ABSTRACT 1
The purpose of this study was to examine anti-inflammatory effect of ethanolic extract of 2
Antrodia salmonea (EAS) in the lipopolysaccharide (LPS)-stimulated RAW246.7
3
macrophages and the carrageenan (Carr)-induced edema paw model, and to clarify its possible 4
molecular mechanisms. Inhibitory effects of EAS were examined on cells proliferation, 5
nitric oxide (NO) production, expression of inducible nitric oxide synthase (iNOS) and 6
cyclooxygenase-2 (COX-2) proteins, and the activity of antioxidant enzymes. Our data 7
demonstrated that EAS inhibited NO production, and expression of iNOS and COX-2 8
proteins in LPS-stimulated RAW246.7 cells. EAS can also significantly reduce paw edema, 9
content of NO, TNF- and malondialdehyde (MDA), expression of iNOS and COX-2 10
proteins, and neutrophil infiltration within the tissues stimulated by Carr. The 11
anti-inflammatory mechanisms of EAS might be related to the decrease of inflammatory 12
cytokine and increase of antioxidant enzymes activities, which would result in reduction of 13
iNOS, COX-2 and MDA and subsequently inflammatory responses. 14
15
Keywords: herbal medicine, anti-inflammation, Antrodia salmonea, carrageenan, 16
INTRODUCTION 17
The acute inflammatory response is a series of local cellular and vascular responses that 18
occurs immediately following tissue damage, and this complex biological response is a 19
protective mechanism of organisms to remove the injurious stimuli, such as pathogens, 20
irritants or physical injury, from the tissues and to initiate the healing process. However, 21
chronic inflammation has been reported to involve in the development of several diseased 22
conditions or disorders such as Alzheimer disease (1), asthma (2), atherosclerosis (3), 23
autoimmune diseases (4), cancers (5) and rheumatoid arthritis (6), which may lead to 24
progressive destruction of the tissue, fibrosis, and necrosis, etc (7, 8). 25
Numerous molecules have been mentioned to contribute the local tissue destruction 26
during chronic inflammation (9-11). Of these, inducible nitric oxide synthase (iNOS), a 27
member of the NOS protein family, catalyzes the formation of nitric oxide (NO) from 28
L-arginine (12). NO can activate guanylate cyclase to induce smooth muscle relaxation in the 29
normal physiological condition. High-output NO produced by the activated macrophage via 30
iNOS has been found to play a major role as antimicrobial molecule (13). However, highly 31
level of NO have the opportunity to react with superoxide resulting in peroxynitrite formation 32
and cell toxicity, which are found to play important roles in inflammation and carcinogenesis. 33
The expression of COX-2 (cyclooxygenase 2) has also been mentioned to implicate the 34
response for the prostaglandin biosynthesis involved in inflammation and pain, and clinical 35
application of highly selective inhibitors of COX-2 has been demonstrated to provide 36
effective anti-inflammatory activity with marked reduction in gastrointestinal toxicity as 37
compared to traditional NSAIDs (non-steroidal anti-inflammatory drugs) (14). Similarly, 38
tumor necrosis factor-alpha (TNF-), an endotoxin-induced glycoprotein, is a critical 39
modulator of host immune response to infection, but inappropriate or excessive production 40
can be harmful. Receiving anti-TNF- antibody and oral administration of soluble TNF 41
receptors have been demonstrated to control the inflammatory conditions (11). 42
The medical fungus Antrodia salmonea, a newly identified species of the genus Antrodia, 43
grow on the empty rotten trunk of Cunninghamia konishii in Taiwan (15). The fruiting body 44
of A. salmonea has been used in the food remedy of diarrhea, abdominal pain, hypertension, 45
itchy skin, and liver cancer and is also used as a detoxicant in Taiwan folk medicine (16). To 46
date, there were several newly compounds were isolated from the basidiomata of A. salmonea, 47
whose in vitro studies displayed anti-oxidative effect (17) and anti-inflammatory activities in 48
activated inflammatory cells (18). Thereby, we designed the in vivo study to examine whether 49
the ethanolic extract from fruiting body of A. salmonea has potential effects against 50
