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Gastroprotective and safety effects of WIN-34B, a novel treatment for osteoarthritis, compared to NSAIDs
Journal of Ethnopharmacology 137 (2011) 1011–1017
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Gastroprotective and safety effects of WIN-34B, a novel treatment for osteoarthritis, compared to NSAIDs Jeong-Eun Huh a , Won-Il Lee b , Byung-Kwan Seo b , Yong-Hyeon Baek b , Jae-Dong Lee c , Do-Young Choi c , Dong-Suk Park b,∗ a
Oriental Medicine Research Center for Bone & Joint, Disease Kyung Hee University, 149, Sangil-dong, Gangdong-gu, Seoul 134-727, Republic of Korea Department of Acupuncture & Moxibustion, East-West Neo Medical Center, Kyung Hee University, 149, Sangil-dong, Gangdong-gu, Seoul 134-727, Republic of Korea c Department of Acupuncture & Moxibustion, College of Oriental Medicine, Kyung Hee University, 1 Hoegidong, Dongdaemungu, Seoul 130-701, Republic of Korea b
a r t i c l e
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Article history: Received 22 March 2011 Received in revised form 17 June 2011 Accepted 6 July 2011 Available online 18 July 2011 Keywords: Lonicera japonica Thunb. Anemarrhena asphodeloides Bunge Gastric ulcer Prostaglandin E2 Leukotriene B4
a b s t r a c t Ethnopharmacological relevance: The dried flowers of Lonicera japonica, also known as Japanese honeysuckle, and the dried root of Anemarrhena asphodeloides, the component herbs of WIN-34B, are traditionally used in Eastern medicine to treat various inflammatory conditions including arthritis. Objective: To study the acute and chronic toxicities of WIN-34B and to compare its effects on gastric mucosa with those of diclofenac, a widely used NSAID, and celecoxib, a selective COX-2 inhibitor. Materials and methods: To investigate acute toxicity, we orally administered a single dose of 5000 mg/kg WIN-34B to rats. To investigate chronic toxicity, we orally administered 500, 1000 or 2000 mg/kg WIN34B to rats daily for 13 weeks. To assess its effects on gastric mucosa, rats received either a single dose or repeated doses of WIN-34B (400, 1000, or 2000 mg/kg), diclofenac (10, 40, or 80 mg/kg), celecoxib (100 or 1000 mg/kg), or vehicle, after which samples of gastric mucosa were assessed grossly and histologically. We also measured tissue activity of myeloperoxidase and synthesis of eicosanoids, including prostaglandin E2 (PGE2 ) and leukotriene B4 (LTB4 ). To further assess its effects, we administered WIN-34B to rats either intraperitoneally or orally, measured gastric injury scores using a rat model of diclofenac-induced gastric injury, and measured eicosanoid synthesis. Results: WIN-34B showed no signs of acute or chronic toxicity in terms of general behavior, gross appearance of the internal organs, blood chemistry, or mortality. WIN-34B did not cause significant gastric mucosal damage after single or repeated doses. In contrast, diclofenac and celecoxib both caused gastric damage. In terms of eicosanoid synthesis, WIN-34B significantly suppressed LTB4 synthesis while both diclofenac and celecoxib increased LTB4 synthesis. WIN-34B slightly reduced PGE2 production, while both diclofenac and celecoxib significantly reduced PGE2 production. In a rat model of diclofenac-induced gastric injury, WIN-34B significantly suppressed LTB4 synthesis and restored PGE2 release. Conclusions: These results demonstrate that WIN-34B did not cause acute or chronic toxicity in male or female rats. In addition, WIN-34B did not cause significant gastric mucosal damage, instead appearing to protect the mucosa from diclofenac-induced gastric damage through the regulation of PGE2 and LTB4 . Crown Copyright © 2011 Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Osteoarthritis (OA) is the most common disorder of joint degeneration and can be caused or promoted by joint overuse, aging, and mutations in cartilage genes (Poole, 1999; Ameye and Chee, 2006). In 2005, 26.9 million people suffered from OA in the United States alone, which represents a 30% increase over the past decade (Lawrence et al., 1998, 2008). The most common treatment for OA is non-steroidal anti-inflammatory drugs (NSAIDs), which act through non-selective inhibition of cyclooxygenase (COX)-1 and
∗ Corresponding author. Tel.: +82 24407702. E-mail addresses: [email protected], [email protected] (D.-S. Park).
COX-2. NSAIDs also prevent the up-regulation of prostaglandin E2 (PGE2 ) by COX-1. Although PGE2 contributes to the increased secretion of pro-inflammatory cytokines, it also plays a role in the protection of gastric mucosa. Suppression of PGE2 activity leads to weakened gastric mucosa, and eventually leads to focal injury to the upper and lower gastrointestinal tract (Shorrock and Rees, 1988; Patrignani et al., 1994; Strong et al., 2000; Panzer and Uguccioni, 2004; Sostres et al., 2010). More selective COX-2 inhibitors have been developed that reduce gastrointestinal side effects while retaining their analgesic and anti-inflammatory effects in people with OA (Silverstein et al., 2000; Hooper et al., 2004). Unfortunately, studies have found that selective COX-2 inhibitors increase the risk of life-threatening cardiovascular disorders such as myocardial infarction, heart failure, and sudden cardiac
0378-8741/$ – see front matter. Crown Copyright © 2011 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.07.025
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death. These risks have led to the ban of rofecoxib and valdecoxib in the United States and Europe, and the limitation of celecoxib (Hermann et al., 2005; Grosser et al., 2006). Given the serious side effects of both NSAIDs and selective COX-2 inhibitors, there is a pressing need for less toxic OA treatments. WIN-34B is an extract from a mixture of dried Lonicera japonica flowers and Anemarrhena asphodeloides root that was created based on extensive clinical experience demonstrating excellent anti-nociceptive and anti-inflammatory properties. Similar effects have been reported in several animal models (Kang et al., 2010), as has in vitro evidence of cartilage protection and anti-inflammatory properties suggested by our preliminary data. Both Lonicera japonica Thunb. and Anemarrhena asphodeloides root are traditionally used in Eastern medicine to treat inflammatory diseases. Lonicera japonica has been found to have anti-angiogenic, anti-nociceptive, and anti-inflammatory effects (Yoo et al., 2008). Other in vivo testing showed that neither a single dose nor 28 days of oral administration induced acute or subacute side effects (Thanabhorn et al., 2006). In addition, Anemarrhena asphodeloides has also exhibited antifungal, antiviral, and anti-inflammatory effects (Park et al., 2003; Bae et al., 2007; Kim et al., 2009). Finally, our team recently found WIN-34B to have analgesic and anti-inflammatory effects equal or superior to those of diclofenac, celecoxib, and JoinsTM in an in vivo model, suggesting its use as a new medication for OA (Kang et al., 2010). In this study, the toxicity of WIN-34B was evaluates in acute and chronic animal models. Next, its effects on healthy gastric mucosa and on diclofenac-induced ulcers was investigates in rats. Finally, the underlying mechanisms of these effects of WIN-34B was elucidates compare to those of diclofenac and celecoxib.
