Antinociceptive Activities and the Mechanisms of Anti-Inflammation of Asiatic Acid in Mice
Shyh-Shyun Huang,1 Chuan-Sung Chiu,1, 2 Hsien-Jung Chen,3 Wen-Chi Hou,4 Ming-Jyh Sheu,5 Ying-Chih Lin,6
Pei-Hsin Shie,1 and Guan-Jhong Huang1 1 School of Chinese Pharmaceutical Sciences and ChineseMedicine Resources, College of Pharmacy, China
Medical University, Taichung 404, Taiwan
2Nursing Department, Hsin Sheng College of Medical Care and Management, Taoyuan 325, Taiwan
3Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan
4Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan
5 School of Pharmacy, China Medical University, Taichung 404, Taiwan
6Department of Optometry, Jen-Teh Junior College of Medicine, Nursing, and Management,Miaoli 356, Taiwan
Correspondence should be addressed to Guan-Jhong Huang, [email protected]
Received 27 August 2010; Revised 17 January 2011; Accepted 8 February 2011
Copyright © 2011 Shyh-Shyun Huang et al. This is an open access article distributed under the Creative Commons
Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Asiatic acid (AA), a pentacyclic triterpene compound in the medicinal plant Centella asiatica, was evaluated for
antinociceptive
and anti-inflammatory efects. Treatment of male ICR mice with AA significantly inhibited the numbers of acetic
writhing responses and the formalin-induced pain in the late phase. In the anti-inflammatory test, AA decreased the paw
edema at
the 4th and 5th h after λ-carrageenan (Carr) administration and increased the activities of catalase (CAT), superoxide
dismutase
(SOD), and glutathione peroxidase (GPx) in the liver tissue. AA decreased the nitric oxide (NO), tumor necrosis factor-α
(TNF-α), and interleukin-1β (IL-1β) levels on serum level at the 5th
h after Carr injection. Western blotting revealed that AA decreased
Carr-induced inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and nuclear factor-κB (NF-κB)
expressions at the
5th h in the edema paw. An intraperitoneal (i.p.) injection treatment with AA also diminished neutrophil infiltration into
sites of
inflammation as did indomethacin (Indo). The anti-inflammatory mechanisms of AA might be related to the
decrease in the level
of MDA, iNOS, COX-2, and NF-κB in the edema paw via increasing the activities of CAT, SOD, and GPx in the liver.
1. Introduction
Triterpenes are biosynthesized in plants by the cyclization of squalene, and are widely distributed in the plant kingdom.
Moreover, their biological activities have attracted much attention. Many triterpenoids have shown promising efects when applied as anti-inflammatory agents [1]. In particular,
AA is a member of the ursane-type triterpenoids and is derived from the medicinal plant Centella asiatica, which is used as amedicine in tropical regions [2]. AA has been found
to prevent UVA-mediated photoaging, to inhibit β-amyloidinduced
and glutamate-induced neurotoxicity, and to possess antiulcer and antihepatofibric activities [3]. In addition, it
has been reported to exhibit a cytotoxic efect on liver, colon,
mouse model of focal cerebral ischemia [5]. Carr-induced paw edema is a useful model to assess vascular changes associated with inflammation. Subplantar
injections of Carr in mice induce a biphasic edema. The first phase peaks at 3 h, and the delayed phase peaks at
48 h after Carr injection. In the early phase, there is a difuse cellular infiltrate with polymorphonuclear leukocytes
(PMNs) whereas the infiltrate of the delayed phase is composed by macrophages, eosinophils, and lymphocytes
[6]. The inflammatory efect induced by Carr could be associated with free radical. Free radical, prostaglandin and
NO will be released when administrating with Carr for 1∼ 5 h. The edema efect was raised to maximum at the third
h, and its MDA production was due to free radical attack plasma membrane [6]. Thus, inflammatory efect would result in the accumulation of MDA. Therefore, in this paper,
we examined the analgesic efects of AA on nociception 2 Evidence-Based Complementary and AlternativeMedicine
Figure 1: Chemical structure of asiatic acid (AA).
induced by acetic acid and formalin. We also evaluated the anti-inflammatory efects of AA on paw edema induced by Carr in mice, and we detected the levels of MDA, NO,
TNF-α, iNOS, and COX-2 in either paw edema or serum. Also,
the activities of CAT, SOD, and GPx in the liver at the fifth h after Carr injection were investigated to understand the relationship between the anti-inflammatory mechanism of
the AA and antioxidant enzymes.