inflammatory response in the lipopolysaccharide (LPS)-stimulated RAW246.7 cells and the 51
carrageenan (Carr)-induced edema paw model, and to clarify its possible molecular 52
mechanisms, which will help us to further evaluate the clinical therapeutic potential or food 53
remedy of A. salmonea on anti-inflammation. 54
MATERIALS AND METHODS 55
Chemicals. Lipopolysaccharide (LPS) from Escherichia coli (serotype 0127:B8), 56
carrageenan (Carr), indomethacin (Indo) and other chemicals were purchased from 57
Sigma-Aldrich (St. Louis, MO, USA). TNF-α was purchased from Biosource International 58
Inc (Camarillo, CA, USA). Anti-iNOS, anti-COX-2, anti-β-actin antibody (Santa Cruz 59
Biotechnology, CA, USA) and a protein assay kit (Bio-Rad Lab, Watford, Herts, UK) were 60
obtained as indicated. Polyvinylidene fluoride (PVDF) membrane (Immobilon-P® ) was 61
obtained from Millipore Corp (Bedford, MA, USA). 62
Preparation of ethanolic extract of A. salmonea (EAS). The fruiting body of A. 63
salmonea was purchased from the Ji pin mushroom store (Nantou, Taiwan), and identified by
64
Drs. Yu-Cheng Dai (Institute of Applied Ecology, Chinese Academy of Science, China) and 65
Sheng-Hua Wu (Department of Botany, National Museum of Natural Science, Taiwan). 66
Dried sample of A. salmonea (100 g) was macerated with 1L ethanol for 24 h at room 67
temperature. Filtration and collection of the extract was done three times. The filtrates were 68
collected, concentrated with a vacuum evaporator until the volume was below 10 mL and then 69
freeze-dried. The yield obtained was 4.2 % (w/w). 70
Cell culture. A murine macrophage cell line RAW264.7 (BCRC No. 60001) was 71
purchased from the Bioresources Collection and Research Center (BCRC) of the Food 72
Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in 73
culture dishes containing Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich) 74
supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) in a CO2 incubator (5%
75
CO2 in air) at 37°C and subcultured every 3 days at a dilution of 1:5 using 0.05%
76
trypsin–0.02% EDTA in Dulbecco's phosphate-buffered saline (DPBS) without Ca2+ and 77
Mg2+ ions. 78
Mice model of Carr-induced paw edema. Twenty-four male ICR mice were obtained 79
from the BioLASCO Taiwan Co., Ltd. The animals housed in Plexiglas cages with free access 80
to food and water, and maintained at a constant temperature of 22 ± 1°C and relative humidity 81
of 55 ± 5 % with a photocycle of 12-h light/dark. The experimental procedures were 82
performed according to the National Institutes of Health (NIH) Guide for the Care and Use of 83
Laboratory Animals. In addition, all tests were conducted under the guidelines of the 84
International Association for the Study of Pain (19). After a 2-week adaptation period, the 85
mice (about 18-25 g) were randomly assigned to four groups (n = 6) for further experiments. 86
The control group receives normal saline, and the other three groups include a Carr alone, 87
Carr + Indo (a positive control), and EAS administered groups (Carr+ EAS). The 88
Carr-induced hind paw edema model was used for determination of anti-inflammatory activity 89
(20). Animals were treated with normal saline, Indo or EAS (12.5, 25, and 50 mg/kg) with 90
intraperitoneal injection, 30 min prior to injection of 1% Carr (50 μL) in the plantar side of 91
right hind paws of the mice. The paw volume was measured immediately after Carr injection 92
and at 1, 2, 3, 4, and 5 h intervals after the administration of the edematogenic agent using a 93
Plethysmometer (model 7159; Ugo Basile, Varese, Italy). The degree of swelling induced was 94
evaluated by the ratio A/B, where A is the volume of the right hind paw after Carr treatment, 95
and B is the volume of the right hind paw before Carr treatment. Finally, the animals were 96
sacrificed and all of right hind paw were dissected and stored at -80 ºC. Also, blood were 97
withdrawn and kept at -80 ºC. 98
MTT cell viability assay. RAW264.7 cells (2 × 105) were cultured in 96-well plate 99
containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. 100
Then cells were pre-treated with several concentrations (12.5, 25, 50, 100, and 200 g/mL) of 101
