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Akt and Nrf-2 signaling pathways mediates the anti-inflammatory activity of Schisandrin in Porphyromonas gingivalis LPS-stimulated macrophages
Immunology Letters 139 (2011) 93–101
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Upregulation of heme oxygenase-1 via PI3K/Akt and Nrf-2 signaling pathways mediates the anti-inflammatory activity of Schisandrin in Porphyromonas gingivalis LPS-stimulated macrophages Sun Young Park a,c , Da Jung Park b , Young Hun Kim a , YoungHee Kim c , Sun Gun Kim b , Kwang Jae Shon d , Young-Whan Choi b,∗ , Sang-Joon Lee e,∗∗ a
Bio-IT Fusion Technology Research Institute, Pusan National University, Busan 609-735, Republic of Korea Department of Horticultural Bioscience, Pusan National University, Miryang 627-706, Republic of Korea c Department of Molecular Biology, Pusan National University, Busan 609-735, Republic of Korea d Center for Research Facilities, Pusan National University, Busan 609-735, Republic of Korea e Department of Microbiology, Pusan National University, Busan 609-735, Republic of Korea b
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Article history: Received 11 February 2011 Received in revised form 17 May 2011 Accepted 20 May 2011 Available online 30 May 2011 Keywords: Schisandrin Porphyromonas gingivalis LPS Pro-inflammatory cytokine NF-B Heme oxygenase-1 Nrf-2
a b s t r a c t The lipopolysaccharide (LPS) of Porphyromonas gingivalis is thought to induce periodontitis. In this study, we isolated Schisandrin from the dried fruits of Schisandra chinensis and examined the anti-inflammatory effect of Schisandrin in macrophages stimulated with LPS from P. gingivalis. First, Schisandrin inhibited LPS-induced pro-inflammatory cytokines, including TNF-␣, IL-1, and IL-6. And Schisandrin suppressed the nuclear translocation and activity of NF-B and phosphorylation of IB␣ in LPS-stimulated RAW 264.7 cells. Next, the presence of a selective inhibitor of HO-1 (SnPP) and a siRNA specific for HO-1 inhibited Schisandrin-mediated anti-inflammatory activity. Furthermore, Schisandrin induced HO-1 expression of RAW 264.7 cells through Nrf-2, PI3K/Akt, and ERK activation. Therefore, these results suggest that the anti-inflammatory effects of Schisandrin on P. gingivalis LPS-stimulated RAW 264.7 cells may be due to a reduction of NF-B activity and induction of the expression of HO-1, leading to TNF-␣, IL-1, and IL-6 down-regulation. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Periodontitis is the most common oral disease worldwide; it induces chronic inflammatory destruction of the periodontal tissues and alveolar bone [1]. Periodontal studies suggest that Porphyromonas gingivalis is a causative factor for periodontitis and may result in the development and progression of periodontal disease [2,3]. P. gingivalis possesses a number of virulence factors such as lipopolysaccharide (LPS), which is a potent stimulator of the innate host defense immune system. LPS from P. gingivalis interacts with Toll-like receptors of the innate immune system, which induces pro-inflammatory response in host cells. Periodontal pathogens, including LPS from P. gingivalis, trigger enhanced and uncontrolled secretion of pro-inflammatory cytokines (TNF-␣, IL-1, and IL-6) in host immune cells and thereby play a role in the development and progression of periodontitis [4–6].
∗ Corresponding author. Tel.: +82 55 350 5522; fax: +82 55 350 5529. ∗∗ Corresponding author. Tel.: +82 51 510 2268; fax: +82 51 514 1778. E-mail addresses: [email protected] (Y.-W. Choi), [email protected] (S.-J. Lee). 0165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2011.05.007
Furthermore, the transcription factor NF-B plays a role in regulating pro-inflammatory cytokine gene expression in macrophages stimulated with LPS from P. gingivalis. Therefore, inhibition of proinflammatory cytokine production and NF-B activity in LPS from P. gingivalis infected macrophages may suppress the inflammatory response and thereby improve chronic periodontitis [7,8]. Heme oxygenase-1 (HO-1) is an inducible rate-limiting enzyme that catalyzes heme degradation, in which biliverdin/bilirubin, carbon monoxide (CO), and free iron are released [9,10]. Recently, HO-1 was shown to be activated by various phytochemicals, which are reportedly involved in beneficial immune responses [11–13]. Phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK), and transcription factors such as activator protein-1, nuclear factor-kappa B (NF-B), and NF-E2-related factor-2 (Nrf2) signaling pathways are the predominant cascades that participate in HO-1 expression. A recent study indicated that HO-1 expression via the PI3K/Akt and MAPK signaling pathway induces an anti-inflammatory response [14–16]. Schisandrin is a natural compound derived from the fruit of Schisandra chinensis. In traditional medicine, the beneficial and therapeutic effects of Schisandrin include anti-inflammatory, antitumor, and hepatoprotective biological properties [17–19]. Several
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mechanisms for Schisandrin-mediated anti-inflammatory effects have been proposed, including the regulation of pro-inflammatory mediators (iNOS and COX-2) by inhibition of p38 and JNK phosphorylation and NF-B activation [20]. Although various studies have been conducted on the anti-inflammatory activity of Schisandrin, the molecular signaling pathway by which Schisandrin exerts anti-inflammatory effects on HO-1 expression and on periodontitis have not yet been reported. In this study, we showed the anti-inflammatory potential of Schisandrin by using LPS from P. gingivalis-stimulated RAW 26.7 cells. 2. Materials and methods 2.1. Materials The fruits of S. chinensis (Turcz.) Baill were collected in September 2005 in Moonkyong, Korea. A voucher specimen (Accession no. SC-PDRL-1) was deposited in the Herbarium of Pusan National University. The plant was identified by one of the authors [21]; 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) and other reagents not referred were purchased from Sigma (St. Louis, MO, USA). The protoporphyrin IX (SnPP), HO-1 and Nrf-2 siRNA, and antibodies against HO-1, Nrf-2, NF-B, and TBP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). LPS (phenol extracted from P. gingivalis) was purchased from InvivoGen (San Diego, CA, USA). SB203580 (specific inhibitor of p38), PD98059 (specific inhibitor of ERK1/2), and SP600125 (specific inhibitor of JNK) were purchased from AG Scientific (San Diego, CA, USA). 2.2. Isolation of Schisandrin
well (final concentration was 62.5 g/mL). After 3-h incubation at 37 ◦ C and 5% CO2 , the supernatant was removed and the formed formazan crystals in viable cells were solubilized with 150 L DMSO. The absorbance of the contents in each well was then measured at 570 nm using a microplate reader (Wallac 1420). 2.5. Transient transfection and dual luciferase assay RAW 264.7 cells were transfected with a B-luc reporter plasmid (consisted of 3 B concatamers from the Ig␥ chain) and ARE reporter plasmid (Stratagene, Grand Island, NY) using FuGENE-HD reagent (Roche Applied Science) according to manufacturer’s instructions. The Renilla luciferase control plasmid pRL-CMV (Promega) was cotransfected as an internal control for transfection efficiency. Twenty-four hours after transfection, the cells were incubated with the indicated reagents for 1 h and then treated with LPS (1 g/mL) for 24 h. Luciferase activity was assayed with the dual-luciferase assay kit (Promega) according to manufacturer’s instructions. Luminescence measurements were obtained with a microplate luminometer (Wallac 1420). 2.6. Immunofluorescence confocal microscopy RAW 264.7 cells were cultured directly on glass cover-slips in a dish of diameter 35 mm. The cells were fixed with 3.5% paraformaldehyde in PBS for 10 min at room temperature and permeabilized with 100% MeOH for 10 min. To investigate the cellular localization of NF-B and Nrf-2, the cells were treated with a polyclonal antibody (1:100) against NF-B and Nrf-2 for 2 h. After the cells were extensively washed with PBS, they were incubated with a secondary FITC-conjugated donkey anti-rabbit IgG antibody diluted at 1:1000 in PBS for 1 h at room temperature. Nuclei were stained with 1 g/mL of DAPI and then analyzed by confocal microscopy using a Zeiss LSM 510 Meta microscope.
The dried fruits of S. chinensis (2.5 kg) were ground to a fine powder and successively extracted at room temperature with n-hexane, CHCl3 , and MeOH. The hexane extract (308 g) was evaporated in vacuo and chromatographically separated on a silica gel (40 m; Baker, Phillipsburg, NJ, USA) column (100 cm × 10 cm) with a step gradient (0%, 5%, and 20%) of EtOAc in hexane and 5% MeOH in CHCl3 to obtain 38 fractions as described previously [22]. Fraction 38 (KH38, 7237 mg) was separated on a Sephadex column (105 cm × 3 cm) with methanol to obtain 2 fractions. Next, fraction 1 (KH38IA, 2958 mg) was separated on a silica gel column (100 cm × 3 cm) with 10% acetone in CHCl3 to yield Schisandrin (KH38IAPG, 2001 mg). Pure Schisandrin was identified by GC–MS (Varian Saturn 2000, California, USA) with a helium carrier gas, at a flow rate of 1.0 mL/min. The structure of Schisandrin isolated from S. chinensis fruits was identified by the 1 H, 13 C, DEPT NMR spectra in CDCl3 and compared with previously reported spectral data [21,23,24].