2. Materials and methods 2.1. Plant materials The dried flowers of Lonicera japonica and the dried root of Anemarrhena asphodeloides from Song Lim Pharmaceutical Company (Seoul, Korea) were purchased and identified by the Korea Pharmaceutical Trading Association (Seoul, Korea). Voucher specimens of Lonicera japonica Thunb. (No. OA-LOJ-15) and Anemarrhena asphodeloides Bunge (No. OA-ANA-11) were analyzed by HPLC analysis and deposited in the Central Research Institute, WhanIn Pharm. Co. Ltd. (Suwon, Korea).
2.2. Standardized extract of WIN-34B WIN-34B was prepared by extracting a mixture of 2 kg of dried Lonicera japonica flowers and 1 kg of Anemarrhena asphodeloides root (2:1, w/w) with 10 L of 50% (v/v) ethanol for 4 h at 85 ◦ C. After the extracted solution was filtered and evaporated in vacuo, the resulting concentrate was dissolved in 225 ml distilled water and partitioned with 195 ml n-butanol. The n-butanol layer was evaporated in vacuo and lyophilized for complete removal of the residual solvent, resulting in a 7% yield of 11 g brown powder. We standardized WIN-34B for quality control according to a previous report (Kang et al., 2010), which we then analyzed by HPLC to find the standard compounds, mangiferin and chlorogenic acid. All drugs freshly prepared on each day of administration. WIN-34B was dissolved in 0.5% carboxymethylcellulose at the highest concentration; lower concentrations were prepared by serial dilution. Diclofenac and celecoxib were also suspended in 0.5% carboxymethylcellulose to the required concentrations.
2.3. Animals Male Sprague–Dawley rats were obtained from the animal experimental center (SLC, Shizoka, Japan) and housed them individually in the East-West Neo Medical Center at Kyung Hee University (Seoul, Korea). We performed animal experiments according to international guidelines. On the day of dosing, rats weighed between 200 and 225 g and were housed in groups of 3 in suspended polypropylene cages with wire grid floors. The room temperature and relative humidity controls were set at 21 ◦ C and 50%, respectively. Prior to experiments, the rats were deprived of food, but not water, for 18 h. 2.4. Toxicity testing 2.4.1. Toxicity of single-dose of WIN-34B The single-dose of toxicity on 4 groups of rats, each consisting of 5 males and 5 females, was tested. Each rat received a single oral dose of 5000 mg/kg WIN-34B. All animals were observed for symptoms and/or mortality continuously for 14 days. All animals were weighed immediately before administration, and at 1, 3, 7, and 14 days after. At 14 days, we anesthetized the rats with ether, opened their abdomens, cut the postcaval veins and abdominal arteries until they exsanguinated, and visually examined the body surface and internal organs. 2.4.2. Toxicity of 13-week WIN-34B treatment Long-term toxicity of WIN-34B was performed in 4 groups of rats. The control group (15 male and 15 female rats) received no WIN-34B. A group of 10 male and 10 female rats received 500 mg/kg WIN-34B, a group of 10 male and 10 female rats received 1000 mg/kg, and a group of 15 male and 15 female rats received 2000 mg/kg. All groups orally received 10 ml/kg of WIN-34B or 0.5% CMC-Na solution (control) once daily for 13 weeks. We performed ocular exams, urine tests, blood tests, gross pathological examinations, biopsies, and autopsies, and recorded clinical symptoms, mortality, food/water intake, body weight, and organ weights. For recovery testing, we observed 5 male and 5 female rats from the control and 2000 mg/kg dose groups for an additional 4 weeks after WIN-34B administration. 2.5. Effects on gastric mucosa 2.5.1. Administration of test materials The rats divided into 2 groups of 9–12 rats each; 1 group received a single dose and the other group received five doses. Each group of received oral WIN-34B (400, 1000, 2000 mg/kg, n = 12), diclofenac (10, 40, 80 mg/kg, n = 9), celecoxib (100, 1000 mg/kg, n = 9), or vehicle. In the cases of repeated administration, test materials were administered once daily. 2.5.2. Quantification of gastric damage Rats given a single dose were sacrificed 8 h after treatment and the stomach was removed. Rats given multiple doses were sacrificed 8 h after the fifth administration. Any grossly visible lesions were measured in order to calculate the gastric damage score, where grade 1 corresponded to >3 pinpoint ulcers, grade 2 to 1–5 ulcers <2 mm in diameter, grade 3 to ≥6 ulcers <2 mm in diameter, grade 4 to 1 large ulcer, grade 5 to 2–4 large ulcers, grade 6 to 5–7 large ulcers, and grade 7 to >8 large ulcers. An individual unaware of the treatments performed the injury assessments. The stomach was then fixed in neutral buffered formalin and sections of the tissue were stained with H&E for subsequent histological evaluation.
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Fig. 1. Quantification of gastric damage after single and repeated doses of WIN-34B, diclofenac, celecoxib, and vehicle in rats. (A) Macroscopic appearance of the stomach after a single dose of WIN-34B, diclofenac, celecoxib, or vehicle in rats. All medications were administered orally. The stomachs were harvested for assessment 8 h after treatment: (a) vehicle-treated, (b) celecoxib (100 mg/kg)-treated, (c) diclofenac (80 mg/kg)-treated, (d) WIN-34B (400 mg/kg)-treated, (e) WIN-34B (1000 mg/kg)-treated, and (f) WIN-34B (2000 mg/kg)-treated animals. (B) Gastric damage score caused by oral administration of WIN-34B, diclofenac, and celecoxib in rats. All medications were administered in a single oral dose. The stomachs were harvested for assessment 8 h after treatment. Values are expressed as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences from the control group. (C) Macroscopic appearance of the stomach after repeated doses of WIN-34B, diclofenac, or celecoxib in rats. All medications were administered orally, once daily for 5 days. Stomachs were harvested for assessment 8 h after the fifth dose: (a) WIN-34B (100 mg/kg)-treated, (b) diclofenac (40 mg/kg)-treated, and (c) celecoxib (100 mg/kg)-treated animals.
