Dig Dis Sci (2014) 59:2927–2934 DOI 10.1007/s10620-014-3370-5
ORIGINAL ARTICLE
Protective Effects of Garlic Extract, PMK-S005, Against Nonsteroidal Anti-inflammatory Drugs–Induced Acute Gastric Damage in Rats Yoon Jeong Choi • Nayoung Kim • Ju Yup Lee • Ryoung Hee Nam Hyun Chang • Ji Hyung Seo • Kyu Keun Kang • Hee Jin Kim • Yun Jin Choi • Hye Seung Lee • Dong Ho Lee
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Received: 29 June 2014 / Accepted: 15 September 2014 / Published online: 5 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Background PMK-S005 is synthetic s-allyl-L-cysteine (SAC), a sulfur-containing amino acid, which was initially isolated from garlic. The antioxidant and anti-inflammation activities of SAC have been demonstrated in diverse experimental animal models. Aims The purpose of this study was to investigate the gastroprotective effects of PMK-S005 against NSAIDsinduced acute gastric damage in rats. Methods Eight-week SD rats were pretreated with PMKS005 (1, 5, or 10 mg/kg) or rebamipide (50 mg/kg) 1 h before administration of NSAIDs including aspirin (200 mg/kg), diclofenac (80 mg/kg), and indomethacin
Electronic supplementary material The online version of this article (doi:10.1007/s10620-014-3370-5) contains supplementary material, which is available to authorized users. Y. J. Choi N. Kim (&) J. Y. Lee R. H. Nam H. Chang J. H. Seo K. K. Kang H. J. Kim Y. J. Choi D. H. Lee Department of Internal Medicine, Seoul National University Bundang Hospital, 82, Gumi-ro, 173 Beon-gil, Bundang-gu, Seongnam, Gyeonggi-do 463-707, Korea e-mail: [email protected] Y. J. Choi e-mail: [email protected] J. Y. Lee e-mail: [email protected] R. H. Nam e-mail: [email protected] H. Chang e-mail: [email protected] J. H. Seo e-mail: [email protected]
(40 mg/kg). After 4 h, the gross ulcer index, histological index, and gastric mucus level were determined. Myeloperoxidase (MPO), TNF-a, IL-1b, PGE2, and LTB4 levels were estimated in the gastric mucosal tissue by ELISA. Protein expressions of cPLA2, COX-1, and COX-2 were assessed by Western blot analysis. Results Pretreatment with PMK-S005 significantly attenuated the NSAIDs-induced gastric damage and increased the gastric mucus level. In addition, PMK-S005 attenuated increases in MPO, TNF-a, and IL-1b production. The expressions of cPLA2 and COX-2 induced by NSAIDs were decreased by PMK-S005 pretreatment. PMK-S005 did not cause suppression of PGE2 synthesis induced by NSAIDs, but LTB4 production was significantly suppressed by PMK-S005. The effects of PMK-S005 were consistently maximized at a concentration of 5 mg/kg, which were frequently superior to those of rebamipide. H. J. Kim e-mail: [email protected] Y. J. Choi e-mail: [email protected] D. H. Lee e-mail: [email protected] N. Kim D. H. Lee Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea H. S. Lee Department of Pathology, Seoul National University Bundang Hospital, 82, Gumi-ro, 173 Beon-gil, Bundang-gu, Seongnam, Gyeonggi-do 463-707, Korea e-mail: [email protected]
K. K. Kang e-mail: [email protected]
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Conclusions These results strongly suggest that PMKS005 can be a useful gastroprotective agent against acute gastric mucosal damage by suppressing proinflammatory cytokines, down-regulating cPLA2, COX-2 and LTB4 expression, and increasing the synthesis of mucus. Keywords s-allyl-L-cysteine (SAC) Gastroprotection Anti-inflammatory agent Rat NSAID
Introduction Gastric ulcer is the most common disorder of the gastrointestinal tract, with increasing incidence and prevalence. Gastric ulcer is predominantly caused by infection with Helicobacter pylori (H. pylori) or other environmental factors such as stress, smoking, alcohol consumption, and the ingestion of nonsteroidal anti-inflammatory drugs (NSAIDs) [1, 2]. NSAIDs including aspirin are widely used as anti-inflammatory and analgesic agents. However, gastrointestinal toxicity associated with NSAIDs has emerged as an important medical problem [3]. Currently, the expanding use of NSAIDs in the elderly and its complications like acute gastric erosion, ulceration, bleeding, and perforation have become more frequent in old patients [4–7], and the mortality rate in the elderly due to ulcer bleeding and perforation has rapidly increased [8, 9]. Imbalances between the aggressive (acid and pepsin secretion, NSAIDs, or H. pylori) and defensive factors (mucus secretion, blood flow, nitric oxide, and prostaglandins) have