The RAW 264.7 cells were washed 3 times with cold PBS and the cell pellets were suspended in hypotonic buffer (Active Motif, California, USA) and incubated for 15 min on ice. Detergent was added to the cell extract (Active Motif, California, USA), incubated on ice for 1 min, and centrifuged at 13,000 rpm for 1 min at 4 ◦ C. After collection of cytosolic proteins from the supernatant, nuclear proteins were extracted by addition of complete lysis buffer B (Active Motif, California, USA) for 30 min at 4 ◦ C with occasional vortexing. After centrifugation at 13,000 rpm for 5 min at 4 ◦ C, supernatants were collected and stored at −70 ◦ C.
2.3. Cell culture
2.8. Transient transfection of siRNA
The murine macrophage RAW 264.7 cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) supplemented with 10% FBS, and incubated at 37 ◦ C in a humidified atmosphere of 5% CO2 . To maintain the exponential growth stage, the cells were passaged every 2–3 day. The cell number was assessed by standard cell counting using procedures with a hemacytometer.
RAW 264.7 cells were transfected with siRNA using the Xtreme GENE siRNA Transfection Reagent (Roche Applied Science), according to the manufacturer instructions. Commercially available mouse HO-1 and Nrf-2 (Santa Cruz, Heidelberg, Germany) specific siRNAs and negative control siRNAs (Santa Cruz, Heidelberg, Germany) were used in transfections. In brief, X-treme GENE siRNA Transfection Reagent (10 L) was added to 100 L serumfree medium containing 2 g of each siRNA oligo, incubated for 20 min at RT. Gene silencing was measured after 72 h by western blot.
2.4. Cell viability assay (MTT assay) The cytotoxicity of Schisandrin was assessed using the microculture tetrazolium (MTT)-based colorimetric assay. Cells were incubated in 24-well plates at a density of 5 × 105 cells per well. Five microliters of MTT solution (5 mg/mL) were added to each
2.7. Preparation of nuclear extract
2.9. Western blot analysis Cells were harvested in ice-cold lysis buffer containing 1% Triton X-100, 1% deoxycholate, and 0.1% sodium dodecyl sulfate
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Fig. 1. Chemical structure of Schisandrin (A) isolated from Schisandra chinensis: gas chromatography (GC) profiles of the Schisandrin (B). GC analysis showed that the isolated Schisandrin were >96% pure.
Fig. 2. The effects of Schisandrin on the production of pro-inflammatory cytokines LPS-induced RAW 264.7 cells. Cells were incubated with various concentrations of Schisandrin for 1 h before LPS (1 g/mL) exposure for 24 h. We measured TNF-␣ (A), IL-1 (B), and IL-6 (C) in the culture supernatant by ELISA. (D) Effect of Schisandrin on cell viability. Cells were treated with indicated concentration of Schisandrin in the absence or presence of LPS for 24 h. Cell viabilities were determined by MTT assay. Each bar represents the mean ± SE from 3 independent experiments in each group. * P < 0.05 vs. the LPS-treated group.
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(SDS). The protein content of the cell lysates was then determined using Bradford reagent (Bio-Rad; Hercules, CA, USA). The protein in each sample (30 g total proteins) was resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane, and exposed to the appropriate antibodies. The proteins were visualized by an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA) using horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies. Images were acquired using an ImageQuant 350 analyzer (Amersham Biosciences). 2.10. Real-time reverse transcription (RT)-polymerase chain reaction (PCR) Total cellular RNA was isolated using RNA spin mini RNA isolation kits (GE Healthcare) according to manufacturer instructions. One microgram of total RNA was reverse-transcribed using Maxime RT PreMix (Intron Biotechnology) and anchored oligodT15 -primers. Real-time RT-PCR was performed in a Chromo4TM instrument (BIO-RAD) with the SYBR Green Master Mix (Applied Biosystems, CA). The relative quantity of target mRNA was determined using the comparative threshold (Ct ) method by normalizing target mRNA Ct values to those for GAPDH (Ct ). The real-time PCR cycling conditions were as follows: 95 ◦ C for 5 min, followed by 40 cycles for 30 s at 95 ◦ C, 20 s at 55 ◦ C, and 30 s at 72 ◦ C, followed by fluorescence measurement. The primer sequences are as follows: HO-1-sense (5 -ACAGGTTGACAGAAGAGGCTAA3 ), HO-1-antisense (5 -AACAGGAAGCTGAGAGTGAGG-3 ), GAPDHsense (5 -AGGTGGTCTCCTCTGACTTC-3 ), and GAPDH-antisense (5 -TACCAGGAAATGAGCTTGAC-3 ). 2.11. Statistical analysis Data are expressed as mean ± standard error (SE). Each experiment was repeated at least 3 times. Statistical analysis was performed with SPSS software, version 16.0, to determine significant differences. We used either one- or two-way ANOVA followed by Dunn’s post hoc tests for analyses. Values of * P < 0.05 were considered statistically significant. 3. Results 3.1. Isolation of Schisandrin from S. chinensis In order to prepare Schisandrin for the study of their antiinflammatory effects, various compounds were extracted from S. chinensis by n-hexane, EtOAc and MeOH. Subsequently, Schisandrin that were extracted in large quantities compared to the other lignans were harvested. The structure of Schisandrin was determined by GC–MS and NMR analysis, and identified as Schisandrin [23] (Fig. 1). Schisandrin that was more than 96% in chromatographic verification was used in these experiments to determine their anti-inflammatory effects. Fig. 3. Inhibitory effects of Schisandrin on NF-B transactivation and nuclear translocation in LPS induced RAW 264.7 cells. (A) Cells were co-transfected with B-luc reporter and control Renilla luciferase plasmid pRL-CMV for 24 h. Next, the cells were incubated with the indicated concentrations of Schisandrin for 1 h, and then stimulated with LPS (1 g/mL) for 24 h. Equal amounts of cell extracts were assayed for dual-luciferase activity. B-luciferase activity was normalized to control Renilla luciferase expression. Each bar represents the mean ± SE from 3 independent experiments in each group. * P < 0.05 vs. the LPS-treated group. (B) Nuclear translocation of NF-B was assessed by confocal microscopy. RAW 264.7 cells were pretreated with Schisandrin (40 M) for 1 h and stimulated with LPS (1 g/mL) for 1 h. Fixed cells were stained with DAPI and anti-NF-B p65 antibody and FITC-conjugated anti-rabbit IgG antibody. (C) Nuclear translocation of NF-B was confirmed by western blot. Nuclear extracts were prepared and analyzed by western blot. Cytosolic
3.2. Inhibition of pro-inflammatory cytokine production by Schisandrin in RAW 264.7 cells stimulated with LPS from P. gingivalis LPS, an outer membrane component of P. gingivalis, was shown to play a role in the pathogenesis of periodontitis. LPS from
extracts were analyzed by western blot with phosphorylated (p)-IB-␣ antibody. Histone H1 and ␣-tubulin detected by western blot were used as protein loading controls for each lane.
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P. gingivalis can induce macrophages to produce enormous quantities of pro-inflammatory cytokines (TNF-␣, IL-1, and IL-6) [3]. We first examined whether Schisandrin can modulate proinflammatory cytokine production in macrophages stimulated with LPS from P. gingivalis. Exposure of RAW 264.7 cells to LPS increased pro-inflammatory cytokine production. However, treatment of RAW 264.7 cells with Schisandrin clearly inhibited production of pro-inflammatory cytokines in a dose-dependent manner (Fig. 2). LPS increased TNF-␣ production by 7.1-fold. However, Schisandrin at 40 M reduced TNF-␣ production by 1.6-fold. IL-1 and IL-6 were also significantly inhibited by Schisandrin. To confirm that the inhibitory activity of Schisandrin was not due to the direct cytotoxicity of Schisandrin on RAW 264.7 cells, we examined the toxicity of Schisandrin in RAW 264.7 cells. MTT assays revealed no toxicity of Schisandrin or LPS at the doses utilized in this study (Fig. 2D). These results showed that Schisandrin evidently inhibited the production of pro-inflammatory cytokines without affecting cell viability.