2.Methods
2.1. Chemicals. Asiatic acid (Figure 1), Carr, and
indomethacin (Indo) were obtained from Sigma (St. Louis, MO, USA). Acetic acid was purchased from Merck
(Darmstadt, Germany). Formalin was purchased from Nihon Shiyaku Industries (Japan). TNF-α and IL-1β were purchased from Biosource International Inc. (Camarillo, CA, USA). Anti-iNOS, anti-COX-2, anti-NF-κB (p50), and
anti-β-actin antibody (Santa Cruz, USA) and a protein assay kit (Bio-Rad Laboratories Ltd., Watford, Herts, UK)
membrane (Immobilon-P) was obtained from Millipore Corp. (Bedford, MA, USA).
2.2. Animals. 6–8 weeks male ICR mice were obtained from
the BioLASCO Taiwan Co., Ltd. The animals were kept in plexiglass cages at a constant temperature of 22 ± 1◦C,
relative humidity 55 ± 5% with a 12-hour dark-light cycle for at least 2 week before the experiment. They were given food and water ad libitum. All experimental procedures were
performed according to the NIH Guide for the Care and Use of Laboratory Animals. All tests were conducted under the guidelines of the International Association for the Study of
Pain [7].
After a 2-week adaptation period, male ICR mice (18– 25 g) were randomly assigned to five groups (n = 6) of the animals in acetic acid-induced writhing (1%, 0.1mL/10 g
i.p.) and formalin-induced licking (5%, 20 μL/per mice i.p.) experiments. These include a pathological model group (received acetic acid or formalin), a positive control (acetic
acid or formalin + Indo), and the AA-administered groups (acetic acid or formalin + AA: 1, 5, and 10mg/kg). In the Carr-induced edema experiment, there were randomly
assigned to six groups (n = 6) of the animals in the study. The control group receives normal saline (i.p.). The other
five groups include Carr-treated, positive control (Carr + Indo), and AA-administered groups (Carr + AA: 1, 5, and
10mg/kg).
2.3. Acetic Acid-Induced Writhing Response. The test was
performed as described by Chang et al. [8]. Writhing was induced by an intraperitoneal (i.p.) injection of 0.1mL/10 g
acetic acid solution (10mL/kg). Positive control animals were pretreated with Indo (10mg/kg, i.p.) 25min before acetic acid. Each AA-administered group was pretreated
with 1mg/kg, 5mg/kg, or 10mg/kg (dissolved in 0.5% carboxymethylcellulose) i.p. 25min before acetic acid. Five minutes after the i.p. injection of acetic acid, the number of
writhing and stretching was recorded.
2.4. Formalin Test. The antinociceptive activity of the drugs
was determined using the formalin test [8]. Twenty microliters
of 5% formalin was injected into the dorsal surface of the right hind paw of mice 30min after i.p. administration of AA (1, 5, and 10mg/kg), or Indo. The mice were observed
for 30min after the injection of formalin, and the amount of time spent licking the injected hind paw was recorded. The first 5min after formalin injection are referred to as the early
phase and the period between 15min and 40min as the late phase. The total time spent licking or biting the injured paw (pain behavior) wasmeasured with a stop watch. The activity
was recorded in 5-minute intervals.
2.5. λ-Carrageenin-Induced Edema. A Carr-induced hind
paw edema model was used for determination of antiinflammatory
activity [8]. Animals were i.p. treated with AA
(1, 5, and 10mg/kg), Indo, or normal saline, 30min prior to injection of 1% Carr (50 μL) in the plantar side of right hind
paws of the mice. Paw volume was measured immediately after Carr injection and at 1-, 2-, 3-, 4-, and 5-hour intervals
after the administration of the edematogenic agent using a plethysmometer (model 7159, Ugo Basile, Varese, Italy). The degree of swelling induced was evaluated by the ratio
a/b, where a is the volume of the right hind paw after Carr
treatment, and b is the volume of the right hind paw before Carr treatment. Indo was used as a positive control. After 5 hrs, the animals were sacrificed; the Carr-induced edema
feet were dissected and stored at −80◦C. Also, blood was withdrawn and kept at −80◦C. The protein concentration of the sample was determined by the Bradford dye-binding
assay (Bio-Rad, Hercules, CA).
2.6. MDA Assay. MDA from Carr-induced edema foot was
evaluated by the thiobarbituric acid reacting substances (TRARS) method [8]. Briefly, MDA reacted with thiobarbituric acid in the acidic high temperature and formed a redcomplex
TBARS. The absorbance of TBARS was determined at 532 nm.