been implicated in the etiology of ulcer disease. One of the main therapeutic approaches for this pathological condition is the suppression of acid secretion by proton pump inhibitors (PPIs) or H2 antagonists as well as the eradication of H. pylori by antibiotics. However, ulcers frequently relapse in spite of maintenance therapy with this drug and long-term treatment with anti-secretory agents can cause serious side effects, such as osteoporosis and hypergastrinemia, resulting in hyperplasia of enterochromaffin-like cells and the development of carcinoids in the gastric mucosa [10–13]. Our group suggested that the lower part of rat gastric mucosa was found to be replaced by connective tissue with accumulation of oxidative products as the rats get old [14]. In addition, impairment of apoptosis, angiogenesis, and sensory neuron activity via the activation of Egr-1 and PTEN increased the susceptibility of gastric mucosa to injury during aging [14]. This explains the increase in incidence of benign gastric ulcers in spite of a significant decrease in H. pylori positivity with increased consumption of NSAIDs in the elderly population [15]. As the elderly patients usually take aspirin or NSAIDs continuously in spite of its side effects, there is a growing need for gastroprotective agents, especially phytochemicals,
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which might inhibit gastric acid secretion, with antiinflammatory or antioxidant action without causing any side effect [16, 17]. One of the well-known sources of phytochemicals that have been studied is garlic and its derivative. PMK-S005 is a synthetic s-allyl-L-cysteine (SAC), which is a sulfur-containing amino acid, naturally found in garlic. SAC has been suggested to possess strong antioxidant and antiinflammatory activities in several studies [18, 19], but its gastroprotective effect has not been investigated. Therefore, in this study, the gastroprotective effects of a synthetic SAC, named PMK-S005, were evaluated in a rat model of gastric mucosal damage induced by three types of NSAIDs. In addition, the molecular mechanisms underlying its gastroprotective effect with respect to pro-inflammatory cytokines, mucin synthesis, as well as the arachidonic acid pathway were comprehensively examined.
Materials and Methods Animals Seven-week-old male Sprague–Dawley rats (Orient Co. Ltd., Seoul, Korea) were housed in a cage maintained at 23 °C, 12/12-h light/dark cycle under specific pathogenfree conditions. After 1 week of adaptation, 8-week-old rats weighing 250–300 g were used for the experiments. All of the experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University Bundang Hospital (IACUC number: BA1304-127-033-01). The rats were starved but allowed water for 12 h prior to the experiments. NSAIDs-Induced Gastric Mucosal Injury Model Either PMK-S005 (1, 5, or 10 mg/kg) or rebamipide (50 mg/kg) was administered orally to rats, which had been fasted for 12 h, 1 h prior to the induction of gastric damage. Acute gastric lesions were induced by intragastric administration of aspirin (200 mg/kg) suspended in 0.2 N HCl, diclofenac (80 mg/kg) suspended in 0.5 % carboxymethylcellulose (CMC) or indomethacin (40 mg/kg) suspended in 0.5 % CMC with 10 % ethanol. After 4 h, the animals were killed and the gastric lesions were scored. Gross Ulcer Index and Histological Index After killing the animals, the isolated stomachs were cut open along the greater curvature and washed in ice-cold saline. To investigate the degree of gross mucosal damage, the mucosal sides of the stomachs were photographed using a digital camera and part of the mucosa was immediately fixed with 10 % formalin solution. The gross damage of the
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gastric mucosa was assessed by three gastroenterologists, who were blinded to the treatments, using a gross ulcer index [20]. In addition, the mucosal damage was graded by assigning an index of histological injury [21]. Measurement of Adherent Mucus and Hexosamine The concentration of adherent gastric mucosal mucus was expressed as the concentration of Alcian blue adhering to the gastric mucosal surface (lg/g tissue) [22]. The excised stomach was extensively washed with ice-cold saline, and gastric mucosa was scraped with a slide glass and frozen at -70 °C until use. Finally, hexosamine appropriately obtained from hydrolyzed mucin was assayed using the method described by Neuhaus and Letzring [23].