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3.3. Inhibition of nucleus translocation and activity of NF-B by Schisandrin LPS from P. gingivalis has been reported to activate transcription factors such as NF-B to regulate inflammation. A binding site for NF-B is present in the promoter regions of the pro-inflammatory cytokine genes [7,8]. To elucidate the effect of Schisandrin on NFB transactivity, we examined the activity of luciferase reporter gene driven by NF-B binding consensus concatamers (B-Luc). As shown in Fig. 3A, treatment of RAW 264.7 cells with LPS increased NF-B promoter activity by 7.9-fold. Meanwhile, treatment with 20 M Schisandrin reduced LPS-induced NF-B promoter activity by 2-fold. We next examined the effect of Schisandrin on nucleus translocation of NF-B in LPS-stimulated RAW 264.7 cells. The nuclear levels of NF-B were measured by immunocytochemical and western blot analysis. NF-B was decreased in the nucleus by Schisandrin in LPS-stimulated RAW 264.7 cells (Fig. 3B). In addition, nuclear translocation of NF-B was accompanied by
Fig. 4. Effects of HO-1 disruption on Schisandrin-mediated anti-inflammatory response in LPS-stimulated RAW 264.7 cells. Cells were pretreated with 40 M of Schisandrin in the presence of SnPP for 1 h and then stimulated with LPS (1 g/mL) for 16 h. We measured TNF-␣ (A), IL-1 (B), and IL-6 (C) in the culture supernatant by ELISA. (D) Cells were transfected with si-control and si-HO-1 by using X-treme GENE according to manufacturer’s instructions. Seventy-two hours after transfection, the cells were treated with 40 M Schisandrin for 1 h, then stimulated with LPS (1 g/mL) for 16 h, and the expression of iNOS and COX-2 protein was examined by western blot.
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phosphorylation and degradation of IB␣. Schisandrin inhibits the phosphorylation of IB␣ in LPS stimulated RAW 264.7 cells. These data suggested that Schisandrin inhibit pro-inflammatory cytokine production through inhibition of NF-B transactivity and nuclear translocation. 3.4. Involvement of HO-1 in anti-inflammatory activity of Schisandrin Schisandrin has been shown to reduce pro-inflammatory mediators and NF-B activation in RAW 264.7 cells. In this study, we examined whether treatment with SnPP (a potent HO-1 inhibitor) inhibited Schisandrin-mediated suppression of P. gingivalis LPSstimulated pro-inflammatory cytokines (TNF-␣, IL-1, and IL-6). SnPP alone did not affect pro-inflammatory cytokine production. Because pharmacological inhibitors exert non-specific effects, we applied an HO-1 small interfering (si) RNA system to knock down HO-1 function. RAW 264.7 cells were transfected with si HO-1 or si control. As shown in Fig. 4D, decreased HO-1 expression blocked Schisandrin-mediated suppression of P. gingivalis LPS-stimulated pro-inflammatory mediators (iNOS and COX-2); whereas, transfection with control siRNA showed no affects. We further examined the effect of Schisandrin on expression of HO-1 mRNA and protein by real time RT-PCR and western blot. We first investigated the effect of Schisandrin on HO-1 mRNA expression. As shown Fig. 5A, HO-1 mRNA levels were gradually unregulated with the increase in concentrations of Schisandrin. Furthermore, HO-1 protein levels were significantly increased by Schisandrin in a dose-dependent manner (Fig. 5B). To confirm that Schisandrininduced HO-1 expression in RAW 264.7 cells is mediated by transcription and translation, we used actinomycin D (Act. D), an inhibitor of DNA-dependent RNA polymerase, and cycloheximide (CHX), an inhibitor of ribosomal protein synthesis. Co-treatment with Schisandrin and Act. D or CHX reduced the HO-1 expression (Fig. 5C). Taken together, these data indicated the involvement of HO-1 signals in Schisandrin-induced anti-inflammatory activity in P. gingivalis LPS-stimulated RAW 264.7 cells. 3.5. Schisandrin augments HO-1 expression via Nrf-2 nuclear translocation The promoter region of HO-1 gene contains binding sites for transcription factor Nrf-2 [25]. In the present study, we examined the effect of Schisandrin on nuclear accumulation of Nrf-2 in RAW 264.7 cells. RAW 264.7 cells were treated for different lengths of time with 40 M Schisandrin, and the nuclear extracts were analyzed by western blot for Nrf-2 nuclear accumulation. As shown in Fig. 6A, the Nrf-2 nuclear accumulation increased for 0.5–2 h. Furthermore, the amount of nuclear Nrf-2 was increased by Schisandrin in dose-dependent manner. The immunocytochemical data show that Schisandrin induced Nrf-2 nucleus translocation in RAW 264.7 cells (Fig. 6B). To elucidate the effect of Schisandrin on Nrf-2 transactivity, we examined the activity of the luciferase reporter gene driven by Nrf-2 bind to antioxidant response elements (AREs). As shown in Fig. 6C, treatment of RAW 264.7 cells with Schisandrin increased ARE promoter activity by 3.8-fold. To confirm the requirement of Nrf-2 for Schisandrin-induced HO-1 expression, we examined the transient transfection of siRNA for
Fig. 5. Effects of Schisandrin on HO-1 expression in RAW 264.7 cells. Cells were cultured with increasing concentrations of Schisandrin for 6 h (A) or 40 M of Schisandrin for the indicated times. Relative HO-1 mRNA expression (2−Ct ) was determined by real-time PCR and calculated by subtracting the Ct value for GAPDH from the Ct value for HO-1. The mRNA relative content was indicated as fold change over control. (B) Cells were incubated for 12 h with the indicated concentrations of
Schisandrin and incubated for various times with 40 M Schisandrin. Total cellular extracts were prepared and examined by western blot. ␣-Tubulin detected by western blot was used as a protein loading control for each lane. (C) RAW 264.7 cells were treated with 40 M Schisandrin for 12 h in the presence of Act D or CHX. Subsequently, we determined HO-1 protein expression by western blot. Each bar represents the mean ± SE from 3 independent experiments in each group. * P < 0.05 with respect to each control group.
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Fig. 6. Effects of Schisandrin on Nrf-2 activation in RAW 264.7 cells. (A) Cells were incubated with 40 M Schisandrin for the indicated lengths of time, and were incubated with the indicated concentration of Schisandrin for 1 h. The nuclear extracts were prepared and examined by western blot. Western blot detection of histone H1 was used as a protein loading control for each lane. (B) RAW 264.7 cells were treated with 40 M Schisandrin for 1 h. Fixed cells were stained with DAPI, anti-Nrf-2 antibody, and FITC-conjugated anti-rabbit IgG antibody, and examined by confocal microscopy. (C) Cells were transfected with the antioxidant response elements (ARE)-luciferase construct and then treated with indicated concentrations of Schisandrin. Equal amounts of cell extract were assayed for dual-luciferase activity. Expression from the renilla luciferase control was used to normalize ARE-luciferase activity. Each bar represents the mean ± SE from 3 independent experiments in each group. (D) The cells were transfected with si-control and s-Nrf-2 using X-treme GENE, according to manufacturer’s instructions. 24 h after transfection, we treated the cells with 40 M Schisandrin for 12 h and examined the expression of HO-1 protein by western blot. (E) Band intensities estimated by using Image Quant TL Software, normalized to respective loading controls, and expressed as percentages relative to normalized levels of si-control only transfected cells.
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Nrf-2 in RAW 264.7 cells. After exposure to siRNA of Nrf-2, the expression of HO-1 was decreased in Schisandrin-treated RAW 264.7 cells, whereas control siRNA did not affect. These results indicate that Nrf-2 is markedly regulated by Schisandrin in HO-1 expression. 3.6. Schisandrin mediates HO-1 expression through PI3K/Akt, ERK, and MAP kinase To further elucidate whether Schisandrin induces HO-1 expression and Nrf-2 accumulation through PI3K/Akt and MAPK signaling pathways, we used pharmacological inhibitors (PI3K/Akt: LY294002, ERK: PD98059, JNK: SB203580 and p38: SP600125). We
Fig. 7. Effects of Schisandrin on HO-1 expression and Nrf-2 accumulation in the nucleus via PI3/Akt and ERK signals in RAW 264.7 cells. (A) RAW 264.7 cells were treated with 40 M Schisandrin for 12 h in the presence of LY294002 (PI3K/Akt inhibitor, 20 M), PD98059 (ERK inhibitor, 20 M), SB203580 (p38 inhibitor, 20 M), or SP600125 (JNK inhibitor, 20 M). Subsequently, HO-1 and Nrf-2 protein expression was determined by western blot. (B) The cells were treated with 40 M Schisandrin for the indicated lengths of time and were subjected to western blot for Akt, phosphorylated Akt, ERK, phosphorylated ERK, JNK, phosphorylated JNK, p38, and phosphorylated p38 expression.
