# Antinociceptive Activities and the Mechanisms of Anti-Inflammation of Asiatic Acid in Mice

Authors: Shyh-Shyun Huang; Chuan-Sung Chiu; Hsien-Jung Chen; Wen-Chi Hou; Ming-Jyh Sheu; Ying-Chih Lin; Pei-Hsin Shie; Guan-Jhong Huang
Journal: Evidence-Based Complementary and Alternative Medicine
 (2011)

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## Abstract

Asiatic acid (AA), a pentacyclic triterpene compound in the medicinal plantCentella asiatica, was evaluated for antinociceptive and anti-inflammatory effects. Treatment of male ICR mice with AA significantly inhibited the numbers of acetic acid-induced 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.


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## Body

## 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 effects 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 a medicine in tropical regions [2]. AA has been found to prevent UVA-mediated photoaging, to inhibit β-amyloid-induced and glutamate-induced neurotoxicity, and to possess antiulcer and antihepatofibric activities [3]. In addition, it has been reported to exhibit a cytotoxic effect on liver, colon, and breast cancer cells [4] and to be neuroprotective in a 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 diffuse cellular infiltrate with polymorphonuclear leukocytes (PMNs) whereas the infiltrate of the delayed phase is composed by macrophages, eosinophils, and lymphocytes [6]. The inflammatory effect 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 effect was raised to maximum at the third h, and its MDA production was due to free radical attack plasma membrane [6]. Thus, inflammatory effect would result in the accumulation of MDA. Therefore, in this paper, we examined the analgesic effects of AA on nociception induced by acetic acid and formalin. We also evaluated the anti-inflammatory effects 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 (Figure1), 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) were obtained as indicated. Poly (vinylidene fluoride) membrane (Immobilon-P) was obtained from Millipore Corp. (Bedford, MA, USA).Figure 1
Chemical structure of asiatic acid (AA).


### 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 of22±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%, 0.1 mL/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 10 mg/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 10 mg/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 0.1 mL/10 g acetic acid solution (10 mL/kg). Positive control animals were pretreated with Indo (10 mg/kg, i.p.) 25 min before acetic acid. Each AA-administered group was pretreated with 1 mg/kg, 5 mg/kg, or 10 mg/kg (dissolved in 0.5% carboxymethylcellulose) i.p. 25 min 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 30 min after i.p. administration of AA (1, 5, and 10 mg/kg), or Indo. The mice were observed for 30 min after the injection of formalin, and the amount of time spent licking the injected hind paw was recorded. The first 5 min after formalin injection are referred to as the early phase and the period between 15 min and 40 min as the late phase. The total time spent licking or biting the injured paw (pain behavior) was measured 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 anti-inflammatory activity [8]. Animals were i.p. treated with AA (1, 5, and 10 mg/kg), Indo, or normal saline, 30 min 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, USA).


### 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 red-complex TBARS. The absorbance of TBARS was determined at 532 nm.


### 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 5 min 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 10 min of color development at room temperature, the absorbance was measured at 540 nm with a Micro-Reader (Molecular Devices, Orleans Drive, Sunnyvale, CA, USA). 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, USA) 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 10 mM H2O2 in 20 mM of phosphate buffer (pH 7.0) was monitored by measuring the absorbance at 240 nm. The activity was calculated using a molar absorption coefficient, 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 0.1 mM Tris buffer (pH 7.2) and the absorbance at 340 nm 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 10 mM CHAPS, 1 mM phenylmethylsulphonyl fluoride (PMSF), 5μg/mL, aprotinin, 1 μM pepstatin, and 10 μM leupeptin. The homogenates were centrifuged at 12,000 g for 20 min, 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-buffered saline-Tween (TBST; 20 mM Tris, 500 mM 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. The membranes 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 of Western blot analysis were quantified by measuring the relative intensity compared to the control using Kodak Molecular Imaging Software (Version 4.0.5, Eastman Kodak Company, Rochester, NY, USA) 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 (1.85% formaldehyde, 1% acetic acid) for 1 week at room temperature, dehydrated by graded ethanol, and embedded in paraffin (Sherwood Medical). Sections (thickness 5μm) were deparaffinized with xylene and stained with H & 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 (10 mg/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 Scheffé's multiple range test). Statistical significance is expressed as *P<.05, **P<.01, and ***P<.001.


