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Anti-inflammatory activity of ethanol extract from Geranium sibiricum Linne
Journal of Ethnopharmacology 126 (2009) 90–95
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Research paper
Anti-inflammatory activity of ethanol extract from Geranium sibiricum Linne Jae-Uoong Shim a,1 , Phil-Sun Oh a,1 , Kye-Taek Lim b,∗ a Molecular Biochemistry Laboratory, Biotechnology Research Institute & Center for the Control of Animal Hazards Using Biotechnology (BK21), Chonnam National University, 300 Yongbong-Dong, Gwangju City 500-757, South Korea b Molecular Biochemistry Laboratory, Biotechnology Research Institute, Chonnam National University, 300 Yongbong-Dong, Gwangju City 500-757, South Korea
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Article history: Received 23 May 2009 Received in revised form 3 August 2009 Accepted 4 August 2009 Available online 13 August 2009 Keywords: Geranium sibiricum Linne (GSL) MAPK Nuclear factor (NF)-B Activator protein (AP)-1 Interleukin (IL)-1 Human mast cells (HMC-1)
a b s t r a c t Ethnopharmacological relevance: Geranium sibiricum (Geraniaceae) Linne (GSL) is used to heal various disorders of the diarrhea and the intestinal inflammation as an herbal agent in East Asia. Aims of the study: The purpose of the present study is to determine whether the ethanol (EtOH) extract of GSL regulates the inflammatory reaction stimulated by phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI) in human mast cells (HMC-1). Materials and methods: Western blot was used for activation of mitogen activated protein kinase (MAPK), transcription factors, induced nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 proteins. EMSA was for DNA binding activity. RT-PCR was used for gene expression. Results: EtOH extract of GSL (EGS) inhibits the expression of extracellular signal-regulated kinase (ERK), one of a MAPK, nuclear transcription factors involving nuclear factor (NF)-B and Activator protein (AP)1, COX-2 and iNOS. The results indicated that EGS decreased gene expression of interleukin (IL)-1 and COX-2 in PMACI stimulated HMC-1 cells. Conclusion: Hence, we speculate that EGS can use as a potent anti-inflammatory agent for inflammatory allergic diseases. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Allergic reaction is a hypersensitive response caused by immunoglobulin (Ig)E-dependent mast cell activation. In response to IgE receptor (FcRI) cross-linking or other stimuli, mast cells initiate exocytosis of the contents of secretory granules. These include vasoactive amines, arachidonic acid metabolites, chemokines, and cytokines (Marshall, 2004; Rivera and Gilfillan, 2006). These proinflammatory mediators such as nitric oxide (NO), cyclooxygenase (COX)-2, and interleukin (IL)-1 are responsible for activating mast cells in allergic inflammation and the hypersensitive response (Kawata et al., 1995; Moon et al., 1998; Bochenek et al., 2004; Swindle and Metcalfe, 2007). MAPK and transcription factors, such as nuclear factor (NF)B and activator protein (AP)-1 are well-known mediators in the expression of many pro-inflammatory genes (Davis, 1993; Nishida and Gotoh, 1993; Hecker et al., 1997; Cobb and Goldsmith, 2000). MAPK is serine and/or threonine kinases that transfer mitotic signals to the nucleus. They are activated by extracellular stimuli, including growth factor and various cytokines, and are implicated
∗ Corresponding author. Tel.: +82 62 530 2115; fax: +82 62 530 0285. E-mail addresses: [email protected], [email protected] (K.-T. Lim). 1 Jae-Uoong Shim and Phil-Sun Oh contributed equally to this study. 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.08.004
in the modulation of cytokine expression. The phosphorylation of MAPK eventually results in the direct or indirect activation of transcriptional factors. Nuclear factor (NF)-B is redox-sensitive transcription factor that plays a serious role in the expression of cytokines such as IL-1, which mediate response to inflammatory stimuli in the allergic inflammatory responses (Mukaida, 2000). For these reasons, NF-B is a pivotal target of anti-inflammatory treatment. Activator protein 1 (AP-1), another redox-sensitive transcription factor, is also a key component in the regulation of pro-inflammatory genes, such as IL-1 and COX-2 (Lee et al., 2004). AP-1 is involved in cellular proliferation and transformation, and is a possible molecular target of chemotherapy for cancer and inflammatory diseases (Shaulian and Karin, 2002). Many plant extracts has inhibition effects of inflammation in different cells. Epigallocatechin gallate (EGCG) isolated from green tea reduce the susceptibility to cholesterol gallstone formation through the regulation of inflammation (Shan et al., 2008). Mulberry extract supplements ameliorate the inflammation-related hematological parameters in carrageenan-induced arthritic rats (Kim and Park, 2006). In these concepts, plant-originated extracts have distinctive different bioactive characters. Geranium sibiricum Linne (GSL), a widely spread herb, has been used for healing of diarrhea and intestinal inflammation in traditional folk medicine, in Korea. Many classic texts of oriental medicine showed the name of GSL as a medicine for heal-
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ing of bacteria, intestinal inflammation, dermatitis, diarrhea, and cancer (Guo et al., 1987; Lee et al., 1993; Cho, 2006). Recent studies have reported that a number of phenolic compounds isolated from various Geranium species have antinociceptive and anti-inflammatory effects in inflammatory animal model systems (Küpeli et al., 2007; Pokharel et al., 2007). Among phenolic compounds, 4-hydroxykobusin isolated from Geranium thunbergii inhibits the production of nitric oxide and expression of inducible nitric oxide synthase (iNOS) via transcriptional activations of NF-B and AP-1 in macrophage cells (Pokharel et al., 2007). From these standpoints, it is strongly suggested that Geranium species may possess anti-inflammatory activity. So far, Geranium sibiricum Linne has not been reported its bioactivity, such as mechanism to remedy intestinal problems (diarrhea and intestinal inflammation). Therefore, we investigated to verify EGS prevents inflammation through inhibition of intracellular ROS, MAPK pathway, transcription factors, and proinflammatory cytokines in the inflammation-related signal transduction pathway using phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 (PMACI)-treated human mast cells (HMC-1).