found that preincubation of RAW 264.7 cells with LY294002 or PD98059 suppressed Schisandrin-mediated HO-1 expression and Nrf-2 nuclear accumulation (Fig. 7A). Furthermore, exposure of RAW 264.7 cells to Schisandrin increased phosphorylation of Akt and ERK (Fig. 7B). However, phosphorylation of JNK and p38 was not affected by Schisandrin in RAW 264.7 cells. These results indicate that Schisandrin induces HO-1 expression and Nrf-2 nuclear accumulation by modulation of the PI3K/Akt and ERK signaling pathways. 4. Discussion Although many studies have indicated that the fruits of S. chinensis contain many pharmacologically active lignans, which have detoxificant, antioxidant, anticarcinogenic, antihepatotoxic, and anti-inflammatory properties [26], their precise mechanism of action remains to be elucidated. We identified a Schisandrin from S. chinensis. Schisandrin is a component of S. chinensis that possesses antitumor, antioxidant, and anti-inflammatory biological activity [17–20]. Despite the diverse studies conducted to investigate the anti-inflammatory activity of Schisandrin, its potential against periodontitis remains largely undefined. LPS located on the surface of gram-negative bacteria including P. gingivalis is a crucial microbial antigen. LPS from P. gingivalis has a unique form of lipid A, which is different with that of LPS from enterobacteria such as Escherichia coli. Almost LPS of enterobacteria, usually interacts with TLR4 in host immune system. However, some reports showed that a distinct molecular form of LPS from P. gingivalis utilizes TLR2 instead of TLR4 host cell activation. Moreover, recognition of P. gingivalis LPS by TLR2 leads to induction of innate immune responses through activation of many transcription factors and protein kinases and inflammatory cytokines. Our data show that incubation of LPS from P. gingivalis stimulated RAW 264.7 cells with Schisandrin attenuated production of TNF-␣, IL-1, and IL-6, all of which play relevant roles in the inflammatory response. The up-regulation of pro-inflammatory cytokines requires the activation of several transcription factors, including AP-1 and NFB, which are known to translocate into the nucleus and regulate the expression of genes related to inflammatory responses [29]. Consistent with this finding, treatment with Schisandrin diminished NF-B nucleus translocation and transactivation in response to LPS. The induction of anti-oxidant enzyme systems, including HO-1 and NQO1, is important in cellular inflammatory responses. HO-1 plays an important role in anti-oxidant defenses and in the anti-inflammatory response. Induction of HO-1 is the suggested mechanism in treating inflammatory disorders related to periodontitis [30,31]. Therefore, compounds that induce HO-1 may be beneficial in treating periodontitis. In addition, it is known that anti-inflammatory responses are controlled by a complex network of signaling pathways, including HO-1. This study provides evidence regarding a possible connection between HO-1 signals and the Schisandrin-induced anti-inflammatory activity; involvement of HO-1 in the signaling pathways is suggested by using SnPP (HO-1 specific inhibitor) and a siRNA system of HO-1. The inhibitory effects of Schisandrin on the LPS-induced iNOS and COX-2 expression were found to be blocked by silencing HO-1. These data suggest that knockdown of HO-1 inhibits the Schisandrin-induced anti-inflammatory response, and that Schisandrin upregulates anti-inflammatory activity by inducing HO-1 signaling. The Nrf-2 transcription factor plays an essential role in upregulating HO-1 expression. Nrf-2 is located in the cytoplasm as an inactive complex with Keap1 and is activated by phytochemicals, which allows it to migrate from the cytoplasm to the nucleus and to regulate transactivation of antioxidant response element (ARE) related genes. Several mechanisms exist by which phytochemicals mediate HO-1indcution via Nrf-2 activation by directly and
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covalently binding to Keap1 [16,25,30]. Our results also indicate that Schisandrin-mediated Nrf-2 nucleus accumulation leads to the upregulation of HO-1 expression, which is closely associated with the anti- inflammatory response. The function of kinase signaling pathways (PKC, PI3K/AKT, ERK, JNK, and p38) in the regulation of HO-1 and Nrf-2 signals has been well studied [14]. We examined the influence of PI3K/Akt and MAPK inhibitors on RAW 264.7 cells. Both PI3K/Akt and ERK inhibitors had similar effects: inhibition of the Schisandrin-induced HO-1 expression and of Nrf-2 accumulation in the nucleus. Our results suggest that the underlying mechanism of HO-1 regulation and Nrf-2 expression and accumulation in the nucleus by Schisandrin could be occurring via PI3K/Akt and ERK signaling pathway activation. Although multiple studies regarding the regulation of antiinflammatory responses by Schisandrin have been performed, including inhibition of iNOS and COX-2 expression, we provide the first evidence that Schisandrin decreases the biosynthesis of pro-inflammatory cytokines in murine macrophages stimulated with LPS from P. gingivalis. After exposure to Schisandrin, LPSinduced transactivation and nucleus translocation of NF-B was significantly ameliorated. The data also show that Schisandrininduced expression of HO-1 is required for the Schisandrin-induced anti-inflammatory responses in macrophages. In addition, Nrf-2, PI3K/Akt, and ERK activation are related to Schisandrin-induced anti-inflammatory responses and are also required for the Schisandrin-induced expression of HO-1. Our results suggest that overexpression of HO-1 through Nrf-2, PI3K/Akt, and ERK activation mediated the anti-inflammatory response of Schisandrin in LPS-activated murine macrophages. Therefore, these observations suggest an additional anti-inflammatory mechanism for Schisandrin activity in the macrophage; moreover, Schisandrin might be considered as a candidate for further development in the treatment of periodontitis and chronic inflammatory diseases. Conflicts of interest The authors have no financial conflict of interest. Acknowledgements This study was supported by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries (106048031SB010), Republic of Korea and supported by the 2010 Specialization Project Research Grant funded by the Pusan National University. References [1] Kinane DF. Causation and pathogenesis of periodontal disease. Periodontology 2000;25:8–20. [2] Seymour GJ, Ford PJ, Cullinan MP, Leishman S, Yamazaki K. Relationship between periodontal infections and systemic disease. Clin Microbiol Infect 2007;13:3–10. [3] Yoshimura A, Hara Y, Kaneko T, Kato I. Secretion of IL-1 beta, TNF-alpha, IL-8 and IL-1 alpha by human polymorphonuclear leukocytes in response to lipopolysaccharides from periodontopathic bacteria. J Periodontal Res 1997;32:279–86. [4] Moore WEC, Holdeman LV, Cato EP, Smibert RM, Burmeister JA, Ranney RR. Bacteriology of moderate (chronic) periodontitis in mature adult humans. Infect Immun 1983;42:510–5. [5] Kim J, Amar S. Reriodontal disease and systemic conditions: a bidirectional relationship. Odontology 2006;94:10–21. [6] Han SJ, Jeong SY, Nam YJ, Yang KH, Lim HS, Chung J. Xylitol inhibits inflammatory cytokine expression induced by lipopolysaccharide from Porphyromoans gingivalis. Clin Diagn Lab Immunol 2005;12:1285–91.
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[7] Morimoto Y, Kikuchi K, Ito T, Tokuda M, Matsuyama T, Noma S, et al. MK615 attenuated Porphyromonas gingivalis lipopolysaccharide-induced proinflammatory cytokine release via MAPK inactivation in murine macrophagelike RAW 264.7 cells. Biochem Biophys Res Commun 2009;389:90–4. [8] Park SY, Kim YH, Kim EK, Ryu EY, Lee SJ. Heme oxygenase-1 signals are involved in preferential inhibition of pro-inflammatory cytokine release by surfactin in cells activated with Porphyromonas gingivalis lipopolysaccharide. Chem Biol Interact 2010;188:437–45. [9] Otterbein LE, Mantell LL, Choi AM. Carbon monoxide provides protection against hyperoxic lung injury. Am J Physiol 1999;276:688–94. [10] Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol Rev 2006;86:583–650. [11] Chen CY, Jang JH, Li MH, Surh YJ. Resveratrol upregulates heme oxygenase1 expression via activation of NF-E2-related factor 2 in PC12 cells. Biochem Biophys Res Commun 2005;331:993–1000. [12] Joe B, Vijaykumar M, Lokesh BR. Biological properties of curcumincellular and molecular mechanisms of action. Crit Rev Food Sci Nutr 2004;44:97– 111. [13] Scapagnini G, Foresti R, Calabrese V, Giuffrida Stella AM, Green CJ, Motterlini R. Caffeic acid phenethyl ester and curcumin: a novel class of heme oxygenase-1 inducers. Mol Pharmacol 2002;61:554–61. [14] Furukawa Y, Urano T, Minamimura M, Nakajima M, Okuyama S, Furukawa S. 4Methylcatechol-induced heme oxygenase-1 exerts a protective effect against oxidative stress in cultured neural stem/progenitor cells via PI3 kinase/Akt pathway. Biomed Res 2010;31:45–52. [15] Paine A, Eiz-Vesper B, Blasczyk R, Immenschuh S. Signaling to heme oxygenase-1 and its anti-inflammatory therapeutic potential. Biochem Pharmacol 2010;80:1895–903. [16] Surh YJ, Kundu JK, Na HK. Nrf2 as a master redox switch in turning on the cellular signaling involved in the induction of cytoprotective genes by some chemopreventive phytochemicals. Planta Med 2008;74:1526–39. [17] Ko KM, Ip SP, Poon MK, Wu SS, Che CT, Ng KH, et al. Effect of a lignan-enriched fructus schisandrae extract on hepatic glutathione status in rats: protection against carbon tetrachloride toxicity. Planta Med 1995;61:134–7. [18] Panossian A, Wikman G. Pharmacology of Schisandra chinensis Bail: an overview of Russian research and uses in medicine. J Ethnopharmacol 2008;118:183–212. [19] Liu GT. Pharmacological actions and clinical use of fructus schizandrae. Chin Med J (Engl) 1989;102(10):740–9. [20] Guo LY, Hung TM, Bae KH, Shin EM, Zhou HY, Hong YN, et al. Anti-inflammatory effects of schisandrin isolated from the fruit of Schisandra chinensis Baill. Eur J Pharmacol 2008;591:293–9. [21] Choi YW, Takamatsu S, Khan SI, Srinivas PV, Ferreira D, Zhao J, et al. Schisandrene, a dibenzocyclooctadiene lignan from Schisandra chinensis: structure–antioxidant activity relationships of dibenzocyclooctadiene lignans. J Nat Prod 2006;69:356–9. [22] Choi YW, Kim HJ, Park SS, Chung JH, Lee HW, Oh SO, et al. Inhibition of endothelial cell adhesion by the new anti-inflammatory agent alpha-iso-cubebene. Vascul Pharmacol 2009;51:215–24. [23] Ikeya Y, Taguchi H, Yosioka I, Kobayashi H. The constituents of Schizandra chinensis Baill. I. Isolation and structure determination of five new lignans, gomisin A, B, C, F and G, and the absolute structure of schizandrin. Chem Pharm Bull 1979;27:1383–94. [24] Ikeya Y, Taguchi H, Sasaki H, Nakajima K, Yosioka I. The constituents of Schizandra chinensis Baill. VI. 13 C nuclear magnetic resonance spectroscopy of dibenzocyclooctadiene lignans. Chem Pharm Bull 1980;28:2414–21. [25] Srisook K, Kim C, Cha YN. Molecular mechanisms involved in enhancing HO1 expression: de-repression by heme and activation by Nrf2, the “one-two” punch. Antioxid Redox Signal 2005;7:1674–87. [26] Min HY, Park EJ, Hong JY, Kang YJ, Kim SJ, Chung HJ, et al. Antiproliferative effects of dibenzocyclooctadiene lignans isolated from Schisandra chinensis in human cancer cells. Bioorg Med Chem Lett 2008;18:523–6. [29] Park SY, Kim YH. Surfactin inhibits immunostimulatory function of macrophage through blocking NF-kB, MAPK and Akt pathway. Int Immunopharmarcol 2009;9:886–93. [30] Purdom-Dickinson SE, Sheveleva EV, Sun H, Chen QM. Translational control of nrf2 protein in activation of antioxidant response by oxidants. Mol Pharmacol 2007;72:1074–81. [31] Abuarqoub H, Foresti R, Green CJ, Motterlini R. Heme oxygenase-1 mediates the anti-inflammatory actions of 2’-hydroxychalcone in RAW 264.7 murine macrophages. Am J Physiol Cell Physiol 2006;290:1092–9.
