Journal of Cancer Prevention 2014; 19(4): 279-287
Published online December 31, 2014
© Korean Society of Cancer Prevention
Young-Soon Kang1, Min-Ho Han1, Su-Hyun Hong1, Cheol Park2, Hye-Jin Hwang3,4, Byung Woo Kim3,5, Kim Ho Kyoung6, Young Whan Choi7, Cheol Min Kim8, and Yung Hyun Choi1,3
1Department of Biochemistry, Dongeui University College of Korean Medicine, Busan, Korea, 2Department of Molecular Biology, College of Natural Sciences and Human Ecology, Dongeui University, Busan, Korea, 3Blue-Bio Industry RIC and Anti-Aging Research Center, College of Natural Sciences and Human Ecology, Dongeui University, Busan, Korea, 4Department of Food and Nutrition, College of Natural Sciences and Human Ecology, Dongeui University, Busan, Korea, 5Department of Life Science and Biotechnology, College of Natural Sciences and Human Ecology, Dongeui University, Busan, Korea, 6Herbal Medicine Resources Group, Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon, Korea, 7Department of Horticultural Bioscience and Life and Industry Convergence Research Institute, College of Natural Resource and Life Sciences, Pusan National University, Miryang, Korea, 8Department of Biochemistry, Pusan National University School of Medicine, Yangsan, Korea
Correspondence to :
Yung Hyun Choi, Department of Biochemistry, Dongeui University College of Korean Medicine, 52-57 Yangjeong-ro, Busanjin-gu, Busan 614-052, Korea, Tel: +82-51-850-7413, Fax: +82-51-853-4036, E-mail: email@example.com
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Schisandrae Fructus, the dried fruit of To measure the effects of SF on pro-inflammatory mediator and inflammatory cytokine’s expression and production in RAW 264.7 cells, we used the following methods: cell viability assay, Griess reagent assay, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, Western blotting analysis and immunofluorescence staining. Stimulation of the RAW 264.7 cells with LPS caused an elevated production of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin (IL)-1β, which was markedly inhibited by the pretreatment with SF without causing any cytotoxic effects. SF also inhibited the expression of inducible NO synthase, TNF-α, and IL-1β protein and their mRNAs in LPS-stimulated RAW 264.7 cells. Furthermore, SF attenuated LPS-induced nuclear translocation of nuclear factor- SF may be useful for the treatment of various inflammatory diseases.
Schisandrae Fructus, the dried fruit of
To measure the effects of SF on pro-inflammatory mediator and inflammatory cytokine’s expression and production in RAW 264.7 cells, we used the following methods: cell viability assay, Griess reagent assay, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, Western blotting analysis and immunofluorescence staining.
Stimulation of the RAW 264.7 cells with LPS caused an elevated production of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin (IL)-1β, which was markedly inhibited by the pretreatment with SF without causing any cytotoxic effects. SF also inhibited the expression of inducible NO synthase, TNF-α, and IL-1β protein and their mRNAs in LPS-stimulated RAW 264.7 cells. Furthermore, SF attenuated LPS-induced nuclear translocation of nuclear factor-
SF may be useful for the treatment of various inflammatory diseases.
Inflammation refers to the pathological and physiological processes involved in numerous diseases and is a complex process that is modulated by an array of inflammatory factors released by activated macrophages.1,2 The activation of macrophages by inflammatory stimuli is an important part of initiating defensive reactions, and inflammatory mediators including nitric oxide (NO) and pro-inflammatory cytokines are released to enhance the defense capacity. Among them, NO is synthesized
Multiple studies have shown that nuclear factor (NF)-
The mitogen-activated protein kinase (MAPK) pathway in macrophages is one of the most extensively investigated intracellular signaling cascades involved in pro-inflammatory responses.10,11 The MAPKs include extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK) as a group of serine/threonine-specific protein kinases. A major consequence of MAPK phosphorylation is activation of these transcription factors, which serve as immediate or downstream substrates of the kinases. Thus, NF-
Schisandrae Fructus, the dried fruit of
Several studies have been conducted on the anti-inflammatory activity of Schisandrae Fructus, but the detailed molecular signaling pathway by which Schisandrae Fructus exerts its anti-inflammatory effects
Schisandrae Fructus were collected around Mungyeong-city (Gyeongbuk, Korea) and washed three times with tap water before storage at ?20°C. The frozen samples were lyophilized and homogenized using a grinder before extraction. The materials were extracted with 20% ethanol (SF) at room temperature for 24 hours, filtered, and concentrated using a rotary vacuum evaporator (Buchi Rotavapor R-144; B?CHI Labortechnik, Flawil, Switzerland). The extract was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) as a 50 mg/mL stock solution. The stock solution was stored at 4°C and diluted with medium to the desired concentration prior to use.
