2016 Volume 39 Issue 6 Pages 969-976
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.
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.
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. * p<0.05 compared with untreated cells (B). JA-4 cells were treated with SHK or derivatives at the indicated dose with or without LPS at 100 ng/mL at 37°C for 4 h. Cells were observed under an Olympus CKX41 microscope (20×10). Scale bars, 50 µm (C).
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 transferase-mediated 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 DerivativesChopped 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 (55.2 g) was subjected to column chromatography on silica gel (150 g) and eluted successively by using hexane and methanol. The methanol fraction (34.1 g) was rechromatographed on a Sep-Pak C-18 column (Waters, Milford, MA, U.S.A.) (70% CH3CN) followed by preparative HPLC (column: Cosmosil 5C18-AR-II, 20 mm i.d.×250 mm; mobile phase, 70% CH3CN; flow rate, 4 mL/min; detection, UV 515 nm) to give SHK (1) (12.5 mg), β-HIVS (2) (18 mg), ACS (3) (48.8 mg), and IBS (4) (12.5 mg). The four naphthoquinones were identified by their spectral (1H-NMR, 13C-NMR) analyses. lH- and 13C-NMR spectra were recorded on an Agilent VNMRS-400 spectrometer (Santa Clara, CA, U.S.A.) operating at 400 MHz for proton and 100 MHz for carbon, with tetramethylsilane as an internal standard.
Cell CultureCulturing of the JA-4 cell line, an LPS-sensitive subline of the murine macrophage-like cell line J774.1 was performed as described previously.13) In brief, the cells were cultured in Ham’s F-12 medium containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) and antibiotics and maintained in a humidified atmosphere with 5% CO2 at 37°C. Mouse 3T3-L1 cells (Health Science Research Resources Bank, Osaka, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% (v/v) FBS and antibiotics. The cells were maintained in a humidified atmosphere of 5% CO2 at 37°C.
Preparation of Mouse Peritoneal MacrophagesSix-week-old BALB/c female mice were obtained from Japan SLC (Shizuoka, Japan) as specific pathogen-free animals. Mouse peritoneal resident macrophages were collected by washing the peritoneal cavity with 5 mL of ice-cold saline. After centrifugation (2000 rpm for 3 min), the cells were suspended in 10 mL of 30% (v/v) normal saline for 1 min to disrupt other hemolytic cells, followed by the addition of 20 mL of normal saline to recover osmolality. After centrifugation, the cells were seeded at 4×104 cells onto 96-well plates in the culture medium as described above. After incubation at 37°C for 1 h, non-adherent cells were removed by aspiration, and adherent cells were obtained as macrophages; >98% 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 AssayJA-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 BlottingJA-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 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 10% (v/v) glycerol, and 1% (v/v) Protease Inhibitor Cocktail (Nacalai Tesque) in 20 mM N-(2-hydroxyethyl)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 mM 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 FragmentationCells were lysed in Tris–HCl buffer, pH 7.5, containing 5 mM EDTA, 200 mM 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 AssayApproximately 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) LevelLR 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 Synthesis3T3 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 AnalysisQuantitative 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 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 AnalysisResults 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 than two groups. p<0.05 was considered statistically significant. EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan)15) was used to perform all statistical analyses.
Because JA-4 cells have been activated using 100 ng/mL LPS and showed activated macrophage phenotypes16) but without cell damage,17) we examined the effects of SHK derivatives on the macrophage cell line treated with 100 ng/mL LPS. The cells were incubated with various concentrations of SHK derivatives in the presence or absence of 100 ng/mL LPS at 37°C for 4 h. As shown in Fig. 1B, SHK dose-dependently decreased cell viability at >5 µM in the absence of LPS and caused a much greater decrease in cell viability at >2.5 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 (<5 µM), β-HIVS (<10 µM), ACS (<10 µM), or IBS (<5 µM) alone did not affect macrophage viability. However, these low compound doses significantly decreased cell viability in the presence of LPS. Regarding cell morphology, untreated macrophages showed round shape (Fig. 1C), and SHK (2.5 µM), β-HIVS (10 µM), ACS (10 µM), or IBS (5 µM) alone did not cause morphological changes of the macrophages (Fig. 1C). In the presence of LPS, the control cell shape elongated into a spindle shape (Fig. 1C). However, co-treatment with LPS and SHK or its derivatives induced marked changes in the cells with nuclear condensation and swelling (Fig. 1C). Compared with the control cells treated with LPS alone, the cells treated with SHK and its derivatives obviously caused damage and altered cell morphology in the presence of LPS. These results suggest that LPS triggers the cytotoxic effects of SHK derivatives on macrophages.