Evidence-Based Complementary and AlternativeMedicine 3
2.7. Measurement of Nitric Oxide/Nitrite. NO production
was indirectly assessed by measuring the nitrite levels in serum determined by a colorimetric method based on
the Griess reaction [8]. Serum samples were diluted four times with distilled water and deproteinized by adding 1/20
volume of zinc sulfate (300 g/L) to a final concentration of 15 g/L. After centrifugation at 10,000×g for 5min at room temperature, 100 μL supernatant was applied to a microliter plate well, followed by 100 μL of Griess reagent (1% sulfanilamide and 0.1% N-1-naphthylethylenediamine dihydrochloride in 2.5% polyphosphoric acid). After 10min of color development at room temperature, the absorbance
was measured at 540nm with a Micro-Reader (Molecular Devices, Orleans Drive, Sunnyvale, CA). By using sodium nitrite to generate a standard curve, the concentration of
nitrite was measured by absorbance at 540 nm.
2.8. Measurement of Serum TNF-α and IL-1β by ELISA.
Serum levels of TNF-α and IL-1β were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Biosource International Inc., Camarillo, CA) according to the manufacturer’s instruction. TNF-α and IL-1β were determined from a standard curve. The
concentrations were expressed as pg/mL.
2.9. Antioxidant Enzyme Activity Measurements. The
following
biochemical parameters were analyzed to check the hepatoprotective activity of AA by the methods given below.
Total SOD activity was determined by the inhibition of cytochrome c reduction [9]. The reduction of cytochrome
c was mediated by superoxide anions generated by xanthine/
xanthine oxidase system and monitored at 550 nm. One unit of SOD was defined as the amount of enzyme
required to inhibit the rate of cytochrome c reduction by 50%. Total CAT activity was based on that of Aebi [10]. In brief, the reduction of 10mM H2O2 in 20mM of phosphate bufer (pH 7.0) was monitored by measuring the absorbance at 240 nm. The activity was calculated using
a molar absorption coefcient, and the enzyme activity was defined as nmoles of dissipating hydrogen peroxide per mg protein per min. Total GPx activity in cytosol was determined according to Paglia and Valentine’s method [11].
The enzyme solution was added to a mixture containing hydrogen peroxide and glutathione in 0.1mM Tris bufer (pH 7.2) and the absorbance at 340nm was measured. Activity was evaluated from a calibration curve, and the enzyme activity was defined as nmoles of NADPH oxidized
per mg protein per min.
2.10. Western Blot Analysis of iNOS, COX-2, and NF-κB.
Soft tissues were removed from individual mice paws and homogenized in a solution containing 10mM CHAPS, 1mM phenylmethylsulphonyl fluoride (PMSF), 5 μg/mL,
aprotinin, 1 μM pepstatin, and 10μM leupeptin. The homogenates were centrifuged at 12,000 g for 20min, and 30 μg of protein from the supernatants was then separated
on 10% sodium dodecyl sulphate-polyacrylamide gel and transferred to polyvinylidene difluoride membranes. Following transfer, the membrane was blocked for 2 h at
room temperature with 5% skim milk in Tris-bufered saline-Tween (TBST; 20mM Tris, 500mM NaCl, pH 7.5, 0.1% Tween 20). The membranes were then incubated with
mouse monoclonal anti-iNOS, anti-COX-2, or
anti-NF-κB (p50) antibody in 5% skim milk in TBST for 2 h at
room temperature. Themembranes were washed three times with TBST at room temperature and then incubated with a 1 : 2000 dilution of antimouse IgG secondary antibody conjugated to horseradish peroxidase (Sigma, St. Louis,
MO, USA) in 2.5% skim milk in TBST for 1 h at room temperature. The membranes were washed three times and
the immunoreactive proteins were detected by enhanced chemiluminescence (ECL) using hyperfilm and ECL reagent
(Amersham International plc., Buckinghamshire, UK). The results ofWestern blot analysis were quantified bymeasuring
the relative intensity compared to the control using Kodak Molecular Imaging Software (Version 4.0.5, Eastman Kodak
Company, Rochester, NY) and represented in the relative intensities.
2.11. Histological Examination. For histological examination,
biopsies of paws were taken 5 h following the interplanetary injection of Carr. The tissue slices were fixed in
temperature, dehydrated by graded ethanol, and embedded in parafn (Sherwood Medical). Sections (thickness 5 μm) were deparafnized with xylene and stained withH& E stain.
All samples were observed and photographed with Nikon microscopy. Every 3∼5 tissue slices were randomly chosen
from Carr-, Indo-, and AA-treated (10mg/kg) groups. Histological examination of these tissue slices revealed an
excessive inflammatory response with massive infiltration of PMNs by microscope. The numbers of neutrophils were counted in each scope (400x) and thereafter their average
count from 5 scopes of every tissue slice was obtained.