Further reading [27] Gemmell E, Marshall RI, Seymour GJ. Cytokines and prostaglandins in immune homeostasis and tissue destruction in periodontal disease. Periodontology 2000 1997;14:112–43. [28] Ogawa T. Chemical structure of lipid A from Porphyromoans (Bacteroides) gingivalis lipopolysaccharide. FEBS Lett 1993;332:197–201.
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Upregulation of heme oxygenase-1 via PI3K/Akt and Nrf-2 signaling pathways mediates the anti-inflammatory activity of Schisandrin in Porphyromonas gingivalis LPS-stimulated macrophages Sun Young Park a,c , Da Jung Park b , Young Hun Kim a , YoungHee Kim c , Sun Gun Kim b , Kwang Jae Shon d , Young-Whan Choi b,∗ , Sang-Joon Lee e,∗∗ a
Bio-IT Fusion Technology Research Institute, Pusan National University, Busan 609-735, Republic of Korea Department of Horticultural Bioscience, Pusan National University, Miryang 627-706, Republic of Korea c Department of Molecular Biology, Pusan National University, Busan 609-735, Republic of Korea d Center for Research Facilities, Pusan National University, Busan 609-735, Republic of Korea e Department of Microbiology, Pusan National University, Busan 609-735, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 11 February 2011 Received in revised form 17 May 2011 Accepted 20 May 2011 Available online 30 May 2011 Keywords: Schisandrin Porphyromonas gingivalis LPS Pro-inflammatory cytokine NF-B Heme oxygenase-1 Nrf-2
a b s t r a c t The lipopolysaccharide (LPS) of Porphyromonas gingivalis is thought to induce periodontitis. In this study, we isolated Schisandrin from the dried fruits of Schisandra chinensis and examined the anti-inflammatory effect of Schisandrin in macrophages stimulated with LPS from P. gingivalis. First, Schisandrin inhibited LPS-induced pro-inflammatory cytokines, including TNF-␣, IL-1, and IL-6. And Schisandrin suppressed the nuclear translocation and activity of NF-B and phosphorylation of IB␣ in LPS-stimulated RAW 264.7 cells. Next, the presence of a selective inhibitor of HO-1 (SnPP) and a siRNA specific for HO-1 inhibited Schisandrin-mediated anti-inflammatory activity. Furthermore, Schisandrin induced HO-1 expression of RAW 264.7 cells through Nrf-2, PI3K/Akt, and ERK activation. Therefore, these results suggest that the anti-inflammatory effects of Schisandrin on P. gingivalis LPS-stimulated RAW 264.7 cells may be due to a reduction of NF-B activity and induction of the expression of HO-1, leading to TNF-␣, IL-1, and IL-6 down-regulation. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Periodontitis is the most common oral disease worldwide; it induces chronic inflammatory destruction of the periodontal tissues and alveolar bone [1]. Periodontal studies suggest that Porphyromonas gingivalis is a causative factor for periodontitis and may result in the development and progression of periodontal disease [2,3]. P. gingivalis possesses a number of virulence factors such as lipopolysaccharide (LPS), which is a potent stimulator of the innate host defense immune system. LPS from P. gingivalis interacts with Toll-like receptors of the innate immune system, which induces pro-inflammatory response in host cells. Periodontal pathogens, including LPS from P. gingivalis, trigger enhanced and uncontrolled secretion of pro-inflammatory cytokines (TNF-␣, IL-1, and IL-6) in host immune cells and thereby play a role in the development and progression of periodontitis [4–6].
∗ Corresponding author. Tel.: +82 55 350 5522; fax: +82 55 350 5529. ∗∗ Corresponding author. Tel.: +82 51 510 2268; fax: +82 51 514 1778. E-mail addresses: [email protected] (Y.-W. Choi), [email protected] (S.-J. Lee). 0165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2011.05.007
Furthermore, the transcription factor NF-B plays a role in regulating pro-inflammatory cytokine gene expression in macrophages stimulated with LPS from P. gingivalis. Therefore, inhibition of proinflammatory cytokine production and NF-B activity in LPS from P. gingivalis infected macrophages may suppress the inflammatory response and thereby improve chronic periodontitis [7,8]. Heme oxygenase-1 (HO-1) is an inducible rate-limiting enzyme that catalyzes heme degradation, in which biliverdin/bilirubin, carbon monoxide (CO), and free iron are released [9,10]. Recently, HO-1 was shown to be activated by various phytochemicals, which are reportedly involved in beneficial immune responses [11–13]. Phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK), and transcription factors such as activator protein-1, nuclear factor-kappa B (NF-B), and NF-E2-related factor-2 (Nrf2) signaling pathways are the predominant cascades that participate in HO-1 expression. A recent study indicated that HO-1 expression via the PI3K/Akt and MAPK signaling pathway induces an anti-inflammatory response [14–16]. Schisandrin is a natural compound derived from the fruit of Schisandra chinensis. In traditional medicine, the beneficial and therapeutic effects of Schisandrin include anti-inflammatory, antitumor, and hepatoprotective biological properties [17–19]. Several
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mechanisms for Schisandrin-mediated anti-inflammatory effects have been proposed, including the regulation of pro-inflammatory mediators (iNOS and COX-2) by inhibition of p38 and JNK phosphorylation and NF-B activation [20]. Although various studies have been conducted on the anti-inflammatory activity of Schisandrin, the molecular signaling pathway by which Schisandrin exerts anti-inflammatory effects on HO-1 expression and on periodontitis have not yet been reported. In this study, we showed the anti-inflammatory potential of Schisandrin by using LPS from P. gingivalis-stimulated RAW 26.7 cells. 2. Materials and methods 2.1. Materials The fruits of S. chinensis (Turcz.) Baill were collected in September 2005 in Moonkyong, Korea. A voucher specimen (Accession no. SC-PDRL-1) was deposited in the Herbarium of Pusan National University. The plant was identified by one of the authors [21]; 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) and other reagents not referred were purchased from Sigma (St. Louis, MO, USA). The protoporphyrin IX (SnPP), HO-1 and Nrf-2 siRNA, and antibodies against HO-1, Nrf-2, NF-B, and TBP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). LPS (phenol extracted from P. gingivalis) was purchased from InvivoGen (San Diego, CA, USA). SB203580 (specific inhibitor of p38), PD98059 (specific inhibitor of ERK1/2), and SP600125 (specific inhibitor of JNK) were purchased from AG Scientific (San Diego, CA, USA). 2.2. Isolation of Schisandrin
well (final concentration was 62.5 g/mL). After 3-h incubation at 37 ◦ C and 5% CO2 , the supernatant was removed and the formed formazan crystals in viable cells were solubilized with 150 L DMSO. The absorbance of the contents in each well was then measured at 570 nm using a microplate reader (Wallac 1420). 2.5. Transient transfection and dual luciferase assay RAW 264.7 cells were transfected with a B-luc reporter plasmid (consisted of 3 B concatamers from the Ig␥ chain) and ARE reporter plasmid (Stratagene, Grand Island, NY) using FuGENE-HD reagent (Roche Applied Science) according to manufacturer’s instructions. The Renilla luciferase control plasmid pRL-CMV (Promega) was cotransfected as an internal control for transfection efficiency. Twenty-four hours after transfection, the cells were incubated with the indicated reagents for 1 h and then treated with LPS (1 g/mL) for 24 h. Luciferase activity was assayed with the dual-luciferase assay kit (Promega) according to manufacturer’s instructions. Luminescence measurements were obtained with a microplate luminometer (Wallac 1420). 2.6. Immunofluorescence confocal microscopy RAW 264.7 cells were cultured directly on glass cover-slips in a dish of diameter 35 mm. The cells were fixed with 3.5% paraformaldehyde in PBS for 10 min at room temperature and permeabilized with 100% MeOH for 10 min. To investigate the cellular localization of NF-B and Nrf-2, the cells were treated with a polyclonal antibody (1:100) against NF-B and Nrf-2 for 2 h. After the cells were extensively washed with PBS, they were incubated with a secondary FITC-conjugated donkey anti-rabbit IgG antibody diluted at 1:1000 in PBS for 1 h at room temperature. Nuclei were stained with 1 g/mL of DAPI and then analyzed by confocal microscopy using a Zeiss LSM 510 Meta microscope.