## 2.1. Chemicals

Asiatic acid (Figure1), 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) were obtained as indicated. Poly (vinylidene fluoride) membrane (Immobilon-P) was obtained from Millipore Corp. (Bedford, MA, USA).Figure 1
Chemical structure of asiatic acid (AA).


## 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 of22±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%, 0.1 mL/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 10 mg/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 10 mg/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 0.1 mL/10 g acetic acid solution (10 mL/kg). Positive control animals were pretreated with Indo (10 mg/kg, i.p.) 25 min before acetic acid. Each AA-administered group was pretreated with 1 mg/kg, 5 mg/kg, or 10 mg/kg (dissolved in 0.5% carboxymethylcellulose) i.p. 25 min 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 30 min after i.p. administration of AA (1, 5, and 10 mg/kg), or Indo. The mice were observed for 30 min after the injection of formalin, and the amount of time spent licking the injected hind paw was recorded. The first 5 min after formalin injection are referred to as the early phase and the period between 15 min and 40 min as the late phase. The total time spent licking or biting the injured paw (pain behavior) was measured 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 anti-inflammatory activity [8]. Animals were i.p. treated with AA (1, 5, and 10 mg/kg), Indo, or normal saline, 30 min 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, USA).


## 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 red-complex TBARS. The absorbance of TBARS was determined at 532 nm.


## 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 5 min 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 10 min of color development at room temperature, the absorbance was measured at 540 nm with a Micro-Reader (Molecular Devices, Orleans Drive, Sunnyvale, CA, USA). 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, USA) 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 10 mM H2O2 in 20 mM of phosphate buffer (pH 7.0) was monitored by measuring the absorbance at 240 nm. The activity was calculated using a molar absorption coefficient, 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 0.1 mM Tris buffer (pH 7.2) and the absorbance at 340 nm 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 10 mM CHAPS, 1 mM phenylmethylsulphonyl fluoride (PMSF), 5μg/mL, aprotinin, 1 μM pepstatin, and 10 μM leupeptin. The homogenates were centrifuged at 12,000 g for 20 min, 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-buffered saline-Tween (TBST; 20 mM Tris, 500 mM 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. The membranes 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 of Western blot analysis were quantified by measuring the relative intensity compared to the control using Kodak Molecular Imaging Software (Version 4.0.5, Eastman Kodak Company, Rochester, NY, USA) 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 (1.85% formaldehyde, 1% acetic acid) for 1 week at room temperature, dehydrated by graded ethanol, and embedded in paraffin (Sherwood Medical). Sections (thickness 5μm) were deparaffinized with xylene and stained with H & 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 (10 mg/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 Scheffé's multiple range test). Statistical significance is expressed as *P<.05, **P<.01, and ***P<.001.


## 3. Results

### 3.1. Effects of AA on Acetic-Induced Writhing Response

The cumulative amount of abdominal stretching correlated with the level of acetic acid-induced pain (Figure2). AA treatment (1 mg/kg) significantly inhibited the number of writhing in comparison with the normal controls (P<.05). AA (5 or 10 mg/kg) further reduced the number of writhing (P<.01 or P<.001), and AA (10 mg/kg) demonstrates more inhibition than Indo (10 mg/kg).Figure 2
Analagesic effects of AA and indomethacin (Indo) on acetic acid-induced writhing response 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 Scheffé’s multiple range test).


### 3.2. Formalin Test

AA (1 mg/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 10 mg/kg) also significantly (P<.01 or P<.001) inhibited the formalin-induced pain in the late phase.Figure 3
Effects 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 Scheffé’s multiple range test).


### 3.3. Effects of AA onλ-Carrageenan-Induced Mice Paw Edema

As shown in Figure4, Carr induced paw edema. AA (5 or 10 mg/kg) inhibited (P<.01 or P<.001) the development of paw edema induced by Carr after 4 and 5 h of treatment, significantly. Indo (10 mg/kg) significantly decreased the Carr-induced paw edema after 4 and 5 h of treatment (P<.001).Figure 4
Effects 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 (one-way ANOVA followed by Scheffé's multiple range test).