EAS for 1 h and then co-stimulated with 100 ng/mL of LPS for 24 h. After that, the cells were 102
washed twice with DPBS and incubated with 100 L of 0.5 mg/mL MTT 103
(3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide) for 2 h at 37°C, and then the 104
medium was discarded and 100 L of dimethyl sulfoxide (DMSO) was added. After 30 min 105
incubation, absorbance at 570 nm was read using a microplate reader. 106
Measurement of nitric oxide/nitrite. NO production was indirectly assessed by 107
measuring the nitrite levels in the cultured media and serum determined according to previous 108
study (20). The cells were pre-incubated with EAS (0, 12.5, 25, and 50 g/mL) for 1 hr and 109
then co-treated with 100 ng/mL LPS at 37°C for 24 h. Subsequently, 100 L of each collected 110
culture medium was mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% 111
naphthyl ethylenediamine dihydrochloride and 5% phosphoric acid) and incubated at room 112
temperature for 10 min. The absorbance of mixture was measured at 540 nm with a 113
Micro-Reader (Molecular Devices, Orleans Drive, Sunnyvale, CA). Homogenized tissue 114
samples were diluted four times with distilled water and deproteinized by adding 1/20 volume 115
of zinc sulfate (300 mg/mL) to a final concentration of 15 mg/mL. After centrifugation at 116
10,000 ×g for 5 min at room temperature, 100 μL of supernatant was applied into a microtiter 117
plate, followed by 100 μL of Griess reagent. After 10 min of color development at room 118
temperature, the absorbance was measured at 540 nm with a Micro-Reader. By using sodium 119
nitrite to generate a standard curve, the concentration of nitrite was measured by absorbance 120
at 540 nm. 121
Western blot analysis. The stimulated RAW264.7 cells were washed with PBS and 122
lysed in an ice-cold lysis buffer [10% glycerol, 1% Triton X-100, 1mM sodium orthovanadate, 123
1mM EGTA, 10mM sodium fluoride, 1mM sodium pyrophosphate, 20 mM Tris buffer (pH 124
7.9), 100 mM -glycerophosphate, 137 mM sodium chloride, 5 mM EDTA and one protease 125
inhibitor cocktail tablet (Roche, Indianapolis, IN, USA)] on ice for 1 h, followed by 126
centrifugation at 12,000 ×g for 30 min at 4°C. Soft tissues were removed from individual 127
mice paws and homogenized in a solution containing 10 mM CHAPS, 1 mM 128
phenylmethylsulphonyl fluoride (PMSF), 5 g/mL, aprotinin, 1 M pepstatin and 10 M 129
leupeptin. The homogenates were centrifuged at 12,000 ×g for 20 min, and the supernatant 130
was collected for Western blot analysis. Protein concentration was measured by the Bio-Rad 131
protein assay kit with bovine serum albumin as a standard. About 30 g of protein from the 132
supernatants was then separated on 10% sodium dodecylsulphate- polyacrylamide gel 133
(SDS-PAGE) and transferred to PVDF membranes. After transfer, the membrane was blocked 134
for 2 h at room temperature with 5% skim milk in TBST buffer (20 mM Tris, 500 mM NaCl, 135
pH 7.5 and 0.1% Tween 20). The membranes were then incubated with mouse monoclonal 136
anti-iNOS or anti-COX-2 antibody in 5% skim milk in TBST buffer for 2 h at room 137
temperature. The membranes were washed three times with TBST at room temperature and 138
then incubated with a 1:2000 dilution of anti-mouse IgG secondary antibody conjugated to 139
horseradish peroxidase (Sigma-Aldrich) in 2.5% skim milk in TBST for 1 h at room 140
temperature. The membranes were washed three times and the immunoreactive proteins were 141
detected by enhanced chemiluminescence (ECL) using Hyperfilm® ECL reagent (Amersham 142
International, Buckinghamshire, UK). The results of Western blot analysis were quantified by 143
measuring the relative intensity compared to the control using Kodak Molecular Imaging 144
Software Ver.4.0.5 (Eastman Kodak Company, Rochester, NY, USA) and represented in the 145
relative intensities. The results for iNOS and COX-2 were normalized to the band density of 146
internal control (-actin), and the relative proteins expression were calculated according to the 147
values of LPS treated alone group as 100%. 148
Malondialdehyde assay. Malondialdehyde (MDA) from Carr-induced edema foot was 149