The dried fruits of S. chinensis (2.5 kg) were ground to a fine powder and successively extracted at room temperature with n-hexane, CHCl3 , and MeOH. The hexane extract (308 g) was evaporated in vacuo and chromatographically separated on a silica gel (40 m; Baker, Phillipsburg, NJ, USA) column (100 cm × 10 cm) with a step gradient (0%, 5%, and 20%) of EtOAc in hexane and 5% MeOH in CHCl3 to obtain 38 fractions as described previously [22]. Fraction 38 (KH38, 7237 mg) was separated on a Sephadex column (105 cm × 3 cm) with methanol to obtain 2 fractions. Next, fraction 1 (KH38IA, 2958 mg) was separated on a silica gel column (100 cm × 3 cm) with 10% acetone in CHCl3 to yield Schisandrin (KH38IAPG, 2001 mg). Pure Schisandrin was identified by GC–MS (Varian Saturn 2000, California, USA) with a helium carrier gas, at a flow rate of 1.0 mL/min. The structure of Schisandrin isolated from S. chinensis fruits was identified by the 1 H, 13 C, DEPT NMR spectra in CDCl3 and compared with previously reported spectral data [21,23,24].
The RAW 264.7 cells were washed 3 times with cold PBS and the cell pellets were suspended in hypotonic buffer (Active Motif, California, USA) and incubated for 15 min on ice. Detergent was added to the cell extract (Active Motif, California, USA), incubated on ice for 1 min, and centrifuged at 13,000 rpm for 1 min at 4 ◦ C. After collection of cytosolic proteins from the supernatant, nuclear proteins were extracted by addition of complete lysis buffer B (Active Motif, California, USA) for 30 min at 4 ◦ C with occasional vortexing. After centrifugation at 13,000 rpm for 5 min at 4 ◦ C, supernatants were collected and stored at −70 ◦ C.
2.3. Cell culture
2.8. Transient transfection of siRNA
The murine macrophage RAW 264.7 cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) supplemented with 10% FBS, and incubated at 37 ◦ C in a humidified atmosphere of 5% CO2 . To maintain the exponential growth stage, the cells were passaged every 2–3 day. The cell number was assessed by standard cell counting using procedures with a hemacytometer.
RAW 264.7 cells were transfected with siRNA using the Xtreme GENE siRNA Transfection Reagent (Roche Applied Science), according to the manufacturer instructions. Commercially available mouse HO-1 and Nrf-2 (Santa Cruz, Heidelberg, Germany) specific siRNAs and negative control siRNAs (Santa Cruz, Heidelberg, Germany) were used in transfections. In brief, X-treme GENE siRNA Transfection Reagent (10 L) was added to 100 L serumfree medium containing 2 g of each siRNA oligo, incubated for 20 min at RT. Gene silencing was measured after 72 h by western blot.
2.4. Cell viability assay (MTT assay) The cytotoxicity of Schisandrin was assessed using the microculture tetrazolium (MTT)-based colorimetric assay. Cells were incubated in 24-well plates at a density of 5 × 105 cells per well. Five microliters of MTT solution (5 mg/mL) were added to each
2.7. Preparation of nuclear extract
2.9. Western blot analysis Cells were harvested in ice-cold lysis buffer containing 1% Triton X-100, 1% deoxycholate, and 0.1% sodium dodecyl sulfate
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Fig. 1. Chemical structure of Schisandrin (A) isolated from Schisandra chinensis: gas chromatography (GC) profiles of the Schisandrin (B). GC analysis showed that the isolated Schisandrin were >96% pure.
Fig. 2. The effects of Schisandrin on the production of pro-inflammatory cytokines LPS-induced RAW 264.7 cells. Cells were incubated with various concentrations of Schisandrin for 1 h before LPS (1 g/mL) exposure for 24 h. We measured TNF-␣ (A), IL-1 (B), and IL-6 (C) in the culture supernatant by ELISA. (D) Effect of Schisandrin on cell viability. Cells were treated with indicated concentration of Schisandrin in the absence or presence of LPS for 24 h. Cell viabilities were determined by MTT assay. Each bar represents the mean ± SE from 3 independent experiments in each group. * P < 0.05 vs. the LPS-treated group.
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(SDS). The protein content of the cell lysates was then determined using Bradford reagent (Bio-Rad; Hercules, CA, USA). The protein in each sample (30 g total proteins) was resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane, and exposed to the appropriate antibodies. The proteins were visualized by an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA) using horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies. Images were acquired using an ImageQuant 350 analyzer (Amersham Biosciences). 2.10. Real-time reverse transcription (RT)-polymerase chain reaction (PCR) Total cellular RNA was isolated using RNA spin mini RNA isolation kits (GE Healthcare) according to manufacturer instructions. One microgram of total RNA was reverse-transcribed using Maxime RT PreMix (Intron Biotechnology) and anchored oligodT15 -primers. Real-time RT-PCR was performed in a Chromo4TM instrument (BIO-RAD) with the SYBR Green Master Mix (Applied Biosystems, CA). The relative quantity of target mRNA was determined using the comparative threshold (Ct ) method by normalizing target mRNA Ct values to those for GAPDH (Ct ). The real-time PCR cycling conditions were as follows: 95 ◦ C for 5 min, followed by 40 cycles for 30 s at 95 ◦ C, 20 s at 55 ◦ C, and 30 s at 72 ◦ C, followed by fluorescence measurement. The primer sequences are as follows: HO-1-sense (5 -ACAGGTTGACAGAAGAGGCTAA3 ), HO-1-antisense (5 -AACAGGAAGCTGAGAGTGAGG-3 ), GAPDHsense (5 -AGGTGGTCTCCTCTGACTTC-3 ), and GAPDH-antisense (5 -TACCAGGAAATGAGCTTGAC-3 ). 2.11. Statistical analysis Data are expressed as mean ± standard error (SE). Each experiment was repeated at least 3 times. Statistical analysis was performed with SPSS software, version 16.0, to determine significant differences. We used either one- or two-way ANOVA followed by Dunn’s post hoc tests for analyses. Values of * P < 0.05 were considered statistically significant. 3. Results 3.1. Isolation of Schisandrin from S. chinensis In order to prepare Schisandrin for the study of their antiinflammatory effects, various compounds were extracted from S. chinensis by n-hexane, EtOAc and MeOH. Subsequently, Schisandrin that were extracted in large quantities compared to the other lignans were harvested. The structure of Schisandrin was determined by GC–MS and NMR analysis, and identified as Schisandrin [23] (Fig. 1). Schisandrin that was more than 96% in chromatographic verification was used in these experiments to determine their anti-inflammatory effects. Fig. 3. Inhibitory effects of Schisandrin on NF-B transactivation and nuclear translocation in LPS induced RAW 264.7 cells. (A) Cells were co-transfected with B-luc reporter and control Renilla luciferase plasmid pRL-CMV for 24 h. Next, the cells were incubated with the indicated concentrations of Schisandrin for 1 h, and then stimulated with LPS (1 g/mL) for 24 h. Equal amounts of cell extracts were assayed for dual-luciferase activity. B-luciferase activity was normalized to control Renilla luciferase expression. Each bar represents the mean ± SE from 3 independent experiments in each group. * P < 0.05 vs. the LPS-treated group. (B) Nuclear translocation of NF-B was assessed by confocal microscopy. RAW 264.7 cells were pretreated with Schisandrin (40 M) for 1 h and stimulated with LPS (1 g/mL) for 1 h. Fixed cells were stained with DAPI and anti-NF-B p65 antibody and FITC-conjugated anti-rabbit IgG antibody. (C) Nuclear translocation of NF-B was confirmed by western blot. Nuclear extracts were prepared and analyzed by western blot. Cytosolic
3.2. Inhibition of pro-inflammatory cytokine production by Schisandrin in RAW 264.7 cells stimulated with LPS from P. gingivalis LPS, an outer membrane component of P. gingivalis, was shown to play a role in the pathogenesis of periodontitis. LPS from
extracts were analyzed by western blot with phosphorylated (p)-IB-␣ antibody. Histone H1 and ␣-tubulin detected by western blot were used as protein loading controls for each lane.