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2.4. Western blot Nuclear protein extract isolated from HMC-1 cells were separated in a 10% polyacrylamide mini-gel at 100 V for 2 h at room temperature using a Mini-PROTEAN II electrophoresis cell (BioRad). After electrophoresis, the proteins were transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA). The transferred membranes were incubated for 1 h at room temperature in TBS-T solution [10 mM Tris–HCl (pH 7.6), 150 mM NaCl and 0.1% (v/v) Tween-20] containing 5% (w/v) non-fat dry milk. The membranes were subsequently incubated for 2 h at room temperature with rabbit polyclonal antibodies (anti-ERK, anti-pERK, anti-c-Jun, anti-c-Fos, anti-NF-B (p50, p65), anti-COX-2, antiiNOS, and ␣-tubulin, Santa Cruz Biotechnology, CA, USA) in TBS-T solution containing 5% non-fat dry milk. After three washes with TBS-T, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000; Cell Signaling Technology, MA, USA) in TBS-T containing 5% non-fat dry milk. The protein bands were visualized by incubation with enhanced chemiluminescence (Amersham Pharmacia Biotech., England, UK).
2. Materials and methods 2.5. Electrophoretic mobility shift assay (EMSA) 2.1. Chemicals All the plastic materials were purchased from Falcon Labware (Becton-Dickinson, Franklin Lakes, NJ). Ethylene diamine tetra acetate (EDTA, E5134), penicillin G (H0474), streptomycin (H0447), ethylene glycol bis (2-aminoethyl ether)-N, N, N N -tetraacetic acid (EGTA, E4378), nonidet P-40 (NP-40, N0896), phenyl methane sulfonyl fluoride (PMSF, P7626), obtained from Sigma (St. Louis, MO, USA). Iscove’s Modified Dulbecco’s Medium (IMDM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Other chemicals and reagents were of the highest analytical grade available. 2.2. Preparation of EGS The Geranium sibiricum Linne was purchased from Naju traditional market in May 2008, Chonnam province, South Korea. They were identified by Dr. H.T. Lim (Plant taxonomy, Department of Biology, Chonnam National University). A voucher specimen (No. LimKT0080522) was deposited at Molecular Biochemistry Laboratory, Chonnam National University, Gwangju, Korea. The GSL dry matter (1.5 kg) was cut into small pieces and soaked in 99% ethanol (EtOH, 19 L, w/v) for several months in a dark basement. The ethanol extracts were passed through Whatman filter paper (No. 2) in order to remove debris and concentrated with a rotary evaporator (B465; Buchi, Flawil, Switzerland). After the concentrated solution was dried with a freeze-dryer (SFDS06; Sam won, Seoul, Korea), it was lyophilized, and stored at −20 ◦ C. The yield of dried extract from starting crude materials was 83.4 g (5.56%). The lyophilized powder was dissolved in di-distilled water for experiment. The GSL ethanol extracts named EGS in this paper. 2.3. Cell culture HMC-1 cells, a human mast cell line, were provided by H.-M. Kim (College of Oriental Medicine, Kyung Hee University, Seoul, Korea). HMC-1 cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Life Technologies, Grand Island, NY, USA) supplemented with 10−5 M monothioglycerol (Sigma, St Louis, MO, USA), 5% heat-inactivated fetal bovine serum (FBS) and 100 IU/ml penicillin/streptomycin (Life Technologies) at 37 ◦ C in an atmosphere containing 5% CO2 . HMC-1 cells were passaged every 3–4 days.
To make the double stranded oligonucleotide, each strand of NFB and AP-1 oligonucleotide were annealed by heating at 37 ◦ C for 30 min. Then, it was labeled with [␣-32 P]dCTP (0.25 mCi, Amersham Pharmacia Biotech, Buckinghamshire, UK) by klenow polymerase and purified on a QIAquick® Nucleotide Removal Kit according to the manufacturer’s protocol (LRS Laboratory Inc., QIA GEN Distibuter, Seoul, Korea). The following NF-B and AP-1 oligonucleotide sequences were used for probing: NF-B sequence: 5 -AGT TGA GGG GAC TTT CCC AGG C-3 3 -TCA ACT CCC CTG AAA GGG TCC G-5 AP-1 sequence: 5 -TTC CGG CTG ACT CAT CAA GCG-3 3 -AAG GCC GAC TGA GTA GTT CGC-5 The DNA–protein binding reaction was performed by incubation of the NF-B and AP-1 probes and 10 g of nucleic protein extract and 0.5 g/ml poly dI/dC (Sigma Chemical Co., USA) in a binding buffer [0.2 M DTT, 20 mg ml-1 BSA, buffer B (20 mM HEPES, 20% glycerol, 100 mM KCl, 0.5 mM EDTA, 0.25% NP-40), buffer C (20% ficoll 400, 100 mM HEPES, 300 mM KCl)] at room temperature for 1–2 h. The DNA–protein complexes were resolved by applying 4% nondenaturing polyacrylamide gel in 0.5 × TBE (45 mM Tris–borate, 1 mM EDTA). Electrophoresis was carried out at 200 V for 3 h in a cold room. Gels were then dried on 3 M blotting paper (Whatman) and exposed to X-ray film at −70 ◦ C overnight.
2.6. Measurement of nitric oxide production HMC-1 Cells were treated with PMA (30 nM) and A23187 (1 M) (PMACI) in the presence of EGS (50–200 g/ml) for 24 h in the 96 well multiple plate. And then, cells were centrifuged at 1000 × g for 10 min and supernatants were collected. NO production was measured as a function of nitrite (NO2 − ) concentration by the method of Green et al. (1982). Supernatants (50 l) were mixed with 100 l of 0.1% sulfanilamide and 100 l 0.1% N-1-naphthylethylenediamine dihydrochloride in 2.5% polyphosphoric acid (Griess reagent) for 10 min. Absorbance was measured at 540 nm with a MicroReader (Hyperion, Inc., USA). Nitrite was quantified by using sodium nitrate as a standard.