2.12. Statistical Analysis. Data are expressed as mean ±
S.E.M. Statistical evaluation was carried out by one-way analysis of variance (ANOVA followed by Schef´e’s multiple range test). Statistical significance is expressed as ∗P < .05,
∗∗P < .01, and ∗∗∗P < .001. 3. Results
3.1. Efects of AA on Acetic-Induced Writhing Response. The
cumulative amount of abdominal stretching correlated with the level of acetic acid-induced pain (Figure 2). AA treatment
(1 mg/kg) significantly inhibited the number of writhing in comparison with the normal controls (P < .05). AA (5 or
10mg/kg) further reduced the number of writhing (P <
.01 or P < .001), and AA (10mg/kg) demonstrates more
inhibition than Indo (10mg/kg).
3.2. Formalin test. AA (1mg/kg) significantly (P < .05)
inhibited formalin-induced pain in the late phase (Figure 3); however, it did not show any inhibition in the early phase.
The positive control Indo (5 or 10mg/kg) also significantly 4 Evidence-Based Complementary and AlternativeMedicine
Writhing response 0 10 20 30 40 50 ∗∗∗ ∗∗∗
∗ ∗∗ AA Control 10 1 5 10 (mg/kg) Indo 1% acetic acid
Figure 2: Analagesic efects of AA and indomethacin (Indo) on acetic acid-induced writhing response in mice. Each value
is
represented asmean ± S.E.M. ∗P < .05, ∗∗P < .01, and
∗∗∗P < .001
as compared with the pathological model group (Con; one-way
ANOVA followed by Schef´e’s multiple range test). Licking time (s) 0 20 40 60 80 100 120 140 Early phase Late phase AA Control 10 1 5 10 (mg/kg) Indo 5% formalin ∗∗ ∗∗∗ ∗∗∗ ∗
Figure 3: Efects of AA and Indo on the early phase and late phase
in formalin test in mice. Each value is represented as mean
±
S.E.M. ∗P < .05, ∗∗P < .01 and ∗∗∗P < .001 as compared with
the pathological model group (Con; one-way ANOVA followed by
Schef´e’s multiple range test).
(P < .01 or P < .001) inhibited the formalin-induced pain in the late phase.
3.3. Efects of AA on λ-Carrageenan-Induced Mice Paw Edema.
As shown in Figure 4, Carr induced paw edema. AA (5 or 10mg/kg) inhibited (P < .01 or P < .001) the development of paw edema induced by Carr after 4 and 5 h of treatment,
significantly. Indo (10mg/kg) significantly decreased the Time (hr)
0 1 2 3 4 5
Changes of edema volume (mL) 0 0.02 0.04 0.06 0.08 Carr Carr and Indo Carr and AA 1 mg/kg Carr and AA 5 mg/kg Carr and AA 10 mg/kg ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗
Figure 4: Efects of AA and Indo on hind paw edema induced by
Carr in mice. Each value is represented as mean±S.E.M. ∗P
< .05,
∗∗P < .01, and ∗∗∗P < .001 as compared with the Carr
group (oneway
Carr-induced paw edema after 4 and 5 h of treatment (P <
.001).
3.4. Efects of AA on MDA Level. MDA level increased
significantly in the edema paw 5th h after Carr injection (P <
.001). However, MDA level was decreased significantly by
treatment with AA (5mg/kg; P < .001), as well as 10mg/kg Indo (Figure 5).
3.5. Efects of AA on NO Level. In Figure 6(a), the NO level
increased significantly in the edema serum 5th h after Carr injection (P < .001). AA (5 or 10mg/kg) significantly decreased the serum NO level (P < .01 or P < .001). The inhibitory potency was similar to that of Indo (10mg/kg) at
the fifth h after induction.
3.6. Efects of AA on TNF-α and 1β Levels. TNF-α and
IL-1β levels increased significantly in serum at the fifth h after Carr injection (P < .001). However, AA (5 or 10mg/kg) decreased the TNF-α and IL-1β levels in serum at the fifth
h after Carr injection (P < .01 or P < .001), as well as 10 mg/kg Indo (Figure 6(b) and 6(c)).
3.7. Efects of AA on Activities of Antioxidant Enzymes. The
acute inflammatory response is associated with the production
of reactive oxygen species (ROS) such as superoxide anions, hydrogen peroxide, and peroxynitrite. In a number of
pathophysiological conditions associated with inflammation or oxidant stress, these ROS have been proposed to mediate
cell damage in the liver [1]. At the fifth h following the intrapaw injection of Carr, liver tissues were analyzed for
the biochemical parameters such as CAT, SOD, and GPx activities (Table 1). CAT, SOD, and GPx activities in liver Evidence-Based Complementary and AlternativeMedicine 5
Tissue MDA concentration (nmol/mg protein) 0 0.3 0.6 0.9 1.2 Control − Indo 1 5 10 (mg/kg) Carr
∗∗ ∗∗∗ ### ∗ AA ∗∗
Figure 5: Efects of AA and Indo on the tissueMDA concentration
of paw in mice. Normal control received 0.9% normal saline. Animals treated with AA (1, 5, and 10 mg/kg) and Indo were
assayed
for their ability in inhibiting MDA production in the right hind paws. The right hind paw tissues were dissected at the 5th h.
