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International Immunopharmacology 35 (2016) 61–69
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Hydrangenol inhibits lipopolysaccharide-induced nitric oxide production in BV2 microglial cells by suppressing the NF-κB pathway and activating the Nrf2-mediated HO-1 pathway Hee-Ju Kim a, Chang-Hee Kang a,b, Rajapaksha Gedara Prasad Tharanga Jayasooriya a, Matharage Gayani Dilshara a, Seungheon Lee a, Yung Hyun Choi c, Yong Taek Seo d, Gi-Young Kim a,⁎ a
Department of Marine Life Sciences, Jeju National University, Jeju 63243, Republic of Korea Nakdonggang National Institute of Biological Resource, Sangju-si, Gyeongsangbuk-do 37242, Republic of Korea c Department of Biochemistry, College of Oriental Medicine, Dong-Eui University, Busan 47227, Republic of Korea d Consilience Medical Center, Dankook University Hospital, Chungcheongnam-do 31116, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 3 November 2015 Received in revised form 15 March 2016 Accepted 17 March 2016 Available online xxxx Keywords: Hydrangenol Nitric oxide Nuclear factor-κB Heme oxygenase-1 Nuclear factor erythroid 2-related factor 2
a b s t r a c t We previously demonstrated the anti-inflammatory effect of water extract of Hydrangea macrophylla in lipopolysaccharide (LPS)-stimulated macrophage cells. Here, we investigated whether hydrangenol, a bioactive component of H. macrophylla, attenuates the expression of nitric oxide (NO) and its associated gene, inducible NO synthase (iNOS), in LPS-stimulated BV2 microglial cells. Our data showed that low dosages of hydrangenol inhibited LPS-stimulated NO release and iNOS expression without any accompanying cytotoxicity. Hydrangenol also suppressed LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) subunits, consequently inhibiting DNA-binding activity of NF-κB. Additionally, the NF-κB inhibitors, pyrrolidine dithiocarbamate (PDTC) and PS-1145, significantly attenuated LPS-induced iNOS expression, indicating that hydrangenolinduced NF-κB inhibition might be a key regulator of iNOS expression. Furthermore, our data showed that hydrangenol suppresses NO production by inducing heme oxygenase-1 (HO-1). The presence of cobalt protoporphyrin, a specific HO-1 inducer, potently suppressed LPS-induced NO production. Hydrangenol also promoted nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and subsequently increased its binding activity at the specific antioxidant response element sites. Additionally, transient knockdown of Nrf2 significantly downregulated hydrangenol-induced HO-1 expression, indicating that hydrangenol-induced Nrf2 is an upstream regulator of HO-1. Taken together, these data suggest that hydrangenol attenuates NO production and iNOS expression in LPS-stimulated BV2 microglial cells by inhibiting NF-κB activation and by stimulating the Nrf2-mediated HO-1 signaling pathway. Therefore, hydrangenol is a promising therapeutic agent for treatment of LPS-mediated inflammatory diseases. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The intrinsic function of microglial cells is to protect the central nervous system by removing dead cells and tissue debris through phagocytosis [1]. In response to injury or infection, microglia become readily activated and secrete proinflammatory mediators such as nitric oxide (NO), cyclooxgenase-2 (COX-2), interleukin-1 beta (IL-1β), and IL-6, which are essential to regulate cellular signaling involved in protection against organ injury, such as ischemic damage [2]. Nevertheless, recent studies showed that excessive and abnormal production of proinflammatory mediators result in systemic inflammatory syndrome, severe tissue damage, atherosclerosis, and septic shock [3,4]. Among the proinflammatory mediators secreted by microglia, NO is well known to ⁎ Corresponding author. E-mail address: [email protected] (G.-Y. Kim).
http://dx.doi.org/10.1016/j.intimp.2016.03.022 1567-5769/© 2016 Elsevier B.V. All rights reserved.
aggravate cerebral ischemia [1]. Normally, NO plays a role in neuroprotective and pathophysiological processes by regulating resting blood flow [5]; however, excessive NO release promotes early blood-brain barrier disruption [6], as well as causes oxidative injury in microglia, but not in astrocytes [7]. In addition, the NO-regulating gene, inducible NO synthase (iNOS), stimulates peroxynitrite formation, which promote protein radicals in microglia-mediated neurodegenerative disorders [8]. Li et al., demonstrated that silencing iNOS expression protected against neurodegeneration of nigrostriatal dopaminergic neurons in an animal model of Parkinson's disease [9]. As a result, many researchers have attempted to identify phytochemicals that suppress the aberrant NO production and iNOS expression, and determine their therapeutic characteristics in the context of neurodegenerative diseases. Nuclear factor-κB (NF-κB) is a transcription factor known to directly regulate many proinflammatory genes such as iNOS [10]. During inflammatory responses, inhibitor of κB (IκB) is degraded through its
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phosphorylation and ubiquitination; consequently, free NF-κB is released and translocated to the nucleus to transcribe proinflammatory genes such as iNOS [11]. Therefore, NF-κB is a good strategic target for modulating inflammatory responses and diseases. So far, many researchers have reported a variety of natural and designed molecules, including proteasome inhibitors, small molecules, active polypeptides, and flavonoids, which target NF-κB, leading to suppress iNOSmediated inflammatory diseases [10,12]. Additionally, recent studies reported that heme oxygenase-1 (HO-1) is induced in most tissues by a variety of oxidative stimuli and is involved in the protection against different types of oxidant-induced tissue damage and cellular injury; however, HO-2 is constitutively expressed [10,11]. It has been also shown that HO-1 has important immune-modulatory and anti-inflammatory functions through regulation of the proinflammatory mediators, NO and iNOS [12]. Nuclear transcription factor erythroid 2-related factor-2 (Nrf2) is a redox-sensitive transcription factor responsible for the induction of antioxidant enzymes. Recent reports found that Nrf2 regulates major immune-modulatory and anti-inflammatory properties via HO-1 induction [13,14]. Currently, Hydrangea macrophylla is used in two medicinal parts, as an oral antipyretic and as an alternative sweetener for diabetic patients [15]. Additionally, there has been growing interest in H. macrophylla since it contains febrifugine and its analogues, which target the cytoplasmic prolyl-tRNA synthetase of the malaria parasites [16,17]. Furthermore, our previous study showed that water extract from processed H. macrophylla leaves possesses anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages [18]. The present study investigated the inhibition of NO production and iNOS expression by hydrangenol, isolated from H. macrophylla, via suppression of NF-κB activity in LPS-stimulated BV2 microglial cells. In addition, antagonistic mechanism of hydrangenol is associated with the induction of Nrf2-mediated HO-1 expression.
2. Materials and methods 2.1. Reagents and antibodies Rabbit anti-human antibodies against iNOS, p65, p50, C-23, HO-1, and Nrf2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin antibody, LPS, and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO). Peroxidase-labeled goat anti-rabbit immunoglobulin was purchased from KOMA Biotechnology (Seoul, Republic of Korea). Pyrrolidine dithiocarbamate (PDTC) and PS-1145 were purchased from Calbiochem (San Diego, CA). Cobalt protoporphyrin (CoPP) was purchased from Tocris Bioscience (Bristol, UK). Dulbecco's Modified Eagle's medium (DMEM), fetal bovine serum (FBS), and antibiotic mixtures were obtained from WelGENE Inc. (Daegu, Republic of Korea). Other chemicals were purchased as Sigma grades. Hydrangenol was isolated in our previous study [16] and the structure of hydrangenol is illustrated in Fig. 1A.