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2.7. RNA isolation and reverse transcription polymerase chain reaction (RT-PCR) Total RNA was isolated from HMC-1 cells using TRIZOL® Reagent according to the manufacture’s protocol (Invitrogen life Technologies, Carlsbad, CA). Reverse transcription polymerase chain reaction (RT-PCR) was performed using the SuperScriptTM OneStep RT-PCR System (Invitrogen, Carlsbad, CA). Briefly, RT-PCR mixture (50 l) containing 1 g of total RNA, 10 M of gene-specific sense and antisense primers, 1 l of SuperScriptTM II RT/ Platinum® Taq Mix, and 25 l of 2X Reaction Mix was assembled on ice. The following primer sequences of IL-1 and GAPDH were used: IL-1 (332 bp) sense (5 -TTG ACG GAC CCC AAA AGA TG-3 ) and antisense (5 -AGA AGG TGC TCA TGT CCT CA-3 ). The amplifications were conducted with 35 cycles for IL-1 (20 s at 95 ◦ C, 40 s at 60 ◦ C, 30 s at 72 ◦ C) and the RT-PCR amplification products were mixed with 2 l loading buffer and separated on a 2% agarose gel. The gels were stained with 5 g/ml ethidium bromide and photographed. 2.8. Statistical analysis All experiments were done in triplicate, and data were expressed as the means ± SE. A one-way analysis of variance (ANOVA) and Duncan test were used to detect significant differences by multiple comparisons (SPSS program, ver. 11.0). 3. Results 3.1. Effects of EGS on MAPK inhibition in PMACI stimulated HMC-1 cells We studied whether or not EGS modulates the ERK MAPK pathway in PMACI stimulated HMC-1 cells. The band intensity of phospho ERK MAPK in whole cell protein extracts was much stronger in (Fig. 1, lane 2) compared with treated with PMACI alone than in control group (Fig. 1, lane 1). Upon increasing concentration of the EGS (25, 50, 100 g/ml), however, its band intensities were gradually attenuated by 0.5, 0.8, 1.1 in PMACI-treated groups (Fig. 1, lanes 3–5). 3.2. Effects of EGS on inhibition of NF-B and AP-1 in PMACI stimulated HMC-1 cells Results in Western blot showed that whether or not EGS modulates the p50 and p65, which is a kind of NF-B subunits and c-jun and c-fos, which is a kind of AP-1 subunit in PMACI stimulated HMC1 cells. The intensity of NF-B and AP-1 band expression in nuclear
Fig. 2. Effects of EGS on activation of NF-B and AP-1 in PMACI stimulated HMC-1 cells. Lane 1, control; lane 2, PMACI alone; lane 3, 25 g/ml EGS + PMACI; lane 4, 50 g/ml EGS + PMACI; lane 5, 100 g/ml EGS + PMACI. ␣-Tubulin was used as an internal control.
extracted protein extracts was much stronger in (Fig. 2, lane 2), which treated with PMACI only than in control group (Fig. 2, lane 1). Upon increasing concentration of the EGS (25, 50, 100 g/ml), however, its band intensities were gradually attenuated by 0.3, 0.8, 1.6 in c-jun, 0.3, 0.8, 1.6 in c-fos, 1.0, 1.7, 2.4 in p65, and 1.0, 1.5, 1.9 in p50, in PMACI-treated groups (Fig. 2, lanes 3–5). 3.3. Effects of EGS on DNA-binding activities of NF-B and AP-1 in PMACI stimulated HMC-1 cells We further studied that EGS inhibits PMACI-induced NF-B and AP-1 activation in HMC-1 cells (Fig. 3). When the mice were treated with PMACI, NF-B complex had maximal DNA-binding activity (Fig. 3A, lane 2). However, the DNA binding activities of NF-B complex intensity were gradually reduced by 0.3, 0.8 in 50 and 100 g/ml of EGS in a dose-dependent manner (Fig. 3A, lanes 3 and 4). Similarly, the DNA-binding activity of AP-1 complex in nuclear colonic protein extracts was higher in Fig. 3B, which treated with PMACI alone (lane 2) than in the control group (Fig. 3B, lane 1). By contrast, treatment of PMACI in presence of 50, 100 g/ml of EGS markedly suppressed the DNA-binding activity of AP-1 complex intensity by 0.2, 0.3 (Fig. 3B, lanes 3 and 4). 3.4. Effects of EGS on COX-2 and iNOS inhibition in PMACI stimulated HMC-1 cells
Fig. 1. Effects of EGS on activation of ERK in PMACI stimulated HMC-1 cells. Lane 1, control; lane 2, PMACI alone; lane 3, 25 g/ml EGS + PMACI; lane 4, 50 g/ml EGS + PMACI; lane 5, 100 g/ml EGS + PMACI. ␣-Tubulin was used as an internal control.
We further examined the inhibitory effects of EGS on PMACIinduced iNOS and COX-2 proteins activation using Western blot
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Fig. 3. Effects of EGS on DNA-binding activities of NF-B and AP-1 in PMACI stimulated HMC-1 cells. Lane 1, free probe alone (no nuclear extracts); lane 2, control; lane 3, PMACI alone; lane 4, 50 g/ml EGS + PMACI; lane 5, 100 g/ml EGS + PMACI.
with whole cell protein extracts. As shown in Fig. 4, EGS was shown to have dose-dependent blocking activities on the activation of pro-inflammatory proteins in PMACI stimulated HMC-1 cells. Namely, iNOS and COX-2 proteins were activated after treatment with PMACI (Fig. 4, lane 2). Upon pretreatment with the EGS (25, 50, 100 g/ml), however, the intensities of the iNOS and COX-2 bands markedly reduced by 1.1, 1.9, and 2.2 in iNOS and 0.2, 0.3, 0.7 in COX-2, compared to PMACI treatment alone (Fig. 4, lanes 3 and 4).
3.5. Effects of EGS on NO production in PMACI stimulated HMC-1 cells As shown in Fig. 5, NO production in PMACI stimulated HMC1 cells were decreased a little compared with control. However, production of NO was much increased by 2.38, 3.17 M in 12 h and 3.06, 3.28 M in 24 h by 100, 200 g/ml of EGS treated group compared with PMACI only treated HMC-1 cells.
Fig. 5. Effects of EGS on NO production in PMACI stimulated HMC-1 cells. Each bar represents the mean ± SE of triplicate experiments. Single asterisk (*) represents significant difference compared to PMACI treatment alone and EGS (100 and 200 g/ml) in the presence of PMACI, p < 0.05.
3.6. Effects of EGS on inflammatory cytokine production in PMACI stimulated HMC-1 cells Results in the Fig. 6 showed that IL-1 mRNAs in the control group not treated with PMACI or EGS were constitutively expressed (Fig. 6 lane 1), whereas the levels of these mRNAs were elevated after treatment with PMACI (Fig. 6, lane 2). However, the intensity of IL-1 mRNAs was obviously down-regulated by 0.7 in treatment with 50 g/ml of EGS, compared to PMACI treatment alone (Fig. 6, lane 3). 4. Discussion
Fig. 4. Effects of EGS on activation of iNOS and COX-2 in PMACI stimulated HMC-1 cells. Lane 1, control; lane 2, PMACI alone; lane 3, 25 g/ml EGS + PMACI; lane 4, 50 g/ml EGS + PMACI. 100 g/ml EGS + PMACI. ␣-Tubulin was used as an internal control.
