Antinociceptive and Anti-Inflammatory Effects of Zerumbone against Mono-Iodoacetate-Induced Arthritis

The fresh rhizome of Zingiber zerumbet Smith (Zingiberaceae) is used as a food flavoring and also serves as a folk medicine as an antipyretic and for analgesics in Taiwan. Zerumbone, a monocyclic sesquiterpene was isolated from the rhizome of Z. zerumbet and is the major active compound. In this study, the anti-inflammatory and antinociceptive effects of zerumbone on arthritis were explored using in vitro and in vivo models. Results showed that zerumbone inhibited inducible nitric oxide (NO) synthase (iNOS), cyclooxygenase (COX)-2 expressions, and NO and prostaglandin E2 (PGE2) production, but induced heme oxygenase (HO)-1 expression in a dose-dependent manner in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. When zerumbone was co-treated with an HO-1 inhibitor (tin protoporphyrin (SnPP)), the NO inhibitory effects of zerumbone were recovered. The above results suggest that zerumbone inhibited iNOS and COX-2 through induction of the HO-1 pathway. Moreover, matrix metalloproteinase (MMP)-13 and COX-2 expressions of interleukin (IL)-1β-stimulated primary rat chondrocytes were inhibited by zerumbone. In an in vivo assay, an acetic acid-induced writhing response in mice was significantly reduced by treatment with zerumbone. Furthermore, zerumbone reduced paw edema and the pain response in a mono-iodoacetate (MIA)-induced rat osteoarthritis model. Therefore, we suggest that zerumbone possesses anti-inflammatory and antinociceptive effects which indicate zerumbone could be a potential candidate for osteoarthritis treatment.

. Anti-inflammatory action of zerumbone on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells after treatment for 6 h. (A) Inducible nitric oxide (NO) synthase (iNOS), cyclooxygenase (COX)-2, and heme oxygenase (HO)-1 protein expressions; (B) Quantitational and statistical analysis of protein expressions, * p < 0.05 compared to control; (C) prostaglandin E2 (PGE2) production level, * p < 0.05, the zerumbone group compared to the control group; (D) NO production inhibition of zerumbone co-treated with tin protoporphyrin (SnPP), * p < 0.05, the zerumbone group compared to the group co-treated with SnPP. C: control, cells were pretreated with vehicle and LPS (500 ng/mL) B: blank, the cells incubated with vehicle alone. Data are summarized and expressed as Mean ± SEM of three individual experiments (n = 3).

Inhibition of COX-2 and MMP-13 Expressions by Zerumbone in IL-1β-stimulated PRCs
In arthritis, COX-2 promotes the production of prostaglandins which are important mediators of inflammatory pain and regulate catabolic processes in the cartilage. Also, MMPs are important factors in chondrolytic processes that contribute to degenerative changes in OA cartilage. The inhibitory effects of zerumbone on COX-2 and MMP-13 were evaluated using IL-1β-stimulated primary rat chondrocytes (PRCs). PRCs were treated with IL-1β (10 ng/mL) in the presence or absence of zerumbone at various concentrations (0.5~4 μM). Zerumbone significantly downregulated COX-2 and MMP-13 expressions by IL-1β-induced PRCs (Figure 2A,B). Quantitational and statistical analysis of protein expressions, * p < 0.05 compared to control; (C) prostaglandin E 2 (PGE 2 ) production level, * p < 0.05, the zerumbone group compared to the control group; (D) NO production inhibition of zerumbone co-treated with tin protoporphyrin (SnPP), * p < 0.05, the zerumbone group compared to the group co-treated with SnPP. C: control, cells were pretreated with vehicle and LPS (500 ng/mL) B: blank, the cells incubated with vehicle alone. Data are summarized and expressed as Mean˘SEM of three individual experiments (n = 3).

Inhibition of COX-2 and MMP-13 Expressions by Zerumbone in IL-1β-stimulated PRCs
In arthritis, COX-2 promotes the production of prostaglandins which are important mediators of inflammatory pain and regulate catabolic processes in the cartilage. Also, MMPs are important factors in chondrolytic processes that contribute to degenerative changes in OA cartilage. The inhibitory effects of zerumbone on COX-2 and MMP-13 were evaluated using IL-1β-stimulated primary rat chondrocytes (PRCs). PRCs were treated with IL-1β (10 ng/mL) in the presence or absence of zerumbone at various concentrations (0.5~4 µM). Zerumbone significantly downregulated COX-2 and MMP-13 expressions by IL-1β-induced PRCs (Figure 2A,B).