Induction of Apoptotic Cell Death in Macrophages Treated with SHK Combined with LPSOn the basis of the results of the cell viability assay and observation of cell morphology, we expected that the cytotoxic effects of SHK on JA-4 cells would be similar to those of its derivatives in the presence of LPS. Therefore, we examined the effect of SHK on LPS-treated macrophages to further investigate the cytotoxic mechanisms. We examined the time–course of induction of cytotoxicity in SHK/LPS-treated macrophages. By 1 h after incubation, no significant changes were observed, but at 2 h and later, the cell viability decreased (Fig. 2A). We next determined if the cytotoxicity caused by SHK and LPS was accompanied by apoptosis. TUNEL staining revealed that the cells co-treated with LPS and SHK became TUNEL-positive (Fig. 2B). Fragmentation of the cells’ DNA was detected ≥3 h after treatment with combined SHK/LPS (Fig. 2C). To explore the detailed molecular mechanisms underlying SHK/LPS-induced macrophage apoptosis, we investigated the role of caspases in this apoptotic pathway. SDS-PAGE and Western blot analysis showed that simultaneous addition of SHK with LPS induced activation of caspases-3 and -7 and cleavage of both ≥2 h (Fig. 2D). In addition, addition of a broad-spectrum caspase inhibitor, Z-ASP-CH2-DCB, for 30 min prior to treatment with SHK and LPS resulted in significant reduction in the decrease in cell viability (Fig. 2E). Collectively, these results suggest that simultaneous addition of SHK with LPS induces apoptotic cell death of JA-4 cells through a caspase-dependent pathway.
JA-4 cells were treated with 2.5 µM SHK or 100 ng/mL LPS, or both, for the indicated times. The cell viability was estimated by WST-1 assay (A). Apoptosis was detected by TUNEL stain and DNA fragmentation assay as described in Materials and Methods (B and C). Caspases and cleaved caspases-3 and -7 were detected by Western blotting. N, none; L, LPS; S, SHK (D). JA-4 cells were treated with or without Z-ASP-CH2-DCB at 50 µM for 30 min and then with 2.5 µM SHK and 100 ng/mL LPS for 4 h. The cell viability was measured by WST-1 assay (E). All data are presented as the mean±S.D., n=3. * p<0.05 compared with untreated cells.
We previously reported that pretreatment of JA-4 cells with low doses of LPS resulted in downregulation of TNF-α secretion as well as LPS- and cycloheximide-induced macrophage cell death.18) On the basis of those results, we determined if pretreatment of low-dose LPS restored the decreased cell viability induced by LPS and SHK (Fig. 3A, upper). As shown in Fig. 3B, pretreatment with low doses of LPS (10 ng/mL) resulted in significant restoration of the decreased cell viability. Next, we examined the time of SHK addition to LPS-treated JA-4 cells (Fig. 3A, lower). As shown in Fig. 3C, the maximal cytotoxic effects were observed when SHK was added before or simultaneously with LPS addition. On the other hand, when SHK was added at 30 min after LPS addition, the cytotoxic effect of SHK was almost completely eliminated (Fig. 3C). These results suggest that SHK acts in an early stage of LPS signaling and needs to be added before the anti-apoptotic effect of LPS on macrophages is complete.
Experimental schemes for experiments of LPS tolerance and the time course of SHK addition or LPS tolerance (A). JA-4 cells were pretreated with or without 10 ng/mL LPS at 37°C for 90 min, washed three times in the culture media, and then reincubated with 100 ng/mL LPS or 2.5 µM SHK, or both, at 37°C for 4 h. The cell viability was assayed by WST-1. Tolerance (−); pretreated without LPS, tolerance (+); pretreated with LPS (B). SHK at 2.5 µM was added to the cells 30 or 15 min prior to 100 ng/mL LPS addition, simultaneously at 0, 15, or 30 min after LPS addition, and then the cells were incubated at 37°C for 4 h. The cell viability was quantified by WST-1 assay at 4 h after LPS addition (C). All data are presented as the mean±S.D., n=3. * p<0.05 compared with untreated cells.