Mast cells are broadly distributed throughout mammalian tissues and play an important role in a wide variety of biological responses. Typically, mast cells have been considered not only in the association of immediate-type hypersensitivity, but also in late reactions like inflammatory responses, which are mast cell dependent (Metzger et al., 1986; Kemp and Lockey, 2002). And they play a critical role in allergic inflammation through the immunoglobulin E (IgE) dependent release of including histamine, tryptase, chymase, heparin and some inflammatory cytokines (Mekori and Metcalfe, 2000; Galli et al., 2005). Through release their proinflammatory
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Fig. 6. Effects of EGS on expression of IL-1 mRNA in PMACI stimulated HMC-1 cells. Each bar represents the means ± SE of triplicate experiments. (#) Represents significant difference compared to the control and PMACI treatment alone, p < 0.05. Single asterisk (*) represents significant difference compared to the PMACI treatment alone and 100 g/ml EGS treatment, p < 0.01.
mediators, mast cells actively participate in the pathogenesis of these intestinal diseases (Walls et al., 2001). The results of this study were revealed interesting inflammation inhibition effects of EGS in PMACI stimulated HMC-1 cells with intracellular signal pathways and secretion of proinflammatory mediators, such as inducible nitric oxide synthase (iNOS), interleukin-1 (IL-1). PMACI is well known as an inducer of HMC-1 cells. The results of in this experiment showed that PMACI stimulates ERK mitogen activated protein kinase (MAPK) (Fig. 1). Among MAPK family, ERK plays a role in the survival of the cells induced by various growth factors and linked to inflammatory pathways (Cobb and Goldsmith, 1995; Zhang and Stanimirovic, 2002). And EGS showed the dramatically decreasing effect on phospho-ERK, one of the MAPK phosphorylation. However, MAPKs p38 and JNK, except ERK are not showed remarkable decrease compared with PMACI stimulation. The phosphorylation of ERK activates transcription factors and some downstream protein kinases. It suggests that PMACI induced ERK stimulate the IKK␣ phosphorylation for inflammation development and EGS can modulate the upper state of ERK pathway. NF-B and AP-1 are redox-sensitive transcription factors which germane to ERK stimulation. Activated these transcription factors head for nuclear and activate many inflammatory mediator genes (Collart et al., 1990; Azzolina et al., 2003). In this study, the results from Western blotting, we carried out EGS inhibits activation of transcription factors’ subunits such as p50 and p65 in NF-B, and c-Jun and c-Fos in AP-1, against PMACI stimulation. To further understand the activation of transcription factors, we evaluated the inhibitory effects of the EGS on DNA binding activities of NFB and AP-1 in PMACI stimulated HMC-1 cells. The results in this experiment showed that PMACI stimulates both the DNA-binding activities of NF-B and AP-1. Data indicate that the increasing DNA binding activities of NF-B and AP-1 by PMACI treatment were decreased in the presence of EGS in a dose-dependent manner. This finding suggests that EGS interferes with PMACI stimulation to activate NF-B and AP-1.
We confirmed the Western blot to elucidate the regulation of PMACI-induced iNOS and COX-2, which are sensitive prime components of intracellular signaling pathways responsible for inflammatory events, activation by EGS. Our findings in this study show that EGS suppressed iNOS activity, and subsequently it reduces the COX-2 activity in HMC-1 cells. Although we have determined the iNOS expression, production of NO was decreased PMACI treated only and increased EGS dose dependently. Interestingly, NO is an important multiplayer, participate in cell death and survival, invasion of pathogen, and inflammation (Beckman et al., 1990). On the other hand, NO inhibits degranulation, cytokine release and mediator expression of HMC-1 cells (Coleman, 2002). These results suggest that although iNOS remains high for inflammation environment, NO is decreased for cytokine release, and cytokine expression in HMC-1 cells. It may responsible for inhibition of iNOS activation, through arginine deletion, inhibition of L-arginine analogs, and inhibition of cofactors (Schoonover et al., 2000). We observed that PMACI stimulated HMC-1 cells release IL-1, pro-inflammatory cytokine. Interleukin-1 gives an inflammatory signal to immune cells such as T cells, B cells, and macrophages and causes lymphocyte activation, macrophage stimulation, pyrexia, and negative acutephase proteins like a transcortin, which upregulates inflammation. The results showed the expression of IL-1 is clearly decreased by EGS compared with PMACI treated only. Taken together, our finding revealed that Geranium sibiricum Linne has also an anti-inflammatory activity like other Geranium species (Küpeli et al., 2007; Pokharel et al., 2007). In conclusion, our results indicate that EGS has an antiinflammatory potential due to activated human mast cells (HMC-1) through modulation of pro-inflammatory cytokine IL-1 expression and NO production. Hence, we speculate that it is applicable in the prevention and treatment of inflammatory diseases. Further study remains to modulate the expression of cytokines like a TNF-␣ and IL-6 by EGS at the molecular level. Acknowledgement This study was financially supported by Biotechnology Research Institute, Chonnam National University in 2009. References Azzolina, A., Bongiovanni, A., Lampiasi, N., 2003. Substance P induces TNF alpha and IL-6 production through NF kappa B in peritoneal mast cells. Biochimica et Biophysica Acta 1643, 75–83. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., Freeman, B.A., 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proceedings of the National Academy of Sciences of the United States of America 87, 1620–1624. Bochenek, G., Nizankowska, E., Gielicz, A., Swierczynska, M., Szczeklik, A., 2004. Plasma 9alpha,11beta-PGF2, a PGD2 metabolite, as a sensitive marker of mast cell activation by allergen in bronchial asthma. Thorax 59, 459–464. Cho, T.D., 2006. Korean Herbs, 1st ed. Daewon, p. 288. Cobb, M.H., Goldsmith, E.J., 1995. How MAPK are regulated. The Journal of Biological Chemistry 270, 14843–14846. Cobb, M.H., Goldsmith, E.J., 2000. Dimerization in MAP-kinase signaling. Trends in Biochemical Sciences 25, 7–9. Coleman, J.W., 2002. Nitric oxide: a regulator of mast cell activation and mast cellmediated inflammation. Clinical and Experimental Immunology 129, 4–10. Collart, M.A., Baeuerle, P., Vassalli, P., 1990. Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B. Molecular and Cellular Biology 10, 1498–1506. Davis, R.J., 1993. The mitogen-activated protein kinase signal transduction pathway. The Journal of Biological Chemistry 268, 14553–14556. Galli, S.J., Kalesnikoff, J., Grimbaldeston, M.A., Piliponsky, A.M., Williams, C.M., Tsai, M., 2005. Mast cells as “tunable” effector and immunoregulatory cells: recent advances. Annual Review of Immunology 23, 749–786. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N] Nitrate in biological fluids. Analytical Biochemistry 126, 131–138. Guo, J.S., Wang, S.X., Li, X., Zhu, T.R., 1987. Studies on the Antibacterial Constituents of Geranium-Sibiricum L. Yaoxue Xuebao 22, 28–32.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Research paper
