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ISSN : 1976-7447(Print)
ISSN : 2287-7363(Online)
Journal of Biomedical Research Vol.13 No.4 pp.331-338
DOI : https://doi.org/10.12729/jbr.2012.13.4.331

Anti-oxidant and anti-inflammatory effects of Fraxinus rhynchophylla on lipopolysaccharide (LPS)-induced murine Raw 264.7 cells

Gon-Sup Kim1 *, Gyeong-Eun Hong1, Hyeon-Soo Park1, Jin-A Kim2, Arulkumar Nagappan1, Jue Zhang3, Sang-rim Kang4, Chung-kil Won1, Jae-Hyeon Cho1, Eun-Hee Kim5
1Research institute of Life Science and College of Veterinary Medicine, Gyeongsang National University
2Korea National Animal Research Resource Center and Korea National Animal Bio-resource Bank, 3Key Laboratory of Nuclear Medicine Jiangsu Institute of Nuclear Medicine, Wuxi, 4Department of Biological Engineering, School of Natural Science, Kyonggi University, 5Department of Nursing Science, International University of Korea
(Received 20 Nov. 2012, Accepted 20 Dec. 2012)

Abstract

Fraxinus rhynchophylla (Oleaceae), a deciduous tree, is known to have properties that include anti-inflammatory, convergence, febricide, antiblenophthalmia, antidiarrhea, antileukorrhea, and so forth. In addition, it has been used for traditional herbal medicine in East Asian countries, including Korea. In this study, we investigated the antioxidant and anti-inflammatory effects of Fraxinus rhynchophylla ethanol extract (FRE) in lipopolysaccharide (LPS)-induced murine macrophage Raw 264.7 cells with FRE pretreatment. We performed DPPH-assay, Western blot, and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). FRE showed 85% free radical scavenging activity at concentrations of 80 µg/ml. Results of this study also showed that FRE down-regulates Cox-2 and iNOS expression in mRNA and protein level. In conclusion, crude ethanol extract of Fraxinus rhynchophylla exhibited antioxidant and anti-inflammatory activities, and it may potentially provide a valuable source of natural herbal agent to inhibit inflammation.

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Introduction

 In the Eastern Asia, plants as medicines have been used to treat human diseases for centuries. These days, medicinal plants have aroused concern at therapy for various diseases because of their good therapeutic properties and low toxicity [1]. Fraxinus rhynchophylla is distributed throughout Asia, Europe, and North America [2]. According to “DONGYIBOGAM”, Fraxinus possessed anti-diarrhea, antibacterial, anti-inflammatory and analgesic properties [3]. F. rhynchophylla has been used for medicinally such as anti-inflammatory, febricide, anti-blenophthalmia, anti-diarrhea, anti-leukorrhea [4].

 The harmful effect of free radicals causing potential biological damage is termed oxidative and nitrosative stress. Free radicals can be defined as molecules or molecular fragments containing one or more unpaired electrons in atomic or molecular orbitals [5]. Oxygen free radicals are superoxide, hydroxyl, peroxyl, alkoxyl and hydroperoxyl radicals. Nitrogen free radicals are nitric oxide (NO) and nitrogen dioxide [6]. Generation of oxidative stress is regarded as oncogenic risk factor because of two major effects. One is oxidative DNA damage caused by radicals and the other is associated with chronic pro-inflammatory signaling, leading to induction of proto-oncogenes [7]. Harmful effects of free radicals and oxidative stress can be reduced by regular consumption of foods and beverages which exhibit antioxidant activity [8]. The recognized dietary antioxidants are vitamin C, vitamin E, selenium, and carotenoids.

Inflammation is the physiologic response to injury caused by wounding, chemical damage, or infection [9]. Lipopolysaccharide (LPS), which is derived from the outer membrane of Gram-negative bacteria, is one of the critical factors contributing to the pathogenesis of sepsis and can induce inflammatory responses. Inflammation is a complex process mediated by inflammatory and immune cells such as macrophages and monocytes [10]. Inflammation is a tissue response to injury characterized by increased blood flow to the tissue causing increased temperature, redness, swelling, and pain [11]. Acute inflammation is part of the defense response, but  chronic inflammation has been found to mediate a wide variety of diseases, including cardiovascular diseases, cancer, diabetes, arthritis, Alzheimer’s disease, pulmonary diseases, and autoimmune diseases [12]. Activated inflammatory cells (neutrophils, eosinophils, mononuclear phagocytes and macrophages) secrete increased amounts of NO, prostaglandin E2 (PGE2) and cytokines [13].