evaluated by the thiobarbituric acid reacting substance (TRARS) method (20).Briefly, MDA 150
reacted with thiobarbituric acid in the acidic high temperature and formed a red-complex 151
TBARS. The absorbance of TBARS was determined at 532 nm. 152
Measurement of Serum TNF-α. Serum levels of TNF- were determined using a
153
commercially available ELISA kit (Biosource International Inc., Camarillo, CA) according to 154
the manufacturer's instruction. The concentration of serum TNF- was presented as pg/mL 155
and determined according to the regression equation of the standard curve. 156
Histological examination. The biopsies of mice hind paws were immediately taken 157
following 5 h treatment with the interplanetary injection of Carr. The tissue slices were fixed 158
in (1.85% formaldehyde, 1% acetic acid) for 1 week at room temperature, dehydrated by 159
graded ethanol and embedded in Paraffin (Sherwood Medical). Tissue sections (5 μm 160
thickness) were deparaffinized with xylene and stained with hematoxylin and eosin for cell 161
counting. All samples were observed and photographed with BH-2 Olympus microscopy. 162
The excessive inflammatory response was illustrated as massive infiltration of 163
ploymorphonuclear leukocytes (PMNs). The observation of tissue slices (3-5 slides) were 164
randomly chosen from every groups, and the number of neutrophils were counted from five 165
scopes (400 ×) on each tissue slice to obtain average value. 166
HPLC analysis of EAS. HPLC was performed according to the minor modification of 167
previous studies (21,22). Before analysis by HPLC, EAS was filtered through a 0.2 µm 168
Millipore filter, and then total volume of 20 L was loaded into HPLC column. Besides, 169
external standards were prepared as concentration of 100 g/mL in HPLC grade-methanol 170
and used to calculate the concentration of examined compounds. Reverse phase HPLC was 171
performed on a HITACHI HPLC system (Tokyo, Japan) equipped with HITACHI L-7100 172
pump, HITACHI L-7400 UV detector and HITACHI L-7200 autosampler. Separations were 173
accomplished on LiChroCART 250-4 C18 HPLC-cartridge (5 m; Merck, Whitehouse 174
Station, NJ, USA). The separation conditions of HPLC analysis for examined compounds 175
were described in Table 1. 176
Measurement of antioxidant enzymes activity. The following biochemical parameters 177
were analyzed to check the hepatoprotective activity of EAS by the methods given below. 178
Total superoxidase dismutase (SOD) activity was determined by the inhibition of cytochrome 179
c reduction (23). The reductionof cytochrome c was mediated by superoxide anions generated
180
by the xanthine/xanthine oxidase system and monitored at 550 nm. One unit of SOD was 181
defined as the amount of enzyme requiredto inhibit the rate of cytochrome c reduction by 182
50%. Total catalase (CAT) activity was measured according to previous study (24). In brief, 183
the reduction of 10 mM hydrogen peroxide in 20 mM of phosphate buffer (pH 7.0) was 184
monitored by measuring the absorbance at 240 nm. The activity was calculated using a molar 185
absorption coefficient, and the enzyme activity was defined as nanomoles of dissipating 186
hydrogen peroxide per milligram protein per minute. Total glutathione peroxidase (GPx) 187
activity in cytosol was determined according to Paglia and Valentine's method (25). The 188
enzyme solution was added to a mixture containing hydrogen peroxide and glutathione in 0.1 189
mM Tris buffer (pH 7.2) and the absorbance at 340 nm was measured. Activity was evaluated 190
from a calibration curve, and the enzyme activity was defined as nanomoles of NADPH 191
oxidized per milligram protein per minute. 192
193
Statistical analysis. Data are expressed as mean ± standard error of the mean (S.E.M). 194
Statistical evaluation was carried out by one-way analysis of variance (ANOVA, Scheffe's 195
post-hoc test). A value of p < 0.05 was regarded as being statistically significant. 196 197 198 199 200 201 202 203 204 205
RESULTS 206
Effect of EAS on cell viability of RAW246.7 cells. The growth inhibitory effect of EAS on 207
RAW264.7 cell viability was determined by a MTT assay (Fig. 1). Cells were pre-treated with 208