### 3.4. Effects 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 (5 mg/kg; P<.001), as well as 10 mg/kg Indo (Figure 5).Figure 5
Effects of AA and Indo on the tissue MDA 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 Scheffé's multiple range test).


### 3.5. Effects of AA on NO Level

In Figure6(a), the NO level increased significantly in the edema serum 5th h after Carr injection (P<.001). AA (5 or 10 mg/kg) significantly decreased the serum NO level (P<.01 or P<.001). The inhibitory potency was similar to that of Indo (10 mg/kg) at the fifth h after induction.Effects 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 Scheffé’s multiple range test).
(a)(b)(c)


### 3.6. Effects of AA on TNF-α and IL-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 10 mg/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. Effects 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 tissue were significantly decreased by Carr administration. CAT, SOD, and GPx activity were increased significantly after treatment with 10 mg/kg AA and 10 mg/kg Indo (P<.01; Table 1).Table 1
Effects of AA and Indo on the liver CAT, SOD, and GPx activities in mice.
GroupsCatalase (U/mg protein)SOD (U/mg protein)GPx (U/mg protein)Control5.12 ± 0.2124.39 ± 0.183.23 ± 0.18Carr3.46 ± 0.32###17.56 ± 0.31###1.96 ± 0.14###Carr + Indo4.53 ± 0.25**22.13 ± 0.26**2.76 ± 0.29**Carr + AA (1 mg/Kg)3.84 ± 0.1719.47 ± 0.152.14 ± 0.19Carr + 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 Scheffé’s multiple range test).


### 3.8. Effects of AA onλ-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 effect 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 AA (10 mg/kg) in Carr-induced paw edema for 5 h inhibited 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 10 mg/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 (10 mg/kg) was better than Indo (10 mg/kg).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 10 mg/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 different experiments performed in triplicate.**P<.01 and ***P<.001 were compared with Carr-alone group.
(a)(b)


### 3.9. Histological Examination

Paw biopsies of Carr model animals showed marked cellular infiltration in the connective tissue. The infiltrates accumulated between collagen fibers and into intercellular spaces. Paw biopsies of animals treated with AA (10 mg/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 (Figure8). Neutrophils were notably increased with Carr treatment (P<.001). Indo and AA (10 mg/kg) could significantly decrease the neutrophils numbers as compared to the Carr-treated group (P<.001) (Figure 8(e)).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<.001 as compared with the control group. ***P<.001 compared with Carr group. Scale bar = 100 μm.
(a)(b)(c)(d)(e)


## 3.1. Effects of AA on Acetic-Induced Writhing Response

The cumulative amount of abdominal stretching correlated with the level of acetic acid-induced pain (Figure2). AA treatment (1 mg/kg) significantly inhibited the number of writhing in comparison with the normal controls (P<.05). AA (5 or 10 mg/kg) further reduced the number of writhing (P<.01 or P<.001), and AA (10 mg/kg) demonstrates more inhibition than Indo (10 mg/kg).Figure 2
Analagesic effects of AA and indomethacin (Indo) on acetic acid-induced writhing response 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 Scheffé’s multiple range test).


## 3.2. Formalin Test

AA (1 mg/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 10 mg/kg) also significantly (P<.01 or P<.001) inhibited the formalin-induced pain in the late phase.Figure 3
Effects 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 Scheffé’s multiple range test).


## 3.3. Effects of AA onλ-Carrageenan-Induced Mice Paw Edema

As shown in Figure4, Carr induced paw edema. AA (5 or 10 mg/kg) inhibited (P<.01 or P<.001) the development of paw edema induced by Carr after 4 and 5 h of treatment, significantly. Indo (10 mg/kg) significantly decreased the Carr-induced paw edema after 4 and 5 h of treatment (P<.001).Figure 4
Effects 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 (one-way ANOVA followed by Scheffé's multiple range test).