Anti-inflammatory activity of ethanol extract from Geranium sibiricum Linne Jae-Uoong Shim a,1 , Phil-Sun Oh a,1 , Kye-Taek Lim b,∗ a Molecular Biochemistry Laboratory, Biotechnology Research Institute & Center for the Control of Animal Hazards Using Biotechnology (BK21), Chonnam National University, 300 Yongbong-Dong, Gwangju City 500-757, South Korea b Molecular Biochemistry Laboratory, Biotechnology Research Institute, Chonnam National University, 300 Yongbong-Dong, Gwangju City 500-757, South Korea
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Article history: Received 23 May 2009 Received in revised form 3 August 2009 Accepted 4 August 2009 Available online 13 August 2009 Keywords: Geranium sibiricum Linne (GSL) MAPK Nuclear factor (NF)-B Activator protein (AP)-1 Interleukin (IL)-1 Human mast cells (HMC-1)
a b s t r a c t Ethnopharmacological relevance: Geranium sibiricum (Geraniaceae) Linne (GSL) is used to heal various disorders of the diarrhea and the intestinal inflammation as an herbal agent in East Asia. Aims of the study: The purpose of the present study is to determine whether the ethanol (EtOH) extract of GSL regulates the inflammatory reaction stimulated by phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI) in human mast cells (HMC-1). Materials and methods: Western blot was used for activation of mitogen activated protein kinase (MAPK), transcription factors, induced nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 proteins. EMSA was for DNA binding activity. RT-PCR was used for gene expression. Results: EtOH extract of GSL (EGS) inhibits the expression of extracellular signal-regulated kinase (ERK), one of a MAPK, nuclear transcription factors involving nuclear factor (NF)-B and Activator protein (AP)1, COX-2 and iNOS. The results indicated that EGS decreased gene expression of interleukin (IL)-1 and COX-2 in PMACI stimulated HMC-1 cells. Conclusion: Hence, we speculate that EGS can use as a potent anti-inflammatory agent for inflammatory allergic diseases. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Allergic reaction is a hypersensitive response caused by immunoglobulin (Ig)E-dependent mast cell activation. In response to IgE receptor (FcRI) cross-linking or other stimuli, mast cells initiate exocytosis of the contents of secretory granules. These include vasoactive amines, arachidonic acid metabolites, chemokines, and cytokines (Marshall, 2004; Rivera and Gilfillan, 2006). These proinflammatory mediators such as nitric oxide (NO), cyclooxygenase (COX)-2, and interleukin (IL)-1 are responsible for activating mast cells in allergic inflammation and the hypersensitive response (Kawata et al., 1995; Moon et al., 1998; Bochenek et al., 2004; Swindle and Metcalfe, 2007). MAPK and transcription factors, such as nuclear factor (NF)B and activator protein (AP)-1 are well-known mediators in the expression of many pro-inflammatory genes (Davis, 1993; Nishida and Gotoh, 1993; Hecker et al., 1997; Cobb and Goldsmith, 2000). MAPK is serine and/or threonine kinases that transfer mitotic signals to the nucleus. They are activated by extracellular stimuli, including growth factor and various cytokines, and are implicated
∗ Corresponding author. Tel.: +82 62 530 2115; fax: +82 62 530 0285. E-mail addresses: [email protected], [email protected] (K.-T. Lim). 1 Jae-Uoong Shim and Phil-Sun Oh contributed equally to this study. 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.08.004
in the modulation of cytokine expression. The phosphorylation of MAPK eventually results in the direct or indirect activation of transcriptional factors. Nuclear factor (NF)-B is redox-sensitive transcription factor that plays a serious role in the expression of cytokines such as IL-1, which mediate response to inflammatory stimuli in the allergic inflammatory responses (Mukaida, 2000). For these reasons, NF-B is a pivotal target of anti-inflammatory treatment. Activator protein 1 (AP-1), another redox-sensitive transcription factor, is also a key component in the regulation of pro-inflammatory genes, such as IL-1 and COX-2 (Lee et al., 2004). AP-1 is involved in cellular proliferation and transformation, and is a possible molecular target of chemotherapy for cancer and inflammatory diseases (Shaulian and Karin, 2002). Many plant extracts has inhibition effects of inflammation in different cells. Epigallocatechin gallate (EGCG) isolated from green tea reduce the susceptibility to cholesterol gallstone formation through the regulation of inflammation (Shan et al., 2008). Mulberry extract supplements ameliorate the inflammation-related hematological parameters in carrageenan-induced arthritic rats (Kim and Park, 2006). In these concepts, plant-originated extracts have distinctive different bioactive characters. Geranium sibiricum Linne (GSL), a widely spread herb, has been used for healing of diarrhea and intestinal inflammation in traditional folk medicine, in Korea. Many classic texts of oriental medicine showed the name of GSL as a medicine for heal-
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ing of bacteria, intestinal inflammation, dermatitis, diarrhea, and cancer (Guo et al., 1987; Lee et al., 1993; Cho, 2006). Recent studies have reported that a number of phenolic compounds isolated from various Geranium species have antinociceptive and anti-inflammatory effects in inflammatory animal model systems (Küpeli et al., 2007; Pokharel et al., 2007). Among phenolic compounds, 4-hydroxykobusin isolated from Geranium thunbergii inhibits the production of nitric oxide and expression of inducible nitric oxide synthase (iNOS) via transcriptional activations of NF-B and AP-1 in macrophage cells (Pokharel et al., 2007). From these standpoints, it is strongly suggested that Geranium species may possess anti-inflammatory activity. So far, Geranium sibiricum Linne has not been reported its bioactivity, such as mechanism to remedy intestinal problems (diarrhea and intestinal inflammation). Therefore, we investigated to verify EGS prevents inflammation through inhibition of intracellular ROS, MAPK pathway, transcription factors, and proinflammatory cytokines in the inflammation-related signal transduction pathway using phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 (PMACI)-treated human mast cells (HMC-1).
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2.4. Western blot Nuclear protein extract isolated from HMC-1 cells were separated in a 10% polyacrylamide mini-gel at 100 V for 2 h at room temperature using a Mini-PROTEAN II electrophoresis cell (BioRad). After electrophoresis, the proteins were transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA). The transferred membranes were incubated for 1 h at room temperature in TBS-T solution [10 mM Tris–HCl (pH 7.6), 150 mM NaCl and 0.1% (v/v) Tween-20] containing 5% (w/v) non-fat dry milk. The membranes were subsequently incubated for 2 h at room temperature with rabbit polyclonal antibodies (anti-ERK, anti-pERK, anti-c-Jun, anti-c-Fos, anti-NF-B (p50, p65), anti-COX-2, antiiNOS, and ␣-tubulin, Santa Cruz Biotechnology, CA, USA) in TBS-T solution containing 5% non-fat dry milk. After three washes with TBS-T, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000; Cell Signaling Technology, MA, USA) in TBS-T containing 5% non-fat dry milk. The protein bands were visualized by incubation with enhanced chemiluminescence (Amersham Pharmacia Biotech., England, UK).