EAS at the concentrations (0, 12.5, 25, 50, 100, and 200 g/mL) for 1 h and then co-incubated 209
with 100 ng/mL of LPS for further 24 h. Our results showed that 100 ng/mL of LPS did not 210
change cell viability of RAW246.7 cells. Pre-treatment of EAS at concentration of 100 and 211
200 g/mL can significantly inhibit cell viability of RAW264.7 macrophages with presence 212
of LPS. However, Lower concentration of EAS (12.5, 25, and 50 g/mL) showed no effects 213
in cell viability in the presence of 100 ng/mL LPS incubation for 24 h. 214
Effect of EAS on LPS-induced NO production in RAW246.7 cells. Various 215
concentration (0, 12.5, 25, and 50 g/mL) of EAS were used on RAW246.7 cells to test 216
whether EAS can reverse LPS-induced dramatically accumulation of NO (Fig. 2). The results 217
revealed that 100 ng/mL LPS can evidently increase NO production as compared with control 218
group (p< 0.001), and this effect can be markedly suppressed in a dose-dependent manner by 219
pre-treatment of EAS (25 and 50 g/mL) as compared to those in LPS treated only group. 220
EAS did not interfere with the reaction between nitrite and Griess reagents at 100 ng/mL (data 221
not shown). 222
Effect of EAS on LPS-induced iNOS and COX-2 proteins expression in RAW246.7 223
cells. Pre-incubation of EAS (0, 12.5, 25, and 50 g/mL) were tested on RAW246.7 cells to 224
examine whether EAS can reduce protein expression of inflammation-associated molecules 225
triggered by LPS (Fig. 3). The experimental results suggested that 100 ng/mL of LPS can 226
significantly stimulated protein expression of iNOS and COX-2 (p<0.001), and pre-treatment 227
of EAS at concentration of 25 and 50 g/mL can obviously down-regulate expression of these 228
LPS-stimulated proteins as compared to LPS treated only group (p < 0.05 and p < 0.001). 229
Effects of EAS in Carr-induced mice paw edema. Carr-induced paw edema model was 230
used to evaluate the in vivo anti-inflammatory effect of EAS (Fig. 4). The results showed that 231
Carr injection will stimulate local inflammation and then induce edema of paw tissues. Indo, a 232
common clinical NSAIDs, was used as positive control to indicate that pre-treatment of 10 233
mg/kg Indo can effectively reduce paw edema after 3th h Carr stimulation (p<0.01). 234
Similarly, pre-treatment of EAS (25 and 50 mg/kg) can also markedly decrease paw edema 235
after 3th h Carr stimulation, as same as the result of Indo + Carr group (p<0.001). 236
Effects of EAS on the NO, TNF-, and MDA Levels. In Fig.5A, the NO level increased 237
significantly in the edema serum at the 5th h after Carr injection (p<0.001), which can be 238
markedly reversed by EAS as concentration more than 12.5 mg/kg (p<0.05), and the 239
inhibitory potency of EAS (50 mg/kg) was similar to that of Indo (10 mg/kg) at 5th h after 240
induction. Likewise, both TNF-α and MDA level were increased significantly in the edema 241
paw at the 5th h after Carr injection (p<0.001), and this effect was decreased significantly by 242
treatment with EAS as well as 10 mg/kg Indo (Fig.5B and 5C). 243
Effects of EAS on Carr-induced iNOS and COX-2 proteins expression in edema paw. 244
Our results showed that EAS (50 mg/kg) can obviously inhibit (p<0.001) iNOS and COX-2 245
proteins expression in edema paw as compared to Carr-treated alone group (Fig. 6). The 246
experiments showed an average of 67.6% and 57.4% down-regulation of iNOS and COX-2 247
protein, respectively, after treatment with EAS at 50 mg/kg compared with the Carr-induced 248
alone (Fig.6B). In addition, the protein expression showed an average of 53.6% and 51.1% 249
down-regulation of iNOS and COX-2 protein after treatment with Indo at 10 mg/kg compared 250
with the Carr-induced alone (Fig. 6B). The potency of EAS (50 mg/kg) on down-regulating 251
the expression of iNOS and COX-2 proteins was similar to that of Indo (10 mg/kg). 252
Histological examination. Paw biopsies of Carr model animals showed marked cellular 253
infiltration in the connective tissue, and the infiltrates accumulated between collagen fibers 254
and into intercellular spaces (Fig. 7B). Paw biopsies of animals treated with EAS (50 mg/kg) 255