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Fig. 2. Microscopic appearance of gastric mucosa after a single dose of WIN-34B, diclofenac, celecoxib, or vehicle in rats. All medications were administered orally. Stomachs were harvested for H&E staining and assessment 8 h after treatment: (a) vehicle-treated, (b) WIN-34B (400 mg/kg)-treated, (c) WIN-34B (1000 mg/kg)-treated, (d) WIN-34B (2000 mg/kg)-treated, (e) celecoxib (100 mg/kg)-treated, (f) celecoxib (1000 mg/kg)-treated, (g) diclofenac (40 mg/kg)-treated, and (h) diclofenac (80 mg/kg)-treated animals.
2.5.3. Myeloperoxidase (MPO) activity Samples weighing 100–150 mg were homogenized in 0.5% hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer (pH 6.0). Suspensions were centrifuged at 9000 × g for 30 min and the resulting supernatant was assayed using an MPO ELISA kit (Assay Designs, MI, USA).
2.5.4. Measurement of gastric eicosanoid production Samples of the corpus region of the rats’ stomachs were excised prior to formalin fixation, finely minced with scissors, and then suspended in 1 ml of 10 mM sodium phosphate buffer (pH 7.4). The samples were incubated at 37 ◦ C for 20 min and then centrifuged at 9000 × g. The supernatant was frozen for subsequent measurement of PGE2 (R&D Systems Inc., MN, USA) and LTB4 (Assay Designs, MI, USA) concentrations by ELISA.
2.6. Gastro-protective activity of WIN-34B To evaluate the possible protective or therapeutic effects of WIN-34B in a rat model of diclofenac-induced gastric injury, we employed 2 experimental methods in rats that received either a single dose or a series of 5 doses of WIN-34B. To induce gastric mucosal lesions, rats received diclofenac 40 mg/kg. Subsequently, the control group received the vehicle and the test groups received WIN-34B. In the first experimental method, we investigated the protective effects of a single intraperitoneal dose of WIN-34B (100 or 400 mg/kg) administered simultaneously with oral diclofenac, intended to cause gastric injury. We sacrificed rats 8 h after simultaneous administration and removed their stomachs. In the second experimental method, rats received the test material orally 8 h after diclofenac administration and then daily for 4 days. We euthanized the rats 24 h after the fourth administration. Their stomachs were removed and samples of the corpus region were excised prior to formalin fixation. Gastric damage was scored as described above. Again, the samples were frozen for subsequent measurement of PGE2 and LTB4 concentrations by ELISA.
2.7. Statistical analysis Data are expressed as mean ± standard error of the mean. Differences between groups were assessed by repeated analysis of variance followed by the Dunnett multiple comparison test. The levels of MPO, LTB4 , and PGE2 were compared between groups with the unpaired t-test. Prism software 4 (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analysis and graphing. Differences were considered significant at a p-value less than 0.05. 3. Results 3.1. Effect of WIN-34B on acute and chronic toxicity To evaluate the acute toxicity of a single oral dose of WIN-34B, we administered 5.0 g/kg body weight to male and female rats. This dose had no apparent effects on mortality, clinical signs, body weight, or gross findings in either sex, suggesting that the lethal dose of WIN-34B is higher than 5.0 g/kg in rats (data not shown). Furthermore, in terms of chronic toxicity, no rat that received WIN-34B at 1000 or 2000 mg/kg for 13 weeks expired. For the 13 weeks of treatment and 4 additional weeks of recovery, no notable abnormalities were found in clinical symptoms, food/water intake, biochemical tests of urine and blood, gross pathological examinations, or autopsies (data not shown). 3.2. Effect of WIN-34B on gastric damage To assess its gastrointestinal effects, rats received single or repeated oral doses of WIN-34B, and gastric mucosal damage was compared with that induced by diclofenac and celecoxib administered in an identical manner. We found that a single dose of diclofenac 80 mg/kg caused gastric mucosal bleeding and large ulcer, and a single dose of celecoxib 1000 mg/kg caused gastric mucosal inflammation. Conversely, a single dose of 400, 1000, or 2000 mg/kg WIN-34B did not cause any damage to the gastric mucosa, such as inflammation or bleeding (Fig. 1A). Analysis of gastric mucosal damage scores after a single dose showed that both diclofenac (10, 40, and 80 mg/kg) and celecoxib (100 and
J.-E. Huh et al. / Journal of Ethnopharmacology 137 (2011) 1011–1017
Fig. 3. Effects of single and repeated doses of WIN-34B, diclofenac, celecoxib, or vehicle on gastric myeloperoxidase activity. Each column is expressed as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences from the control group.
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Fig. 4. Effects of single and repeated doses of WIN-34B, diclofenac, celecoxib, or vehicle on gastric leukotriene B4 activity. Each column is expressed as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences from the control group.
1000 mg/kg) resulted in significantly increased damage. In contrast, WIN-34B resulted in significantly increased damage only at the 2000 mg/kg single dose, which was not statistically different from the damage induced in the control group (Fig. 1B). On histological examination, H&E staining of the gastric tissue after a single dose showed no significant abnormalities in the control or WIN-34B groups (Fig. 2). As expected, rats that received diclofenac showed severe inflammation and hemorrhage that worsened in a dose-dependent manner, as did rats that received celecoxib, albeit to a lesser degree (Fig. 2). Similarly, in repeated dose testing, diclofenac 40 mg/kg resulted in severe intestinal stenosis and celecoxib 100 mg/kg resulted in gastric mucosal inflammation, while WIN-34B caused no apparent abnormalities (Fig. 1C). 3.3. Effect of WIN-34B on myeloperoxidase activity MPO activity was not increased by either single or repeated administration of WIN-34B at 400, 1000, or 2000 mg/kg (Fig. 3). However, diclofenac at 10, 40, and 80 mg/kg showed a dosedependent increase in MPO activity after a single dose, which was more pronounced after repeated doses (Fig. 3). MPO activity was increased after both single and repeated doses of celecoxib 1000 mg/kg, although not after the 100 mg/kg dose (Fig. 3).
Fig. 5. Effects of single and repeated doses of WIN-34B, diclofenac, celecoxib, or vehicle on gastric prostaglandin E2 activity. Each column is expressed as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences from the control group.
was significantly inhibited by both oral and intraperitoneal WIN34B (Fig 7A). Similarly, the PGE2 level in diclofenac-injured gastric mucosa was significantly lower than that of normal gastric mucosa, while both the oral and intraperitoneal WIN-34B restored normal levels of PGE2 in a dose-dependent manner (Fig. 7B).
3.4. Effect of WIN-34B on gastric eicosanoid production 4. Discussion The WIN-34B 400, 1000, and 2000 mg/kg doses resulted in dose-dependent suppression of gastric LTB4 activity (Fig. 4). In contrast, LTB4 activity was significantly increased in rats that received diclofenac at 10, 40, and 80 mg/kg and celecoxib at 1000 mg/kg. Neither the single dose of celecoxib nor repeated treatment increased LTB4 activity (Fig. 4). It was notable that the change in LTB4 activity after repeated doses of diclofenac and celecoxib was significantly higher/lower than the change after single doses (Fig. 4). This pattern is opposite to the changes observed in gastric PGE2 release. Gastric PGE2 release was markedly inhibited after single and repeated doses of both diclofenac and celecoxib (Fig. 5). In contrast, PGE2 release was only insignificantly inhibited after single and repeated doses of WIN-34B (Fig. 5).