2. Materials and methods 2.5. Electrophoretic mobility shift assay (EMSA) 2.1. Chemicals All the plastic materials were purchased from Falcon Labware (Becton-Dickinson, Franklin Lakes, NJ). Ethylene diamine tetra acetate (EDTA, E5134), penicillin G (H0474), streptomycin (H0447), ethylene glycol bis (2-aminoethyl ether)-N, N, N N -tetraacetic acid (EGTA, E4378), nonidet P-40 (NP-40, N0896), phenyl methane sulfonyl fluoride (PMSF, P7626), obtained from Sigma (St. Louis, MO, USA). Iscove’s Modified Dulbecco’s Medium (IMDM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Other chemicals and reagents were of the highest analytical grade available. 2.2. Preparation of EGS The Geranium sibiricum Linne was purchased from Naju traditional market in May 2008, Chonnam province, South Korea. They were identified by Dr. H.T. Lim (Plant taxonomy, Department of Biology, Chonnam National University). A voucher specimen (No. LimKT0080522) was deposited at Molecular Biochemistry Laboratory, Chonnam National University, Gwangju, Korea. The GSL dry matter (1.5 kg) was cut into small pieces and soaked in 99% ethanol (EtOH, 19 L, w/v) for several months in a dark basement. The ethanol extracts were passed through Whatman filter paper (No. 2) in order to remove debris and concentrated with a rotary evaporator (B465; Buchi, Flawil, Switzerland). After the concentrated solution was dried with a freeze-dryer (SFDS06; Sam won, Seoul, Korea), it was lyophilized, and stored at −20 ◦ C. The yield of dried extract from starting crude materials was 83.4 g (5.56%). The lyophilized powder was dissolved in di-distilled water for experiment. The GSL ethanol extracts named EGS in this paper. 2.3. Cell culture HMC-1 cells, a human mast cell line, were provided by H.-M. Kim (College of Oriental Medicine, Kyung Hee University, Seoul, Korea). HMC-1 cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Life Technologies, Grand Island, NY, USA) supplemented with 10−5 M monothioglycerol (Sigma, St Louis, MO, USA), 5% heat-inactivated fetal bovine serum (FBS) and 100 IU/ml penicillin/streptomycin (Life Technologies) at 37 ◦ C in an atmosphere containing 5% CO2 . HMC-1 cells were passaged every 3–4 days.
To make the double stranded oligonucleotide, each strand of NFB and AP-1 oligonucleotide were annealed by heating at 37 ◦ C for 30 min. Then, it was labeled with [␣-32 P]dCTP (0.25 mCi, Amersham Pharmacia Biotech, Buckinghamshire, UK) by klenow polymerase and purified on a QIAquick® Nucleotide Removal Kit according to the manufacturer’s protocol (LRS Laboratory Inc., QIA GEN Distibuter, Seoul, Korea). The following NF-B and AP-1 oligonucleotide sequences were used for probing: NF-B sequence: 5 -AGT TGA GGG GAC TTT CCC AGG C-3 3 -TCA ACT CCC CTG AAA GGG TCC G-5 AP-1 sequence: 5 -TTC CGG CTG ACT CAT CAA GCG-3 3 -AAG GCC GAC TGA GTA GTT CGC-5 The DNA–protein binding reaction was performed by incubation of the NF-B and AP-1 probes and 10 g of nucleic protein extract and 0.5 g/ml poly dI/dC (Sigma Chemical Co., USA) in a binding buffer [0.2 M DTT, 20 mg ml-1 BSA, buffer B (20 mM HEPES, 20% glycerol, 100 mM KCl, 0.5 mM EDTA, 0.25% NP-40), buffer C (20% ficoll 400, 100 mM HEPES, 300 mM KCl)] at room temperature for 1–2 h. The DNA–protein complexes were resolved by applying 4% nondenaturing polyacrylamide gel in 0.5 × TBE (45 mM Tris–borate, 1 mM EDTA). Electrophoresis was carried out at 200 V for 3 h in a cold room. Gels were then dried on 3 M blotting paper (Whatman) and exposed to X-ray film at −70 ◦ C overnight.
2.6. Measurement of nitric oxide production HMC-1 Cells were treated with PMA (30 nM) and A23187 (1 M) (PMACI) in the presence of EGS (50–200 g/ml) for 24 h in the 96 well multiple plate. And then, cells were centrifuged at 1000 × g for 10 min and supernatants were collected. NO production was measured as a function of nitrite (NO2 − ) concentration by the method of Green et al. (1982). Supernatants (50 l) were mixed with 100 l of 0.1% sulfanilamide and 100 l 0.1% N-1-naphthylethylenediamine dihydrochloride in 2.5% polyphosphoric acid (Griess reagent) for 10 min. Absorbance was measured at 540 nm with a MicroReader (Hyperion, Inc., USA). Nitrite was quantified by using sodium nitrate as a standard.
