Vol. 39, No. 6969 Biol. Pharm. Bull. 39, 969–976 (2016)
Regular Article
Simultaneous Addition of Shikonin and Its Derivatives with Lipopolysaccharide Induces Rapid Macrophage Death Atsushi Koike,a Makio Shibano,b Hideya Mori,a Kiyoko Kohama,a Ko Fujimori,a and Fumio Amano*,a a
Laboratory of Biodefense & Regulation, Osaka University of Pharmaceutical Sciences; 4–20–1 Nasahara, Takatsuki, Osaka 569–1094, Japan: and b Laboratory of Pharmacognosy, Osaka University of Pharmaceutical Sciences; 4–20–1 Nasahara, Takatsuki, Osaka 569–1094, Japan. Received November 27, 2015; accepted February 21, 2016 Macrophages play pivotal roles in inflammatory responses. Previous studies showed that various natural products exert antiinflammatory effects by regulating macrophage activation. Recent studies have shown that shikonin (SHK) and its derivatives (β-hydroxyisovalerylshikonin, acetylshikonin, and isobutylshikonin), which are 1,4-naphthoquinone pigments extracted from the roots of Lithospermum erythrorhizon, have various pharmacological, including antiinflammatory and antitumor, effects. Even though there have been many studies on the antiinflammatory activities of SHK derivatives, only a few have described their direct effects on macrophages. We investigated the effects of SHK derivatives on lipopolysaccharide (LPS)-treated macrophages. Low doses of SHK derivatives induced significant macrophage cytotoxicity (mouse macrophage-like J774.1/JA-4 cells and mouse peritoneal macrophages) in the presence of LPS. SHK activated caspases-3 and -7, which led to DNA fragmentation, but this cytotoxicity was prevented through a pan-caspase inhibitor in LPS-treated JA-4 cells. Maximal cytotoxic effects were achieved when SHK was added immediately before LPS addition. These results indicate that SHK derivatives induce caspase-dependent apoptotic cell death of LPS-treated macrophages and suggest that SHK acts during an early stage of LPS signaling. Key words
macrophage cell death; shikonin; lipopolysaccharide; antiinflammatory effect
Macrophages are involved in a variety of host defence systems against a myriad of potentially pathogenic infectious agents in the environment. It is well known that macrophages are activated by bacterial lipopolysaccharide (LPS) and play pivotal roles in inflammatory responses characterized by the expression of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and in the production of reactive oxygen species. Previous studies have shown that various natural products exert anti-inflammatory effects by regulating the activation of macrophages.1–4) Recently, shikonin (SHK) (Fig. 1A), one of the active components of Lithospermi radix (LR), which is a traditional herbal medicine made from the dried root of Lithospermum erythrorhizon SIEB. et ZUCC, has been reported to produce multiple pharmacological effects including antioxidant,5) antitumor,6) antifungal,7) antimicrobial,8) antiviral,9) and anti-obesity10) effects. Some of the latest research has focused on SHK’s beneficial effect in anti-inflammatory treatment.11,12) These reports stated that SHK may exhibit anti-inflammatory effects in various inflammatory stages through regulation of macrophage activation. However, there have been a limited number of reports on the direct cytotoxic effects of SHK on macrophages. Furthermore, the effects of ester derivatives of SHK, such as β-hydroxyisovalerylshikonin (β-HIVS), acetylshikonin (ACS), and isobutylshikonin (IBS), on macrophages in LR (Fig. 1A) are largely unknown. The aim of this study was to investigate the effects of SHK derivatives on LPS-treated macrophages, particularly on the induction of macrophage cell damage, and the timing of SHK addition to the cells. We also discuss new pharmacological effects of SHK on LPS-treated macrophages.