Cell culture and cell viability assay
RAW 264.7 murine macrophages were maintained in Dulbecco’s modified Eagle’s medium (DMEM; WelGENE Inc., Daegu, Korea) supplemented with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37°C in a humidified atmosphere containing 5% CO2. Cell viability was analyzed by incubating the cells (2 × 105 cells/mL) with the indicated concentrations of SF 1 hour before treatment with LPS (1.0 μg/mL) for 24 hours. Cell viability was determined by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma-Aldrich) assay. Briefly, after treatments, the medium was removed and cells were incubated with 0.5 mg/mL MTT solution. Following incubation for 2 hours at 37°C and 5% CO2, the MTT solution was removed, and the cells were dissolved in DMSO. Optical density of the samples was measured at 540 nm using a microplate reader (Dynatech MR-7000; Dynatech Laboratories, Chantilly, VA, USA).
Nitrite quantification assay
Nitrite accumulation, an indicator of NO synthesis, was measured in culture media based on a diazotization reaction using the Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride; Sigma- Aldrich). The RAW 264.7 cells were seeded into a 96-well plate at the density of 5 × 104 cells/mL. After incubation, the cells were pretreated with various concentrations of SF with or without 100 ng/mL LPS (from
Enzyme-linked immunospecific assay assay for cytokines
RAW 264.7 macrophage cells were treated with SF in the absence or presence of LPS. After 24 hours incubation, the conditioned medium was collected. The production of TNF-α or IL-1β in the conditioned media was determined by using enzyme-linked immunosorbent assay (ELISA) kits (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.
Reverse transcriptase-polymerase chain reaction analysis
RAW 264.7 cells were treated with SF in the absence or presence of LPS for 6 hours. Total RNA was extracted from cells using a Trizol reagent kit (Invitrogen, Gaithersburg, MD, USA) according to the manufacturer’s instructions. Aliquots of 2 μg of total RNA from each sample were reverse-transcribed into cDNA with 10,000 U of reverse transcriptase and 0.5 μg/μL oligo-(dT)15 primer (Promega, Madison, WI, USA). The cDNA was amplified by polymerase chain reaction (PCR) using the primer sequences. The conditions for PCR amplification were as follows: iNOS, TNF-α, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 94°C, 1 minute, 55°C, 1 minute, 72°C, 1 minute for a total of 32 cycles; IL-1β, 94°C, 30 seconds, 64°C, 30 seconds, 72°C, 1 minute for a total of 30 cycles. PCR products were resolved on a 1% agarose gel and visualized with ultraviolet light after staining with ethidium bromide (EtBr; Sigma-Aldrich). The specific primers used were: mouse iNOS (forward 5′-CCC TTC CGA AGT TTC TGG CAG CAG C-3′and reverse), IL-1β (forward 5′-TGC AGA GTT CCC CAA CTG GTA CAT C-3′ and reverse 5′-GTG CTG CCT AAT GTC CCC TTG AAT C-3′), TNF-α (forward 5′-ACA AGC CTG TAG CCC ACG-3′ and reverse 5′-TCC AAA GTA GAC CTG CCC-3′), and GAPDH (forward 5′-TGG CAC AGT CAA GGC TGA GA-3′ and reverse 5′-CTT CTG AGT GGC AGT GAT GG-3′).Western blot analysis
RAW 264.7 cells were incubated with SF for 1 hour prior to LPS treatment. After 24 hours incubation, the cells were collected, washed twice with cold phosphate buffered saline (PBS), and then lysed in a lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM EGTA, 1 mM NaVO3, 10 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 25 μg/mL aprotinin, and 25 μg/mL leupeptin]. In a parallel experiment, nuclear and cytosolic proteins were prepared using nuclear extraction reagents (Pierce, Rockford, IL, USA) according to the manufacturer’s protocol. Aliquots of the lysates (30 to 50 μg of protein) were separated on 10% to 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Amersham Biosciences, Piscataway, NJ, USA) with a glycine transfer buffer (192 mM glycine, 25 mM Tris-HCl [pH 8.8], and 20% MeOH [v/v]). Non-specific sites on the membrane were blocked by incubating the membrane in the blocking solution containing 5% non-fat dry milk in Tris-buffered saline Tween (TBS-T, 20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20) for 1 hour at room temperature and incubated with primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. After thoroughly washing with TBS-T, the corresponding horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) were applied for 1 hour at room temperature. The final washed membrane was reacted with an enhanced chemiluminescence (ECL) reagent (Amersham Biosciences) and exposed to films to detect the immunoblots.