In this study, we found that even high doses of WIN-34B did not cause toxicity or gastric injury when orally administered to
3.5. Gastro-protective effects of WIN-34B in a rat model of diclofenac-induced gastric ulcers The single oral dose of WIN-34B 400 mg/kg significantly decreased gastric mucosal damage scores, as did repeated intraperitoneal administration at both the 100 and 400 mg/kg doses (Fig. 6). In this model, the expected increase in LTB4 synthesis
Fig. 6. Gastroprotective effects of WIN-34B on diclofenac-induced gastric ulcers. Each column is expressed as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences from the control group.
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Fig. 7. Effects of WIN-34B on diclofenac-induced gastric eicosanoid release. (A) Effects of WIN-34B on diclofenac-induced gastric LTB4 synthesis. (B) Effects of WIN34B on diclofenac-induced gastric PGE2 release. Each column is expressed as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences from the control group.
rats, in contrast to diclofenac and celecoxib, which caused inflammation and hemorrhage. In addition, both single and multiple doses of WIN-34B effectively suppressed MPO activity and reduced gastric mucosal LTB4 synthesis with no effect on PGE2 release. Furthermore, in a rat model of diclofenac-induced gastric ulcers, WIN-34B exhibited gastroprotective effects by reducing LTB4 levels and restoring PGE2 production. PGE2 and PGI2 are important factors in maintaining gastric mucosal integrity by protecting it from both endogeneous and exogeneous factors. The suppression of PG synthesis by NSAIDs, especially by inhibiting COX-1, injures the mucosa’s protective system and leads to gastrointestinal injury (Allen et al., 1993; Wallace and Granger, 1996). Surprisingly, we found that celecoxib caused gastric damage and increased MPO activity and LTB4 synthesis as well (Figs. 1–4). These effects were more pronounced with repeated doses of diclofenac and high-dose celecoxib (1000 mg/kg) (Figs. 1C, 3 and 4). Several studies have demonstrated that celecoxib can worsen gastric ulcers and delay their healing (Sun et al., 2000; Laudanno et al., 2001; Gotta, 2002). Another study found that celecoxib worsened gastric damage in aspirin-treated rats (Fiorucci et al., 2001). In our study, we found that single and repeated doses of WIN-34B significantly altered gastric PGE2 release in rats, appearing to restore PGE2 production in a rat model of diclofenac-induced gastric ulcers (Figs. 5 and 7). These results indicate that WIN-34B does not damage gastric mucosa and may exert gastroprotective effects in rats. Our data also demonstrate that WIN-34B significantly suppressed gastric mucosal LTB4 synthesis in a rat model of diclofenac-induced gastric ulcers (Figs. 4 and 7). LTB4 is a potent
pro-inflammatory mediator involved in inflammatory diseases such as atherosclerosis (Lewis et al., 1990; Mehrabian and Allayee, 2003). Leukotrienes have been found to stimulate pepsin secretion, which reduces mucosal blood flow and interferes with gastric emptying. Lipoxygenase products impair gastric mucosal integrity and exacerbate the damaging effects of noxious agents. These findings suggest a place for leukotriene inhibitors in the treatment of gastric ulcers. A previous report found that LTB4 synthesis increased in patients with rheumatoid arthritis or osteoarthritis who used NSAIDs for over 3 months (Hudson et al., 1993). LTB4 is a robust leukocyte activity stimulator that may adhere to vascular endothelial cells, inducing chemotactic and chemokinetic responses and leading to gastric mucosal damage (Bray et al., 1981; Wallace and Keenan, 1990; Sala et al., 1998). Cysteinyl-leukotrienes (LTC4 , LTD4 , and LTE4 ) damage the gastric mucosa by inducing mucosal capillary damage, gastric vascular contraction, disintegration of the gastro-protective layer, and increased gastric acid secretion (Peskar, 1991). A study on the herbal arthritis medication JoinsTM found it to suppress gastric LTB4 synthesis without causing mucosal injury and control PGE2 activity in a model of diclofenacinduced gastric ulcers (Kim et al., 2005a,b). The study identified a promising new arthritis treatment that prevents gastric damage. The effects of PGE2 and LTB4 on gastric mucosa and the inflammatory process have spurred much research in the direction of dual 5-LOX/COX inhibitors (Martel-Pelletier et al., 2003). Early studies focused on selective 5-LOX inhibitors, which did not have sufficient therapeutic effects on inflammatory conditions (McMillan and Walker, 1992; Steinhilber, 1999). This caused a shift in interest toward inhibiting the two major metabolic pathways of arachidonic acid, 5-LOX and COX. Although the early dual blockers such as tepoxalin did not cause gastric injury, they exhibited hepatotoxicity (Wong et al., 1992). Licofelone showed anti-inflammatory and analgesic effects similar to those of NSAIDs and selective COX-2 inhibitors without gastrointestinal side effects (Alvaro-Gracia, 2004; Cicero and Laghi, 2007). Later dual blockers did not exhibit hepatotoxicity in preclinical or clinical studies (Laufer et al., 1994, 1995; Knight et al., 1996). Dual 5-LOX/COX inhibitors also inhibit PGE2 and therefore may have comparatively stronger anti-inflammatory and analgesic effects (Charlier and Michaux, 2003; Martel-Pelletier et al., 2003). Of note, PGE2 has been found to exhibit gastroprotective effects in a model of acetic acid-induced gastric ulcers (Alvaro-Gracia, 2004; Cicero and Laghi, 2007). In our study, oral or intraperitoneal WIN-34B prevented gastric damage, reduced LTB4 synthesis, and restored PGE2 release in a rat model of diclofenac-induced gastric damage (Figs. 6 and 7). We have previously reported that WIN-34B exhibits similar or better anti-nociceptive and anti-inflammatory effects in animal models of osteoarthritis (Kang et al., 2010). It has been hypothesized that WIN-34B acts through selective inhibition of COX-2 and 5-LOX. Thus, further research is warranted that compares WIN-34B and dual blockers with respect to their anti-inflammatory, analgesic, gastro-protective, and adverse effects. In conclusion, we found no evidence of toxicity or gastric damage in rats treated with oral WIN-34B. In addition, our data suggest that WIN-34B possesses gastroprotective effects in a rat model of diclofenac-induced mucosal damage. These results support the safety and therapeutic usefulness of WIN-34B.