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P. gingivalis can induce macrophages to produce enormous quantities of pro-inflammatory cytokines (TNF-␣, IL-1, and IL-6) [3]. We first examined whether Schisandrin can modulate proinflammatory cytokine production in macrophages stimulated with LPS from P. gingivalis. Exposure of RAW 264.7 cells to LPS increased pro-inflammatory cytokine production. However, treatment of RAW 264.7 cells with Schisandrin clearly inhibited production of pro-inflammatory cytokines in a dose-dependent manner (Fig. 2). LPS increased TNF-␣ production by 7.1-fold. However, Schisandrin at 40 M reduced TNF-␣ production by 1.6-fold. IL-1 and IL-6 were also significantly inhibited by Schisandrin. To confirm that the inhibitory activity of Schisandrin was not due to the direct cytotoxicity of Schisandrin on RAW 264.7 cells, we examined the toxicity of Schisandrin in RAW 264.7 cells. MTT assays revealed no toxicity of Schisandrin or LPS at the doses utilized in this study (Fig. 2D). These results showed that Schisandrin evidently inhibited the production of pro-inflammatory cytokines without affecting cell viability.
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3.3. Inhibition of nucleus translocation and activity of NF-B by Schisandrin LPS from P. gingivalis has been reported to activate transcription factors such as NF-B to regulate inflammation. A binding site for NF-B is present in the promoter regions of the pro-inflammatory cytokine genes [7,8]. To elucidate the effect of Schisandrin on NFB transactivity, we examined the activity of luciferase reporter gene driven by NF-B binding consensus concatamers (B-Luc). As shown in Fig. 3A, treatment of RAW 264.7 cells with LPS increased NF-B promoter activity by 7.9-fold. Meanwhile, treatment with 20 M Schisandrin reduced LPS-induced NF-B promoter activity by 2-fold. We next examined the effect of Schisandrin on nucleus translocation of NF-B in LPS-stimulated RAW 264.7 cells. The nuclear levels of NF-B were measured by immunocytochemical and western blot analysis. NF-B was decreased in the nucleus by Schisandrin in LPS-stimulated RAW 264.7 cells (Fig. 3B). In addition, nuclear translocation of NF-B was accompanied by
Fig. 4. Effects of HO-1 disruption on Schisandrin-mediated anti-inflammatory response in LPS-stimulated RAW 264.7 cells. Cells were pretreated with 40 M of Schisandrin in the presence of SnPP for 1 h and then stimulated with LPS (1 g/mL) for 16 h. We measured TNF-␣ (A), IL-1 (B), and IL-6 (C) in the culture supernatant by ELISA. (D) Cells were transfected with si-control and si-HO-1 by using X-treme GENE according to manufacturer’s instructions. Seventy-two hours after transfection, the cells were treated with 40 M Schisandrin for 1 h, then stimulated with LPS (1 g/mL) for 16 h, and the expression of iNOS and COX-2 protein was examined by western blot.
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phosphorylation and degradation of IB␣. Schisandrin inhibits the phosphorylation of IB␣ in LPS stimulated RAW 264.7 cells. These data suggested that Schisandrin inhibit pro-inflammatory cytokine production through inhibition of NF-B transactivity and nuclear translocation. 3.4. Involvement of HO-1 in anti-inflammatory activity of Schisandrin Schisandrin has been shown to reduce pro-inflammatory mediators and NF-B activation in RAW 264.7 cells. In this study, we examined whether treatment with SnPP (a potent HO-1 inhibitor) inhibited Schisandrin-mediated suppression of P. gingivalis LPSstimulated pro-inflammatory cytokines (TNF-␣, IL-1, and IL-6). SnPP alone did not affect pro-inflammatory cytokine production. Because pharmacological inhibitors exert non-specific effects, we applied an HO-1 small interfering (si) RNA system to knock down HO-1 function. RAW 264.7 cells were transfected with si HO-1 or si control. As shown in Fig. 4D, decreased HO-1 expression blocked Schisandrin-mediated suppression of P. gingivalis LPS-stimulated pro-inflammatory mediators (iNOS and COX-2); whereas, transfection with control siRNA showed no affects. We further examined the effect of Schisandrin on expression of HO-1 mRNA and protein by real time RT-PCR and western blot. We first investigated the effect of Schisandrin on HO-1 mRNA expression. As shown Fig. 5A, HO-1 mRNA levels were gradually unregulated with the increase in concentrations of Schisandrin. Furthermore, HO-1 protein levels were significantly increased by Schisandrin in a dose-dependent manner (Fig. 5B). To confirm that Schisandrininduced HO-1 expression in RAW 264.7 cells is mediated by transcription and translation, we used actinomycin D (Act. D), an inhibitor of DNA-dependent RNA polymerase, and cycloheximide (CHX), an inhibitor of ribosomal protein synthesis. Co-treatment with Schisandrin and Act. D or CHX reduced the HO-1 expression (Fig. 5C). Taken together, these data indicated the involvement of HO-1 signals in Schisandrin-induced anti-inflammatory activity in P. gingivalis LPS-stimulated RAW 264.7 cells. 3.5. Schisandrin augments HO-1 expression via Nrf-2 nuclear translocation The promoter region of HO-1 gene contains binding sites for transcription factor Nrf-2 [25]. In the present study, we examined the effect of Schisandrin on nuclear accumulation of Nrf-2 in RAW 264.7 cells. RAW 264.7 cells were treated for different lengths of time with 40 M Schisandrin, and the nuclear extracts were analyzed by western blot for Nrf-2 nuclear accumulation. As shown in Fig. 6A, the Nrf-2 nuclear accumulation increased for 0.5–2 h. Furthermore, the amount of nuclear Nrf-2 was increased by Schisandrin in dose-dependent manner. The immunocytochemical data show that Schisandrin induced Nrf-2 nucleus translocation in RAW 264.7 cells (Fig. 6B). To elucidate the effect of Schisandrin on Nrf-2 transactivity, we examined the activity of the luciferase reporter gene driven by Nrf-2 bind to antioxidant response elements (AREs). As shown in Fig. 6C, treatment of RAW 264.7 cells with Schisandrin increased ARE promoter activity by 3.8-fold. To confirm the requirement of Nrf-2 for Schisandrin-induced HO-1 expression, we examined the transient transfection of siRNA for
Fig. 5. Effects of Schisandrin on HO-1 expression in RAW 264.7 cells. Cells were cultured with increasing concentrations of Schisandrin for 6 h (A) or 40 M of Schisandrin for the indicated times. Relative HO-1 mRNA expression (2−Ct ) was determined by real-time PCR and calculated by subtracting the Ct value for GAPDH from the Ct value for HO-1. The mRNA relative content was indicated as fold change over control. (B) Cells were incubated for 12 h with the indicated concentrations of
Schisandrin and incubated for various times with 40 M Schisandrin. Total cellular extracts were prepared and examined by western blot. ␣-Tubulin detected by western blot was used as a protein loading control for each lane. (C) RAW 264.7 cells were treated with 40 M Schisandrin for 12 h in the presence of Act D or CHX. Subsequently, we determined HO-1 protein expression by western blot. Each bar represents the mean ± SE from 3 independent experiments in each group. * P < 0.05 with respect to each control group.
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Fig. 6. Effects of Schisandrin on Nrf-2 activation in RAW 264.7 cells. (A) Cells were incubated with 40 M Schisandrin for the indicated lengths of time, and were incubated with the indicated concentration of Schisandrin for 1 h. The nuclear extracts were prepared and examined by western blot. Western blot detection of histone H1 was used as a protein loading control for each lane. (B) RAW 264.7 cells were treated with 40 M Schisandrin for 1 h. Fixed cells were stained with DAPI, anti-Nrf-2 antibody, and FITC-conjugated anti-rabbit IgG antibody, and examined by confocal microscopy. (C) Cells were transfected with the antioxidant response elements (ARE)-luciferase construct and then treated with indicated concentrations of Schisandrin. Equal amounts of cell extract were assayed for dual-luciferase activity. Expression from the renilla luciferase control was used to normalize ARE-luciferase activity. Each bar represents the mean ± SE from 3 independent experiments in each group. (D) The cells were transfected with si-control and s-Nrf-2 using X-treme GENE, according to manufacturer’s instructions. 24 h after transfection, we treated the cells with 40 M Schisandrin for 12 h and examined the expression of HO-1 protein by western blot. (E) Band intensities estimated by using Image Quant TL Software, normalized to respective loading controls, and expressed as percentages relative to normalized levels of si-control only transfected cells.