2.2. Cell culture and viability BV2 microglial cells were cultured in DMEM media containing 5% FBS and antibiotic mixtures, and incubated at 37 °C and 5% CO2 conditions. MTT assay was performed to determine relative cell viability. Briefly, BV2 microglial cells (1 × 105 cells/ml) were treated with various concentrations (0–100 μM) of hydrangenol for 2 h before treatment with LPS (500 ng/ml). In a parallel experiment, 500 μM hydrogen peroxide (H2O2) were treated 2 h prior to LPS treatment as a apoptotic positive control. After 24-h incubation, the cells were incubated with MTT solution (0.5 mg/ml) for 15 min at 37 °C. Supernatant was removed and the formation of formazan was observed by monitoring the signal
Fig. 1. Effect of hydrangenol on the viability of BV2 microglial cells. (A) Chemical structure of hydrangenol. BV2 microglial cells (1 × 105 cells/ml) were incubated with the indicated concentrations of hydrangenol (0–100 μM) for 2 h before treatment with LPS (500 ng/ml) for 24 h. (B) Cell viability was determined using an MTT assay. (C) In a parallel experiment, 40 μM hydrangenol was treated 24 h before 500 ng/ml LPS treatment. As a positive cell death-inducing control, 500 μM hydrogen peroxide (H2O2) was treated instead of hydrangenol. The percentage of annexin V+-cell population (top panel) and sub-G1 DNA content (bottom panel) are indicated in each panel. Each value indicates the means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a one-way ANOVA test (a, p b 0.05 vs. untreated control).
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at 540 nm using a microplate reader (Thermo Electron Corporation, Marietta, OH). 2.3. Flow cytometric analysis BV2 microglial cells were pretreated with hydrangenol for 2 h and then administered with 500 ng/ml LPS for 24 h. After harvesting, the cells were washed two times with phosphate buffer saline (PBS) and fixed with 1 U/ml RNase A (DNase free) and 10 μg/ml propidium iodide (PI, Sigma) for 1 h at room temperature in the dark. For annexin V staining, the live cells were incubated with annexin V-FITC (R&D systems, Minneapolis, MN) according manufacturer's instructions. A FACSCalibur flow cytometer (Becton Dickenson, San Jose, CA) was used to analyze the level of apoptotic cells containing sub-G1 DNA content and annexin V-FITC+ population. 2.4. NO production BV2 microglial cells (1 × 105 cells/ml) were dispensed on to 24 well plates and pretreated with the indicated concentrations of hydrangenol 2 h prior to stimulation with 500 ng/ml LPS for 24 h. Supernatants were collected and assayed for NO production by Griess reaction. Briefly, the samples were mixed with equal volume of Griess reagent (1% sulfanilamide in 5% acetic acid and 0.1% naphthylethylenediamine dihydrochloride) and then incubated at room temperature for 10 min. The absorbance was measured at 540 nm on a microplate reader. Sodium nitrite dilution series were used as a standard to determine the nitrite concentration in the supernatants. 2.5. Isolation of total RNA and RT-PCR Total RNA was extracted using an easy-BLUE kit (iNtRON Biotechnology, Sungnam, Republic of Korea) according to the manufacturer's instruction. One microgram of RNA was reverse-transcribed using moloney murine leukemia virus (MMLV) reverse transcriptase (Bioneer, Daejeon, Republic of Korea). cDNA was amplified by PCR using the specific primers, iNOS (forward 5′-CCTCCTCCACCCTACCAA GT-3′ and reverse 5′-CACCCAAACTGCTTCAGTCA-3′), HO-1 (forward 5′-TCGCCAGAAAGCTGAGTATAA-3′ and reverse 5′-ATTGCCAGTGCCAC CACCAAGTTCAAG-3′), and β-actin (forward 5′-TGTGATGGTGGGAATG GGTC-3′ and reverse 5′-TTTGATGTCACGCACGATTT-3′). The following PCR conditions were applied: iNOS and HO-1, 25 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s and extended at 72 °C for 30 s; β-actin, 23 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s and extended at 72 °C for 30 s. 2.6. Western blot analysis BV2 microglial cells were seeded at the density of 1 × 105 cells/ml and treated with the indicated compounds. After 24 h-incubation, total cell extracts were prepared using a PROPREP protein extraction kit (iNtRON Biotechnology). Briefly, the PROPREP solution was treated to the cells on the ice for 30 min and lysates were centrifuged at 14,000 ×g at 4 °C for 10 min to obtain the supernatants. In a parallel experiment, cytoplasmic and nuclear extracts were prepared from the cells using NE-PER nuclear and cytosolic extraction reagents (Pierce, Rockford, IL). The supernatants were collected and protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). The samples were stored at −80 °C or immediately used for Western blot analysis. The proteins were separated on SDSpolyacrylamide gels and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Finally, proteins were detected using an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL).
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2.7. Electrophoretic mobility shift assay (EMSA) BV2 microglial cells were seeded at the density of 1 × 105 cells/ml and treated with the indicated compounds for 30 min. Total cells were suspended with cytosolic extraction reagent and incubated at 4 °C for 30 min. Then, the pellets were resuspended with NE-PER nuclear reagent for EMSA. Synthetic complementary NF-κB (5′-AGTTGAGGGGAC TTTCCCAGGC-3′) binding oligonucleotides (Santa Cruz Biotechnology) and anti-oxidant response elements (ARE) consensus (5′TMANNRTGAYNNNGCRWWWW-3′) were 3′-biotinylated using the biotin 3′-end DNA labeling kit (Pierce) according to the manufacturer's instructions and annealed for 30 min at room temperature. Nuclear extracts and 3′-labeled complementary oligonucleotides were incubated with NE-PER-binding buffer for 1 h. Assays were loaded onto native 4% polyacrylamide gels pre-electrophoresed for 1 h in 0.5 × Tris borate/EDTA before being transferred onto a positively charged nylon membrane (HybondTM-N +) in 0.5 × Tris borate/EDTA at 100 V for 30 min. The transferred DNAs were cross-linked to the membrane at 120 mJ/cm 2 . Horseradish peroxidase-conjugated streptavidin was used according to the manufacturer's instructions to detect the transferred DNA. 2.8. Transient knockdown of Nrf2 BV2 microglial cells were seeded on a 24-well plate at a density of 1 × 105 cells/ml and transfected Nrf2-specific silencing RNA (siRNA, Santa Cruz Biotechnology) for 24 h. For each transfection, 450 μl growth medium was added to 20 nM siRNA duplex with the transfection reagent G-Fectin (Genolution Pharmaceuticals Inc., Seoul, Republic of Korea) and the entire mixture was added gently to the cells. 2.9. Statistical analysis The images were visualized with Chemi-Smart 2000 (Vilber Lourmat, Marine, Cedex, France). Images were captured using ChemiCapt (Vilber Lourmat) and transported into Photoshop. All bands were shown a representative obtained in three independent experiments and quantified by Scion Imaging software (http://www.scioncorp. com). Statistical analyses were conducted using SigmaPlot software (version 12.0). Values were presented as mean ± S.E. of three experiments. Significant differences between the groups were determined by one-way or two-way ANOVA followed with Bonferroni's test. Statistical significance was regarded at a and b, p b 0.05. 3. Results 3.1. Hydrangenol has no influence on the viability of BV2 microglial cells In order to first determine the effect of hydrangenol on cell viability, we pretreated BV2 microglial cells with hydrangenol for 2 h before LPS treatment for 24 h. MTT data showed that cell viability was not significantly altered by hydrangenol at concentrations up to 40 μM at 24 h. However, treatment with concentrations above 50 μM hydrangenol gradually decreased the viability of BV2 microglial cells in a dosedependent manner (Fig. 1B). These results suggested that concentrations of hydrangenol below 40 μM were non-toxic to BV2 microglial cells. Thus, further experiments were carried out with hydrangenol at concentrations below 40 μM. Next, flow cytometric analysis performed to evaluate hydrangenol cytotoxicity in more detail. For the comparative positive control, 500 μM H2O2 were treated for 24 h to induced apoptosis. Data obtained using annexin V staining showed that of the H2O2-treated BV2 microglial cells, 38 ± 5% were annexin V+ or apoptotic; however, a minor population of hydrangenol-pretreated BV2 microglial cells were found to be annexin V+, thereby suggesting that hydrangenol has no influence on BV2 microglial cell apoptosis (Fig. 1C, top). In a parallel experiment, we also analyzed hydrangenol
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cytotoxicity based on the amount of DNA in the sub-G1 phase. Apoptotic cell death was not detected in any of the panels of cells treated with hydrangenol, contrary to that observed for the H2O2-treated positive control group (Fig. 1C, bottom). Taken together, these data indicate that hydrangenol (≤ 40 μM) has no influence on the viability of BV2 microglial cells. 3.2. Hydrangenol inhibits NO production and iNOS expression in LPSstimulated BV2 microglial cells In order to analyze the suppressive effect of hydrangenol on NO production, BV2 microglial cells were pretreated with different concentrations of hydrangenol for 2 h, followed by treatment with LPS for 24 h, before determination of the levels of NO in the culture media by Griess assay. LPS markedly increased NO production in BV2 microglial cells (18.9 ± 2.7 μM; Fig. 2A). On the other hand, hydrangenol significantly decreased NO production in LPS-stimulated BV2 microglial cells in a dose-dependent manner (13.1 ± 2.2 μM, 10.9 ± 1.5 μM, and 8.8 ± 2.1 μM at 10 μM, 20 μM, and 40 μM hydrangenol, respectively). In particular, 20 μM and 40 μM hydrangenol significantly decreased the LPSinduced NO production; however, this downregulation did not reach the level observed for untreated control (4.2 ± 1.2 μM). Additionally, we investigated whether hydrangenol regulates LPS-stimulated iNOS mRNA expression. Western blot analysis showed that treatment with LPS markedly increased iNOS protein expression at 24 h and this was significantly suppressed by pretreatment with hydrangenol in a concentration-dependent manner (Fig. 2B). Consistent with the
decrease of iNOS protein expression, hydrangenol significantly attenuated LPS-induced upregulation of iNOS expression (Fig. 2C). These results demonstrate that hydrangenol possesses significant inhibitory effects on the LPS-induced iNOS expression and NO production.