Acknowledgements This work was supported by a grant from Kyung Hee University in 2011 (KHU-20110063) and by a grant of the Oriental
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Gastroprotective and safety effects of WIN-34B, a novel treatment for osteoarthritis, compared to NSAIDs Jeong-Eun Huh a , Won-Il Lee b , Byung-Kwan Seo b , Yong-Hyeon Baek b , Jae-Dong Lee c , Do-Young Choi c , Dong-Suk Park b,∗ a
Oriental Medicine Research Center for Bone & Joint, Disease Kyung Hee University, 149, Sangil-dong, Gangdong-gu, Seoul 134-727, Republic of Korea Department of Acupuncture & Moxibustion, East-West Neo Medical Center, Kyung Hee University, 149, Sangil-dong, Gangdong-gu, Seoul 134-727, Republic of Korea c Department of Acupuncture & Moxibustion, College of Oriental Medicine, Kyung Hee University, 1 Hoegidong, Dongdaemungu, Seoul 130-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 22 March 2011 Received in revised form 17 June 2011 Accepted 6 July 2011 Available online 18 July 2011 Keywords: Lonicera japonica Thunb. Anemarrhena asphodeloides Bunge Gastric ulcer Prostaglandin E2 Leukotriene B4
a b s t r a c t Ethnopharmacological relevance: The dried flowers of Lonicera japonica, also known as Japanese honeysuckle, and the dried root of Anemarrhena asphodeloides, the component herbs of WIN-34B, are traditionally used in Eastern medicine to treat various inflammatory conditions including arthritis. Objective: To study the acute and chronic toxicities of WIN-34B and to compare its effects on gastric mucosa with those of diclofenac, a widely used NSAID, and celecoxib, a selective COX-2 inhibitor. Materials and methods: To investigate acute toxicity, we orally administered a single dose of 5000 mg/kg WIN-34B to rats. To investigate chronic toxicity, we orally administered 500, 1000 or 2000 mg/kg WIN34B to rats daily for 13 weeks. To assess its effects on gastric mucosa, rats received either a single dose or repeated doses of WIN-34B (400, 1000, or 2000 mg/kg), diclofenac (10, 40, or 80 mg/kg), celecoxib (100 or 1000 mg/kg), or vehicle, after which samples of gastric mucosa were assessed grossly and histologically. We also measured tissue activity of myeloperoxidase and synthesis of eicosanoids, including prostaglandin E2 (PGE2 ) and leukotriene B4 (LTB4 ). To further assess its effects, we administered WIN-34B to rats either intraperitoneally or orally, measured gastric injury scores using a rat model of diclofenac-induced gastric injury, and measured eicosanoid synthesis. Results: WIN-34B showed no signs of acute or chronic toxicity in terms of general behavior, gross appearance of the internal organs, blood chemistry, or mortality. WIN-34B did not cause significant gastric mucosal damage after single or repeated doses. In contrast, diclofenac and celecoxib both caused gastric damage. In terms of eicosanoid synthesis, WIN-34B significantly suppressed LTB4 synthesis while both diclofenac and celecoxib increased LTB4 synthesis. WIN-34B slightly reduced PGE2 production, while both diclofenac and celecoxib significantly reduced PGE2 production. In a rat model of diclofenac-induced gastric injury, WIN-34B significantly suppressed LTB4 synthesis and restored PGE2 release. Conclusions: These results demonstrate that WIN-34B did not cause acute or chronic toxicity in male or female rats. In addition, WIN-34B did not cause significant gastric mucosal damage, instead appearing to protect the mucosa from diclofenac-induced gastric damage through the regulation of PGE2 and LTB4 . Crown Copyright © 2011 Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Osteoarthritis (OA) is the most common disorder of joint degeneration and can be caused or promoted by joint overuse, aging, and mutations in cartilage genes (Poole, 1999; Ameye and Chee, 2006). In 2005, 26.9 million people suffered from OA in the United States alone, which represents a 30% increase over the past decade (Lawrence et al., 1998, 2008). The most common treatment for OA is non-steroidal anti-inflammatory drugs (NSAIDs), which act through non-selective inhibition of cyclooxygenase (COX)-1 and
∗ Corresponding author. Tel.: +82 24407702. E-mail addresses: [email protected], [email protected] (D.-S. Park).
COX-2. NSAIDs also prevent the up-regulation of prostaglandin E2 (PGE2 ) by COX-1. Although PGE2 contributes to the increased secretion of pro-inflammatory cytokines, it also plays a role in the protection of gastric mucosa. Suppression of PGE2 activity leads to weakened gastric mucosa, and eventually leads to focal injury to the upper and lower gastrointestinal tract (Shorrock and Rees, 1988; Patrignani et al., 1994; Strong et al., 2000; Panzer and Uguccioni, 2004; Sostres et al., 2010). More selective COX-2 inhibitors have been developed that reduce gastrointestinal side effects while retaining their analgesic and anti-inflammatory effects in people with OA (Silverstein et al., 2000; Hooper et al., 2004). Unfortunately, studies have found that selective COX-2 inhibitors increase the risk of life-threatening cardiovascular disorders such as myocardial infarction, heart failure, and sudden cardiac
0378-8741/$ – see front matter. Crown Copyright © 2011 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.07.025
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death. These risks have led to the ban of rofecoxib and valdecoxib in the United States and Europe, and the limitation of celecoxib (Hermann et al., 2005; Grosser et al., 2006). Given the serious side effects of both NSAIDs and selective COX-2 inhibitors, there is a pressing need for less toxic OA treatments. WIN-34B is an extract from a mixture of dried Lonicera japonica flowers and Anemarrhena asphodeloides root that was created based on extensive clinical experience demonstrating excellent anti-nociceptive and anti-inflammatory properties. Similar effects have been reported in several animal models (Kang et al., 2010), as has in vitro evidence of cartilage protection and anti-inflammatory properties suggested by our preliminary data. Both Lonicera japonica Thunb. and Anemarrhena asphodeloides root are traditionally used in Eastern medicine to treat inflammatory diseases. Lonicera japonica has been found to have anti-angiogenic, anti-nociceptive, and anti-inflammatory effects (Yoo et al., 2008). Other in vivo testing showed that neither a single dose nor 28 days of oral administration induced acute or subacute side effects (Thanabhorn et al., 2006). In addition, Anemarrhena asphodeloides has also exhibited antifungal, antiviral, and anti-inflammatory effects (Park et al., 2003; Bae et al., 2007; Kim et al., 2009). Finally, our team recently found WIN-34B to have analgesic and anti-inflammatory effects equal or superior to those of diclofenac, celecoxib, and JoinsTM in an in vivo model, suggesting its use as a new medication for OA (Kang et al., 2010). In this study, the toxicity of WIN-34B was evaluates in acute and chronic animal models. Next, its effects on healthy gastric mucosa and on diclofenac-induced ulcers was investigates in rats. Finally, the underlying mechanisms of these effects of WIN-34B was elucidates compare to those of diclofenac and celecoxib.