Analgesic Effects of Zerumbone on the Acetic-Acid-Induced Writhing Response
The acetic writhing test is widely used for analgesic screening for potential peripheral analgesic effects of compounds. Figure 3 shows the cumulative amount of abdominal stretching responses of acetic acid-induced pain. A higher dosage (50 mg/kg) of zerumbone treatment significantly inhibited the number of writhing instances compared to the controls (p < 0.05). The inhibition by zerumbone was similar to that produced by morphine ( Figure 3). This result indicates that the analgesic effect of zerumbone might be mediated by its peripheral effect.

Analgesic Effects of Zerumbone on the Acetic-Acid-Induced Writhing Response
The acetic writhing test is widely used for analgesic screening for potential peripheral analgesic effects of compounds. Figure 3 shows the cumulative amount of abdominal stretching responses of acetic acid-induced pain. A higher dosage (50 mg/kg) of zerumbone treatment significantly inhibited the number of writhing instances compared to the controls (p < 0.05). The inhibition by zerumbone was similar to that produced by morphine ( Figure 3). This result indicates that the analgesic effect of zerumbone might be mediated by its peripheral effect.

Analgesic Effects of Zerumbone on the Acetic-Acid-Induced Writhing Response
The acetic writhing test is widely used for analgesic screening for potential peripheral analgesic effects of compounds. Figure 3 shows the cumulative amount of abdominal stretching responses of acetic acid-induced pain. A higher dosage (50 mg/kg) of zerumbone treatment significantly inhibited the number of writhing instances compared to the controls (p < 0.05). The inhibition by zerumbone was similar to that produced by morphine ( Figure 3). This result indicates that the analgesic effect of zerumbone might be mediated by its peripheral effect.

Analgesic and Anti-Inflammatory Effects of Zerumbone on MIA-Induced OA
Since zerumbone demonstrated anti-inflammatory effects in vitro, an MIA-induced OA model was used to further evaluate its effect on paw-edema in vivo. Edema was induced by an MIA injection in normal saline into the right-hind ankle of each rat on day 1. Paw volumes were first measured before the MIA injection on day 1 before the MIA injection as the baseline and then measured again on day 4. The increase in paw volume was calculated by the difference in paw volume on days 1 and 4. The swelling volume of the paw significantly increased in the control group. Both zerumbone and indomethacin markedly reduced paw edema compared to the control group. Further, a high dose (5 mg/kg) of zerumbone exhibited a greater suppressive effect than low-dose treatment ( Figure 4). was used to further evaluate its effect on paw-edema in vivo. Edema was induced by an MIA injection in normal saline into the right-hind ankle of each rat on day 1. Paw volumes were first measured before the MIA injection on day 1 before the MIA injection as the baseline and then measured again on day 4. The increase in paw volume was calculated by the difference in paw volume on days 1 and 4. The swelling volume of the paw significantly increased in the control group. Both zerumbone and indomethacin markedly reduced paw edema compared to the control group. Further, a high dose (5 mg/kg) of zerumbone exhibited a greater suppressive effect than low-dose treatment ( Figure 4).
Measurement of weight bearing in MIA-induced animal is an indicator of disease progression OA and reveal the efficacy of anti-inflammatory compounds [11]. Changes in the hind-limb weight distribution between the right (MIA-induced side) and left limbs were utilized as an index of joint discomfort in the osteoarthritic ankle. Weight distributions of rats on day 6 were assessed with an incapacitance tester which determined the distribution ratio of hind-limb weighting. Zerumbone at 1 and 5 mg/kg was orally administered daily, and Figure 5 indicates that a statistically significant difference existed between the imbalanced rate in control subjects and the imbalanced rates with zerumbone at both the high and low doses. The pharmacological activity of zerumbone were observed that reduced joint discomfort in this model which implied the therapies ability to intervention by a commonly utilized therapeutic agent.  Measurement of weight bearing in MIA-induced animal is an indicator of disease progression OA and reveal the efficacy of anti-inflammatory compounds [11]. Changes in the hind-limb weight distribution between the right (MIA-induced side) and left limbs were utilized as an index of joint discomfort in the osteoarthritic ankle. Weight distributions of rats on day 6 were assessed with an incapacitance tester which determined the distribution ratio of hind-limb weighting. Zerumbone at 1 and 5 mg/kg was orally administered daily, and Figure 5 indicates that a statistically significant difference existed between the imbalanced rate in control subjects and the imbalanced rates with zerumbone at both the high and low doses. The pharmacological activity of zerumbone were observed that reduced joint discomfort in this model which implied the therapies ability to intervention by a commonly utilized therapeutic agent.