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2.7. RNA isolation and reverse transcription polymerase chain reaction (RT-PCR) Total RNA was isolated from HMC-1 cells using TRIZOL® Reagent according to the manufacture’s protocol (Invitrogen life Technologies, Carlsbad, CA). Reverse transcription polymerase chain reaction (RT-PCR) was performed using the SuperScriptTM OneStep RT-PCR System (Invitrogen, Carlsbad, CA). Briefly, RT-PCR mixture (50 l) containing 1 g of total RNA, 10 M of gene-specific sense and antisense primers, 1 l of SuperScriptTM II RT/ Platinum® Taq Mix, and 25 l of 2X Reaction Mix was assembled on ice. The following primer sequences of IL-1 and GAPDH were used: IL-1 (332 bp) sense (5 -TTG ACG GAC CCC AAA AGA TG-3 ) and antisense (5 -AGA AGG TGC TCA TGT CCT CA-3 ). The amplifications were conducted with 35 cycles for IL-1 (20 s at 95 ◦ C, 40 s at 60 ◦ C, 30 s at 72 ◦ C) and the RT-PCR amplification products were mixed with 2 l loading buffer and separated on a 2% agarose gel. The gels were stained with 5 g/ml ethidium bromide and photographed. 2.8. Statistical analysis All experiments were done in triplicate, and data were expressed as the means ± SE. A one-way analysis of variance (ANOVA) and Duncan test were used to detect significant differences by multiple comparisons (SPSS program, ver. 11.0). 3. Results 3.1. Effects of EGS on MAPK inhibition in PMACI stimulated HMC-1 cells We studied whether or not EGS modulates the ERK MAPK pathway in PMACI stimulated HMC-1 cells. The band intensity of phospho ERK MAPK in whole cell protein extracts was much stronger in (Fig. 1, lane 2) compared with treated with PMACI alone than in control group (Fig. 1, lane 1). Upon increasing concentration of the EGS (25, 50, 100 g/ml), however, its band intensities were gradually attenuated by 0.5, 0.8, 1.1 in PMACI-treated groups (Fig. 1, lanes 3–5). 3.2. Effects of EGS on inhibition of NF-B and AP-1 in PMACI stimulated HMC-1 cells Results in Western blot showed that whether or not EGS modulates the p50 and p65, which is a kind of NF-B subunits and c-jun and c-fos, which is a kind of AP-1 subunit in PMACI stimulated HMC1 cells. The intensity of NF-B and AP-1 band expression in nuclear
Fig. 2. Effects of EGS on activation of NF-B and AP-1 in PMACI stimulated HMC-1 cells. Lane 1, control; lane 2, PMACI alone; lane 3, 25 g/ml EGS + PMACI; lane 4, 50 g/ml EGS + PMACI; lane 5, 100 g/ml EGS + PMACI. ␣-Tubulin was used as an internal control.
extracted protein extracts was much stronger in (Fig. 2, lane 2), which treated with PMACI only than in control group (Fig. 2, lane 1). Upon increasing concentration of the EGS (25, 50, 100 g/ml), however, its band intensities were gradually attenuated by 0.3, 0.8, 1.6 in c-jun, 0.3, 0.8, 1.6 in c-fos, 1.0, 1.7, 2.4 in p65, and 1.0, 1.5, 1.9 in p50, in PMACI-treated groups (Fig. 2, lanes 3–5). 3.3. Effects of EGS on DNA-binding activities of NF-B and AP-1 in PMACI stimulated HMC-1 cells We further studied that EGS inhibits PMACI-induced NF-B and AP-1 activation in HMC-1 cells (Fig. 3). When the mice were treated with PMACI, NF-B complex had maximal DNA-binding activity (Fig. 3A, lane 2). However, the DNA binding activities of NF-B complex intensity were gradually reduced by 0.3, 0.8 in 50 and 100 g/ml of EGS in a dose-dependent manner (Fig. 3A, lanes 3 and 4). Similarly, the DNA-binding activity of AP-1 complex in nuclear colonic protein extracts was higher in Fig. 3B, which treated with PMACI alone (lane 2) than in the control group (Fig. 3B, lane 1). By contrast, treatment of PMACI in presence of 50, 100 g/ml of EGS markedly suppressed the DNA-binding activity of AP-1 complex intensity by 0.2, 0.3 (Fig. 3B, lanes 3 and 4). 3.4. Effects of EGS on COX-2 and iNOS inhibition in PMACI stimulated HMC-1 cells
Fig. 1. Effects of EGS on activation of ERK in PMACI stimulated HMC-1 cells. Lane 1, control; lane 2, PMACI alone; lane 3, 25 g/ml EGS + PMACI; lane 4, 50 g/ml EGS + PMACI; lane 5, 100 g/ml EGS + PMACI. ␣-Tubulin was used as an internal control.
We further examined the inhibitory effects of EGS on PMACIinduced iNOS and COX-2 proteins activation using Western blot
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Fig. 3. Effects of EGS on DNA-binding activities of NF-B and AP-1 in PMACI stimulated HMC-1 cells. Lane 1, free probe alone (no nuclear extracts); lane 2, control; lane 3, PMACI alone; lane 4, 50 g/ml EGS + PMACI; lane 5, 100 g/ml EGS + PMACI.
with whole cell protein extracts. As shown in Fig. 4, EGS was shown to have dose-dependent blocking activities on the activation of pro-inflammatory proteins in PMACI stimulated HMC-1 cells. Namely, iNOS and COX-2 proteins were activated after treatment with PMACI (Fig. 4, lane 2). Upon pretreatment with the EGS (25, 50, 100 g/ml), however, the intensities of the iNOS and COX-2 bands markedly reduced by 1.1, 1.9, and 2.2 in iNOS and 0.2, 0.3, 0.7 in COX-2, compared to PMACI treatment alone (Fig. 4, lanes 3 and 4).
3.5. Effects of EGS on NO production in PMACI stimulated HMC-1 cells As shown in Fig. 5, NO production in PMACI stimulated HMC1 cells were decreased a little compared with control. However, production of NO was much increased by 2.38, 3.17 M in 12 h and 3.06, 3.28 M in 24 h by 100, 200 g/ml of EGS treated group compared with PMACI only treated HMC-1 cells.
Fig. 5. Effects of EGS on NO production in PMACI stimulated HMC-1 cells. Each bar represents the mean ± SE of triplicate experiments. Single asterisk (*) represents significant difference compared to PMACI treatment alone and EGS (100 and 200 g/ml) in the presence of PMACI, p < 0.05.
3.6. Effects of EGS on inflammatory cytokine production in PMACI stimulated HMC-1 cells Results in the Fig. 6 showed that IL-1 mRNAs in the control group not treated with PMACI or EGS were constitutively expressed (Fig. 6 lane 1), whereas the levels of these mRNAs were elevated after treatment with PMACI (Fig. 6, lane 2). However, the intensity of IL-1 mRNAs was obviously down-regulated by 0.7 in treatment with 50 g/ml of EGS, compared to PMACI treatment alone (Fig. 6, lane 3). 4. Discussion
Fig. 4. Effects of EGS on activation of iNOS and COX-2 in PMACI stimulated HMC-1 cells. Lane 1, control; lane 2, PMACI alone; lane 3, 25 g/ml EGS + PMACI; lane 4, 50 g/ml EGS + PMACI. 100 g/ml EGS + PMACI. ␣-Tubulin was used as an internal control.
