Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
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ent-Kaurane and ent-Pimarane Diterpenes from Siegesbeckia pubescens Inhibit Lipopolysaccharide-Induced Nitric Oxide Production in BV2 Microglia
Mina LeeSeung Hyun KimHee Kyoung LeeYekyung ChoJimmy KangSang Hyun Sung
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2014 年 37 巻 1 号 p. 152-157

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Abstract

The extract of Siegesbeckia pubescens herb and its chemical constituents were tested for the ability to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV2 microglia. The methanol extract and the 90% MeOH fraction of S. pubescens effectively attenuated lipopolysaccharide-induced nitric oxide production. Several steps of chromatography yielded eight ent-kaurane diterpenes (18) and one ent-pimarane diterpene (9) from the 90% MeOH fraction. Among these compounds, compounds 29 showed significant inhibitory effect on lipopolysaccharide-induced nitric oxide production in BV2 microglia. Compounds 3 and 9 concentration-dependently decreased the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), supported by quantitative real time polymerase chain reaction (PCR) and Western blot analysis. These results suggest that ent-kaurane and ent-pimarane diterpenes isolated from S. pubescens are expected to be potential candidates against neuroinflammation-related disease.

Brain inflammation has an important role in various neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), amylotrophic lateral sclerosis and so on. There are two types of glial cells participating in brain inflammation, microiglia and astrocytes. Among them, microglia cells are particularly sensitive to changes in their microenvironment and readily become activated in response to immunologic challenge and injury.1) Activated microglia produce a variety of proinflammatory and neurotoxic factors including nitric oxide (NO).2) Overproduction of NO appears to contribute to neuronal cell death through oxidative damage on cellular lipids, proteins and DNA.3) In addition, microglial activation affects the initiation and progression of several neurodegenerative diseases. A number of anti-inflammatory substances have been reported as attractive pharmacological targets that suppress microglial activation or overproduction of inflammatory cytokines in central nervous system (CNS) diseases, and attenuate neuronal degeneration.4) Thus, regulating microglial activation could be a potent therapeutic strategy to enhance various neuroinflammatory and neurodegenerative diseases.

Siegesbeckia pubescens (Compositae) is an annual herb indigenous to the mountainous regions of Korean. The aerial part and root of this plant have been used to treat rheumatoid arthritis and hypertension in Korean traditional medicine.5) Pharmacological studies of S. pubescens have shown cartilage protective, anti-inflammatory and analgesic activities, and an inhibitory activity on immediate hypersensitivity.68) Regarding the chemical composition, a series of ent-pimarane and ent-kaurane diterpenes have been isolated from this plant.9) Although these diterpenes are best known for their cytotoxic or cardiovascular effects,10,11) there have also some reports indicating their anti-inflammatory activity. Tedanol, a ent-pimarane diterpene isolated from Tedania ignis, significantly reduced carrageenan-induced inflammation in mice.12) Isodon species have been known to contain various anti-inflammatory ent-kaurane diterpenes and some of them have been demonstrated to inhibit nuclear factor-kappa B (NF-κB) activation.1316) In addition, ent-kaurane diterpenes isolated from Rabdosia rubescens or Croton tonkinensis, showed anti-inflammatory activities in primary cultured rat microglia or RAW264.7 macrophages.17,18) Moreover, Park et al. found that siegeskaurolic acid, an ent-kaurane diterpene isolated from S. pubescens, significantly inhibited lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 cells through down-regulation of the NF-κB binding activity.19) Kirenol isolated from Herba Siegesbeckiae also reduces pro-inflammatory cytokines secretion, increase anti-inflammatory cytokines production of CII-specific lymphocytes from CIA rats, and exerts a potent anti-arthritic effect in collagen-induced arthritis by modifying the balance of T cells.20) These previous studies suggest the possible presence of anti-inflammatory ent-pimarane or ent-kaurane diterpenes in S. pubescens. However, anti-neuroinflammatory diterpenes of S. pubescens have not been investigated yet. Thus, we used LPS-stimulated BV2 microglia as a screening system to identify anti-neuroinflammatory diterpenes. We previously reported that ent-kaurane diterpene, ent-16αH,17-hydroxy-kauran-19-oic acid, isolated from S. pubescens regenerates epidermal tissue mainly through epithelial growth factor receptor (EGFR) and increases the proliferation, migration, and tube formation of vasculogenic progenitor cells (VPCs).21,22) Further investigation on S. pubescens showed the anti-neuroinflammatory activity of the total methanol extract and fractions of CH2Cl2, n-hexane, and 90% MeOH. Among these fractions, the 90% MeOH fraction showed the most significant inhibitory activity against LPS-induced NO production in BV2 microglia. Therefore, in the present study, we attempted to isolate the anti-neuroinflammatory diterpenes from the 90% MeOH fraction of S. pubescens leaves. The isolated compounds were evaluated for their effects on NO production and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated BV2 cells. In present study, we first described the anti-neuroinflammatory activity and therapeutic potential for the treatment of neuroinflammatory diseases of kaurane and pimarane type diterpene from S. pubescens.