Analgesic and Anti-Inflammatory Effects of Zerumbone on MIA-Induced OA
Since zerumbone demonstrated anti-inflammatory effects in vitro, an MIA-induced OA model was used to further evaluate its effect on paw-edema in vivo. Edema was induced by an MIA injection in normal saline into the right-hind ankle of each rat on day 1. Paw volumes were first measured before the MIA injection on day 1 before the MIA injection as the baseline and then measured again on day 4. The increase in paw volume was calculated by the difference in paw volume on days 1 and 4. The swelling volume of the paw significantly increased in the control group. Both zerumbone and indomethacin markedly reduced paw edema compared to the control group. Further, a high dose (5 mg/kg) of zerumbone exhibited a greater suppressive effect than low-dose treatment (Figure 4).
Measurement of weight bearing in MIA-induced animal is an indicator of disease progression OA and reveal the efficacy of anti-inflammatory compounds [11]. Changes in the hind-limb weight distribution between the right (MIA-induced side) and left limbs were utilized as an index of joint discomfort in the osteoarthritic ankle. Weight distributions of rats on day 6 were assessed with an incapacitance tester which determined the distribution ratio of hind-limb weighting. Zerumbone at 1 and 5 mg/kg was orally administered daily, and Figure 5 indicates that a statistically significant difference existed between the imbalanced rate in control subjects and the imbalanced rates with zerumbone at both the high and low doses. The pharmacological activity of zerumbone were observed that reduced joint discomfort in this model which implied the therapies ability to intervention by a commonly utilized therapeutic agent.

Discussion
Chronic joint pain, such as with OA, is one the most common types of pain and is predicted to become the fourth leading cause of disability worldwide by 2020 [12]. OA is a loss of cartilage that initiates chondrocyte activation, and collagenases, such as MMPs and inflammatory factors, are released from the matrix. This regenerative process results in local activation of an inflammatory response. Without an ideal cure, medication management toward pain and symptom relief in clinical such as acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), and glucosamine sulfate [13]. It was believed that the anti-inflammatory and analgesic effects of NSAIDs are due to the inhibition of prostaglandin synthesis [14]. However, NSAIDs are associated with a spectrum of side effects such as gastric ulceration, renal insufficiency, and prolonged bleeding times [15]. Thus, finding new and efficient pharmacological agents from medicinal plants for treating OA might be a solution. Several studies reported that zerumbone possesses remarkable antimicrobial, antihyperglycemic, antiallergic, anti-inflammatory, and chemopreventive activities [1]. Essential oil from the rhizome of Z. zerumbet exhibited a significant antinociceptic effects on acetic acid-induced writing test in a dose-dependent manner [16]. Perimal et al. demonstrated that zerumbone possesses significant peripheral and central antinociceptive effects [17,18]. However, previous research used an intraperitoneal injection to deliver zerumbone to experimental animals. Our present results revealed that oral administration of zerumbone also produced similar antinociceptive activities. In the present study, anti-inflammatory effects of zerumbone were assessed, and its suppression of COX-2 expression by LPS-induced RAW 264.7 cells was via a mechanism that might involve the upregulation of HO-1. HO-1 is known as an inducible isozyme with major anti-inflammatory effects mediated by the catalytic breakdown of proinflammatory free heme and production of the anti-inflammatory compounds, carbon monoxide and bile pigments [19]. Also, LPS-induced COX-2 and PGE 2 expressions were inhibited by HO-1 through interruption of the toll-like receptor 4 (TLR4)/MyD88/nuclear factor (NF)-κB pathway [20]. In addition, OA cartilage expresses elevated levels of COX-2, with consequent increases in PGE 2 production which contribute to synovial inflammation. These data suggest that the mechanism of zerumbone against OA may be an attenuation of COX-2/prostaglandin production by chronically inflamed tissues.
Numerous studies showed that the L-arginine/NO/cyclic guanosine monophosphate cascade participates in nociceptive processes [21]. Substances capable of inhibiting NO donors increase the analgesic effects of opioid receptor agonists during peripheral inflammation [22]. Articular chondrocytes stimulated with cytokines and/or endotoxin in vitro release various inflammatory mediators such as NO and PGE 2 [23]. NO releasing was proposed that cause cartilage damage from OA-affected patient cartilage ex vivo [24]. Another research study demonstrated that NO induced chondrocyte death signaling including PGE 2 production via COX-2 induction. Our results demonstrated that zerumbone was a potent NOS inhibitor, and significantly reduced NO production by LPS-stimulated macrophages. It not only involves the anti-inflammatory effects but possibly also the antinociceptive activity.
On the other hand, proinflammatory cytokines (such as IL-1β) damage cartilage via the synthesis and secretion of MMPs, which lead to matrix degradation. Rousset et al. identified a mechanism by which the induced expression of HO-1 downregulates ROS production by NADPH oxidase in human chondrocytes, that consequently reduces MMP-1 secretion and cell death, two main features of OA [25]. We found that zerumbone suppressed IL-1β-induced MMP-13 expression, which suggests that it might be influenced by downregulation of HO-1 as well.
Animal models of joint pain are mostly induced by an intra-articular injection of irritants or cartilage-degenerating agents. We use acetic acid-induced writhing mice and MIA-induced OA rats to evaluate the analgesic properties of zerumbone. Pain induced by acetic acid is caused by increased prostaglandins level which can be quantitatively determined in peritoneal fluid. For this reason, excessive prostaglandin production through COX are highly associated to the pain response [26]. This work showed that the efficacy to reduce the abdominal writhing of high dose zerumbone was comparable to morphine. Furthermore, the analgesic effect of zerumbone might be correlated to down-regulation of COX/prostaglandins signaling pathway.
The most obvious clinical symptoms of OA is joint swelling which is formed with inflammation of a synovial tissue and synovial fluid accumulation [27]. In the present study, we emphasized that the strong anti-inflammatory activity of zerumbone was mediated by HO-1. Decreases in PGE 2 and NO production were also observed when macrophages were treated with zerumbone during the LPS challenge. Taken together, our results suggest that zerumbone not only produced a dose-dependent inhibition of the mice pain response in MIA-induced weight bearing imbalance, but was also able to reduce edema formation of mice.
In conclusion, this is the first known report demonstrating that zerumbone from Z. zerumbet has a markedly analgesic effect on MIA-induced OA in rats. We provided in vitro and in vivo evidence regarding the anti-inflammatory effectiveness relative to OA by zerumbone which exhibited inhibitory effects on NO and PGE 2 production via HO-1, iNOS, and COX-2 modulation. Also, MMP production by IL-1β-induced PRCs was downregulated by the expression of COX-2, suggesting the potential chondroprotective activity of zerumbone. These pharmacological properties suggested that zerumbone is an ideal nutritional supplement for arthritis-associated inflammation.