2. Materials and methods 2.1. Plant materials The dried flowers of Lonicera japonica and the dried root of Anemarrhena asphodeloides from Song Lim Pharmaceutical Company (Seoul, Korea) were purchased and identified by the Korea Pharmaceutical Trading Association (Seoul, Korea). Voucher specimens of Lonicera japonica Thunb. (No. OA-LOJ-15) and Anemarrhena asphodeloides Bunge (No. OA-ANA-11) were analyzed by HPLC analysis and deposited in the Central Research Institute, WhanIn Pharm. Co. Ltd. (Suwon, Korea).
2.2. Standardized extract of WIN-34B WIN-34B was prepared by extracting a mixture of 2 kg of dried Lonicera japonica flowers and 1 kg of Anemarrhena asphodeloides root (2:1, w/w) with 10 L of 50% (v/v) ethanol for 4 h at 85 ◦ C. After the extracted solution was filtered and evaporated in vacuo, the resulting concentrate was dissolved in 225 ml distilled water and partitioned with 195 ml n-butanol. The n-butanol layer was evaporated in vacuo and lyophilized for complete removal of the residual solvent, resulting in a 7% yield of 11 g brown powder. We standardized WIN-34B for quality control according to a previous report (Kang et al., 2010), which we then analyzed by HPLC to find the standard compounds, mangiferin and chlorogenic acid. All drugs freshly prepared on each day of administration. WIN-34B was dissolved in 0.5% carboxymethylcellulose at the highest concentration; lower concentrations were prepared by serial dilution. Diclofenac and celecoxib were also suspended in 0.5% carboxymethylcellulose to the required concentrations.
2.3. Animals Male Sprague–Dawley rats were obtained from the animal experimental center (SLC, Shizoka, Japan) and housed them individually in the East-West Neo Medical Center at Kyung Hee University (Seoul, Korea). We performed animal experiments according to international guidelines. On the day of dosing, rats weighed between 200 and 225 g and were housed in groups of 3 in suspended polypropylene cages with wire grid floors. The room temperature and relative humidity controls were set at 21 ◦ C and 50%, respectively. Prior to experiments, the rats were deprived of food, but not water, for 18 h. 2.4. Toxicity testing 2.4.1. Toxicity of single-dose of WIN-34B The single-dose of toxicity on 4 groups of rats, each consisting of 5 males and 5 females, was tested. Each rat received a single oral dose of 5000 mg/kg WIN-34B. All animals were observed for symptoms and/or mortality continuously for 14 days. All animals were weighed immediately before administration, and at 1, 3, 7, and 14 days after. At 14 days, we anesthetized the rats with ether, opened their abdomens, cut the postcaval veins and abdominal arteries until they exsanguinated, and visually examined the body surface and internal organs. 2.4.2. Toxicity of 13-week WIN-34B treatment Long-term toxicity of WIN-34B was performed in 4 groups of rats. The control group (15 male and 15 female rats) received no WIN-34B. A group of 10 male and 10 female rats received 500 mg/kg WIN-34B, a group of 10 male and 10 female rats received 1000 mg/kg, and a group of 15 male and 15 female rats received 2000 mg/kg. All groups orally received 10 ml/kg of WIN-34B or 0.5% CMC-Na solution (control) once daily for 13 weeks. We performed ocular exams, urine tests, blood tests, gross pathological examinations, biopsies, and autopsies, and recorded clinical symptoms, mortality, food/water intake, body weight, and organ weights. For recovery testing, we observed 5 male and 5 female rats from the control and 2000 mg/kg dose groups for an additional 4 weeks after WIN-34B administration. 2.5. Effects on gastric mucosa 2.5.1. Administration of test materials The rats divided into 2 groups of 9–12 rats each; 1 group received a single dose and the other group received five doses. Each group of received oral WIN-34B (400, 1000, 2000 mg/kg, n = 12), diclofenac (10, 40, 80 mg/kg, n = 9), celecoxib (100, 1000 mg/kg, n = 9), or vehicle. In the cases of repeated administration, test materials were administered once daily. 2.5.2. Quantification of gastric damage Rats given a single dose were sacrificed 8 h after treatment and the stomach was removed. Rats given multiple doses were sacrificed 8 h after the fifth administration. Any grossly visible lesions were measured in order to calculate the gastric damage score, where grade 1 corresponded to >3 pinpoint ulcers, grade 2 to 1–5 ulcers <2 mm in diameter, grade 3 to ≥6 ulcers <2 mm in diameter, grade 4 to 1 large ulcer, grade 5 to 2–4 large ulcers, grade 6 to 5–7 large ulcers, and grade 7 to >8 large ulcers. An individual unaware of the treatments performed the injury assessments. The stomach was then fixed in neutral buffered formalin and sections of the tissue were stained with H&E for subsequent histological evaluation.
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Fig. 1. Quantification of gastric damage after single and repeated doses of WIN-34B, diclofenac, celecoxib, and vehicle in rats. (A) Macroscopic appearance of the stomach after a single dose of WIN-34B, diclofenac, celecoxib, or vehicle in rats. All medications were administered orally. The stomachs were harvested for assessment 8 h after treatment: (a) vehicle-treated, (b) celecoxib (100 mg/kg)-treated, (c) diclofenac (80 mg/kg)-treated, (d) WIN-34B (400 mg/kg)-treated, (e) WIN-34B (1000 mg/kg)-treated, and (f) WIN-34B (2000 mg/kg)-treated animals. (B) Gastric damage score caused by oral administration of WIN-34B, diclofenac, and celecoxib in rats. All medications were administered in a single oral dose. The stomachs were harvested for assessment 8 h after treatment. Values are expressed as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences from the control group. (C) Macroscopic appearance of the stomach after repeated doses of WIN-34B, diclofenac, or celecoxib in rats. All medications were administered orally, once daily for 5 days. Stomachs were harvested for assessment 8 h after the fifth dose: (a) WIN-34B (100 mg/kg)-treated, (b) diclofenac (40 mg/kg)-treated, and (c) celecoxib (100 mg/kg)-treated animals.
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Fig. 2. Microscopic appearance of gastric mucosa after a single dose of WIN-34B, diclofenac, celecoxib, or vehicle in rats. All medications were administered orally. Stomachs were harvested for H&E staining and assessment 8 h after treatment: (a) vehicle-treated, (b) WIN-34B (400 mg/kg)-treated, (c) WIN-34B (1000 mg/kg)-treated, (d) WIN-34B (2000 mg/kg)-treated, (e) celecoxib (100 mg/kg)-treated, (f) celecoxib (1000 mg/kg)-treated, (g) diclofenac (40 mg/kg)-treated, and (h) diclofenac (80 mg/kg)-treated animals.