showed a reduction in Carr-induced inflammatory response (Fig. 7D). Actually inflammatory 256
cells were reduced in number and confined to near the vascular areas, and intercellular spaces 257
did not show any cellular infiltrations (Fig.7D). Collagen fibers were regular in shape and 258
showed a reduction of intercellular spaces. Moreover, the hypoderm connective tissue was not 259
damaged (Fig. 7D). In Fig. 7E, neutrophils increased with Carr treatment (p<0.001). As Indo 260
and EAS (50 mg/kg) could significantly decrease the neutrophils numbers as compared to the 261
Carr-treated group (p<0.001). 262
Effects of EAS on activities of antioxidant enzymes. At 5th h after the intrapaw injection 263
of Carr, paw tissues were analyzed for the biochemical parameters such as CAT, SOD, and 264
GPx activities (Table 1). CAT, SOD, and GPx activities in paw tissue were decreased 265
significantly by Carr administration. CAT, SOD, and GPx activity were increased 266
significantly after treated with 25 mg/kg EAS (p < 0.05) and 10 mg/kg Indo (p < 0.01). 267
Qualification of extraction procedure of EAS. Some of the reference compounds 268
(adenosine and zhankuic acid A) within EAS were identified by HPLC to be as indicator 269
compounds for quality check of extraction procedure of each batch (Fig. 8). Using HPLC 270
quantification, the content of adenosine and zhankuic acid A were calculated to be 16.3 and 271
11.5 mg/g of EAS, respectively (Table 2).
272
273
Discussion 274
In the present study, we demonstrated anti-inflammatory activities of EAS in both in vitro 275
and in vivo experimental systems, using LPS-stimulated RAW264.7 macrophages and a 276
mouse model of topical inflammation respectively. Dual inhibitory activities against iNOS 277
and COX-2 as shown in in vitro assays appear to confer on EAS a potent in vivo efficacy in 278
mouse Carr-induced paw edema, comparable with a potent and well known COX inhibitor, 279
indomethacin, suggesting its potential therapeutic usage as a novel topical anti-inflammatory 280
source of health food. 281
Previous studies reported that some bioactive compounds have been identified within 282
extract of the basidiomata of A. salmonea and displayed potential with anti-inflammatory 283
effect (17,18). Our experimental data showed that at least two bioactive compounds, including 284
adenosine and zhankuic acid A, have been identified in the ethanolic extract of fruiting body 285
of A. salmonea (Fig. 8). Of these, adenosine levels rise during inflammation and modulate 286
inflammatory responses by the interaction with their receptors (26). Activation of A2 and A3 287
adenosine receptors (AR) has been evidenced to provide anti-inflammatory effects (27-31). 288
A3AR is considered to be expressed in macrophage cells (32). Lee et al. noticed that a novel 289
A3AR agonist, thio-Cl-IB-MECA, can inhibit the LPS-stimulated expression of 290
pro-inflammatory markers including iNOS, interleukin-1beta (IL-1), and TNF- thought 291
suppressing phosphatidylinositol 3-kinase (PI3 kinase)/Akt and NF-kB signaling pathways 292
(27). Stimulating A3AR can alter the cytokine milieu by decreasing TNF- (33). Similarly, 293
A3 receptor agonist, IB-MECA, inhibited the production of interleukin-12 and 294
interferon-gamma (IFN) and prevented lethality in endotoxemic mice (31). It also has been 295
reported that IC51, an adenosine kinase inhibitor, stimulated the extracellular adenosine 296
release and reduced the LPS/ IFN-mediated production of NO, and induction of iNOS and 297
TNF- gene expression (34). Bouma et al. evidenced that adenosine acts via A2AR as well as 298
A3AR to inhibit neutrophil degranulation and neutrophil-mediated tissue injury (35). Our 299
experimental results showed that EAS significantly reduce serum level of TNF- (Fig. 5B) as 300
well as the level of iNOS protein (Fig. 3 and 6) and subsequently NO production (Fig. 2 and 301
5A) in in vitro and in vivo assays, which might be caused by the pharmacological effect of 302
adenosine. On the other hand, zhankuic acid A isolated from A. camphorate extract has also 303
been reported to provide anti-inflammatory effect through inhibiting ROS production and 304