Zerumbone
Zerumbone ((2E,6E,10E)-2,6,9,9-tetramethylcycloundeca-2,6,10-trien-1-one), with an Mw of 218 ( Figure 6), was isolated and purified from rhizomes of Z. zerumbet, as in our previous study [28]. The purity of zerumbone was determined by high-performance liquid chromatography (HPLC), and was shown to exceed 99.0%. The most obvious clinical symptoms of OA is joint swelling which is formed with inflammation of a synovial tissue and synovial fluid accumulation [27]. In the present study, we emphasized that the strong anti-inflammatory activity of zerumbone was mediated by HO-1. Decreases in PGE2 and NO production were also observed when macrophages were treated with zerumbone during the LPS challenge. Taken together, our results suggest that zerumbone not only produced a dose-dependent inhibition of the mice pain response in MIA-induced weight bearing imbalance, but was also able to reduce edema formation of mice.
In conclusion, this is the first known report demonstrating that zerumbone from Z. zerumbet has a markedly analgesic effect on MIA-induced OA in rats. We provided in vitro and in vivo evidence regarding the anti-inflammatory effectiveness relative to OA by zerumbone which exhibited inhibitory effects on NO and PGE2 production via HO-1, iNOS, and COX-2 modulation. Also, MMP production by IL-1β-induced PRCs was downregulated by the expression of COX-2, suggesting the potential chondroprotective activity of zerumbone. These pharmacological properties suggested that zerumbone is an ideal nutritional supplement for arthritis-associated inflammation.

Primary Chondrocyte Culture
Primary rat chondrocytes (PRCs) were obtained after the cartilage tissue of a rat knee joint was sequentially digested with pronase (10 g/L, Roche, Indianapolis, IN, USA) for 30 min and collagenase type IV (1 g/L, Sigma, St. Louis, MO, USA) for 6 h, as described previously [29]. Experiments were performed with 3 rd passage cells. Monolayer cultures were established in 60-mm Petri dishes at a concentration of 6 × 10 6 cells/mL in DMEM supplemented with 10% FBS, 100 μg/mL streptomycin, and 100 IU/mL penicillin (Gibco BRL, Grand Island, NY, USA). PRCs were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. Experiments were performed with 3rd passage cells.