Mast cells are broadly distributed throughout mammalian tissues and play an important role in a wide variety of biological responses. Typically, mast cells have been considered not only in the association of immediate-type hypersensitivity, but also in late reactions like inflammatory responses, which are mast cell dependent (Metzger et al., 1986; Kemp and Lockey, 2002). And they play a critical role in allergic inflammation through the immunoglobulin E (IgE) dependent release of including histamine, tryptase, chymase, heparin and some inflammatory cytokines (Mekori and Metcalfe, 2000; Galli et al., 2005). Through release their proinflammatory
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Fig. 6. Effects of EGS on expression of IL-1 mRNA in PMACI stimulated HMC-1 cells. Each bar represents the means ± SE of triplicate experiments. (#) Represents significant difference compared to the control and PMACI treatment alone, p < 0.05. Single asterisk (*) represents significant difference compared to the PMACI treatment alone and 100 g/ml EGS treatment, p < 0.01.
mediators, mast cells actively participate in the pathogenesis of these intestinal diseases (Walls et al., 2001). The results of this study were revealed interesting inflammation inhibition effects of EGS in PMACI stimulated HMC-1 cells with intracellular signal pathways and secretion of proinflammatory mediators, such as inducible nitric oxide synthase (iNOS), interleukin-1 (IL-1). PMACI is well known as an inducer of HMC-1 cells. The results of in this experiment showed that PMACI stimulates ERK mitogen activated protein kinase (MAPK) (Fig. 1). Among MAPK family, ERK plays a role in the survival of the cells induced by various growth factors and linked to inflammatory pathways (Cobb and Goldsmith, 1995; Zhang and Stanimirovic, 2002). And EGS showed the dramatically decreasing effect on phospho-ERK, one of the MAPK phosphorylation. However, MAPKs p38 and JNK, except ERK are not showed remarkable decrease compared with PMACI stimulation. The phosphorylation of ERK activates transcription factors and some downstream protein kinases. It suggests that PMACI induced ERK stimulate the IKK␣ phosphorylation for inflammation development and EGS can modulate the upper state of ERK pathway. NF-B and AP-1 are redox-sensitive transcription factors which germane to ERK stimulation. Activated these transcription factors head for nuclear and activate many inflammatory mediator genes (Collart et al., 1990; Azzolina et al., 2003). In this study, the results from Western blotting, we carried out EGS inhibits activation of transcription factors’ subunits such as p50 and p65 in NF-B, and c-Jun and c-Fos in AP-1, against PMACI stimulation. To further understand the activation of transcription factors, we evaluated the inhibitory effects of the EGS on DNA binding activities of NFB and AP-1 in PMACI stimulated HMC-1 cells. The results in this experiment showed that PMACI stimulates both the DNA-binding activities of NF-B and AP-1. Data indicate that the increasing DNA binding activities of NF-B and AP-1 by PMACI treatment were decreased in the presence of EGS in a dose-dependent manner. This finding suggests that EGS interferes with PMACI stimulation to activate NF-B and AP-1.
We confirmed the Western blot to elucidate the regulation of PMACI-induced iNOS and COX-2, which are sensitive prime components of intracellular signaling pathways responsible for inflammatory events, activation by EGS. Our findings in this study show that EGS suppressed iNOS activity, and subsequently it reduces the COX-2 activity in HMC-1 cells. Although we have determined the iNOS expression, production of NO was decreased PMACI treated only and increased EGS dose dependently. Interestingly, NO is an important multiplayer, participate in cell death and survival, invasion of pathogen, and inflammation (Beckman et al., 1990). On the other hand, NO inhibits degranulation, cytokine release and mediator expression of HMC-1 cells (Coleman, 2002). These results suggest that although iNOS remains high for inflammation environment, NO is decreased for cytokine release, and cytokine expression in HMC-1 cells. It may responsible for inhibition of iNOS activation, through arginine deletion, inhibition of L-arginine analogs, and inhibition of cofactors (Schoonover et al., 2000). We observed that PMACI stimulated HMC-1 cells release IL-1, pro-inflammatory cytokine. Interleukin-1 gives an inflammatory signal to immune cells such as T cells, B cells, and macrophages and causes lymphocyte activation, macrophage stimulation, pyrexia, and negative acutephase proteins like a transcortin, which upregulates inflammation. The results showed the expression of IL-1 is clearly decreased by EGS compared with PMACI treated only. Taken together, our finding revealed that Geranium sibiricum Linne has also an anti-inflammatory activity like other Geranium species (Küpeli et al., 2007; Pokharel et al., 2007). In conclusion, our results indicate that EGS has an antiinflammatory potential due to activated human mast cells (HMC-1) through modulation of pro-inflammatory cytokine IL-1 expression and NO production. Hence, we speculate that it is applicable in the prevention and treatment of inflammatory diseases. Further study remains to modulate the expression of cytokines like a TNF-␣ and IL-6 by EGS at the molecular level. Acknowledgement This study was financially supported by Biotechnology Research Institute, Chonnam National University in 2009. References Azzolina, A., Bongiovanni, A., Lampiasi, N., 2003. Substance P induces TNF alpha and IL-6 production through NF kappa B in peritoneal mast cells. Biochimica et Biophysica Acta 1643, 75–83. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., Freeman, B.A., 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proceedings of the National Academy of Sciences of the United States of America 87, 1620–1624. Bochenek, G., Nizankowska, E., Gielicz, A., Swierczynska, M., Szczeklik, A., 2004. Plasma 9alpha,11beta-PGF2, a PGD2 metabolite, as a sensitive marker of mast cell activation by allergen in bronchial asthma. Thorax 59, 459–464. Cho, T.D., 2006. Korean Herbs, 1st ed. Daewon, p. 288. Cobb, M.H., Goldsmith, E.J., 1995. How MAPK are regulated. The Journal of Biological Chemistry 270, 14843–14846. Cobb, M.H., Goldsmith, E.J., 2000. Dimerization in MAP-kinase signaling. Trends in Biochemical Sciences 25, 7–9. Coleman, J.W., 2002. Nitric oxide: a regulator of mast cell activation and mast cellmediated inflammation. Clinical and Experimental Immunology 129, 4–10. Collart, M.A., Baeuerle, P., Vassalli, P., 1990. Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B. Molecular and Cellular Biology 10, 1498–1506. Davis, R.J., 1993. The mitogen-activated protein kinase signal transduction pathway. The Journal of Biological Chemistry 268, 14553–14556. Galli, S.J., Kalesnikoff, J., Grimbaldeston, M.A., Piliponsky, A.M., Williams, C.M., Tsai, M., 2005. Mast cells as “tunable” effector and immunoregulatory cells: recent advances. Annual Review of Immunology 23, 749–786. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N] Nitrate in biological fluids. Analytical Biochemistry 126, 131–138. Guo, J.S., Wang, S.X., Li, X., Zhu, T.R., 1987. Studies on the Antibacterial Constituents of Geranium-Sibiricum L. Yaoxue Xuebao 22, 28–32.
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