3.3. Hydrangenol inhibits LPS-induced iNOS expression by suppressing NFκB activation and its nuclear translocation In order to examine the activity of NF-κB against iNOS expression, we conducted an EMSA and Western blot analysis. EMSA data confirmed that LPS treatment significantly increased the specific DNAbinding activity of NF-κB at 30 min; however, pretreatment with hydrangenol completely suppressed LPS-induced NF-κB activity (Fig. 3A). Additionally, we investigated whether hydrangenol regulates nuclear translocation of NF-κB subunits, p50 and p65, in LPS-treated BV2 microglial cells. Western blot analysis showed that LPS significantly increased the total amount of p50 and p65 in the nuclear extracts at 30 min (Fig. 3B, top) and relatively decreased p50 and p65 content in the cytosolic extracts (Fig. 3B, bottom), indicating that LPS promotes NF-κB activity by inducing nuclear translocation of the NF-κB subunits. The data also revealed that hydrangenol decreased LPS-induced nuclear translocation of p50 and p65 and sustained p50 and p65 levels in the cytosol. Next, we tested the functional effects of attenuating NF-κB activity by NF-κB inhibitors, PDTC and PS-1145. Both NF-κB inhibitors significantly decreased the expression levels of LPS-induced iNOS expression (Fig. 3C), suggesting that hydrangenol-mediated NF-κB inhibition is an
Fig. 2. Preventive effect of hydrangenol on LPS-induced NO production and iNOS expression in BV2 microglial cells. (A) BV2 microglial cells (1 × 105 cells/ml) were incubated with the indicated concentrations of hydrangenol for 2 h before LPS treatment (500 ng/ml) for 24 h. The amounts of NO were determined by Griess reaction and a standard curve was constructed using NaNO2. (B and C) In a parallel experiment, the cells (1 × 105 cells/ml) were incubated with the indicated concentrations of hydrangenol for 2 h before LPS (500 ng/ml) treatment for 24 h (Western blot analysis) and 6 h (PT-PCR). Cell lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against iNOS (B). Total RNA was isolated and RT-PCR analysis of iNOS was performed (C). β-Actin was used as an internal control for Western blot analyses and RTPCR. Each value indicates means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. untreated control and b, p b 0.05 vs. LPS-treated group).
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Fig. 3. Effect of hydrangenol on NF-κB activity in LPS-stimulated BV2 microglial cells. BV2 microglial cells were pre-incubated with the indicated concentrations of hydrangenol for 2 h before stimulation with LPS (500 ng/ml) for 30 min. (A) Nuclear extracts were assayed for NF-κB activity by electrophoretic mobility shift assay (EMSA). (B) The nuclear (top) and cytoplasmic (bottom) extracts were prepared to determine the expression levels of p65 and p50 using Western blot analysis. C-23 and β-actin were used as internal controls for the nuclear and cytosol extracts, respectively. (C) In a parallel experiment, the cells (1 × 105 cells/ml) were incubated with pyrrolidine dithiocarbamate (PDTC; 40 μM) and PS-1145 (20 μM) for 2 h before treatment with LPS (500 ng/ml) for 6 h. Total RNA was isolated and RT-PCR analysis of iNOS was performed. β-Actin was used as an internal control for RT-PCR. Each value indicates means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. LPS-treated group).
important factor contributing to the observed antagonistic effect on LPS-stimulated iNOS expression in BV2 microglial cells.
3.5. Hydrangenol-induced Nrf2 is an important regulator for HO-1 induction
3.4. Hydrangenol decreases NO release by inducing HO-1 expression
Since Nrf2 potentially activates antioxidant stress-related proteins such as HO-1, we investigated whether hydrangenol regulates Nrf2mediated HO-1 induction in LPS-stimulated BV2 microglial cells. According to EMSA data, Nrf2 activity significantly increased with hydrangenol treatment in a dose-dependent manner (Fig. 5A). Additionally, Western blot analysis confirmed that hydrangenol decreased Nrf2 levels in the cytoplasmic extract and gradually increased Nrf2 levels in the nuclear extract. This indicates that hydrangenol promotes the specific DNA-binding activity of Nrf2 by inducing nuclear translocation of Nrf2 (Fig. 5B). Next, we investigated whether hydrangenol-induced Nrf2 regulates the expression of HO-1 and subsequent production of NO using Nrf2 siRNA (siNrf2). Transient knockdown of Nrf2 significantly decreased hydrangenol-induced HO-1 mRNA expression (Fig. 5C). Finally, we investigated the production of NO in the condition of Nrf2 knockdown. Production of NO was 6.5 ± 0.5 μM in the control; however,
We examined the effects of hydrangenol on HO-1 expression in BV2 microglial cells because HO-1 activity is associated with the antiinflammatory response. According to Western blot analysis, hydrangenol induced HO-1 expression in a dose-dependent manner at 24 h (Fig. 4A). Similar to the expression of HO-1 protein, significant HO-1 mRNA expression was observed with hydrangenol at 6 h (Fig. 4B). Then, we assessed the level of NO release in the presence of the HO-1 inducer, CoPP. Pretreatment with CoPP significantly decreased LPS-induced NO release (17.8 ± 2.3 μM, 12.4 ± 1.2 μM, and 8.7 ± 1.5 μM at 0 μM, 2.5 μM, and 5.0 μM CoPP, respectively), indicating that one of the major roles of HO-1 is to decrease LPS-induced NO release (Fig. 4C). These data indicate that in the presence of hydrangenol, increased levels of HO-1 downregulate LPS-induced inflammatory response by reducing NO release.