## 3.4. Effects 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 (5 mg/kg; P<.001), as well as 10 mg/kg Indo (Figure 5).Figure 5
Effects of AA and Indo on the tissue MDA 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 Scheffé's multiple range test).


## 3.5. Effects of AA on NO Level

In Figure6(a), the NO level increased significantly in the edema serum 5th h after Carr injection (P<.001). AA (5 or 10 mg/kg) significantly decreased the serum NO level (P<.01 or P<.001). The inhibitory potency was similar to that of Indo (10 mg/kg) at the fifth h after induction.Effects 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 Scheffé’s multiple range test).
(a)(b)(c)


## 3.6. Effects of AA on TNF-α and IL-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 10 mg/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. Effects 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 tissue were significantly decreased by Carr administration. CAT, SOD, and GPx activity were increased significantly after treatment with 10 mg/kg AA and 10 mg/kg Indo (P<.01; Table 1).Table 1
Effects of AA and Indo on the liver CAT, SOD, and GPx activities in mice.
GroupsCatalase (U/mg protein)SOD (U/mg protein)GPx (U/mg protein)Control5.12 ± 0.2124.39 ± 0.183.23 ± 0.18Carr3.46 ± 0.32###17.56 ± 0.31###1.96 ± 0.14###Carr + Indo4.53 ± 0.25**22.13 ± 0.26**2.76 ± 0.29**Carr + AA (1 mg/Kg)3.84 ± 0.1719.47 ± 0.152.14 ± 0.19Carr + 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 Scheffé’s multiple range test).


## 3.8. Effects of AA onλ-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 effect 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 AA (10 mg/kg) in Carr-induced paw edema for 5 h inhibited 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 10 mg/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 (10 mg/kg) was better than Indo (10 mg/kg).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 10 mg/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 different experiments performed in triplicate.**P<.01 and ***P<.001 were compared with Carr-alone group.
(a)(b)


## 3.9. Histological Examination

Paw biopsies of Carr model animals showed marked cellular infiltration in the connective tissue. The infiltrates accumulated between collagen fibers and into intercellular spaces. Paw biopsies of animals treated with AA (10 mg/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 (Figure8). Neutrophils were notably increased with Carr treatment (P<.001). Indo and AA (10 mg/kg) could significantly decrease the neutrophils numbers as compared to the Carr-treated group (P<.001) (Figure 8(e)).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<.001 as compared with the control group. ***P<.001 compared with Carr group. Scale bar = 100 μm.
(a)(b)(c)(d)(e)


## 4. Discussion

We have evaluated the putative analgesic and anti-inflammatory activities of AA to clarify the pain and inflammation relieving effects. Two different analgesic testing methods were employed with the objective of identifying possible peripheral and central effects of the test substances. The acetic writhing test is normally used to study the peripheral analgesic effects 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 10 mg/kg) exhibited an antinociceptive effect in acetic acid-induced writhing response (Figure 2). This effect may be due to inhibition of the synthesis of the arachidonic acid metabolites [13].Thein vivomodel 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 5 min) corresponding to acute neurogenic pain, and a second phase (lasting from 15 to 30 min after injection of formalin) corresponding to inflammatory pain responses [14]. Therefore, the test can be used to clarify the possible mechanism of an antinociceptive effect 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 effect of AA on the nociceptive response in the late phase of the formalin test suggested that the anti-nociceptive effect 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 paws was maximal the 3th h after injection. Statistical analysis revealed that AA (10 mg/kg) and Indo significantly inhibited the development of edema at the fourth hour after treatment (P<.001; Figure 4). They both showed anti-inflammatory effects 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 the mechanism 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 10 mg/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 Carr-induced 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 10 mg/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 effect of AA. The inhibitory effects of AA and asiaticoside on the LPS-induced 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 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 effect 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 effect was raised to the maximum at the third hour. MDA production is due to free radical attacking plasma membrane. Thus, inflammatory effect would result in the accumulation of MDA. 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 effect 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.Figure 9
The proposed mechanism of AA inλ-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.In conclusion, these results suggested that AA possessed analgesic and anti-inflammatory effects. The anti-inflammatory 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.



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*Source: 895857-2011-04-20.xml*