MATERIALS AND METHODS Reagents Escherichia coli (055 : B5) LPS obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.); Ham’s F-12 and fetal bovine sera from Life Technologies (Carlsbad, CA, U.S.A.); penicillin–streptomycin mixed solution, HPLC-grade acetonitrile, reagent-grade hexane, methanol, ethanol, and CDCl3 from Nacalai Tesque (Kyoto, Japan); Wakogel C-200 from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); cell proliferation assay reagent (WST-1) from Roche Diagnostics GmbH (Mannheim, Germany); and Z-Asp-CH2-dichlorobenzen (DCB) from the Peptide Institute (Osaka, Japan), were used in this study. Recombinant murine interferon-γ (IFN-γ) was a generous gift from TORAY (Tokyo, Japan). Primary antibodies, including rabbit anti-caspase-3, cleaved caspase-3, caspase-7, and cleaved caspase-7, and a second antibody, anti-rabbit immunoglobulin G (IgG) antibody conjugated with horseradish peroxidase (HRP) antibody, were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.). An in situ cell detection kit for terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay was obtained from Roche. An herbal medicine, Lithospermi radix, was purchased from Uchida Wakanyaku Ltd. (Tokyo, Japan) and Tochimoto Tenkaido Co., Ltd. (Osaka, Japan). A voucher specimen was deposited at the herbarium of Osaka University of Pharmaceutical Sciences. A traditional Japanese ointment medicine, “Shiunko,” was also obtained from Uchida Wakanyaku. Extraction, Isolation, and Identification of SHK Derivatives Chopped LR (100 g) was extracted into ethanol (200 mL×5) for 20 min with sonication. The combined ethanol extracts were concentrated to dryness in vacuo. The residue
To whom correspondence should be addressed. e-mail:
[email protected] * © 2016 The Pharmaceutical Society of Japan
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Fig. 1.
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Effects of Simultaneous Addition of SHK Derivatives and LPS on JA-4 Cells
Chemical structures of SHK derivatives (A). JA-4 cells were treated with SHK derivatives with or without LPS at 37°C for 4 h, as described in Materials and Methods. Cell viability was quantified by WST-1 assay. All data are presented as the mean±S.D., n=3. * p98% of the macrophages were compared, as previously described.14) All animal experiments complied with the approved animal care protocols of the Osaka University of Pharmaceutical Sciences. Cell Toxicity Assay JA-4 cells were seeded at 4×104 cells per well onto 96-well plates (Iwaki, Asahi Techno Glass, Shizuoka, Japan) and incubated at 37°C for 3 h. The cells or the above-described mouse peritoneal macrophages were then incubated with various concentrations of SHK derivatives (0, 1, 2.5, 5, 10, or 20 µM) with or without 100 ng/mL LPS for 4 h. 3T3 cells were seeded at 1×104 cells per well onto 96-well plates and incubated over night at 37°C. The cells were then incubated with various concentrations of SHK (0, 1, 2.5, 5, or 10 µM) with or without 100 or 1000 ng/mL LPS for 4 h. Cell proliferation reagent (WST-1) was used to measure cell toxicity according to the manufacturer’s protocol. Absorbance was measured at 450 nm with subtraction at 620 nm by using a MultiSkan FC (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.). Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)/Western Blotting JA-4 cells were seeded in a 60-mm dish (Corning #430556) at 2×106 cells/5 mL. The cells were incubated at 37°C for 3 h and then treated with SHK at 2.5 µM and/or LPS at 100 ng/mL for the indicated times. The cells were lysed in ice-cold lysis buffer containing 1% (v/v) Triton X-100, 2 m M ethylenediaminetetraacetic acid (EDTA), 150 m M NaCl, 10% (v/v) glycerol, and 1% (v/v) Protease Inhibitor Cocktail (Nacalai Tesque) in 20 m M N-(2hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES)– NaOH buffer, pH 7.5. The cell lysates were sonicated at 4°C, then centrifuged at 10000 rpm (9100×g) for 1 min at 4°C, and the resultant supernatants were used as the cell extracts. Protein concentrations were measured using Pierce BCA Protein Assay Reagent (Thermo Fisher Scientific). For SDS-PAGE/ Western blotting, 25-µg aliquots of the cell extracts were treated with SDS-sample buffer, containing 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 4% (v/v) glycerol, and 0.01% (w/v) bromophenol blue in 40 m M Tris–HCl buffer (pH 6.8) and then boiled at 100°C for 5 min. The samples were thereafter separated by SDS-PAGE using 5–20% gradient gels (ATTO, Tokyo, Japan), and then transferred onto Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, U.S.A.) for Western blotting. The blots were reacted with a primary antibody, anti-caspase-3 (1 : 1000), anti-cleaved caspase-3 (1 : 1000), anti-caspase-7 (1 : 1000), or anti-cleaved caspase-7 (1 : 1000), followed by reaction with a secondary antibody, anti-rabbit IgG antibody conjugated with HRP (1 : 1000). Chemiluminescence was generated by using Pierce Western