MATERIALS AND METHODS

Plant Material

The S. pubescens herbs were purchased from the Kyungdong traditional herbal market (Seoul, Korea) and authenticated by Dr. Jong Hee Park, a professor at Pusan National University. A voucher specimen (EC-0168) has been deposited at the Herbarium of the Medicinal Plant Garden, College of Pharmacy, Seoul National University.

Isolation of NO Production Inhibitory Compounds

The extraction and fractionation methodology for the S. pubescens herb (5 kg) used in this study was previously described in a peer review journals.21,22) The 90% MeOH fraction, which showed the most potent inhibitory activity was chromatographed on silica gel column using a CHCl3–MeOH gradient as the mobile phase to yield eleven fractions (M1–M11). Compound 1 (20 mg) was obtained from M3 by silica gel column chromatography (CC) and then recrystallized with MeOH. M4 was sequentially rechromatographed on a silica gel column and Sephadex LH-20 CC to yield compound 2 (2 mg). Compound 3 (890 mg) was obtained by recrystallization of M5 using MeOH. Silica gel CC followed by HPLC (Inertsil ODS-3, 5 µm, 4.6×250 mm, H2O : MeOH=60 : 40→20 : 80, 1 mL/min) of M6 yielded sixteen subfractions (M6-4-1–M6-4-16). Sephadex LH-20 CC and following HPLC (Inertsil ODS-3, 5 µm, 4.6×250 mm, H2O : MeOH=60 : 40→20 : 80, 1 mL/min) of M6-4-8 yielded compound 4 (35 mg). M7 was divided by silica gel CC and rechromatographed on Sephadex LH-20. Compound 5 (606 mg) was purified from one of the resulting subfractions of M7 using Sephadex LH-20 CC. Compound 6 (961 mg) was obtained from M8 by recrystallization with MeOH. Compound 7 (216 mg) was purified by MeOH recrystallization of M9-9 which was obtained from M9 by silica gel CC. M11 was divided into fifteen fractions (M11-1–M11-15) by silica gel CC. Compounds 8 (1030 mg) and 9 (15 mg) were obtained from M10 and M11-11 by recrystallization with MeOH, respectively.

Evaluation of the Inhibitory Effect on NO Production in LPS-Stimulated BV2 Microglia

The BV2 mouse microglia cell line originally developed by Dr. Bocchini at University of Perugia (Perugia, Italy)23) was generously provided by Dr. Sun-yeou Kim at Gachon University (Incheon, Korea). The cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) with penicillin (100 IU/mL) and streptomycin (10 mg/mL) at 37°C in a humidified atmosphere of 95% air–5% CO2. Test fractions and compounds were dissolved in dimethyl sulfoxide (DMSO) (final concentration in cultures <0.1%). To remove any traces of phenol red, the cell cultures were washed and the medium was replaced with phenol red-free DMEM. Then, BV2 microglia cells (2×105 cells/well in 96 well plates) were treated with test samples for 1 h before exposure to 100 ng/mL of LPS. After incubation, NO production (NP) was measured by the Griess reaction and sodium nitrite was used as the standard. Relative NP (%) was calculated as (NP of sample treated-NP of control)/(NP of LPS-treated-NP of control)×100 (%).24) Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are expressed as the percentage cell viability relative to vehicle-treated control cultures; 100×[optical density (OD) of LPS-treated or LPS+sample-treated cultures]/[OD of control cultures].24)