RAW 264.7 cells were cultured directly on glass coverslips in 6-well plates for 24 hours to detect NF-
All results are presented as the mean ± the standard deviation and are representative of three or more independent experiments. Data were compared by using Student’s
Concentrations of SF that would not induce cell toxicity were determined by treating RAW 264.7 cells with various concentrations of SF in the presence or absence of LPS (100 ng/mL) for 24 hours, and monitoring the cell viability with the MTT assay. The assay data showed no significant changes in cell viability, indicating that SF was not cytotoxic at dosage up to 500 μg/mL (Fig. 1). Therefore, 500 μg/mL SF was selected as the optimal dose for studying the anti-inflammatory effects of SF in LPS-stimulated RAW 264.7 cells.
SF inhibits lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages
The possibility that SF has anti-inflammatory properties was investigated by determining the effects of SF on the level of NO in the culture media of RAW 264.7 cells after a 24 hours treatment with 100 ng/mL LPS and SF. Treatment of RAW 264.7 cells with LPS resulted in a dramatic increase in NO production. Treatment with 500 μg/mL SF significantly inhibited this production of NO (Fig. 2A). The possibility that this inhibitory effect of SF on NO production occurred
Schisandrae Fructus ethanol extract prevents lipopolysaccharide-induced tumor necrosis factor-α and I interleukin-1β release in RAW 264.7 macrophages
The effects of SF on LPS-induced inflammatory related cytokine production in RAW 264.7 cells were studied by evaluating the production of TNF-α and IL-1β induced by LPS by ELISA. Figure 3A shows that incubation of RAW 264.7 cells with LPS caused a marked enhancement of TNF-α and IL-1β release. Treatment with 500 μg/mL SF significantly blocked the production of IL-1β and showed non-significant decreases of TNF-α production when compared with LPS-treated cells. The effects of SF on the expression of pro-inflammatory cytokines following LPS treatment were examined by RT-PCR and Western blot analyses. Figure 3B and 3C show that LPS alone significantly elevated the expression of TNF-α and IL-1β. The expression of TNF-α and IL-1β at both mRNA and protein levels was significantly reduced by the pretreatment with SF.
Schisandrae Fructus ethanol extract attenuates lipopolysaccharide-induced nuclear translocation of nuclear factor-
We also used western blotting to determine whether SF would block LPS-stimulated degradation of I
Schisandrae Fructus ethanol extract inhibits the lipopolysaccharide-induced activation of itogen-activated protein kinases
We explored the molecular mechanism underlying the anti-inflammatory effects of SF by determining its effects on the activation of MAPKs by Western blot analysis using phosphor-specific MAPK antibodies. Stimulation of RAW 264.7 cells with LPS led to rapid activation of ERK, JNK, and p38, with peak levels of each phospho-MAPK occurring 1 hour after addition of LPS. SF pretreatment significantly inhibited phosphorylation of MAPKs in LPS-stimulated RAW 264.7 cells (Fig. 5). These results indicated that the inhibitory effects of SF on iNOS, TNF-α, and IL-1β expressions were possibly mediated
During the progress of inflammation, macrophages actively participate in inflammatory responses by releasing pro-inflammatory mediators and cytokines, which play a key role in the pathogenesis of many acute and chronic inflammatory diseases. NO, a major iNOS-derived product at inflammatory sites, is induced during the response to various stimulants, and can trigger the development of inflammatory diseases.3?5
Pro-inflammatory cytokines are also produced in response to inflammatory stimuli and play a key role in the inflammatory cascade. Among them, IL-1β is mainly released from macrophages after LPS treatment and is an important component in the initiation and enhancement of inflammatory response. TNF-α is also a pivotal pro-inflammatory cytokine, and excessive production of NO by LPS also up-regulates TNF-α synthesis in macrophages. Therefore, we analyzed the effect of SF on the accumulation of NO and pro-inflammatory cytokines, including TNF-α, and IL-1β, in LPS-stimulated RAW 264.7 macrophages. Our results support a significant inhibition of LPS-induced NO production by SF
Much evidence implicates the transcription factor NF-
In addition to the NF-
In conclusion, our findings indicate that SF acted as an anti-inflammatory agent in RAW 264.7 macrophages
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