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Fig. 4. Effect of hydrangenol-induced HO-1 expression on NO production in BV2 microglial cells. (A) BV2 microglial cells (1 × 105 cells/ml) were pretreated with the indicated concentrations of hydrangenol for 24 h. Equal amounts of cell lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against HO-1. (B) Total RNA was isolated at 6 h and an RT-PCR analysis of HO-1 was performed. β-Actin was used as an internal control for Western blot analysis and RT-PCR. Each value indicates means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. LPS-treated group). (C) In a parallel experiment, BV2 microglial cells were pretreated with the indicated concentrations of CoPP, which is an HO-1 inducer, for 2 h and then incubated with LPS (500 ng/ml) for 24 h. The amount of NO production in the medium was measured by Griess reaction. Each value indicates means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. respective CPT -treated group and b, p b 0.05 vs. LPS alone-treated group).
it increased two-fold when treated with siNrf2 (14.8 ± 1.2 μM), and LPS-induced NO production significantly increased in this state (22.1 ± 2.3 μM; Fig. 5D). This suggests that Nrf2 is an upstream regulator of HO-1 expression. The data indicate that Nrf2-mediated HO1 activation is an axis of the hydrangenol-mediated anti-inflammatory response in BV2 microglial cells. 3.6. Hydrangenol responds to treatment effect in LPS-mediated NO production Finally, we assessed whether hydrangenol influences curative effect in LPS-mediated NO production. LPS was incubated 2 h before treatment with hydrangenol and NO production was measured at 24 h. Treatment effect of hydrangenol only appeared at 40 μM and the effect was weaker than that of preventive effect shown in Fig. 2A (Fig. 6). Given our result, hydrangenol possesses weak treatment effect in LPSmediated NO production. 4. Discussion Since the discovery that leaf extract of H. macrophylla possesses antimalarial activity [19], many scientists have isolated active compounds from the plant. The processed leaves of H. macrophylla have also been recognized to possess nutritive value in experimental models of diabetes, in addition to having a pleasant sweet taste [20]. Recently, we demonstrated that the water extract of H. macrophylla leaves inhibits the
production of proinflammatory mediators in RAW264.7 macrophage cells, and isolated two bioactive compounds, hydrangenol and phyllodulcin from this extract [18]. In the current in vitro study, we investigated whether one of the bioactive compounds, hydrangenol, has potential as a pharmaceutical candidate for inflammatory response. The second candidate, phyllodulcin, is still undergoing research. The present study showed that hydrangenol attenuates NO production and the expression of its associated gene, iNOS. Moreover, hydrangenol showed weak inhibitory effects when given post-LPS administration. In addition, we found that hydrangenol regulates iNOS expression and NO release by suppressing NF-κB activity and by inducing Nrf2-mediated upregulation of HO-1 expression (Fig. 7). Nevertheless, we need future studies whether hydrangenol suppresses LPS-induced inflammation in primary microglia, and ultimately, in in vivo models, and long-term treatment of hydrangenol influences cytotoxicity. An accumulated number of studies have shown that NO and iNOS are involved in the pathogenesis of neurodegenerative disorders through various harmful pathways such as oxidative injury in microglia and early blood-brain barrier disruption [5–7]. Moreover, iNOS plays a pivotal role in NO production by oxidative deamination, and inhibition of iNOS significantly reverses neuroinflammatory responses in animal models [8,9]. The presented data confirmed that hydrangenol downregulates NO production and iNOS expression. These data indicate that hydrangenol may be a good preventive agent against LPS-mediated inflammatory responses. Additionally, hydrangenol weakly inhibited post-LPS treatment-induced NO production, which means that
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Fig. 5. Effect of hydrangenol-induced Nrf2 in LPS-stimulated BV2 microglial cells. BV2 microglial cells (1 × 105 cells/ml) were incubated with the indicated concentrations of hydrangenol for 2 h before LPS stimulation for 30 min. (A) Nuclear extracts were prepared to analyze antioxidant response element (ARE)-binding of Nrf2 by electrophoretic mobility shift assay (EMSA). Statistical significance was determined by a one-way ANOVA test (a, p b 0.05 vs. untreated control). (B) For Western blot analysis, equal amounts of nuclear (top) and cytosolic (bottom) lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against Nrf2. C-23 and β-actin were used as nuclear and cytosol internal controls, respectively. Statistical significance was determined by a one-way ANOVA test (a, p b 0.05 vs. untreated control). (C) BV2 microglial cells were transiently transfected with Nrf2 siRNA (siNrf2) and then treated with or without hydrangenol (40 μM) for 24 h. Total RNA was isolated at 6 h and RT-PCR analysis of HO-1 was performed. β-Actin was used as an internal control for RT-PCR. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. siNrf2-treated group). (D) BV2 microglial cells were transiently transfected with siNrf2 for 24 h and then treated with 40 μM hydrangenol in the presence or absence of LPS (500 ng/ml). The amount of NO production in the medium was measured using the Griess reaction. Each value indicates means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. untreated control and b, p b 0.05 vs. respective siNrf2-untreated group).
hydrangenol shows treatable influence in LPS-induced NO production. Some recent research indicates that NO is essential for neuroprotection
Fig. 6. Treatment effect of hydrangenol on LPS-induced NO production in BV2 microglial cells. BV2 microglial cells (1 × 105 cells/ml) were incubated with 500 ng/ml LPS 2 h before treatment with the indicated concentrations of hydrangenol for 24 h. The amounts of NO were determined by Griess reaction and a standard curve was constructed using NaNO2. Each value indicates means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. LPS-untreated group).
and development of the brain [21,22]. In particular, Gulati and Singh demonstrated that pretreatment with a nonselective NOS inhibitor, LNAME, significantly diminished NO-mediated neuroprotective effects [21]. Additionally, NO-mediated S-nitrosylation of proteins is a prerequisite for neuronal differentiation and maturation [22]. Although there is a lot of discrepancy regarding the functions of NO in neuroinflammation, it has been shown that iNOS inhibition is an important pathway in hydrangenol-induced anti-inflammatory response because LPSstimulated iNOS but not neuronal NOS (nNOS) expression has neurotoxic effects [5]. NF-κB is one of the key transcription factors regulating proinflammatory genes such as iNOS in neuroinflammatory diseases [5,6]. Although the role of severe neuroinflammatory processes in diseases such as Alzheimer's are difficult to understand fully, a widely established theory is that the disease causes neuronal cell death through activation of NF-κB gene products [23]. In this regard, the current study also shows that hydrangenol isolated from H. macrophylla may be a good therapeutic agent against neuroinflammatory diseases by suppressing NF-κB activity. Nevertheless, there still exists the enigmatic problem concerning the function of NF-κB, since the NF-κB signaling
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Fig. 7. Scheme of the anti-inflammatory effect of hydrangenol in LPS-stimulated BV2 microglial cells. Hydrangenol inhibits LPS-induced NF-κB activation by suppressing nuclear translocation of its respective subunits, p65 and p50 and simultaneously activates Nrf2 activity to express HO-1. Consequently, hydrangenol-induced HO-1 blocks NO production in LPS-stimulated BV2 microglial cells. LPS, lipopolysaccharide; C.M., cytoplasmic membrane; iNOS, inducible nitric oxide synthase; NO, nitric oxide; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-related factor 2.