2.5.3. Myeloperoxidase (MPO) activity Samples weighing 100–150 mg were homogenized in 0.5% hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer (pH 6.0). Suspensions were centrifuged at 9000 × g for 30 min and the resulting supernatant was assayed using an MPO ELISA kit (Assay Designs, MI, USA).
2.5.4. Measurement of gastric eicosanoid production Samples of the corpus region of the rats’ stomachs were excised prior to formalin fixation, finely minced with scissors, and then suspended in 1 ml of 10 mM sodium phosphate buffer (pH 7.4). The samples were incubated at 37 ◦ C for 20 min and then centrifuged at 9000 × g. The supernatant was frozen for subsequent measurement of PGE2 (R&D Systems Inc., MN, USA) and LTB4 (Assay Designs, MI, USA) concentrations by ELISA.
2.6. Gastro-protective activity of WIN-34B To evaluate the possible protective or therapeutic effects of WIN-34B in a rat model of diclofenac-induced gastric injury, we employed 2 experimental methods in rats that received either a single dose or a series of 5 doses of WIN-34B. To induce gastric mucosal lesions, rats received diclofenac 40 mg/kg. Subsequently, the control group received the vehicle and the test groups received WIN-34B. In the first experimental method, we investigated the protective effects of a single intraperitoneal dose of WIN-34B (100 or 400 mg/kg) administered simultaneously with oral diclofenac, intended to cause gastric injury. We sacrificed rats 8 h after simultaneous administration and removed their stomachs. In the second experimental method, rats received the test material orally 8 h after diclofenac administration and then daily for 4 days. We euthanized the rats 24 h after the fourth administration. Their stomachs were removed and samples of the corpus region were excised prior to formalin fixation. Gastric damage was scored as described above. Again, the samples were frozen for subsequent measurement of PGE2 and LTB4 concentrations by ELISA.
2.7. Statistical analysis Data are expressed as mean ± standard error of the mean. Differences between groups were assessed by repeated analysis of variance followed by the Dunnett multiple comparison test. The levels of MPO, LTB4 , and PGE2 were compared between groups with the unpaired t-test. Prism software 4 (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analysis and graphing. Differences were considered significant at a p-value less than 0.05. 3. Results 3.1. Effect of WIN-34B on acute and chronic toxicity To evaluate the acute toxicity of a single oral dose of WIN-34B, we administered 5.0 g/kg body weight to male and female rats. This dose had no apparent effects on mortality, clinical signs, body weight, or gross findings in either sex, suggesting that the lethal dose of WIN-34B is higher than 5.0 g/kg in rats (data not shown). Furthermore, in terms of chronic toxicity, no rat that received WIN-34B at 1000 or 2000 mg/kg for 13 weeks expired. For the 13 weeks of treatment and 4 additional weeks of recovery, no notable abnormalities were found in clinical symptoms, food/water intake, biochemical tests of urine and blood, gross pathological examinations, or autopsies (data not shown). 3.2. Effect of WIN-34B on gastric damage To assess its gastrointestinal effects, rats received single or repeated oral doses of WIN-34B, and gastric mucosal damage was compared with that induced by diclofenac and celecoxib administered in an identical manner. We found that a single dose of diclofenac 80 mg/kg caused gastric mucosal bleeding and large ulcer, and a single dose of celecoxib 1000 mg/kg caused gastric mucosal inflammation. Conversely, a single dose of 400, 1000, or 2000 mg/kg WIN-34B did not cause any damage to the gastric mucosa, such as inflammation or bleeding (Fig. 1A). Analysis of gastric mucosal damage scores after a single dose showed that both diclofenac (10, 40, and 80 mg/kg) and celecoxib (100 and
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Fig. 3. Effects of single and repeated doses of WIN-34B, diclofenac, celecoxib, or vehicle on gastric myeloperoxidase activity. Each column is expressed as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences from the control group.
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Fig. 4. Effects of single and repeated doses of WIN-34B, diclofenac, celecoxib, or vehicle on gastric leukotriene B4 activity. Each column is expressed as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences from the control group.
1000 mg/kg) resulted in significantly increased damage. In contrast, WIN-34B resulted in significantly increased damage only at the 2000 mg/kg single dose, which was not statistically different from the damage induced in the control group (Fig. 1B). On histological examination, H&E staining of the gastric tissue after a single dose showed no significant abnormalities in the control or WIN-34B groups (Fig. 2). As expected, rats that received diclofenac showed severe inflammation and hemorrhage that worsened in a dose-dependent manner, as did rats that received celecoxib, albeit to a lesser degree (Fig. 2). Similarly, in repeated dose testing, diclofenac 40 mg/kg resulted in severe intestinal stenosis and celecoxib 100 mg/kg resulted in gastric mucosal inflammation, while WIN-34B caused no apparent abnormalities (Fig. 1C). 3.3. Effect of WIN-34B on myeloperoxidase activity MPO activity was not increased by either single or repeated administration of WIN-34B at 400, 1000, or 2000 mg/kg (Fig. 3). However, diclofenac at 10, 40, and 80 mg/kg showed a dosedependent increase in MPO activity after a single dose, which was more pronounced after repeated doses (Fig. 3). MPO activity was increased after both single and repeated doses of celecoxib 1000 mg/kg, although not after the 100 mg/kg dose (Fig. 3).
Fig. 5. Effects of single and repeated doses of WIN-34B, diclofenac, celecoxib, or vehicle on gastric prostaglandin E2 activity. Each column is expressed as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences from the control group.
was significantly inhibited by both oral and intraperitoneal WIN34B (Fig 7A). Similarly, the PGE2 level in diclofenac-injured gastric mucosa was significantly lower than that of normal gastric mucosa, while both the oral and intraperitoneal WIN-34B restored normal levels of PGE2 in a dose-dependent manner (Fig. 7B).
3.4. Effect of WIN-34B on gastric eicosanoid production 4. Discussion The WIN-34B 400, 1000, and 2000 mg/kg doses resulted in dose-dependent suppression of gastric LTB4 activity (Fig. 4). In contrast, LTB4 activity was significantly increased in rats that received diclofenac at 10, 40, and 80 mg/kg and celecoxib at 1000 mg/kg. Neither the single dose of celecoxib nor repeated treatment increased LTB4 activity (Fig. 4). It was notable that the change in LTB4 activity after repeated doses of diclofenac and celecoxib was significantly higher/lower than the change after single doses (Fig. 4). This pattern is opposite to the changes observed in gastric PGE2 release. Gastric PGE2 release was markedly inhibited after single and repeated doses of both diclofenac and celecoxib (Fig. 5). In contrast, PGE2 release was only insignificantly inhibited after single and repeated doses of WIN-34B (Fig. 5).