 NO is synthesized from the amino acid L-arginine by the action of NO synthase (NOS). NO is a signaling molecule with diverse functions, including neurotransmission, vasodilation, immune regulation, and host defense against pathogenic micro-organism [14]. However, a sustained and very high level of NO produced during chronic inflammation can be involved in pathological disorders, including cancer [15]. NOS has three different isoforms; neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). nNOS and eNOS are controlled by Ca2+/calmodulin, but iNOS is up-regulated by inflammatory stimuli. NO produced by iNOS in macrophages can result in oxidative damage [16]. It is also known that iNOS expression is up-regulated in tissues of various chronic inflammatory diseases such as rheumatoid arthritis and asthma [17].

 PGE2 as well as NO is a major indicator of inflammation [18]. PGE2 is a major cyclooxygenase-2 (COX-2) product at inflammatory sites where it contributes to an increase in local blood flow, edema, and pain sensitization [11]. Two forms of cyclooxygenase (COX) isoenzymes are known: cyclooxygenase-1 (COX-1) and COX-2. COX-1 is expressed in most tissues and appears to be responsible for maintaining normal physiological functions. In contrast, COX-2 is induced by growth factors, pro-inflammatory cytokines, and bacterial toxins [19].

Fraxinus rhynchophylla is a traditional medicinal plant in East Asia [20]. To date, numerous studies on the natural chemotherapeutic activities against oxidative stress and inflammatory responses are still scarce. In this study, we used LPS-induced murine macrophage Raw 264.7 cell and the crude ethanol extract of F. rhynchophylla (FRE) to examine the antioxidant and anti-inflammatory effects of F. rhynchophylla. 

Materials and Methods

1. Chemicals and reagents

 Escherichia coli O111:B4 LPS and (3-(4, 5-Dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide) (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and antibiotics (streptomycin/penicillin) were obtained from Gibco (Grand Island, NY, USA) and Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Hyclone(Logan, UT, USA). Antibody to COX-2 and iNOS were purchased from Cell Signaling Technology (Danvers, MA, USA).

2. Preparation of crude plant extract

 The dried bark of F. rhynchophylla was purchased from the local market. The 500 g of dried plant materials was extracted with 2 L of 95% ethanol to obtain crude FRE. The extraction was done at room temperature for three days. The liquid of soaked material was collected by filtration and then concentrated under vacuum on a rotary evaporator at 50°C. The 8.4 g of powder was obtained from freeze-dried for complete solvent removal.

3. Cell Culture

 The murine macrophage RAW 264.7 cell line (KCKB 40071) was purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). The RAW 264.7 cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C.

4. DPPH radical scavenging activity

 Free radical scavenging activity of the extract was measured (Blois, 1958) using the stable 1,1-diphenyl-2-picryl hydrazyl radical (DPPH). Various concentrations of extracts (10, 20, 40, and 80 μg/ml) were added, at an equal volume, to methanolic solution of DPPH (100 μM). After 30 min at room temperature, the absorbance was recorded at 517 nm. The DPPH scavenging activity was calculated using following formula:
DPPH scavenging activity (%) = (1−A0/A1) × 100

 Where A0 is the absorbance of the control without sample, and A1 is the absorbance of sample. The experiment was repeated for three times and Vitamin C was used as standard controls.

5. Cytotoxicity assay

 The cell viability was measured using an established MTT-based assay. The cells were seeded in a 6-well plate and incubated for 24 hours. The macrophage cells were treated with different concentration of FRE (10, 20, 40, and 80 μg/ml) and incubated for 18h. 200 μl of a MTT solution (5 mg/mL in a PBS) was added to the wells and incubated for 2 h. After media aspirate, 500 μl of dimethyl sulfoxide (DMSO) was added to each well to dissolve the crystalline deposits on the cells and incubation at RT for 30 min with shaking drastically. The optical density of the cells at 540 nm was measured using an ELISA plate reader.

