μl of each respective standard, samples, and controls were added, and plates were incubated overnight at 4 °C. After five washes, 100 μl of working detector (biotinylated anti-mouse TNF-α polyclonal (1:500) or biotinylated anti-human TNF-α monoclonal antibody (1:500) and avidin-horseradish peroxidase) was added, and plates were incubated for 1 h at room temperature. After a final wash, tetramethylbenzidine and hydrogen peroxide substrate solution was added and incubated for 30 min at room temperature in the dark. Absorbance was read at 450 nm in an ELISA reader after the addition of stop solution. No cross-reactivity was observed between mouse and human TNF-α cytokines. Western Blot Analysis—Mice were bombarded with gold particles alone or pTNFP-Luc DNA-coated gold particles on their shaven abdomens as described above. Transfected skin was untreated, treated with solvent alone, or treated with shikonin, and skin samples were collected at the indicated time points. For isolation of total protein, mouse skin was excised, immediately placed in liquid nitrogen, and pulverized in mortar. The pulverized skin was lysed in 2 ml of ice-cold lysis buffer (150 mm NaCl, 0.5% Triton X-100, 50 mm Tris-HCl (pH 7.4), 20 mm EGTA, 1 mm dithiothreitol, 1 mm Na3VO4, and protease inhibitor mixture tablets) for 10 min. Lysates were centrifuged at 12,000 × g for 20 min, and supernatant containing 30 μg of protein was boiled in SDS sample loading buffer for 10 min before electrophoresis on a 12% NuPAGE BisTris gel (Invitrogen). After electrophoresis for 2 h, proteins in the gel were transferred to polyvinylidene difluoride membrane (Novex, San Diego, CA), and the blots were blocked with 5% nonfat dry milk, PBST buffer (PBS containing 0.1% Tween 20) for 60 min at room temperature. The membranes were incubated overnight at 4 °C with a 1:1000 dilution of phospho-p44/42 mitogen-activated protein kinase (extracellular signal regulated-kinase 1/2 (Erk1/2)) and phospho-NF-κB p65 polyclonal antibodies (Cell Signaling Technology Inc., Beverly, MA). Equal protein loading was assessed using mouse β-actin (Sigma). The blots were rinsed three times with PBST buffer for 5 min each. Washed blots were incubated with a 1:2000 dilution of the horseradish peroxidase-conjugated secondary antibody and then washed again three times with PBST buffer. The transferred proteins were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences). Electrophoretic Mobility Shift Assay—Mice were treated as described for Western blotting, and skin samples were collected after 1 h. For isolation of nuclear protein, mouse skin was excised, immediately placed in liquid nitrogen, and pulverized in mortar. The pulverized skin was lysed in 2 ml of ice-cold hypotonic buffer (10 mm HEPES (pH 7.8), 10 mm KCl, 2 mm MgCl2,1 mm dithiothreitol, 0.1 mm EDTA, and 0.1 mm phenylmethylsulfonyl fluoride for 15 min on ice. To the lysates, 125 μl of 10% Nonidet P-40 solution was added, and the mixture was centrifuged for 2 min at 14,800 × g. The pelleted nuclei were washed once with 400 μl of buffer A plus 25 μl of 10% Nonidet P-40, centrifuged, and resuspended in 150 μl of nuclear extract buffer (50 mm HEPES (pH 7.8), 50 mm KCl, 300 mm NaCl, 0.1 mm EDTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, and 10% glycerol) for 30 min. The mixture was centrifuged for 10 min at 4 °C. The supernatant containing nuclear proteins was collected, and protein concentrations were determined. EMSA was performed using a LightShift chemiluminescent EMSA kit (Pierce) according to the manufacturer's protocol. Briefly double-stranded oligonucleotide corresponding to the -30 bp core promoter element of human TNF-α, 5′-GGACATATAAAGGCAGTTGTTGGCACACCC-3′ (-30 to -1) was end-labeled with biotin (Purigo Biotech, Taipei, Taiwan). Binding reactions were carried out in a total volume of 20 μl containing 1× binding buffer (100 mm Tris, 500 mm KCl, 10 mm dithiothreitol, pH 7.5), 50 ng/μl