RNA Preparation and Quantitative Real-Time Polymerase Chain Reaction (PCR)

Total cellular RNA was extracted from the BV2 microglia cells using the RNease Plus Kit (QIAGEN Korea Ltd., Seoul, Korea) according to the manufacturer’s instructions. cDNA was synthesized with 1 µg of total RNA using QuantiTech Reverse Transcription Kit (QIAGEN Korea Ltd., Seoul, Korea) and then was mixed with QuantiFast SYBR Green PCR master mix (QIAGEN Korea Ltd., Seoul, Korea) and specific primers in a total reaction volume of 20 µL. The PCR specific primers used with the QIAGEN kits for SYBR® Green-based real time RT-PCR were iNOS (NM_010927), COX-2 (NM_011198), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (NM_008084) and obtained from QIAGEN Korea Ltd., Seoul, Korea. The amplification cycles were carried out at 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s. The last cycle was followed by a final extension step at 72°C for 5 min. Quantitative SYBR Green real-time PCR was done with an Applied Biosystems 7300 Real-Time PCR System (Life Technologies Corporation, Carlsbad, CA, U.S.A.) and analyzed with comparative Ct quantification.25) GAPDH was amplified as an internal control. The Ct values of GAPDH were subtracted from the Ct values of the target genes (ΔCt). The ΔCt values of the treated cells were compared with the ΔCt values of the untreated cells.

Protein Extraction and Western Blot Analysis

The BV2 cells were washed twice with cold phosphate buffered saline (PBS), and then lysed in an ice-cold modified radio immuno precipitation assay (RIPA) buffer containing protease inhibitors. The lysate was centrifuged for 20 min at 12000×g at 4°C, and the supernatants were collected. The protein content was determined with the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, U.S.A.). Total proteins (30 µg) were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, U.S.A.). After blocking in TBST (100 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) with 5% non-fat dry milk for 1 h at room temperature, the membrane was incubated overnight at 4°C with 1 : 1000 diluted iNOS (BD Biosciences, San Jose, CA, U.S.A.) and COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), primary antibodies. After incubation with 1 : 2000 diluted horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) for 1 h at room temperature, immunoreactive proteins were visualized by an enhanced chemiluminescent solution (Amersham, Uppsala, Sweden). After normalization to β-actin, density values for the protein bands of interest were expressed as a percentage of the control using the ImageJ software (http://rsweb.nih.gov/ij).

Statistical Analysis

All data were expressed as the means±standard deviation (S.D.). The evaluation of statistical significance was determined by a “one-way ANOVA” test using a computerized statistical package. The data were considered to be statistically significant if the probability had a value of 0.05 or less.

RESULTS AND DISCUSSION

Microglia are important inflammatory cells in the CNS because microglia activation initiate an inflammatory cascade and contributes to the pathogenesis of AD and PD, as well as other neurodegenerative diseases.4) A murine cell line BV2 was generated by infecting primary microglia cell cultures with a v-raf/v-myc oncogene carrying the retrovirus (J2).23) Since BV2 cells retain most of their morphological, phenotypical and functional properties described for freshly isolated microglial cells, such as LPS-stimulated NO production, it has been used as an in vitro screening model to search for anti-neuroinflammatory natural products. Thus we tried to validate the anti-neuroinflammatory effect of S. pubescens and its active constituents, ent-kaurane diterpenes. In this screening system, the methanol extract (36.3±3.4% of the control at 10 µg/mL) of S. pubescens significantly inhibited LPS-induced NO production. The methanol extract was then suspended in H2O and extracted with CH2Cl2. The CH2Cl2 fraction (8.3±4.5% of the control at 10 µg/mL) also showed significant inhibitory activity against NO production. Further fractionation of the CH2Cl2 fraction yielded the n-hexane (10.2±3.2% of the control at 10 µg/mL) and 90% MeOH fractions (4.4±3.6% of the control at 10 µg/mL). Among these fractions, the 90% MeOH fraction showing the most significant inhibitory effect on NO production was used to isolate the active compounds to yield nine diterpenes (19).