6. Western blot analysis

 The RAW 264.7 cells were cultured in 6-well plates at a density of 7 × 105 and incubated with various concentrations of FRE (10, 20, 40 and 80 μg/ml) in the presence or absence of LPS (1 μg/ml) for 18 h at 37°C. And then the RAW 264.7 cells were washed twice with ice-cold PBS and lysed. The total protein concentration was determined with a Bradford assay kit (Bio-Rad, Hercules, CA, USA). Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore). The immunoblot on the membrane was visualizedby enhanced chemiluminescence (GE Healthcare Life Sciences, Buckinghamshire, UK) and developed by x-ray film (Fuji, Tokyo, Japan). Quantification of each band was performed using the Image J program (http://rsb.info.nih.gov).

7. RNA isolation and RT-PCR analysis

 Total RNA was isolated using Trizol reagent (GeneALL Biotechnology, Seoul, Korea). The cell was pretreated with various doses (10, 20 40, and 80 μg/ml) of FRE for 1hr followed by a LPS (1 μg/ml) treatment for 6 hours. One microgram of total RNA was reverse-transcribed into cDNA. PCR primers were used to amplify the mouse COX-2, iNOS2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Table 1 shows sequence of primers used for in this study. The final PCR products were stained with ethidium bromide and electrophoresed using a 1.5% agarose gel. The amount of mRNA was measured using Image J software. The signal intensity of the specific mRNAs was normalized by a comparison with that of GAPDH and was calculated as the relative amounts.

Table 1. Primer design for RT-PCR

8. Statistical Analysis

 The data is presented as the mean ± SD of the results of at least three experiments. Data were analyzed by the paired sample t-test. One way analysis of variance (ANOVA) was used for multiple comparison. A P-value <0.05 was considered significant.

Results

1. Effect of extraction on macrophage RAW 264.7 cell viability

 The cytotoxicity of FRE (10, 20, 40, and 80 μg/mL) on macrophage RAW 264.7 cells were evaluated by a MTT-based viability assay after incubating the cells for 18 h in the absence or presence of LPS (1 μg/mL). The cell viability was not compromised by the concentration of FRE ranging from 10 to 80 mg/mL (Fig. 1). Therefore, concentrations of FRE were made from 10 to 80 μg/mL for the study of their antioxidant and anti-inflammatory effects.

Fig. 1. Effect of Fraxinus rhynchophylla ethanol extract on Raw 264.7 cell viability. Raw 264.7 macrophage cells were pretreated for 1h with the indicated concentrations of F. rhynchophylla extract, followed stimulation with LPS (1 μg/mL) for 18 hr. The viability of Raw 264.7 cells in the (A) absence or (B) presence of LPS was assayed using 3-(4, 5-Dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide.

2. Cell morphology of macrophage RAW 264.7 cells

 Fig. 2 presented the cell morphology of the macrophage RAW 264.7 cells under FRE treatment in the presence or absence of LPS (1 μg/mL). The cells were monitored under optical microscopy (×400) after 18 h incubation. The normal cell morphology generally showed a round and smooth form in whereas LPS-activated RAW 264.7 cell had changed to an irregular and rough form with accelerated spreading and forming pseudopodia. The co-treatment of LPS with FRE reduced the level of cell spreading and pseudopodia formation in dose dependent manner.

Fig. 2. Morphological change in RAW 264.7cells. Morphology of RAW 264.7 macrophage cell visualized by optical microscopy (×400). The cells were pretreated with F. rhynchophylla extract before incubation with LPS for 18 hours. (A) Control, (B) LPS- (1 μg/mL) treated only, and (C) to (F) LPS-treated with F. rhynchophylla extract (10, 20, 40, and 80 μg/mL). The arrow bars → indicate activated Raw 264.7 macrophage cells with LPS-induced and dotted arrows indicate restored Raw 264.7 macrophage cell with plant extract.

3. Radical scavenging activity of F. rhynchophylla

 The antioxidant activities of F. rhynchophylla were evaluated by DPPH free radical scavenging activity. DPPH radical scavenging activities of F. rhynchophylla was 36% at 10 μg/ml, 68% at 20 μg/mL, 81% at 40 μg/mL and 85% at 80 μg/mL, respectively (Fig. 3). The vitamin C used as a positive control showed 91% of radical scavenging activity at various concentrations (10, 20, 40, and 80 μg/mL).

Fig. 3. Antioxidative activity of F. rhynchophylla extract and standard ascorbic acid. Present radical scavenging activity of F. rhynchophylla extract was evaluated using DPPH assay.