To determine if the above-mentioned phenomenon was specific to JA-4 cell line macrophages or if it was common to mouse primary macrophages, we examined the effect of SHK and LPS on BALB/c mouse peritoneal macrophages. Preliminary experiments showed that the peritoneal macrophages treated with SHK alone induced significant cytotoxicity (data not shown) at 2.5 µM, at which concentration it showed little cytotoxic effect on JA-4 cells. We then used lower doses of SHK on the peritoneal macrophages in the presence or absence of 100 ng/mL LPS. The peritoneal macrophages did not show cell damage caused by treatment with SHK alone at doses up to 1 µM (Fig. 4A). However, simultaneous addition of 100 ng/mL LPS with 1 µM SHK significantly decreased the cell viability to 64±7.7%. Consistent with the results of the WST-1 assay, remarkable cell damage was morphologically observed in the cells treated with 1 µM SHK and 100 ng/mL LPS (Fig. 4B). Next, to determine whether this phenomenon is characteristic of macrophages, we examined the effect of SHK and LPS on the other cell type, 3T3 fibroblast cells. Because, it is well known that fibroblast cells also respond to LPS, and expressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expression.19,20) As shown in Fig. 4C, 100 ng/mL LPS and 100 ng/mL LPS plus 10 U/mL IFN-γ, which synergistically enhances the LPS response, up-regulate the expression of iNOS mRNA in 3T3 cells. On the basis of this result, when 3T3 cells were treated with various concentration of SHK in the presence or absence of 100 or 1000 ng/mL LPS, there is no difference between the cytotoxicity of SHK and SHK plus LPS (Fig. 4D). These results show that simultaneous addition of SHK with LPS exerted cytotoxic effects on not only a macrophage-like cell line, JA-4, but also on mouse peritoneal macrophages. Furthermore, this phenomenon might be characteristic of macrophages.
Primary macrophages were treated with various concentrations of SHK with or without 100 ng/mL LPS at 37°C for 4 h. Cell viability was estimated by WST-1 assay (A). Cells were observed under an Olympus CKX41 microscope (40×10) (B). Scale bars, 10 µm. 3T3 cells were treatment with 100 ng/mL LPS, 10 U/mL IFN-γ, or both for 0 or 3 h. The expression levels of iNOS gene were determined by using qRT-PCR, with the data normalized with the internal control GAPDH gene and the results expressed as values relative to those of the LPS plus IFN-γ-treated cells at 3 h (C). 3T3 cells were treated with various SHK with or without 100 or 1000 ng/mL LPS for 4 h. Cell viability was estimated by WST-1 assay (D). All data are presented as the mean±S.D., n=3. * p<0.05 compared with 0 µM SHK-treated cells.
Our present data indicates that the SHK derivatives exhibited cytotoxic effects on macrophages in the presence of LPS. LR and a Japanese traditional ointment medicine, “Shiunko” containing LR extracts, have also been traditionally used widely in Asia to treat various diseases, including sore throat, burns, cuts, and skin diseases. We next determined if endotoxin (LPS) was a contaminant in LR or “Shiunko.” To test endotoxin levels, the samples were boiled, and endotoxin was extracted. As shown in Table 1, both LR and “Shiunko” contained endotoxin, but LR had a much larger amount. These results suggest that treatment with LR or “Shiunko” may have cytotoxic effects on macrophages.
| Lot | Endotoxin (LPS) (ng/g) | Average (ng/g) | |
|---|---|---|---|
| Lithospermi radix | 1 | 3.77×105 | 1.31×105±1.23×104 |
| 2 | 9.66×104 | ||
| 3 | 5.97×104 | ||
| Shiunko | 4 | 4.68 | 1.40±0.63 |
| 5 | 4.11 | ||
| 6 | 1.19 | ||
| 7 | 3.82 | ||
| 8 | 2.56 |
Results are presented as the mean±S.D.
The aim of this study was to examine the direct effects of SHK derivatives on macrophage cell viability. We first found that SHK derivatives induced rapid death in LPS-treated macrophages accompanied by caspase-dependent apoptosis and that SHK derivatives need to be added prior to LPS treatment to cause maximal cytotoxicity.
SHK derivatives were extracted from a traditional medical herb, LR, the dried root of L. erythrorhizon. These compounds have been shown to exert anti-inflammatory, anti-microbial and anti-tumor effects.6,8,11,21) In this study, the effect of SHK derivatives on LPS-treated macrophages was examined by using a JA-4 cell line, an LPS-sensitive subline of a murine macrophage-like cell line, J774.1, and by using murine peritoneal primary macrophages. Induction of cytotoxic effects of SHK derivatives at lower concentrations on the macrophages was observed in the presence of LPS (Figs. 1, 4), which suggested that the cytotoxicity was not cell line specific but rather common to macrophages. Previously, we and others reported that a protein synthesis inhibitor, cycloheximide (CHX), and a p38 mitogen-activated protein kinase (MAPK)-specific inhibitor, SB202190, induced cell death of JA-4 or human macrophage-like U937 cells in the presence of LPS.22–25) These reports also appear to suggest the presence of a mechanism underlying induction of macrophage cell death in the presence of LPS.