poly(dI·dC), 2.5% glycerol, 0.05% Nonidet P-40, 5 mm MgCl2, 5 μg of nuclear proteins, and 20 fmol of labeled probe. A 200-fold excess of unlabeled oligonucleotide (competitor) was added where necessary. Where direct binding of shikonin with the probe was tested, nuclear protein was replaced by shikonin in binding reactions. After a 20-min incubation at room temperature, 5 μl of loading buffer was added, and samples were electrophoresed through a 6% native polyacrylamide gel at 100 V. Electrophoresed binding reactions were transferred to nylon membrane and detected by following the manufacturer's instructions. Statistical Analysis—Results are expressed as mean ± S.D. Statistical differences were assessed with an unpaired, two-tailed Student's t test. Transcriptional Activity of Transgenic Human TNF-α Promoter in Mouse Skin Tissue—To develop an in vivo molecular screening system for the evaluation and identification of anti-inflammatory phytocompounds, we transfected a proinflammatory cytokine human TNF-α promoter (-1049/+48 bp)-luciferase reporter construct (pTNFP-Luc) into mouse skin via particle-mediated gene transfer using a Helios gene gun. Transgenic promoter activity was measured in terms of luciferase activity expressed in relative light units/defined tissue site (3.14 cm2/mouse). We observed a relatively high level of human TNF-α promoter activity, which was 2370-fold higher than the promoterless negative control vector (pGL3-Basic) and was only 8.4-fold lower than that obtained for a constitutively active CMV immediate early gene enhancer/promoter (pCMV-Luc) in mouse skin (Fig. 2), whereas the luciferase activity obtained from skin samples transfected with pIL2P-Luc vector containing the human interleukin-2 cytokine promoter (-1437/+50 bp) was less than 1% of that observed for the human TNF-α promoter (pTNFP-Luc). Induction of Local Inflammatory Response and Endogenous TNF-α Expression in Mouse Skin Tissue by Gene Gun Particle-mediated Physical Injury/Stress—The relatively high level of human TNF-α promoter activity observed in mouse skin tissue led us to suspect that gene gun particle-mediated physical injury/stress may have resulted in its transcriptional activation. To test this hypothesis, mouse skin tissue was bombarded twice to deliver 1 mg of 2-μm gold particles, without any coated DNA, by using the gene gun at 380 p.s.i. helium gas pressure/bombardment. As shown in Fig. 3A, within 1 min of particle bombardment we observed reddening of skin resulting from an acute local inflammatory response (erythema). Skin samples were collected at different time points, and endogenous mouse TNF-α mRNA expression levels were analyzed by reverse transcription-PCR analysis. A time-dependent induction of endogenous TNF-α expression was observed in gold particle-bombarded skin samples (lanes 2-5) as compared with unbombarded control skin (lane 1) (Fig. 3B). As a positive control (lane 6), topical application of 2% croton oil (non-sensitizing contact irritant) also induced clearly detectable endogenous mouse TNF-α mRNA expression. Densitometry analysis showed a 4.6-fold increase in TNF-α mRNA expression level at the 16-h time point (lane 5) after gold particle bombardment as compared with unbombarded control skin (lane 1) (Fig. 3C). We further analyzed the endogenous mouse TNF-α protein production in response to gene gun particle-mediated physical injury/stress by using a mouse-specific ELISA kit. Gold particle-bombarded skin samples showed a 6.0-fold increase in TNF-α protein production as compared with unbombarded control skin (Table I). These results suggested that gene gun particle-mediated physical injury/stress could induce localized inflammatory response, activate TNF-α promoter, and induce its mRNA expression and protein production. Therefore, this indicated that the particle-mediated physical injury/stress-induced human TNF-α promoter a
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