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Nrf-2 in RAW 264.7 cells. After exposure to siRNA of Nrf-2, the expression of HO-1 was decreased in Schisandrin-treated RAW 264.7 cells, whereas control siRNA did not affect. These results indicate that Nrf-2 is markedly regulated by Schisandrin in HO-1 expression. 3.6. Schisandrin mediates HO-1 expression through PI3K/Akt, ERK, and MAP kinase To further elucidate whether Schisandrin induces HO-1 expression and Nrf-2 accumulation through PI3K/Akt and MAPK signaling pathways, we used pharmacological inhibitors (PI3K/Akt: LY294002, ERK: PD98059, JNK: SB203580 and p38: SP600125). We
Fig. 7. Effects of Schisandrin on HO-1 expression and Nrf-2 accumulation in the nucleus via PI3/Akt and ERK signals in RAW 264.7 cells. (A) RAW 264.7 cells were treated with 40 M Schisandrin for 12 h in the presence of LY294002 (PI3K/Akt inhibitor, 20 M), PD98059 (ERK inhibitor, 20 M), SB203580 (p38 inhibitor, 20 M), or SP600125 (JNK inhibitor, 20 M). Subsequently, HO-1 and Nrf-2 protein expression was determined by western blot. (B) The cells were treated with 40 M Schisandrin for the indicated lengths of time and were subjected to western blot for Akt, phosphorylated Akt, ERK, phosphorylated ERK, JNK, phosphorylated JNK, p38, and phosphorylated p38 expression.
found that preincubation of RAW 264.7 cells with LY294002 or PD98059 suppressed Schisandrin-mediated HO-1 expression and Nrf-2 nuclear accumulation (Fig. 7A). Furthermore, exposure of RAW 264.7 cells to Schisandrin increased phosphorylation of Akt and ERK (Fig. 7B). However, phosphorylation of JNK and p38 was not affected by Schisandrin in RAW 264.7 cells. These results indicate that Schisandrin induces HO-1 expression and Nrf-2 nuclear accumulation by modulation of the PI3K/Akt and ERK signaling pathways. 4. Discussion Although many studies have indicated that the fruits of S. chinensis contain many pharmacologically active lignans, which have detoxificant, antioxidant, anticarcinogenic, antihepatotoxic, and anti-inflammatory properties [26], their precise mechanism of action remains to be elucidated. We identified a Schisandrin from S. chinensis. Schisandrin is a component of S. chinensis that possesses antitumor, antioxidant, and anti-inflammatory biological activity [17–20]. Despite the diverse studies conducted to investigate the anti-inflammatory activity of Schisandrin, its potential against periodontitis remains largely undefined. LPS located on the surface of gram-negative bacteria including P. gingivalis is a crucial microbial antigen. LPS from P. gingivalis has a unique form of lipid A, which is different with that of LPS from enterobacteria such as Escherichia coli. Almost LPS of enterobacteria, usually interacts with TLR4 in host immune system. However, some reports showed that a distinct molecular form of LPS from P. gingivalis utilizes TLR2 instead of TLR4 host cell activation. Moreover, recognition of P. gingivalis LPS by TLR2 leads to induction of innate immune responses through activation of many transcription factors and protein kinases and inflammatory cytokines. Our data show that incubation of LPS from P. gingivalis stimulated RAW 264.7 cells with Schisandrin attenuated production of TNF-␣, IL-1, and IL-6, all of which play relevant roles in the inflammatory response. The up-regulation of pro-inflammatory cytokines requires the activation of several transcription factors, including AP-1 and NFB, which are known to translocate into the nucleus and regulate the expression of genes related to inflammatory responses [29]. Consistent with this finding, treatment with Schisandrin diminished NF-B nucleus translocation and transactivation in response to LPS. The induction of anti-oxidant enzyme systems, including HO-1 and NQO1, is important in cellular inflammatory responses. HO-1 plays an important role in anti-oxidant defenses and in the anti-inflammatory response. Induction of HO-1 is the suggested mechanism in treating inflammatory disorders related to periodontitis [30,31]. Therefore, compounds that induce HO-1 may be beneficial in treating periodontitis. In addition, it is known that anti-inflammatory responses are controlled by a complex network of signaling pathways, including HO-1. This study provides evidence regarding a possible connection between HO-1 signals and the Schisandrin-induced anti-inflammatory activity; involvement of HO-1 in the signaling pathways is suggested by using SnPP (HO-1 specific inhibitor) and a siRNA system of HO-1. The inhibitory effects of Schisandrin on the LPS-induced iNOS and COX-2 expression were found to be blocked by silencing HO-1. These data suggest that knockdown of HO-1 inhibits the Schisandrin-induced anti-inflammatory response, and that Schisandrin upregulates anti-inflammatory activity by inducing HO-1 signaling. The Nrf-2 transcription factor plays an essential role in upregulating HO-1 expression. Nrf-2 is located in the cytoplasm as an inactive complex with Keap1 and is activated by phytochemicals, which allows it to migrate from the cytoplasm to the nucleus and to regulate transactivation of antioxidant response element (ARE) related genes. Several mechanisms exist by which phytochemicals mediate HO-1indcution via Nrf-2 activation by directly and
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covalently binding to Keap1 [16,25,30]. Our results also indicate that Schisandrin-mediated Nrf-2 nucleus accumulation leads to the upregulation of HO-1 expression, which is closely associated with the anti- inflammatory response. The function of kinase signaling pathways (PKC, PI3K/AKT, ERK, JNK, and p38) in the regulation of HO-1 and Nrf-2 signals has been well studied [14]. We examined the influence of PI3K/Akt and MAPK inhibitors on RAW 264.7 cells. Both PI3K/Akt and ERK inhibitors had similar effects: inhibition of the Schisandrin-induced HO-1 expression and of Nrf-2 accumulation in the nucleus. Our results suggest that the underlying mechanism of HO-1 regulation and Nrf-2 expression and accumulation in the nucleus by Schisandrin could be occurring via PI3K/Akt and ERK signaling pathway activation. Although multiple studies regarding the regulation of antiinflammatory responses by Schisandrin have been performed, including inhibition of iNOS and COX-2 expression, we provide the first evidence that Schisandrin decreases the biosynthesis of pro-inflammatory cytokines in murine macrophages stimulated with LPS from P. gingivalis. After exposure to Schisandrin, LPSinduced transactivation and nucleus translocation of NF-B was significantly ameliorated. The data also show that Schisandrininduced expression of HO-1 is required for the Schisandrin-induced anti-inflammatory responses in macrophages. In addition, Nrf-2, PI3K/Akt, and ERK activation are related to Schisandrin-induced anti-inflammatory responses and are also required for the Schisandrin-induced expression of HO-1. Our results suggest that overexpression of HO-1 through Nrf-2, PI3K/Akt, and ERK activation mediated the anti-inflammatory response of Schisandrin in LPS-activated murine macrophages. Therefore, these observations suggest an additional anti-inflammatory mechanism for Schisandrin activity in the macrophage; moreover, Schisandrin might be considered as a candidate for further development in the treatment of periodontitis and chronic inflammatory diseases. Conflicts of interest The authors have no financial conflict of interest. Acknowledgements This study was supported by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries (106048031SB010), Republic of Korea and supported by the 2010 Specialization Project Research Grant funded by the Pusan National University. References [1] Kinane DF. Causation and pathogenesis of periodontal disease. Periodontology 2000;25:8–20. [2] Seymour GJ, Ford PJ, Cullinan MP, Leishman S, Yamazaki K. Relationship between periodontal infections and systemic disease. Clin Microbiol Infect 2007;13:3–10. [3] Yoshimura A, Hara Y, Kaneko T, Kato I. Secretion of IL-1 beta, TNF-alpha, IL-8 and IL-1 alpha by human polymorphonuclear leukocytes in response to lipopolysaccharides from periodontopathic bacteria. J Periodontal Res 1997;32:279–86. [4] Moore WEC, Holdeman LV, Cato EP, Smibert RM, Burmeister JA, Ranney RR. Bacteriology of moderate (chronic) periodontitis in mature adult humans. Infect Immun 1983;42:510–5. [5] Kim J, Amar S. Reriodontal disease and systemic conditions: a bidirectional relationship. Odontology 2006;94:10–21. [6] Han SJ, Jeong SY, Nam YJ, Yang KH, Lim HS, Chung J. Xylitol inhibits inflammatory cytokine expression induced by lipopolysaccharide from Porphyromoans gingivalis. Clin Diagn Lab Immunol 2005;12:1285–91.
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Further reading [27] Gemmell E, Marshall RI, Seymour GJ. Cytokines and prostaglandins in immune homeostasis and tissue destruction in periodontal disease. Periodontology 2000 1997;14:112–43. [28] Ogawa T. Chemical structure of lipid A from Porphyromoans (Bacteroides) gingivalis lipopolysaccharide. FEBS Lett 1993;332:197–201.