Then,
the homogenate was centrifuged, and the supernatant was obtained
for the MDA assays. Each value is represented as mean±S.E.M.
###P < .001 as compared with the control group. ∗P < .05,
∗∗P <
.01, and ∗∗∗P < .001 as compared with the Carr group
(one-way
ANOVA followed by Schef´e’s multiple range test). tissue were significantly decreased by Carr administration. CAT, SOD, and GPx activity were increased significantly after
treatment with 10mg/kg AA and 10mg/kg Indo (P < .01; Table 1).
3.8. Efects of AA on λ-Carrageenan-Induced iNOS, COX-2, and NF-κB Protein Expressions in Mice Paw Edema.
Transcription of proinflammatory mediators such as iNOS, COX-2, TNF-α, and IL-1β is regulated by activation of
transcription
factor NF-κB [1] The efect of AA on iNOS, COX-2, and NF-κB protein expression was studied by western blot. Equal amounts of protein (30 μg/lane) were resolved
by SDS-PAGE and then transferred to a nitrocellulose membrane and iNOS, COX-2, and NF-κB were detected using a specific antibody. The results showed that injection of
iNOS, COX-2, and NF-κB proteins expression (Figure 7(a)). The detection of β-actin was also performed in the same blot as an internal control. The intensity of protein bands was analyzed using Kodak Quantity software (Molecular
Imaging Software System, Kodak) in three independent experiments and showed an average of 77.6%, 72.4%, and
62.8% downregulation of iNOS, COX-2, and NF-κB protein, respectively, after the treatment with AA at 10mg/kg
compared
with the Carr-induced one alone (Figure 7(b)). The
protein expression showed an average of 43.6%, 41.1%, and 36.4% downregulation of iNOS, COX-2, and NF-κB protein after treatment with Indo at 10 mg/kg compared with the Carr-induced one alone (Figure 7(b)). The downregulation of iNOS, COX-2, and NF-κB activity of AA (10mg/kg) was
better than Indo (10mg/kg).
3.9. Histological Examination. Paw biopsies of Carr model
animals showedmarked cellular infiltration in the connective tissue. The infiltrates accumulated between collagen fibers and into intercellular spaces. Paw biopsies of animals treated
with AA (10mg/kg) showed a reduction in Carr-induced inflammatory response. Inflammatory cells were actually reduced in number and confined to near the vascular areas.
Intercellular spaces did not show any cellular infiltrations. Collagen fibers were regular in shape and showed a
reduction
of intercellular spaces. Moreover, the hypoderm connective tissue was not damaged (Figure 8). Neutrophils were notably
increased with Carr treatment (P < .001). Indo and AA (10mg/kg) could significantly decrease the neutrophils numbers as compared to the Carr-treated group (P < .001)
(Figure 8(e)).
4. Discussion
We have evaluated the putative analgesic and antiinflammatory
activities of AA to clarify the pain and
inflammation relieving efects. Two diferent analgesic testing
possible peripheral and central efects of the test substances.
The acetic writhing test is normally used to study the peripheral analgesic efects of drugs. Although this test is nonspecific (e.g., anticholinergic, antihistaminic, and other
agents also show activity in the test), it is widely used for analgesic screening [12]. In our study, we found that AA (1, 5, and 10mg/kg) exhibited an antinociceptive efect in acetic
acid-induced writhing response (Figure 2). This efect may be due to inhibition of the synthesis of the arachidonic acid
metabolites [13].
The in vivo model of pain, formalin-induced paw pain, has been well established as a valid model for analgesic
study. It is well known that the formalin test produces a distinct biphasic nociception, a first phase (lasting the first 5min) corresponding to acute neurogenic pain, and a second phase (lasting from 15 to 30min after injection of formalin) corresponding to inflammatory pain responses [14]. Therefore, the test can be used to clarify the possible
mechanism of an antinociceptive efect of a proposed analgesic. Centrally acting drugs such as opioids inhibit both phases equally, but peripherally acting drugs such as aspirin,
Indo, and dexamethasone only inhibit the late phase [15]. The inhibitory efect of AA on the nociceptive response in
the late phase of the formalin test suggested that the antinociceptive
efect of AA could be due to its peripheral action (Figure 3).