The structures of the isolated compounds (19) were identified as methyl ent-16αH-17-hydroxy-kauran-19-oate (1), ent-16βH,17-isobutyryloxy-kauran-19-oic acid (2), ent-16αH,17-hydroxy-kauran-19-oic acid (siegeskaurolic acid) (3), methyl ent-16α,17-dihydroxy-kauran-19-oate (4), ent-16βH,17-isobutyryloxy-18-hydroxy-kauran-19-oic acid (5), ent-16α,17-dihydroxy-kauran-19-oic acid (6), ent-16βH,17,18-dihydroxy-kauran-19-oic acid (7), methyl ent-16β,17,18-trihydroxy-kauran-19-oate (8), and kirenol (9), respectively, from spectral data comparison with those reported in the literature (Fig. 1).9,2630) All the isolated compounds were ent-kaurane diterpenes except for compound 9, an ent-pimarane diterpene. Among the tested compounds, compounds 2-9 showed a significant inhibitory effect on LPS-induced NO production which was comparable to the positive control, ω-nitro-L-arginine methyl ester (NAME) (Table 1). To verify whether the reduced cell numbers from the cytotoxicity of these compounds resulted in the decreased NO production, cell viability was measured using the MTT assay. Among the tested compounds, only compound 1 exhibited a strong cytotoxicity on the BV2 microglia with a relative cell viability of 35.9 and 25.0% at 50 µM and 100 µM, respectively. Other compounds exerted their inhibitory effect on NO production inhibitory effect without any cytotoxicity. As has been noted in previous studies on the anti-rheumatism action of kirenol, compound 9 effectively inhibited LPS-induced NO production.31) Non-substituted kauran-19-oic acids, compounds 3, 4 and 6, generally showed significant inhibitory activity against LPS-induced NO production. Methyl esterification at C-19 of compound 6 increased its inhibitory activity as seen in compound 4. However, compound 1, a methyl ester of compound 3, exerted a strong cytotoxicity. The substitution of an isobutyloxy group at C-17 in compounds 2 and 5 also affected decreasing LPS-induced NO production. Compound 5 with the isobutyloxy group at C-17 exhibited a more inhibitory effect than compound 7 with no isobutyloxy group. The substitutional changes at C-17 and C-19 made significant differences among the isolated ent-kaurane diterpenoids. From the results, it was postulated that the skeleton of ent-kaurane and ent-pimarane diterpenoids might be necessary for the inhibitory activity on NO production in LPS-stimulated BV2 microglia cell. Hence, further investigation using compounds 3 and 9, which exhibited the most potent inhibitory activity (20.5±11.3% and 22.5±5.8% of the control at 100 µM) against LPS-stimulated NO production, was carried out to reveal any anti-neuroinflammatory activity on BV2 microglia cell.

Fig. 1. Structures of the Compounds Isolated from S. pubescens
Table 1. Inhibitory Effect of the Isolated Compounds from S. pubescens on LPS-Induced NO Production in BV2 Microglia
50 µM100 µM
Nitrite (µM)Viability (%)Nitrite (µM)Viability (%)
Control1.6±0.4100.0±0.9
LPS49.6±3.6###101.7±2.7
11.8±3.2***35.9±4.8***0.2±0.4***25.0±5.7***
229.9±5.4**100.0±15.217.8±2.7**103.4±12.3
330.5±5.5*110.6±2.510.9±5.9***98.2±4.5
425.2±6.6*106.9±10.023.1±1.6**105.5±8.9
541.2±5.4*108.7±1.321.0±5.7**110.7±2.2
644.3±3.3*103.8±5.226.2±2.8**102.2±9.2
747.4±5.6104.9±4.033.9±5.2*106.6±7.1
847.4±4.5116.3±6.234.8±7.8*114.3±7.8
932.5±0.9**106.2±5.212.0±3.0***107.9±3.9
NAMEa)31.9±2.2*93.6±5.627.8±1.9**89.2±7.5

BV2 cells were washed with phenol red-free DMEM and incubated with test compounds for 1 h. The cultures were then stimulated by 100 ng/mL of LPS for 24 h. After incubation, NO production and cell viability (%) were measured by the Griess reaction and MTT assay, respectively. Each value represents the mean±S.D. of three experiments. ###p<0.001, compared with untreated control; * p<0.05, ** p<0.01 and *** p<0.001, compared with LPS-treated control. a) NAME (ω-nitro-L-arginine methyl ester) was used as a positive control.