blotting Substrate (Thermo Fisher Scientific) and detected by using an LAS 3000 Mini Image Analyzer (FUJIFILM, Tokyo, Japan). The results were analyzed by using Image J software (Ver. 1.48V). Analysis of DNA Fragmentation Cells were lysed in Tris–HCl buffer, pH 7.5, containing 5 m M EDTA, 200 m M NaCl, 0.2% (v/v) SDS, and 0.1 mg/mL Proteinase K (Nippon Gene, Tokyo, Japan). After incubation at 60°C for 1 h, the proteins were excluded using phenol–chloroform extraction, followed by isolation of DNA using ethanol precipitation. The DNA was further incubated with 10 mg/mL RNase A (Nippon Gene) at 37°C for 30 min to degrade RNA. The purified DNA was analyzed by staining the gel with GelRed Nucleic Acid Gel Stain (Wako Pure Chemical Industries, Ltd.). Terminal dUTP TUNEL Assay Approximately 1.5×103 cells were plated per well of 8-well slide glasses (Thermo Fisher Scientific). After incubation at 37°C for 3 h, the cells were washed twice with Ham’s F-12 medium and then treated with 100 ng/mL LPS and/or 2.5 µM SHK at 37°C for 4 h. The cells were fixed with 3.6% formaldehyde in the culture medium pH 7.4 for 30 min. After washing the fixed cells with phosphate-buffered saline (PBS) without divalent cations [PBS (−)] repeatedly, the cells were permeabilized using 0.1% (v/v) Triton X-100 in PBS (−) at room temperature (r.t.) for 10 min, followed by repeated washing with PBS (−). A TUNEL assay using an in situ cell death detection kit (Roche) was performed according to the manufacturer’s protocol. In brief, the permeabilized cells were treated with TUNEL reaction mixture and incubated in a humidified dark chamber at 37°C for 1 h. The samples were then washed with PBS (−), followed by incubation with 20 ng/mL 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at r.t. After washing with PBS (−), the cells were mounted by using PermaFluor ® mounting medium (Thermo Fisher Scientific). The images were observed under an LSM700 laser-scanning microscope (Carl Zeiss MicroImaging GHBH, Jena, Germany). Quantification of Endotoxin (LPS) Level LR and a Japanese traditional ointment medicine “Shiunko,” were boiled for 20 min, and the supernatants were then centrifuged at 2000 rpm for 3 min at r.t. The resultant supernatants were filtered through 0.45 µL membrane filters (Millipore) and then assayed for endotoxin amounts. The endotoxin level was determined by using an Endospecy ES-24S kit and a Toxicolor DIA kit (Seikagaku, Tokyo, Japan), following the manufacturer’s instructions. RNA Isolation and cDNA Synthesis 3T3 cells were seeded at 5×105 cells per well onto 12-well plates (Costar; Sigma-Aldrich) and incubated over night at 37°C. The cells were then incubated with or without 100 ng/mL LPS and/or 10 units/mL IFN-γ for 3 h. RNA was isolated from the cells by using Tripure Isolation Reagent (Roche) according to the manufacturer’s described protocol. Reverse transcription was done in 10 µL reaction volumes with a ReverTra Ace® qPCR RT Master Mix (TOYOBO, Osaka, Japan) as per our previous studies.13) Quantitative PCR Analysis Quantitative PCR was performed on an applied Biosystems StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific) using Power SYBR Green Master Mix (Thermo Fisher Scientific), and relative quantification (RQ) was calculated by using StepOne™ software V2.2.2, based on the equation RQ=2−ΔΔCt, where
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Ct is the threshold cycle to detect fluorescence. Ct data were normalized to the internal standard β-actin. The following primer sets for qRT-PCR are used; NOS2, 5′-CTT TGC CAC GGA CGA GAC-3′ and 5′-TCA TTG TAC TCT GAG GGC TGA C-3′; β-actin, 5′-CTA AGG CCA ACC GTG AAA AG-3′ and 5′-ACC AGA GGC ATA CAG GGA CA-3′. Statistical Analysis Results are expressed as the mean±standard deviation (S.D.). The significance of differences was analyzed using the Student’s t-test for comparisons between two groups and using the one-way ANOVA with the Tukey–Kramer post hoc test for comparisons among more
Fig. 2.
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than two groups. p2.5 µM in the presence of LPS. Similar results were obtained by addition of the other SHK derivatives, β-HIVS, ACS, and IBS; whereas low doses of SHK (