The injection of Carr in mice produces a typical biphasic edema associated with the production of several
inflammatory
mediators, such as bradykinin, prostaglandins, nitric oxide, and cytokines. The Carr test is highly sensitive to nonsteroidal antiinflammatory drugs, and has long been accepted as a useful phlogistic tool for investigating new
drug
therapies [16]. The degree of swelling of the Carr injected 6 Evidence-Based Complementary and AlternativeMedicine
∗∗∗ Control − Indo 1 5 10 (mg/kg) ∗∗ ∗ Carr ### Nitrite (μM) 0 2 4 6 8 10 12 14 16 AA (a) ∗∗∗ ∗∗∗ ∗∗ ∗ ### Control − Indo 1 5 10 (mg/kg) Carr AA TNF-α (pg/mL) 0 100 200 300 400 500 600 (b) ∗∗∗ ∗∗∗ ∗∗ ∗
### Control − Indo 1 5 10 (mg/kg) Carr AA Interleukin-1β (pg/mL) 0 20 40 60 80 100 120 140 160 (c)
Figure 6: Efects of AA and Indo on Carr-induced (a) NO, (b) TNF-α, and (c) interlukin-1β concentrations of serum at the
5th h in mice.
Normal control received 0.9% normal saline. Animals treated with AA (1, 5, and 10 mg/kg) and Indo were assayed in the
right hind paws.
After 5 h, the animals were sacrificed and blood was withdrawn. Then fresh blood was centrifuged, and the
supernatant was obtained for
measuring NO, TNF-α, and interlukin-1β levels. Each value represents as mean ± S.E.M. ###P < .001 as compared
with the control group.
∗P < .05, ∗∗P < .01, and ∗∗∗P < .001 as compared with
the Carr group (one-way ANOVA followed by Schef´e’s multiple range test).
Table 1: Efects of AA and Indo on the liver CAT, SOD, and GPx activities in mice.
Groups Catalase (U/mg protein) SOD (U/mg protein) GPx (U/mg protein) Control 5.12 ± 0.21 24.39 ± 0.18 3.23 ± 0.18 Carr 3.46 ± 0.32### 17.56 ± 0.31### 1.96 ± 0.14### Carr + Indo 4.53 ± 0.25∗∗ 22.13 ± 0.26∗∗ 2.76 ± 0.29∗∗ Carr + AA (1 mg/Kg) 3.84 ± 0.17 19.47 ± 0.15 2.14 ± 0.19 Carr + AA (5 mg/Kg) 4.36 ± 0.25∗ 21.32 ± 0.19∗ 2.49 ±
0.27∗
Carr + AA (10 mg/Kg) 4.67 ± 0.36∗∗ 23.06 ± 0.33∗∗ 2.93
± 0.14∗∗
Each value is represented as mean ± S.E.M. ###P < .001 as compared with the control. ∗P < .05 and ∗∗P < .01 as
compared with the Carr group (one-way
ANOVA followed by Schef´e’s multiple range test). Evidence-Based Complementary and AlternativeMedicine 7
1% Carr − + + + Indo (10 mg/kg) − − + − AA (10 mg/kg) − − − + iNOS COX-2 NF-κB β-actin (a) ∗∗∗ ∗∗∗ ∗∗∗ ∗∗ ∗∗ ∗∗ Control − 10 10 (mg/kg) Indo AA Carr
iNOS, COX-2, and (% of control) 0 0.2 0.4 0.6 0.8 1 1.2 iNOS COX-2 NF-κB (p50) ###### ### NF-κB (b)
Figure 7: Inhibition of iNOS, COX-2, and NF-κB protein expression by AA induced by Carr in mice paw edema for 5
h. Normal control
received 0.9% normal saline. Animals treated with AA (1, 5, and 10mg/kg) and Indo to injection of Carr right hind paws.
The right hind paw
tissues were taken at the 5th h. Then, the homogenate was centrifuged and tissue suspended and were then prepared
and subjected to western
blotting using an antibody specific for iNOS, COX-2, and
NF-κB. β-actin was used as an internal control. (a)
Representative western blot
from two separate experiments is shown. (b) Relative iNOS, COX-2 and NF-κB protein levels were calculated with
reference to Carr-injected
mice. ###compared with sample of control group. The data were presented as mean ± S.D. for three diferent
experiments performed in
triplicate. ∗∗P < .01 and ∗∗∗P < .001 were compared with Carr-alone group.