In this study, we found that even high doses of WIN-34B did not cause toxicity or gastric injury when orally administered to
3.5. Gastro-protective effects of WIN-34B in a rat model of diclofenac-induced gastric ulcers The single oral dose of WIN-34B 400 mg/kg significantly decreased gastric mucosal damage scores, as did repeated intraperitoneal administration at both the 100 and 400 mg/kg doses (Fig. 6). In this model, the expected increase in LTB4 synthesis
Fig. 6. Gastroprotective effects of WIN-34B on diclofenac-induced gastric ulcers. Each column is expressed as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences from the control group.
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Fig. 7. Effects of WIN-34B on diclofenac-induced gastric eicosanoid release. (A) Effects of WIN-34B on diclofenac-induced gastric LTB4 synthesis. (B) Effects of WIN34B on diclofenac-induced gastric PGE2 release. Each column is expressed as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences from the control group.
rats, in contrast to diclofenac and celecoxib, which caused inflammation and hemorrhage. In addition, both single and multiple doses of WIN-34B effectively suppressed MPO activity and reduced gastric mucosal LTB4 synthesis with no effect on PGE2 release. Furthermore, in a rat model of diclofenac-induced gastric ulcers, WIN-34B exhibited gastroprotective effects by reducing LTB4 levels and restoring PGE2 production. PGE2 and PGI2 are important factors in maintaining gastric mucosal integrity by protecting it from both endogeneous and exogeneous factors. The suppression of PG synthesis by NSAIDs, especially by inhibiting COX-1, injures the mucosa’s protective system and leads to gastrointestinal injury (Allen et al., 1993; Wallace and Granger, 1996). Surprisingly, we found that celecoxib caused gastric damage and increased MPO activity and LTB4 synthesis as well (Figs. 1–4). These effects were more pronounced with repeated doses of diclofenac and high-dose celecoxib (1000 mg/kg) (Figs. 1C, 3 and 4). Several studies have demonstrated that celecoxib can worsen gastric ulcers and delay their healing (Sun et al., 2000; Laudanno et al., 2001; Gotta, 2002). Another study found that celecoxib worsened gastric damage in aspirin-treated rats (Fiorucci et al., 2001). In our study, we found that single and repeated doses of WIN-34B significantly altered gastric PGE2 release in rats, appearing to restore PGE2 production in a rat model of diclofenac-induced gastric ulcers (Figs. 5 and 7). These results indicate that WIN-34B does not damage gastric mucosa and may exert gastroprotective effects in rats. Our data also demonstrate that WIN-34B significantly suppressed gastric mucosal LTB4 synthesis in a rat model of diclofenac-induced gastric ulcers (Figs. 4 and 7). LTB4 is a potent
pro-inflammatory mediator involved in inflammatory diseases such as atherosclerosis (Lewis et al., 1990; Mehrabian and Allayee, 2003). Leukotrienes have been found to stimulate pepsin secretion, which reduces mucosal blood flow and interferes with gastric emptying. Lipoxygenase products impair gastric mucosal integrity and exacerbate the damaging effects of noxious agents. These findings suggest a place for leukotriene inhibitors in the treatment of gastric ulcers. A previous report found that LTB4 synthesis increased in patients with rheumatoid arthritis or osteoarthritis who used NSAIDs for over 3 months (Hudson et al., 1993). LTB4 is a robust leukocyte activity stimulator that may adhere to vascular endothelial cells, inducing chemotactic and chemokinetic responses and leading to gastric mucosal damage (Bray et al., 1981; Wallace and Keenan, 1990; Sala et al., 1998). Cysteinyl-leukotrienes (LTC4 , LTD4 , and LTE4 ) damage the gastric mucosa by inducing mucosal capillary damage, gastric vascular contraction, disintegration of the gastro-protective layer, and increased gastric acid secretion (Peskar, 1991). A study on the herbal arthritis medication JoinsTM found it to suppress gastric LTB4 synthesis without causing mucosal injury and control PGE2 activity in a model of diclofenacinduced gastric ulcers (Kim et al., 2005a,b). The study identified a promising new arthritis treatment that prevents gastric damage. The effects of PGE2 and LTB4 on gastric mucosa and the inflammatory process have spurred much research in the direction of dual 5-LOX/COX inhibitors (Martel-Pelletier et al., 2003). Early studies focused on selective 5-LOX inhibitors, which did not have sufficient therapeutic effects on inflammatory conditions (McMillan and Walker, 1992; Steinhilber, 1999). This caused a shift in interest toward inhibiting the two major metabolic pathways of arachidonic acid, 5-LOX and COX. Although the early dual blockers such as tepoxalin did not cause gastric injury, they exhibited hepatotoxicity (Wong et al., 1992). Licofelone showed anti-inflammatory and analgesic effects similar to those of NSAIDs and selective COX-2 inhibitors without gastrointestinal side effects (Alvaro-Gracia, 2004; Cicero and Laghi, 2007). Later dual blockers did not exhibit hepatotoxicity in preclinical or clinical studies (Laufer et al., 1994, 1995; Knight et al., 1996). Dual 5-LOX/COX inhibitors also inhibit PGE2 and therefore may have comparatively stronger anti-inflammatory and analgesic effects (Charlier and Michaux, 2003; Martel-Pelletier et al., 2003). Of note, PGE2 has been found to exhibit gastroprotective effects in a model of acetic acid-induced gastric ulcers (Alvaro-Gracia, 2004; Cicero and Laghi, 2007). In our study, oral or intraperitoneal WIN-34B prevented gastric damage, reduced LTB4 synthesis, and restored PGE2 release in a rat model of diclofenac-induced gastric damage (Figs. 6 and 7). We have previously reported that WIN-34B exhibits similar or better anti-nociceptive and anti-inflammatory effects in animal models of osteoarthritis (Kang et al., 2010). It has been hypothesized that WIN-34B acts through selective inhibition of COX-2 and 5-LOX. Thus, further research is warranted that compares WIN-34B and dual blockers with respect to their anti-inflammatory, analgesic, gastro-protective, and adverse effects. In conclusion, we found no evidence of toxicity or gastric damage in rats treated with oral WIN-34B. In addition, our data suggest that WIN-34B possesses gastroprotective effects in a rat model of diclofenac-induced mucosal damage. These results support the safety and therapeutic usefulness of WIN-34B.
Acknowledgements This work was supported by a grant from Kyung Hee University in 2011 (KHU-20110063) and by a grant of the Oriental
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