pathway is indispensable in the process of postnatal neurogenesis [24]. Conversely, Nrf2 is a key factor in the oxidative- or stress-induced endogenous defense system of cells. Under stressful conditions, Nrf2 translocates to the nucleus and binds a specific site on the antioxidant response element to promote transcription of cytoprotective genes such as HO-1, which protect against acute cerebral insults and neurodegenerative diseases [25]. In particular, genetic deletion and dysfunction of Nrf2 have been observed in neurodegenerative diseases, whereas upregulated Nrf2 delayed the onset of neuroinflammation and extended survival in a mouse model by inducing HO-1 [26]. Our results indicate that hydrangenol increases the expression of Nrf2-mediated HO-1, which is in turn responsible for the inhibition of NO in LPS-simulated BV2 microglial cells. Interestingly, cross talk between NF-κB and Nrf2 has been reported; the NF-κB subunit, p65, induces ubiquitinationmediated degradation of Nrf2 and consequently diminishes Nrf2 binding to its associated DNA sequence [27]. Nevertheless, research into the activation of the Nrf2 pathway is a promising step towards curing inflammatory diseases, including brain disorders. In summary, we showed that hydrangenol inhibits NO production in LPS-stimulated BV2 microglial cells through suppression of their regulatory genes. In addition, the anti-inflammatory effects of hydrangenol are associated with suppression of NF-κB and enhanced Nrf2-mediated HO1 activation. Post-LPS administration-mediated NO production was also weakly inhibited in response to hydrangenol, suggesting that hydrangenol possesses preventive and curable effect against LPSinduced inflammation by inducing NO production. Taken together, we conclude that hydrangenol has potential as a novel anti-inflammatory drug. Nevertheless, we found preventive effect of hydrangenol in LPSstimulated response, not treatable efficacy. We also need further study to yield clinically relevant a result because our data validated that hydrangenol regulates NO production and iNOS expression in vitro. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment This study was supported by Basic Science Research Program (2015R1D1A1A01060538) through the National Research Foundation of Korea (NRF) funded from the Ministry of Education, Science and Technology of Korea.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Hydrangenol inhibits lipopolysaccharide-induced nitric oxide production in BV2 microglial cells by suppressing the NF-κB pathway and activating the Nrf2-mediated HO-1 pathway Hee-Ju Kim a, Chang-Hee Kang a,b, Rajapaksha Gedara Prasad Tharanga Jayasooriya a, Matharage Gayani Dilshara a, Seungheon Lee a, Yung Hyun Choi c, Yong Taek Seo d, Gi-Young Kim a,⁎ a
Department of Marine Life Sciences, Jeju National University, Jeju 63243, Republic of Korea Nakdonggang National Institute of Biological Resource, Sangju-si, Gyeongsangbuk-do 37242, Republic of Korea c Department of Biochemistry, College of Oriental Medicine, Dong-Eui University, Busan 47227, Republic of Korea d Consilience Medical Center, Dankook University Hospital, Chungcheongnam-do 31116, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 3 November 2015 Received in revised form 15 March 2016 Accepted 17 March 2016 Available online xxxx Keywords: Hydrangenol Nitric oxide Nuclear factor-κB Heme oxygenase-1 Nuclear factor erythroid 2-related factor 2
a b s t r a c t We previously demonstrated the anti-inflammatory effect of water extract of Hydrangea macrophylla in lipopolysaccharide (LPS)-stimulated macrophage cells. Here, we investigated whether hydrangenol, a bioactive component of H. macrophylla, attenuates the expression of nitric oxide (NO) and its associated gene, inducible NO synthase (iNOS), in LPS-stimulated BV2 microglial cells. Our data showed that low dosages of hydrangenol inhibited LPS-stimulated NO release and iNOS expression without any accompanying cytotoxicity. Hydrangenol also suppressed LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) subunits, consequently inhibiting DNA-binding activity of NF-κB. Additionally, the NF-κB inhibitors, pyrrolidine dithiocarbamate (PDTC) and PS-1145, significantly attenuated LPS-induced iNOS expression, indicating that hydrangenolinduced NF-κB inhibition might be a key regulator of iNOS expression. Furthermore, our data showed that hydrangenol suppresses NO production by inducing heme oxygenase-1 (HO-1). The presence of cobalt protoporphyrin, a specific HO-1 inducer, potently suppressed LPS-induced NO production. Hydrangenol also promoted nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and subsequently increased its binding activity at the specific antioxidant response element sites. Additionally, transient knockdown of Nrf2 significantly downregulated hydrangenol-induced HO-1 expression, indicating that hydrangenol-induced Nrf2 is an upstream regulator of HO-1. Taken together, these data suggest that hydrangenol attenuates NO production and iNOS expression in LPS-stimulated BV2 microglial cells by inhibiting NF-κB activation and by stimulating the Nrf2-mediated HO-1 signaling pathway. Therefore, hydrangenol is a promising therapeutic agent for treatment of LPS-mediated inflammatory diseases. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The intrinsic function of microglial cells is to protect the central nervous system by removing dead cells and tissue debris through phagocytosis [1]. In response to injury or infection, microglia become readily activated and secrete proinflammatory mediators such as nitric oxide (NO), cyclooxgenase-2 (COX-2), interleukin-1 beta (IL-1β), and IL-6, which are essential to regulate cellular signaling involved in protection against organ injury, such as ischemic damage [2]. Nevertheless, recent studies showed that excessive and abnormal production of proinflammatory mediators result in systemic inflammatory syndrome, severe tissue damage, atherosclerosis, and septic shock [3,4]. Among the proinflammatory mediators secreted by microglia, NO is well known to ⁎ Corresponding author. E-mail address: [email protected] (G.-Y. Kim).
http://dx.doi.org/10.1016/j.intimp.2016.03.022 1567-5769/© 2016 Elsevier B.V. All rights reserved.
aggravate cerebral ischemia [1]. Normally, NO plays a role in neuroprotective and pathophysiological processes by regulating resting blood flow [5]; however, excessive NO release promotes early blood-brain barrier disruption [6], as well as causes oxidative injury in microglia, but not in astrocytes [7]. In addition, the NO-regulating gene, inducible NO synthase (iNOS), stimulates peroxynitrite formation, which promote protein radicals in microglia-mediated neurodegenerative disorders [8]. Li et al., demonstrated that silencing iNOS expression protected against neurodegeneration of nigrostriatal dopaminergic neurons in an animal model of Parkinson's disease [9]. As a result, many researchers have attempted to identify phytochemicals that suppress the aberrant NO production and iNOS expression, and determine their therapeutic characteristics in the context of neurodegenerative diseases. Nuclear factor-κB (NF-κB) is a transcription factor known to directly regulate many proinflammatory genes such as iNOS [10]. During inflammatory responses, inhibitor of κB (IκB) is degraded through its
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phosphorylation and ubiquitination; consequently, free NF-κB is released and translocated to the nucleus to transcribe proinflammatory genes such as iNOS [11]. Therefore, NF-κB is a good strategic target for modulating inflammatory responses and diseases. So far, many researchers have reported a variety of natural and designed molecules, including proteasome inhibitors, small molecules, active polypeptides, and flavonoids, which target NF-κB, leading to suppress iNOSmediated inflammatory diseases [10,12]. Additionally, recent studies reported that heme oxygenase-1 (HO-1) is induced in most tissues by a variety of oxidative stimuli and is involved in the protection against different types of oxidant-induced tissue damage and cellular injury; however, HO-2 is constitutively expressed [10,11]. It has been also shown that HO-1 has important immune-modulatory and anti-inflammatory functions through regulation of the proinflammatory mediators, NO and iNOS [12]. Nuclear transcription factor erythroid 2-related factor-2 (Nrf2) is a redox-sensitive transcription factor responsible for the induction of antioxidant enzymes. Recent reports found that Nrf2 regulates major immune-modulatory and anti-inflammatory properties via HO-1 induction [13,14]. Currently, Hydrangea macrophylla is used in two medicinal parts, as an oral antipyretic and as an alternative sweetener for diabetic patients [15]. Additionally, there has been growing interest in H. macrophylla since it contains febrifugine and its analogues, which target the cytoplasmic prolyl-tRNA synthetase of the malaria parasites [16,17]. Furthermore, our previous study showed that water extract from processed H. macrophylla leaves possesses anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages [18]. The present study investigated the inhibition of NO production and iNOS expression by hydrangenol, isolated from H. macrophylla, via suppression of NF-κB activity in LPS-stimulated BV2 microglial cells. In addition, antagonistic mechanism of hydrangenol is associated with the induction of Nrf2-mediated HO-1 expression.
2. Materials and methods 2.1. Reagents and antibodies Rabbit anti-human antibodies against iNOS, p65, p50, C-23, HO-1, and Nrf2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin antibody, LPS, and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO). Peroxidase-labeled goat anti-rabbit immunoglobulin was purchased from KOMA Biotechnology (Seoul, Republic of Korea). Pyrrolidine dithiocarbamate (PDTC) and PS-1145 were purchased from Calbiochem (San Diego, CA). Cobalt protoporphyrin (CoPP) was purchased from Tocris Bioscience (Bristol, UK). Dulbecco's Modified Eagle's medium (DMEM), fetal bovine serum (FBS), and antibiotic mixtures were obtained from WelGENE Inc. (Daegu, Republic of Korea). Other chemicals were purchased as Sigma grades. Hydrangenol was isolated in our previous study [16] and the structure of hydrangenol is illustrated in Fig. 1A.