4. Effects of F. rhynchophylla on LPS-induced iNOS mRNA and protein expression

 We investigated the effect of FRE on iNOS, which catalyzes the production of nitric oxide (NO) from L-arginine, by detecting its expressions at mRNA and protein level by RT-PCR and Western blot, respectively. The expression of iNOS mRNA was significantly decreased by cotreatment LPS with FRE comparing with only LPS-induced cells (Fig. 4A). In addition, Fig. 4B shows that protein expression of iNOS also was down-regulated on FRE-pretreated cells. These suggested that FRE may inhibit the expressions of iNOS at both mRNA and protein level in LPS-induced RAW 264.7 cells.

Fig. 4. Suppressive effects of F. rhynchophylla extract on the LPS-induced iNOS expression in mRNA and protein in RAW 264.7 cells. RAW 264.7 cells were pretreated with F. rhynchophylla extract for 1 hour, and then incubated with LPS for 18 hours. The cells were lysed, and the lysates were examined by RT-PCR and immunoblotting for the (A) iNOS mRNA and (B) iNOS protein. The asterisk (*) indicates a significant difference from the control group (P<0.05) and **indicates significant difference from the LPS-treated group (P<0.05).

5. Effects of F. rhynchophylla on LPS-induced COX-2 mRNA and protein expression

 In order to evaluate effect to FRE on COX-2 expressions at mRNA and protein level, RT-PCR and western bolt were carried out. In transcription level, COX-2 mRNA expression was reduced by co-treatment of FRE but not significant (Fig. 5A). In translation level, COX-2 protein expression was significantly attenuated by FRE in concentration-dependent manner (Fig. 5B). These results suggested that F. rhynchophylla can suppress LPS-induced COX-2 expression in both transcription and translation level.

Fig. 5. Suppressive effects of F. rhynchophylla extract on the LPS-induced COX-2 expression in mRNA and protein in RAW 264.7 cells. RAW 264.7 cells were pretreated with F. rhynchophylla extract for 1 hour, and then incubated with LPS for 18 hours. The cells were lysed, and the lysates were examined by RT-PCR and immunoblotting for the (A) COX-2 mRNA and (B) COX-2 protein. The asterisk (*) indicates a significant difference from the control group (P<0.05) and **indicates significant difference from the LPS-treated group (P<0.05).

Discussion

 Fossil records date shows plants have been used as medicines at least to the Middle Paleolithic age some 60,000 years ago [21]. Recently, interest in the natural therapy of medicinal plants is increasing rapidly. Fraxinus rhynchophylla is a traditional medicinal plant used to treat various human diseases such as eye disease, eclampsia, dysentery, asthma, bronchitis and leucorrhea from East Asia [20, 22]. To date, numerous studies have been reported on the natural chemotherapeutic activities of Fraxinus rhynchophylla against oxidant stress or inflammatory responses but there are still scarce. Therefore, the present study examined the suppressive effect of ethanol extract of F. rhynchophylla on the oxidative stress and proinflammatory mediators in macrophage RAW 264.7 cells.

Free radicals are defined as molecules having an unpaired electron. This unpaired electron(s) usually gives a considerable degree of reactivity to the free radical. Radicals derived from oxygen represent the most important class of radical species generated in living systems [23]. However, anti-oxidant enzyme systems, which are superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, and various anti-oxidant chemicals, such as vitamins C and E, uric acid and bilirubin, constantly remove the ROS. Therefore, a balance between the generation of ROS and its removal is made. However, the balance of ROS formation and removal is lost, cells are attacked by ROS, a process referred to as “oxidative stress” [24]. Oxidative stress causes oxidative damages associated with many degenerative diseases such as atherosclerosis, coronary heart diseases and aging [1]. Antioxidants through their scavenging power are useful for the management of those diseases. In the present paper, we have evaluated the free radical scavenger activity of ethanol extract of F. rhynchophylla using the DPPH-assay. Various concentrations (10, 20, 40, 80 μg/ml) of FRE were added, at an equal volume, to methanolic solution of DPPH (100 μM). The experiment was repeated for three times. Vitamine C was used as standard control. DPPH is a stable nitrogen-centered free radical the color of which changes from violet to yellow upon reduction (Fig. 3A). DPPH-assay results showed 85% free radical scavenging activity of FRE at concentration of 80 μg/ml where Vitamin C showed 91% free radical scavenging activity (Fig. 3B). To determine whether FRE causes toxicity on RAW 264.7 cells, the cell viability was tested at various concentrations of FRE in the cells by MTT assay. As shown in the Fig. 1, FRE did not exhibit cytotoxicity at any of the concentrations examined.