Measurement of NO and PGE2 Production
The measurement of NO and PGE2 was assessed as previously described [30]. Briefly, after 24 h of incubation with or without samples and/or LPS (500 ng/mL), cells generated NO and PGE2 in the

Cell Cultures
The murine macrophage cell line, RAW 264.7 was purchased from American Type Culture Collection (Rockville, MD, USA). Cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 µg/mL streptomycin (Gibco BRL, Grand Island, NY, USA) in a humidified incubator containing 5% CO 2 at 37˝C.

Primary Chondrocyte Culture
Primary rat chondrocytes (PRCs) were obtained after the cartilage tissue of a rat knee joint was sequentially digested with pronase (10 g/L, Roche, Indianapolis, IN, USA) for 30 min and collagenase type IV (1 g/L, Sigma, St. Louis, MO, USA) for 6 h, as described previously [29]. Experiments were performed with 3 rd passage cells. Monolayer cultures were established in 60-mm Petri dishes at a concentration of 6ˆ10 6 cells/mL in DMEM supplemented with 10% FBS, 100 µg/mL streptomycin, and 100 IU/mL penicillin (Gibco BRL, Grand Island, NY, USA). PRCs were incubated in a humidified atmosphere of 95% air and 5% CO 2 at 37˝C. Experiments were performed with 3rd passage cells.

Measurement of NO and PGE 2 Production
The measurement of NO and PGE 2 was assessed as previously described [30]. Briefly, after 24 h of incubation with or without samples and/or LPS (500 ng/mL), cells generated NO and PGE 2 in the culture medium. Quantitation of NO production was detected spectrophotometrically at 530 nm after the Griess reaction. The NO inhibition % was calculated using the following equation: NO inhibition (%) = [1´(T/C)]ˆ100%; where T and C represent the mean optical density of LPS-stimulated RAW 264.7 cells with and without samples, respectively. Otherwise, the culture medium was collected after 24 h of incubation with a sample, and PGE 2 concentrations were determined with an enzyme-linked immunosorbent assay (ELISA) kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Measurement of iNOS, COX-2, and MMP-13 Protein Expressions
IL-1β (10 ng/mL) in phosphate-buffered saline (PBS) was used to induce MMP-13 and COX-2 expressions in PRCs. Whole-cell lysates from cells treated with a sample were prepared by washing with PBS and lysing with radioimmunoprecipitation assay (RIPA) buffer. Equal amounts of protein (30 µg) from cell lysates were boiled for 5 min in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and visualized using a BCIP/NBT kit (Gibco BRL, Grand Island, NY, USA). Protein expressions were analyzed with the Azure Biosystem C300 Imaging System. GAPDH expression was used as the internal control to compare with iNOS, COX-2 and HO-1, and the fold was calculated by the control group expression.

Animals
Male SD rats weighing 180~220 g and ICR mice weighing 20~25 g were housed in a controlled environment at 21˘2˝C with sufficient food and water and kept on an alternating 12-h dark and light cycle. The animal experiments were approved by Ethical Regulations on Animal Research of Taipei Medical University (approval no: LAC-100-0043).

Acetic Acid-Induced Writhing Test
Acetic acid-induced writhing in ICR mice was induced according to a previously described method [31]. ICR mice were divided into four groups of six animals per group (n = 6). Rats in the control group were administered 10% DMSO (10 mL/kg i.p.) while animals in the zerumbone groups were administered 10 or 50 mg/kg zerumbone. Thirty minutes after treatment, each mouse was administered 10 mL/kg of 0.6% acetic acid (i.p.) before being assessed for 30 min inside its cage (25ˆ25ˆ30 cm 3 ). The number of times writhing was displayed by each mouse was counted and recorded. Morphine (1 mg/kg, i.p.) served as the reference drug in the positive control group.

MIA-Induced OA Model
Four groups of six male SD rats each were divided as follows: control group, positive control group (PC), and zerumbone groups. OA in male SD rats was induced by an intra-articular injection of 80 µL of 80 mg/mL MIA (Sigma) into a rat's ankle using a 100-µL syringe (Figure 7). Rats were randomly divided into six animals per group. After the MIA injection, the zerumbone groups were orally administered 1 or 5 mg/kg daily for 7 days. This is an effective dosage determined by our previous report [4]. The control group was treated with 10 mg/kg indomethacin daily for 7 days. The change in the paw volume was measured with a plethysmometer (Ugo Basile, Comerio VA, Italy) on days 1 and 4 after the MIA injection. Weight-bearing of both hind limbs was observed by an incapacitance tester with a dual-channel weight averager (Linton Instrumentation, Norfolk, UK) [11]. The weight-bearing force measured by the hind limb was averaged over a 3-s period. Each data point