2.2. Cell culture and viability BV2 microglial cells were cultured in DMEM media containing 5% FBS and antibiotic mixtures, and incubated at 37 °C and 5% CO2 conditions. MTT assay was performed to determine relative cell viability. Briefly, BV2 microglial cells (1 × 105 cells/ml) were treated with various concentrations (0–100 μM) of hydrangenol for 2 h before treatment with LPS (500 ng/ml). In a parallel experiment, 500 μM hydrogen peroxide (H2O2) were treated 2 h prior to LPS treatment as a apoptotic positive control. After 24-h incubation, the cells were incubated with MTT solution (0.5 mg/ml) for 15 min at 37 °C. Supernatant was removed and the formation of formazan was observed by monitoring the signal
Fig. 1. Effect of hydrangenol on the viability of BV2 microglial cells. (A) Chemical structure of hydrangenol. BV2 microglial cells (1 × 105 cells/ml) were incubated with the indicated concentrations of hydrangenol (0–100 μM) for 2 h before treatment with LPS (500 ng/ml) for 24 h. (B) Cell viability was determined using an MTT assay. (C) In a parallel experiment, 40 μM hydrangenol was treated 24 h before 500 ng/ml LPS treatment. As a positive cell death-inducing control, 500 μM hydrogen peroxide (H2O2) was treated instead of hydrangenol. The percentage of annexin V+-cell population (top panel) and sub-G1 DNA content (bottom panel) are indicated in each panel. Each value indicates the means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a one-way ANOVA test (a, p b 0.05 vs. untreated control).
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at 540 nm using a microplate reader (Thermo Electron Corporation, Marietta, OH). 2.3. Flow cytometric analysis BV2 microglial cells were pretreated with hydrangenol for 2 h and then administered with 500 ng/ml LPS for 24 h. After harvesting, the cells were washed two times with phosphate buffer saline (PBS) and fixed with 1 U/ml RNase A (DNase free) and 10 μg/ml propidium iodide (PI, Sigma) for 1 h at room temperature in the dark. For annexin V staining, the live cells were incubated with annexin V-FITC (R&D systems, Minneapolis, MN) according manufacturer's instructions. A FACSCalibur flow cytometer (Becton Dickenson, San Jose, CA) was used to analyze the level of apoptotic cells containing sub-G1 DNA content and annexin V-FITC+ population. 2.4. NO production BV2 microglial cells (1 × 105 cells/ml) were dispensed on to 24 well plates and pretreated with the indicated concentrations of hydrangenol 2 h prior to stimulation with 500 ng/ml LPS for 24 h. Supernatants were collected and assayed for NO production by Griess reaction. Briefly, the samples were mixed with equal volume of Griess reagent (1% sulfanilamide in 5% acetic acid and 0.1% naphthylethylenediamine dihydrochloride) and then incubated at room temperature for 10 min. The absorbance was measured at 540 nm on a microplate reader. Sodium nitrite dilution series were used as a standard to determine the nitrite concentration in the supernatants. 2.5. Isolation of total RNA and RT-PCR Total RNA was extracted using an easy-BLUE kit (iNtRON Biotechnology, Sungnam, Republic of Korea) according to the manufacturer's instruction. One microgram of RNA was reverse-transcribed using moloney murine leukemia virus (MMLV) reverse transcriptase (Bioneer, Daejeon, Republic of Korea). cDNA was amplified by PCR using the specific primers, iNOS (forward 5′-CCTCCTCCACCCTACCAA GT-3′ and reverse 5′-CACCCAAACTGCTTCAGTCA-3′), HO-1 (forward 5′-TCGCCAGAAAGCTGAGTATAA-3′ and reverse 5′-ATTGCCAGTGCCAC CACCAAGTTCAAG-3′), and β-actin (forward 5′-TGTGATGGTGGGAATG GGTC-3′ and reverse 5′-TTTGATGTCACGCACGATTT-3′). The following PCR conditions were applied: iNOS and HO-1, 25 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s and extended at 72 °C for 30 s; β-actin, 23 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s and extended at 72 °C for 30 s. 2.6. Western blot analysis BV2 microglial cells were seeded at the density of 1 × 105 cells/ml and treated with the indicated compounds. After 24 h-incubation, total cell extracts were prepared using a PROPREP protein extraction kit (iNtRON Biotechnology). Briefly, the PROPREP solution was treated to the cells on the ice for 30 min and lysates were centrifuged at 14,000 ×g at 4 °C for 10 min to obtain the supernatants. In a parallel experiment, cytoplasmic and nuclear extracts were prepared from the cells using NE-PER nuclear and cytosolic extraction reagents (Pierce, Rockford, IL). The supernatants were collected and protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). The samples were stored at −80 °C or immediately used for Western blot analysis. The proteins were separated on SDSpolyacrylamide gels and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Finally, proteins were detected using an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL).
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2.7. Electrophoretic mobility shift assay (EMSA) BV2 microglial cells were seeded at the density of 1 × 105 cells/ml and treated with the indicated compounds for 30 min. Total cells were suspended with cytosolic extraction reagent and incubated at 4 °C for 30 min. Then, the pellets were resuspended with NE-PER nuclear reagent for EMSA. Synthetic complementary NF-κB (5′-AGTTGAGGGGAC TTTCCCAGGC-3′) binding oligonucleotides (Santa Cruz Biotechnology) and anti-oxidant response elements (ARE) consensus (5′TMANNRTGAYNNNGCRWWWW-3′) were 3′-biotinylated using the biotin 3′-end DNA labeling kit (Pierce) according to the manufacturer's instructions and annealed for 30 min at room temperature. Nuclear extracts and 3′-labeled complementary oligonucleotides were incubated with NE-PER-binding buffer for 1 h. Assays were loaded onto native 4% polyacrylamide gels pre-electrophoresed for 1 h in 0.5 × Tris borate/EDTA before being transferred onto a positively charged nylon membrane (HybondTM-N +) in 0.5 × Tris borate/EDTA at 100 V for 30 min. The transferred DNAs were cross-linked to the membrane at 120 mJ/cm 2 . Horseradish peroxidase-conjugated streptavidin was used according to the manufacturer's instructions to detect the transferred DNA. 2.8. Transient knockdown of Nrf2 BV2 microglial cells were seeded on a 24-well plate at a density of 1 × 105 cells/ml and transfected Nrf2-specific silencing RNA (siRNA, Santa Cruz Biotechnology) for 24 h. For each transfection, 450 μl growth medium was added to 20 nM siRNA duplex with the transfection reagent G-Fectin (Genolution Pharmaceuticals Inc., Seoul, Republic of Korea) and the entire mixture was added gently to the cells. 2.9. Statistical analysis The images were visualized with Chemi-Smart 2000 (Vilber Lourmat, Marine, Cedex, France). Images were captured using ChemiCapt (Vilber Lourmat) and transported into Photoshop. All bands were shown a representative obtained in three independent experiments and quantified by Scion Imaging software (http://www.scioncorp. com). Statistical analyses were conducted using SigmaPlot software (version 12.0). Values were presented as mean ± S.E. of three experiments. Significant differences between the groups were determined by one-way or two-way ANOVA followed with Bonferroni's test. Statistical significance was regarded at a and b, p b 0.05. 3. Results 3.1. Hydrangenol has no influence on the viability of BV2 microglial cells In order to first determine the effect of hydrangenol on cell viability, we pretreated BV2 microglial cells with hydrangenol for 2 h before LPS treatment for 24 h. MTT data showed that cell viability was not significantly altered by hydrangenol at concentrations up to 40 μM at 24 h. However, treatment with concentrations above 50 μM hydrangenol gradually decreased the viability of BV2 microglial cells in a dosedependent manner (Fig. 1B). These results suggested that concentrations of hydrangenol below 40 μM were non-toxic to BV2 microglial cells. Thus, further experiments were carried out with hydrangenol at concentrations below 40 μM. Next, flow cytometric analysis performed to evaluate hydrangenol cytotoxicity in more detail. For the comparative positive control, 500 μM H2O2 were treated for 24 h to induced apoptosis. Data obtained using annexin V staining showed that of the H2O2-treated BV2 microglial cells, 38 ± 5% were annexin V+ or apoptotic; however, a minor population of hydrangenol-pretreated BV2 microglial cells were found to be annexin V+, thereby suggesting that hydrangenol has no influence on BV2 microglial cell apoptosis (Fig. 1C, top). In a parallel experiment, we also analyzed hydrangenol
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cytotoxicity based on the amount of DNA in the sub-G1 phase. Apoptotic cell death was not detected in any of the panels of cells treated with hydrangenol, contrary to that observed for the H2O2-treated positive control group (Fig. 1C, bottom). Taken together, these data indicate that hydrangenol (≤ 40 μM) has no influence on the viability of BV2 microglial cells. 3.2. Hydrangenol inhibits NO production and iNOS expression in LPSstimulated BV2 microglial cells In order to analyze the suppressive effect of hydrangenol on NO production, BV2 microglial cells were pretreated with different concentrations of hydrangenol for 2 h, followed by treatment with LPS for 24 h, before determination of the levels of NO in the culture media by Griess assay. LPS markedly increased NO production in BV2 microglial cells (18.9 ± 2.7 μM; Fig. 2A). On the other hand, hydrangenol significantly decreased NO production in LPS-stimulated BV2 microglial cells in a dose-dependent manner (13.1 ± 2.2 μM, 10.9 ± 1.5 μM, and 8.8 ± 2.1 μM at 10 μM, 20 μM, and 40 μM hydrangenol, respectively). In particular, 20 μM and 40 μM hydrangenol significantly decreased the LPSinduced NO production; however, this downregulation did not reach the level observed for untreated control (4.2 ± 1.2 μM). Additionally, we investigated whether hydrangenol regulates LPS-stimulated iNOS mRNA expression. Western blot analysis showed that treatment with LPS markedly increased iNOS protein expression at 24 h and this was significantly suppressed by pretreatment with hydrangenol in a concentration-dependent manner (Fig. 2B). Consistent with the
decrease of iNOS protein expression, hydrangenol significantly attenuated LPS-induced upregulation of iNOS expression (Fig. 2C). These results demonstrate that hydrangenol possesses significant inhibitory effects on the LPS-induced iNOS expression and NO production.