paws was maximal the 3th h after injection. Statistical analysis revealed that AA (10mg/kg) and Indo significantly inhibited the development of edema at the fourth hour after
treatment (P < .001; Figure 4). They both showed antiinflammatory
efects in Carr-induced mice edema paw. It
is well known that the third phase of the edema induced by Carr, in which the edema reaches its highest volume, is characterized by the presence of prostaglandins and other
compounds of slow reaction [17], it was found that the injection of Carr into the rat paw induces the liberation of bradykinin, which later induces the biosynthesis of prostaglandin and other autacoids, which are responsible for
the formation of the inflammatory exudates. In addition, the classification of antinociceptive drugs is usually based on their mechanism of action either on the central nervous
system or on the peripheral nervous system [18]. NO plays an important role in Carr-induced paw edema. iNOS is expressed in this model within 4 h after injection
of Carr. The subsequent production of NO maintains the edema. In the studies of themechanism of the inflammation,
L-arginine-NO pathway has been proposed to play an important role in the Carr-induced inflammatory response [19]. Our present results also confirm that the Carr-induced
paw edema model results in the production of NO. The expression of the inducible isoform of NO synthase has been
proposed as an important mediator of inflammation [20]. In our study, the level of NO was decreased significantly by
treatment with 1, 5, and 10mg/kg AA. We suggest that the mechanism of anti-inflammatory of AA may be through the
L-arginine-NO pathway since AA significantly inhibits the NO production (Figure 6(a)).
TNF-α is a major mediator in inflammatory responses, inducing innate immune responses by activating T cells and
macrophages and stimulating secretion of other inflammatory
cytokines [21]. Also, TNF-α is a mediator of Carrinduced inflammatory incapacitation and is able to induce the further release of kinins and leukotrienes, which is suggested to have an important role in the maintenance of long-lasting nociceptive response. IL-1β is also important in the regulation of the inflammatory response. Moreover,
IL-1β increases the expression of adhesion factors on endothelial
cells to enable transmigration of leukocytes and is associated
with hyperalgesia and fever [22]. In this study, we found that AA decreased the TNF-α and IL-1β levels in serum after
Carr injection by treatment with 1, 5, and 10mg/kg AA, significantly (Figures 6(b) and 6(c)).
AA is one of the most common triterpenes and has a variety of pharmacological activities [23]. Nonetheless, little information is available with respect to the molecular mechanisms underlying the anti-inflammatory efect of AA.
The inhibitory efects of AA and asiaticoside on the LPSinduced
proinflammatory molecules, including NO and prostaglandin E2, and found that AA is a more potent
inhibitor than asiaticoside. These results suggest that the anti-inflammatory properties of AA might be the results 8 Evidence-Based Complementary and AlternativeMedicine
Epidermis Dermal region Skeletal muscle fibers 100 μm (a) (b) (c) (d) ∗∗∗ Control − Indo 10 (mg/kg) AA Neutrophil/scope (cell) 0 20 40 60 80 100 ∗∗∗ Carr ### (e)
Figure 8: Histological appearance of the mouse hind footpad after a subcutaneous injection with Carr stained with H & E
stain at the fifth
hour to reveal hemorrhage, edema, and inflammatory cell infiltration in (a) control mice, (b) Carr-treated mice
demonstrating hemorrhage
with moderately extravascular red blood cells and a large amount of inflammatory leukocytemainly neutrophils
infiltration in the subdermis
interstitial tissue of mice, and (c) mice given Indo (10 mg/kg) before Carr. AA significantly shows (d) morphological
alterations (100x) and
(e) the numbers of neutrophils in each scope (400x) compared to subcutaneous injection of Carr only. ###P < .
control group. ∗∗∗P < .001 compared with Carr group. Scale bar = 100 μm. Carrageenan Free radicals NF-κB iNOS NO COX-2 TNF-α, IL-1β Lipid peroxidation CAT SOD GPX MDA
Edema Neutrophil infiltration O2 − ONOO−
Figure 9: The proposed mechanism of AA in λ-carrageenan-(Carr-) injected mice. AA inhibits the production of TNF-α,
free radicals,
and lipid peroxidation, which in turn decreases MDA level, iNOS, COX-2, and NF-κB activation in the paw edema and
increase the CAT,
SOD and GPx activities in the liver. MDA: malondialdehyde; TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β; NO:
nitric oxide; CAT:
catalase; SOD: superoxide dismutase; GPx: glutathione peroxidase; iNOS: inducible nitric oxide synthase; COX-2:
cyclooxygenase-2; NF-κB: nuclear factor-κB.
Evidence-Based Complementary and AlternativeMedicine 9 from the inhibition of iNOS, COX-2, interleukin-6, IL-1β, and TNF-α expression through the downregulation of
nuclear factor-kappa B activation via suppression of IκB kinase and mitogen-activated protein kinase (p38, ERK1/2,
and JNK) phosphorylation in RAW264.7 cells [24]. The Carr-induced inflammatory response has been linked to neutrophils infiltration and the production of neutrophils-derived free radicals as well as the release of other neutrophils-derived mediators [8]. Some researches
demonstrate that the inflammatory efect induced by Carr is associated with free radicals. Free radicals, prostaglandin
and
NO will be released when administrating with Carr for 1– 6 h. The edema efect was raised to the maximum at the
third
hour.MDA production is due to free radical attacking plasma membrane. Thus, inflammatory efect would result in the accumulation ofMDA. GSH is a known oxyradical scavenger.