firm adhesion of neutrophil in isolated peripheral human neutrophils (36), whose 305
pharmacological function might provide one of possible reasons why EAS can decrease 306
serum level of MDA (Fig. 5C), a production of ROS-induced lipid peroxidation. 307
The Carr test is highly sensitive to NSAIDs, and has long been accepted as a useful 308
phlogistic tool for investigating new drug therapies (37). It is well known that the third phase 309
of the edema-induced by Carr, in which the edema reaches its highest volume, is 310
characterized by the presence of prostaglandins and other compounds of slow reaction found 311
that the injection of Carr into the rat paw induces the liberation of bradykinin, which later 312
induces the biosynthesis of prostaglandin and other autacoids, which are responsible for the 313
formation of the inflammatory exudates (38). In the present study, statistical analysis revealed 314
that 10 mg/kg of Indo and 25 mg/kg of EAS significantly inhibited the development of edema 315
4th h after treatment (p<0.001 or p<0.01) (Fig. 4). L-arginine–NO pathway has been proposed 316
to play an important role in the Carr-induced inflammatory response (39), and the expression 317
of the inducible isoform of NO synthase has been proposed as an important mediator of 318
inflammation (40). Our present results confirm that Carr-induced paw edema model results in 319
the production of NO, and the level of NO was decreased significantly by treatment with 12.5, 320
25, and 50 mg/kg EAS (Fig.5A). We suggest the anti-inflammatory mechanism of EAS may 321
be through the L-arginine–NO pathway because EAS significantly inhibits the NO production. 322
TNF- is also a mediator of Carr-induced inflammatory incapacitation, and is able to induce 323
the further release of kinins and leukotrienes, which is suggested to have an important role in 324
the maintenance of long-lasting nociceptive response (41). In this study, we found that EAS 325
obviously decreased the level of serum TNF-α after Carr injection by treatment with 12.5, 25 326
and 50 mg/kg EAS (Fig. 5B). 327
The Carr-induced inflammatory response has been linked to neutrophils infiltration and 328
the production of neutrophils-derived free radicals as well as the release of other 329
neutrophils-derived mediators (41). It has been demonstrate that free radical and NO will be 330
released when administrating with Carr, and increasing free radical might attack plasma 331
membrane and result in the accumulation of MDA. Our study demonstrated that 50 mg/kg 332
EAS the same as Indo can markedly decrease neutrophils infiltration and accumulation of 333
MDA within edema paw after Carr treatment (Fig. 7 and Fig. 5C). In addition, glutathione 334
(GSH) plays an important role against Carr-induced local inflammation (42), and endogenous 335
GSH can reduce MDA production. In the present study, increases of CAT, SOD, and GPx 336
activities were found in the group with EAS treatment (Fig. 5C and Table 1). Thereby, we 337
assume the suppression of MDA production is probably due to the increases of CAT, SOD, 338
and GPx activities. 339
In conclusion, these results suggested that the anti-inflammatory mechanism of EAS 340
may be related to the inhibitions of iNOS and COX-2, and it is associated with the increase 341
in the activities of antioxidant enzymes (CAT, SOD, and GPx). Based on reported 342
bioactivities above, it might be partially explained why EAS can exhibit the 343
anti-inflammatory effect in the LPS-stimulated RAW246.7 macrophages and the 344
-Carr-induced paw edema model. EAS may be used as a pharmacological agent in the 345
prevention or treatment of disease in which free radical formation in a pathogenic factor. 346
347
Acknowledgements 348
The authors want to thank the financial supports from the National Science Council (NSC 349
97-2313-B-039-001-MY3), China Medical University (CMU) (CMU99-S-29 and 350
CMU99-TC-35) and Taiwan Department of Heath Clinical Trial and Research Center of 351
Excellence (DOH100-TD-B-111-004) and the Cancer Research Center of Excellence 352
(DOH100-TD-C-111-005). 353
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