3.3. Hydrangenol inhibits LPS-induced iNOS expression by suppressing NFκB activation and its nuclear translocation In order to examine the activity of NF-κB against iNOS expression, we conducted an EMSA and Western blot analysis. EMSA data confirmed that LPS treatment significantly increased the specific DNAbinding activity of NF-κB at 30 min; however, pretreatment with hydrangenol completely suppressed LPS-induced NF-κB activity (Fig. 3A). Additionally, we investigated whether hydrangenol regulates nuclear translocation of NF-κB subunits, p50 and p65, in LPS-treated BV2 microglial cells. Western blot analysis showed that LPS significantly increased the total amount of p50 and p65 in the nuclear extracts at 30 min (Fig. 3B, top) and relatively decreased p50 and p65 content in the cytosolic extracts (Fig. 3B, bottom), indicating that LPS promotes NF-κB activity by inducing nuclear translocation of the NF-κB subunits. The data also revealed that hydrangenol decreased LPS-induced nuclear translocation of p50 and p65 and sustained p50 and p65 levels in the cytosol. Next, we tested the functional effects of attenuating NF-κB activity by NF-κB inhibitors, PDTC and PS-1145. Both NF-κB inhibitors significantly decreased the expression levels of LPS-induced iNOS expression (Fig. 3C), suggesting that hydrangenol-mediated NF-κB inhibition is an
Fig. 2. Preventive effect of hydrangenol on LPS-induced NO production and iNOS expression in BV2 microglial cells. (A) BV2 microglial cells (1 × 105 cells/ml) were incubated with the indicated concentrations of hydrangenol for 2 h before LPS treatment (500 ng/ml) for 24 h. The amounts of NO were determined by Griess reaction and a standard curve was constructed using NaNO2. (B and C) In a parallel experiment, the cells (1 × 105 cells/ml) were incubated with the indicated concentrations of hydrangenol for 2 h before LPS (500 ng/ml) treatment for 24 h (Western blot analysis) and 6 h (PT-PCR). Cell lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against iNOS (B). Total RNA was isolated and RT-PCR analysis of iNOS was performed (C). β-Actin was used as an internal control for Western blot analyses and RTPCR. Each value indicates means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. untreated control and b, p b 0.05 vs. LPS-treated group).
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Fig. 3. Effect of hydrangenol on NF-κB activity in LPS-stimulated BV2 microglial cells. BV2 microglial cells were pre-incubated with the indicated concentrations of hydrangenol for 2 h before stimulation with LPS (500 ng/ml) for 30 min. (A) Nuclear extracts were assayed for NF-κB activity by electrophoretic mobility shift assay (EMSA). (B) The nuclear (top) and cytoplasmic (bottom) extracts were prepared to determine the expression levels of p65 and p50 using Western blot analysis. C-23 and β-actin were used as internal controls for the nuclear and cytosol extracts, respectively. (C) In a parallel experiment, the cells (1 × 105 cells/ml) were incubated with pyrrolidine dithiocarbamate (PDTC; 40 μM) and PS-1145 (20 μM) for 2 h before treatment with LPS (500 ng/ml) for 6 h. Total RNA was isolated and RT-PCR analysis of iNOS was performed. β-Actin was used as an internal control for RT-PCR. Each value indicates means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. LPS-treated group).
important factor contributing to the observed antagonistic effect on LPS-stimulated iNOS expression in BV2 microglial cells.
3.5. Hydrangenol-induced Nrf2 is an important regulator for HO-1 induction
3.4. Hydrangenol decreases NO release by inducing HO-1 expression
Since Nrf2 potentially activates antioxidant stress-related proteins such as HO-1, we investigated whether hydrangenol regulates Nrf2mediated HO-1 induction in LPS-stimulated BV2 microglial cells. According to EMSA data, Nrf2 activity significantly increased with hydrangenol treatment in a dose-dependent manner (Fig. 5A). Additionally, Western blot analysis confirmed that hydrangenol decreased Nrf2 levels in the cytoplasmic extract and gradually increased Nrf2 levels in the nuclear extract. This indicates that hydrangenol promotes the specific DNA-binding activity of Nrf2 by inducing nuclear translocation of Nrf2 (Fig. 5B). Next, we investigated whether hydrangenol-induced Nrf2 regulates the expression of HO-1 and subsequent production of NO using Nrf2 siRNA (siNrf2). Transient knockdown of Nrf2 significantly decreased hydrangenol-induced HO-1 mRNA expression (Fig. 5C). Finally, we investigated the production of NO in the condition of Nrf2 knockdown. Production of NO was 6.5 ± 0.5 μM in the control; however,
We examined the effects of hydrangenol on HO-1 expression in BV2 microglial cells because HO-1 activity is associated with the antiinflammatory response. According to Western blot analysis, hydrangenol induced HO-1 expression in a dose-dependent manner at 24 h (Fig. 4A). Similar to the expression of HO-1 protein, significant HO-1 mRNA expression was observed with hydrangenol at 6 h (Fig. 4B). Then, we assessed the level of NO release in the presence of the HO-1 inducer, CoPP. Pretreatment with CoPP significantly decreased LPS-induced NO release (17.8 ± 2.3 μM, 12.4 ± 1.2 μM, and 8.7 ± 1.5 μM at 0 μM, 2.5 μM, and 5.0 μM CoPP, respectively), indicating that one of the major roles of HO-1 is to decrease LPS-induced NO release (Fig. 4C). These data indicate that in the presence of hydrangenol, increased levels of HO-1 downregulate LPS-induced inflammatory response by reducing NO release.
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Fig. 4. Effect of hydrangenol-induced HO-1 expression on NO production in BV2 microglial cells. (A) BV2 microglial cells (1 × 105 cells/ml) were pretreated with the indicated concentrations of hydrangenol for 24 h. Equal amounts of cell lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against HO-1. (B) Total RNA was isolated at 6 h and an RT-PCR analysis of HO-1 was performed. β-Actin was used as an internal control for Western blot analysis and RT-PCR. Each value indicates means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. LPS-treated group). (C) In a parallel experiment, BV2 microglial cells were pretreated with the indicated concentrations of CoPP, which is an HO-1 inducer, for 2 h and then incubated with LPS (500 ng/ml) for 24 h. The amount of NO production in the medium was measured by Griess reaction. Each value indicates means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. respective CPT -treated group and b, p b 0.05 vs. LPS alone-treated group).