NO is synthesized endogenously from L-arginine in a reaction catalyzed by a family of nitric oxide synthase (NOS) enzymes.32) LPS-stimulation induces the expression of inducible NOS (iNOS) expression in microglial cells, astrocytes, and macrophages.33,34) Excessive NO production induced by iNOS in activated microglia is correlated with the progression of neurodegenerative disorders and inflammatory diseases.35) COX-2 is another enzyme that has a central role in mediating inflammation by stimulating the biosynthesis of prostaglandins.36) Previous study reported that compounds 3 and 9 affect the inflammation in murine macrophage cell line, RAW264.7.19) In present study, we focused on anti-neuroinflammatory activity of compounds 3 and 9, and investigated their anti-neuroinflammtory actions targeting microglia-mediated neurodegenerative diseases in BV2 microglial cells. Unlike macrophage, Microglial as resident mononuclear phagocytes of the CNS, belonging to the glial system of non-neuronal cells that support and protect neuronal functions play central roles in CNS neuroinflammation.37) Thus, microglia occupy a central position in the defense and maintenance targets in neurological disorders and recovery from brain injury.38) To explore the anti-neuroinflammatory mechanism of compounds 3 and 9, the mRNA expression levels of iNOS and COX-2 were examined by quantitative real time PCR. Real time RT-PCR analysis showed that compounds 3 and 9 significantly down-regulated the mRNA expressions of iNOS and COX-2 in a concentration-dependent manner (Fig. 2A). The protein expressions of iNOS and COX-2 were also measured by western blot. As shown in Fig. 2B, compounds 3 and 9 also significantly decreased LPS-stimulated iNOS and COX-2 induction in a concentration-dependent manner. The inhibition of protein induction had a similar pattern to the mRNA expression except that compound 9 more effectively attenuated LPS-induced expression of iNOS and COX-2 than that of compound 3 at a concentration of 50 µM in the western blot analysis. Taken together, these results suggest that compounds 3 and 9 have anti-neuroinflammatory activity by suppressing LPS-stimulated expression of iNOS and COX-2.

Fig. 2. Effects of Compounds 3 and 9 on LPS-Induced iNOS and COX-2 Expression in BV2 Cells by Quantitative Real-Time RT-PCR (A) and Western Blot (B) Analysis

(A) BV2 microglial cells were pre-treated with compounds 3 and 9 for 1 h, and then exposed to LPS for 6 h. The mRNA expression levels of iNOS and COX-2 were estimated by quantitative real-time RT-PCR analysis. (B) BV2 microglial cells were pre-treated with compounds 3 and 9 for 1 h, and then exposed to LPS for 20 h. Cell lysates were prepared and the iNOS, COX-2, and β-actin protein levels were determined by Western blot analysis. Each value represents the mean±S.D. of three experiments. #p<0.05, ##p<0.01, compared with control; * p<0.05, ** p<0.01, *** p<0.001, compared with LPS-treated control.

There is growing interest in finding natural products that have anti-neuroinflammatory activity. S. pubescens has been mentioned as a possible candidate for various inflammatory diseases. In the present study, nine diterpenes were isolated from S. pubescens by activity-guided fractionation using LPS-stimulated BV2 microglia as a screening system. The significant inhibitory activity of the isolated compounds against LPS-induced NO production suggest they are involved in the anti-neuroinflammatory activity of S. pubescence. In particular, compounds 3 and 9 effectively inhibited some important neuroinflammatory markers such as iNOS and COX-2. These findings are relevant in the further development of therapeutic potential for the treatment of neuroinflammatory diseases.

Acknowledgment

This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF-2010-0012250).

REFERENCES
 
© 2014 The Pharmaceutical Society of Japan
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