Increasing the level of GSH toward favor reduces the production
of MDA. Endogenous GSH plays an important role against Carr-induced local inflammation. In a number of pathophysiological conditions associated with inflammation or oxidant stress, these ROS have been proposed to mediate
cell damage via a number of independent mechanisms including the initiation of lipid peroxidation, the inactivation
of a variety of antioxidant enzymes, and depletion of glutathione. Given the importance of the oxidative status
in the formation of edema, the anti-inflammatory efect exhibited by the drug in this model might be related to its antioxidant properties [8]. In this study, there are significant
increases in CAT, SOD, and GPx activities with AA treatment (Table 1). Furthermore, there are significant decreases in
MDA level with AA treatment (Figure 5). We assume that the suppression of MDA production is probably due to the
increases of CAT, SOD, and GPx activities.
During inflammatory processes, large amounts of the proinflammatory mediators, NO and PGE2, are generated
by inducible iNOS and COX-2, respectively [25]. INOS, is generally not present in resting cells but is induced by
various stimuli, which include bacterial LPS, TNF-α, IL-1β, and interferon-γ [26]. However, COX-2 is induced by
proinflammatory stimuli, including LPS and cytokines in cells in vitro and in inflamed sites in vivo. Furthermore, COX-2 is believed to be the isoform responsible for the production of proinflammatory prostaglandins (PGs) in various models of inflammation [27]. In this study, there are significant decreases in iNOS and COX-2 activities with
AA treatment (Figure 7(a)).We assume that the suppression of NO production is probably due to the decrease of iNOS and COX-2 activities. An inflammatory response implicates macrophages and neutrophils, which secrete a number of mediators (eicosinoids, oxidants, cytokine, and lytic enzymes) responsible for initiation, progression and persistence of acute or chronic state of inflammation [28].
NO is the most important among these mediators and is produced in macrophages by COX-2 and iNOS, respectively [29]. COXs are proinflammatory enzymes that are involved
in arachidonic acid metabolism and influence biological reactions such as tissue repair and immune responses, all
of which are associated with inflammation. COX-1 and COX-2 are the rate-limiting enzymes in the synthesis of PGE2. COX-1 is constitutively expressed and involved in the acute inflammatory response whereas COX-2 is expressed in specific cells (i.e.,macrophages,monocytes, and neutrophils) after stimulation of COX-2-dependent PGE2 is produced by
inflammatory cells and increased in disease [30]. NF-κB is known to be a major transcription factor to regulate the expressions of proinflammatory enzymes and
cytokines, such as iNOS, COX-2, and TNF-α [31]. NF-κB subunits (p65 and/or p50) are normally sequestered in the
cytosol as an inactive complex by binding to inhibitory factor IκB-α in unstimulated cells. Upon stimulation of
proinflammatory signals, including LPS, IκB-α is phosphorylated
by IκB kinase (IKK) and inactivated through
ubiquitin-mediated degradation. The resulting free NF-κB is translocated into the nucleus and acts as a transcription
factor. As shown in Figure 7(a), the treatment with AA blocks the degradation of NF-κB in Carr-induced paw edema. Therefore, these results suggest that AA inhibits the
expression of iNOS and COX-2, and thus NO production through inactivation of NF-κB activation.
NO is also responsible for vasodilatation, the increase in vascular permeability and edema formation at the site
of inflammation [32]. NO along with superoxide (O2
and the products of their interaction, also initiates a wide range of toxic oxidative reactions causing tissue injury [33]. Likewise, the neutrophils produce oxidants and release granular constituents comprised of lytic enzymes performing
an important role in inflammatory injury [34]. In this study, AA inhibition in the release of these mediators is a potential strategy to control inflammation and is implicated
in mechanism of action as shown in Figure 9.
In conclusion, these results suggested that AA possessed analgesic and anti-inflammatory efects. The
antiinflammatory
mechanism of AA may be related to iNOS and
associated with the increase in the activities of antioxidant enzymes (CAT, SOD, and GPx). AA may be used as a pharmacological agent in the prevention or treatment of disease in which free radical formation is a pathogenic factor.
Acknowledgments
The authors wish to thank the financial support from the National Science Council (NSC 97-2313-B-039-001-MY3) and ChinaMedicalUniversity (CMU; CMU95-PH-11, CMU96-113, CMU97-232, and CMU99-S-29). The authors would like to thank Dr. Jefrey Conrad for critically reading the paper. C.-S. Chiu and H.-J. Chen contributed equally to
this paper.
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