Apoptosis and necrosis are cell death pathways that have distinctly different morphological and biochemical features. Treatment with SHK reportedly caused cell death in various cancer cell lines, and a low dose of SHK (<3 µM) induced apoptosis in a human non-small cell lung cancer cell line, A549.26) Consistent with those reports, our time–course study indicated that decreased viability was associated with DNA fragmentation and caspase-3/7 cleavage (Fig. 2). Thus, we conclude that SHK-induced cell death of LPS-treated macrophages involves a caspase-dependent apoptotic pathway. However, because treatment with Z-Asp-CH2-DCB did not completely restore the cytotoxicity, other mechanisms underlying cell death pathways were possible.
Several studies indicate that the anti-inflammatory effects of SHK derivatives due to attenuate the products of activated macrophages such as inflammatory cytokines via inhibition of MAPK, nuclear factor-kappaB (NF-κB) signaling, or proteasome.11,12,21,27) However, we consider that the cytotoxicity of SHK in LPS-treated macrophages may also contribute to the anti-inflammatory effect. Because, in progression of inflammation, it is important that although activated macrophages secretion of inflammatory cytokines, newly activated macrophages participate in amplifying the inflammatory response; our present findings may suggest that SHK derivatives inhibit the aggravation of inflammatory response by inducing cell death of newly activated macrophages. Recently, Marriott et al. suggested that macrophage apoptosis helps to decrease inflammation in pneumonia.28) This report supports our hypothesis that the cytotoxicity of SHK in LPS-treated macrophages may contribute to the anti-inflammatory effect.
As shown in Fig. 3B, pretreatment with low doses of LPS restored the decreased cell viability caused by LPS and SHK. LPS (>1 ng/mL) is known to induce negative regulators of LPS signaling, including inhibitor of κ-Bα, MAPK phosphatase 1, and interleukin receptor-associated kinase M.29) These negative regulators mediate downregulation of LPS signaling. Thus, our results suggest that SHK causes cytotoxicity of macrophages through LPS signaling. Furthermore, the cytotoxicity of SHK toward LPS-treated macrophages was maximal when SHK was added before or simultaneously with, LPS addition. In addition, treatment of SHK 30 min after LPS addition significantly attenuated the cytotoxicity. Therefore, we think that SHK acts only at the early stages of LPS signaling and, thus interrupts it, which leads to cell death of the LPS-treated macrophages. Furthermore, previous studies indicate that the p38 MAPK pathway plays a pivotal role in the induction of apoptosis,30,31) and we also previously show that sustained phosphorylation of p38 MAPK is a candidate as to LPS/CHX-induced cytotoxicity. Treatment with LPS/SHK also induced sustained phosphorylation of p38 MAPK (data not shown). So we expect that the target molecule(s) of SHK is/are associated with p38 MAPK pathway in the cytotoxicity of LPS/SHK. However, to date, we have not identified the target molecule(s). Further study is necessary to elucidate the precise mechanisms underlying SHK-induced cytotoxicity of the macrophages in the presence of LPS.
In this study, we also showed that SHK derivatives may induce macrophage cell death if LPS is present in SHK reagents. Regarding herbal medicine, LR is a useful herb that can be used alone or formulated with other drugs, such as “Shiunko” (formulated with LR, Angelica radix, Oleum Sesame, beeswax, and Adeps suillus) for treatment of burns, cuts, and skin disease in Asia since ancient times.32,33) In this study, we found that both LR and “Shiunko,” in particular LR, were contaminated with significant amounts of endotoxin (LPS). The reason for this could be that LR is made from the roots of L. erythrorhizon, and it is well known that numerous bacteria, both Gram-negative and Gram-positive, and fungi are present in soil. Thus, it seems feasible that treatment with LR and “Shiunko” could cause macrophage cytotoxicity.
To the best of our knowledge, our results provide the first evidence that SHK derivatives induced rapid apoptotic cell death of LPS-treated macrophages. Our study results also provided support for a novel mechanism of macrophage cytotoxicity induced by SHK derivatives and LPS and showed that herbal medicines that contain SHK can cause damage to macrophages and affect their functions.
This work was supported by JSPS KAKENHI Grant number 26860072.
The authors declare no conflict of interest.