 Inflammation is the normal physiological response. Acute inflammation process is a protective [25], but chronic inflammation is connected with diseases, such as cancer [12]. As the inflammation progresses, various types of leukocytes, lymphocytes, and other inflammatory cells are activated. Macrophages play critical roles in the occurrence of various inflammatory diseases by inducing the expression of proinflammatory [13]. LPS is a potent inducer of monocytes and macrophages, which is derived from the outer-membrane component of gram-negative bacteria, and stimulates the production of pro-inflammatory mediators [26]. Macrophage activation was expressed as increased cell size, cytoplasmic spreading. Additional changes in activated macrophages assist in amplifying the immune response [27, 28]. Morphological change of macrophage Raw 264.7 cells induced by the FRE treatment in the presence or absence of LPS was observed under microscopy (×400) (Fig. 2) The cells were round without the LPS treatment but became irregularly shaped showing increased spreading and pseudopodia formation after LPS stimulation. The FRE decreased the degree of cell spreading and pseudopodia formation in a dose-dependent manner.

 During inflammation, activated inflammatory cells (neutrophils, eosinophils, mononuclear phagocytes and macrophages) secrete increased amounts of NO, PGE2 and cytokines which may lead to chronic inflammatory diseases [13]. The large amount of NO produced during chronic inflammation can be involved in pathological disorders, including cancer. NO is a free radical generated through conversion of l-arginine to citrulline, catalyzed by NO synthase (NOS) [11]. There are three different isoforms of NOS; nNOS, eNOS, and iNOS. Inducible nitric oxide synthase (iNOS) is not expressed under normal conditions. Hence, the level of iNOS might reflect the degree of inflammation, and can be used to evaluate the effects of drugs on the inflammatory process [29]. This study examined the effect of FRE on the expression of iNOS at the protein and mRNA levels. The immunoblotting results showed the FRE significantly suppressed the iNOS expression (Fig. 4). Moreover, in mRNA level, iNOS expression was inhibited by FRE in a dose-dependent manner.

PGE2 is another important inflammatory mediator and contributes to an increase in local blood flow, edema, and pain sensitization. PGE2 is produced from arachidonic acid metabolites by the catalysis of cyclooxygenase-2 (COX-2) which is transiently induced by growth factors, pro-inflammatory cytokines, and bacterial toxins. There is another COX enzyme known as COX-1 which appears to be responsible for maintaining normal physiological functions in contradistinction to COX-2 [11]. Triggered-COX-2 could induce production of PGE2. The importance of COX-2 in prostaglandin synthesis and inflammation is emphasized by the observation that down-regulation of COX-2 expression prevents the synthesis of PGE2, and as a result, they inhibit inflammation [30]. Fig. 5 showed that the expression of COX-2 protein and mRNA in LPS-stimulated macrophage RAW 264.7 cells was suppressed by various concentrations of FRE (10, 20, 40, and 80 μg/mL).

In conclusion, the ethanol extract of F. rhynchophylla bark showed antioxidant and anti-inflammatory effect in LPS-induced macrophage RAW 264.7 cell. The DPPH assay showed 85% free radical scavenging activity of FRE at concentration of 80 μg/ml. Also, considerable DPPH radical scavenging difference was found between FRE (85%) and vitamin C (91%) used as control. Moreover, the spreading and pseudopodia formation of RAW 264.7 cells was reduced by co-treatment of FRE with LPS comparing with LPS-induced cell without FRE. This study also revealed that FRE down-regulates the Cox-2 and iNOS expression in mRNA and protein level. These results suggest that crude ethanol extract of Fraxinus rhynchophylla showed antioxidant and anti-inflammatory activities, and it may provide a good possibility for valuable sources of natural therapeutic agents to inhibit the inflammation. 

Acknowledgements

 This work was supported by the National Research Foundation (NRF) of Korea grant funded by the Korean government (MEST) (no. 2012045015) and the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family affairs, Republic of Korea (no. 0820050).

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