it increased two-fold when treated with siNrf2 (14.8 ± 1.2 μM), and LPS-induced NO production significantly increased in this state (22.1 ± 2.3 μM; Fig. 5D). This suggests that Nrf2 is an upstream regulator of HO-1 expression. The data indicate that Nrf2-mediated HO1 activation is an axis of the hydrangenol-mediated anti-inflammatory response in BV2 microglial cells. 3.6. Hydrangenol responds to treatment effect in LPS-mediated NO production Finally, we assessed whether hydrangenol influences curative effect in LPS-mediated NO production. LPS was incubated 2 h before treatment with hydrangenol and NO production was measured at 24 h. Treatment effect of hydrangenol only appeared at 40 μM and the effect was weaker than that of preventive effect shown in Fig. 2A (Fig. 6). Given our result, hydrangenol possesses weak treatment effect in LPSmediated NO production. 4. Discussion Since the discovery that leaf extract of H. macrophylla possesses antimalarial activity [19], many scientists have isolated active compounds from the plant. The processed leaves of H. macrophylla have also been recognized to possess nutritive value in experimental models of diabetes, in addition to having a pleasant sweet taste [20]. Recently, we demonstrated that the water extract of H. macrophylla leaves inhibits the
production of proinflammatory mediators in RAW264.7 macrophage cells, and isolated two bioactive compounds, hydrangenol and phyllodulcin from this extract [18]. In the current in vitro study, we investigated whether one of the bioactive compounds, hydrangenol, has potential as a pharmaceutical candidate for inflammatory response. The second candidate, phyllodulcin, is still undergoing research. The present study showed that hydrangenol attenuates NO production and the expression of its associated gene, iNOS. Moreover, hydrangenol showed weak inhibitory effects when given post-LPS administration. In addition, we found that hydrangenol regulates iNOS expression and NO release by suppressing NF-κB activity and by inducing Nrf2-mediated upregulation of HO-1 expression (Fig. 7). Nevertheless, we need future studies whether hydrangenol suppresses LPS-induced inflammation in primary microglia, and ultimately, in in vivo models, and long-term treatment of hydrangenol influences cytotoxicity. An accumulated number of studies have shown that NO and iNOS are involved in the pathogenesis of neurodegenerative disorders through various harmful pathways such as oxidative injury in microglia and early blood-brain barrier disruption [5–7]. Moreover, iNOS plays a pivotal role in NO production by oxidative deamination, and inhibition of iNOS significantly reverses neuroinflammatory responses in animal models [8,9]. The presented data confirmed that hydrangenol downregulates NO production and iNOS expression. These data indicate that hydrangenol may be a good preventive agent against LPS-mediated inflammatory responses. Additionally, hydrangenol weakly inhibited post-LPS treatment-induced NO production, which means that
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Fig. 5. Effect of hydrangenol-induced Nrf2 in LPS-stimulated BV2 microglial cells. BV2 microglial cells (1 × 105 cells/ml) were incubated with the indicated concentrations of hydrangenol for 2 h before LPS stimulation for 30 min. (A) Nuclear extracts were prepared to analyze antioxidant response element (ARE)-binding of Nrf2 by electrophoretic mobility shift assay (EMSA). Statistical significance was determined by a one-way ANOVA test (a, p b 0.05 vs. untreated control). (B) For Western blot analysis, equal amounts of nuclear (top) and cytosolic (bottom) lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against Nrf2. C-23 and β-actin were used as nuclear and cytosol internal controls, respectively. Statistical significance was determined by a one-way ANOVA test (a, p b 0.05 vs. untreated control). (C) BV2 microglial cells were transiently transfected with Nrf2 siRNA (siNrf2) and then treated with or without hydrangenol (40 μM) for 24 h. Total RNA was isolated at 6 h and RT-PCR analysis of HO-1 was performed. β-Actin was used as an internal control for RT-PCR. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. siNrf2-treated group). (D) BV2 microglial cells were transiently transfected with siNrf2 for 24 h and then treated with 40 μM hydrangenol in the presence or absence of LPS (500 ng/ml). The amount of NO production in the medium was measured using the Griess reaction. Each value indicates means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. untreated control and b, p b 0.05 vs. respective siNrf2-untreated group).
hydrangenol shows treatable influence in LPS-induced NO production. Some recent research indicates that NO is essential for neuroprotection
Fig. 6. Treatment effect of hydrangenol on LPS-induced NO production in BV2 microglial cells. BV2 microglial cells (1 × 105 cells/ml) were incubated with 500 ng/ml LPS 2 h before treatment with the indicated concentrations of hydrangenol for 24 h. The amounts of NO were determined by Griess reaction and a standard curve was constructed using NaNO2. Each value indicates means ± S.E. and is representative of results obtained from three independent experiments. Statistical significance was determined by a two-way ANOVA test (a, p b 0.05 vs. LPS-untreated group).
and development of the brain [21,22]. In particular, Gulati and Singh demonstrated that pretreatment with a nonselective NOS inhibitor, LNAME, significantly diminished NO-mediated neuroprotective effects [21]. Additionally, NO-mediated S-nitrosylation of proteins is a prerequisite for neuronal differentiation and maturation [22]. Although there is a lot of discrepancy regarding the functions of NO in neuroinflammation, it has been shown that iNOS inhibition is an important pathway in hydrangenol-induced anti-inflammatory response because LPSstimulated iNOS but not neuronal NOS (nNOS) expression has neurotoxic effects [5]. NF-κB is one of the key transcription factors regulating proinflammatory genes such as iNOS in neuroinflammatory diseases [5,6]. Although the role of severe neuroinflammatory processes in diseases such as Alzheimer's are difficult to understand fully, a widely established theory is that the disease causes neuronal cell death through activation of NF-κB gene products [23]. In this regard, the current study also shows that hydrangenol isolated from H. macrophylla may be a good therapeutic agent against neuroinflammatory diseases by suppressing NF-κB activity. Nevertheless, there still exists the enigmatic problem concerning the function of NF-κB, since the NF-κB signaling
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Fig. 7. Scheme of the anti-inflammatory effect of hydrangenol in LPS-stimulated BV2 microglial cells. Hydrangenol inhibits LPS-induced NF-κB activation by suppressing nuclear translocation of its respective subunits, p65 and p50 and simultaneously activates Nrf2 activity to express HO-1. Consequently, hydrangenol-induced HO-1 blocks NO production in LPS-stimulated BV2 microglial cells. LPS, lipopolysaccharide; C.M., cytoplasmic membrane; iNOS, inducible nitric oxide synthase; NO, nitric oxide; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-related factor 2.
pathway is indispensable in the process of postnatal neurogenesis [24]. Conversely, Nrf2 is a key factor in the oxidative- or stress-induced endogenous defense system of cells. Under stressful conditions, Nrf2 translocates to the nucleus and binds a specific site on the antioxidant response element to promote transcription of cytoprotective genes such as HO-1, which protect against acute cerebral insults and neurodegenerative diseases [25]. In particular, genetic deletion and dysfunction of Nrf2 have been observed in neurodegenerative diseases, whereas upregulated Nrf2 delayed the onset of neuroinflammation and extended survival in a mouse model by inducing HO-1 [26]. Our results indicate that hydrangenol increases the expression of Nrf2-mediated HO-1, which is in turn responsible for the inhibition of NO in LPS-simulated BV2 microglial cells. Interestingly, cross talk between NF-κB and Nrf2 has been reported; the NF-κB subunit, p65, induces ubiquitinationmediated degradation of Nrf2 and consequently diminishes Nrf2 binding to its associated DNA sequence [27]. Nevertheless, research into the activation of the Nrf2 pathway is a promising step towards curing inflammatory diseases, including brain disorders. In summary, we showed that hydrangenol inhibits NO production in LPS-stimulated BV2 microglial cells through suppression of their regulatory genes. In addition, the anti-inflammatory effects of hydrangenol are associated with suppression of NF-κB and enhanced Nrf2-mediated HO1 activation. Post-LPS administration-mediated NO production was also weakly inhibited in response to hydrangenol, suggesting that hydrangenol possesses preventive and curable effect against LPSinduced inflammation by inducing NO production. Taken together, we conclude that hydrangenol has potential as a novel anti-inflammatory drug. Nevertheless, we found preventive effect of hydrangenol in LPSstimulated response, not treatable efficacy. We also need further study to yield clinically relevant a result because our data validated that hydrangenol regulates NO production and iNOS expression in vitro. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment This study was supported by Basic Science Research Program (2015R1D1A1A01060538) through the National Research Foundation of Korea (NRF) funded from the Ministry of Education, Science and Technology of Korea.
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