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(cPLA2) antibody (1:1,000, Santa Cruz, CA, USA), cyclooxygenase (COX)-1 antibody (1:1,000; Santa Cruz, CA, USA), or cyclooxygenase (COX)-2 antibody (1:1,000; Cayman Chemical, Ann Arbor, MI, USA) at 4 °C overnight. The blots were incubated with secondary antibody (1:4,000; Santa Cruz, CA, USA), and an imaging analyzer was used to measure the band densities. Statistical Analysis Data are expressed as the mean ± standard errors (SEM). Statistical analyses were conducted with GraphPad Prism (GraphPad Software, La Jolla, CA, USA) and SPSS software (version 12.0; SPSS Inc., Chicago, IL, USA). Statistical significance was determined using the Mann–Whitney U test, and P \ 0.05 was considered to be statistically significant.
Measurement of Mucosal Myeloperoxidase (MPO), TNF-a, and IL-1b Results Ten milligrams of scraped mucosa was homogenized for 30 s with a polytron homogenizer in 200 ll of ice-cold lysis buffer (200 mM NaCl, 5 mM EDTA, 10 mM Tris (pH 7.4), 10 % glycerin, 1 mM PMSF, 1 lg/ml leupeptin, and 28 lg/ ml aprotinin). Suspensions were centrifuged at 13,000 rpm for 15 min, and the resulting supernatant was assayed using an MPO ELISA kit (HyCult Biotechnology, Uden, the Netherlands). For TNF-a and IL-1b, the appropriate kits from R&D systems (Minneapolis, MN, USA) were used by following the manufacturer’s instructions. Protein concentration was measured by using Bio-Rad protein assay kit (Bio-Rad laboratories, Hercules, MA, USA). Measurement of Mucosal PGE2 and LTB4 PGE2 and LTB4 levels were measured with a commercial kit (Cayman Chemical, Ann Arbor, MI, USA). Briefly, the scraped mucosa was weighed and homogenized with lysis buffer containing 0.1 M phosphate (pH 7.4), EDTA (1 mM), and indomethacin (10 lM). The samples were then centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant of each sample was used for the determination of PGE2 and LTB4 levels. Western Blotting for cPLA2, COX-1, and COX-2 The gastric mucosa was homogenized with lysis buffer containing 25 mM Tris–HCl (pH 7.4), EGTA (1 mM), DTT (1 mM), leupeptin (10 lg/ml), aprotinin (10 lg/ml), PMSF (1 mM), and Triton X-100 (0.1 %). Briefly, the proteins (each sample, 50 lg) were separated by SDS–PAGE (10 % wt/wt gel) and transferred to nitrocellulose membranes. The membranes were then blocked with dried milk (5 % wt/vol) and subsequently incubated with cytosolic phospholipase A2
Effect of PMK-S005 on Acute Gastric Damage Induced by Aspirin To investigate the gastroprotective effect of PMK-S005, we established an acute gastric damage model using NSAIDs including aspirin, diclofenac, and indomethacin. Administration of aspirin (200 mg/kg) induced multiple hemorrhagic erosions and ulcers, which were prevented by pretreatment with either PMK-S005 or rebamipide (Fig. 1a–h). Quantitatively, the gross ulcer indices were reduced from 10.3 to 0.2 after pretreatment with PMK-S005 (5 mg/kg; P = 0.026), but rebamipide pretreatment did not cause a statistically significant difference (Fig. 1i). In addition, the histological indices were dramatically reduced after pretreatment with 5 mg/kg of PMK-S005 (Fig. 1j). In accordance with that in the aspirin-induced gastric damage model, pretreatment with PMK-S005 ameliorated gastric ulceration in the diclofenac(80 mg/kg) and indomethacin (40 mg/kg)-induced gastric damage model (Supplementary Fig. S1, S4). Effect of PMK-S005 on Hexosamine and Adherent Mucus in Aspirin-Induced Gastric Damage Intragastric administration of aspirin significantly decreased gastric mucosal hexosamine (2.6 mg/g tissues) and adherent mucus concentration (352.1 lg/g tissues) compared to that in the control rat (Fig. 2a, b). However, pretreatment with 5 mg/kg of PMK-S005, before aspirin administration, significantly increased gastric mucosal hexosamine (4.6 mg/g tissues; P = 0.004) and adherent mucus concentration (490.1 lg/g tissues; P = 0.038; Fig. 2a, b). Similar results were observed in the diclofenac and indomethacin-induced gastric damage model (Supplementary Fig. S2, S5).
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Fig. 1 Gross (a–d) and histological findings (e–h) in the aspirininduced gastric damage of the rat stomach. Normal rat stomachs (a, e) are damaged by aspirin administration (200 mg/kg) (b, f), and most of the damage is prevented by pretreatment with 5 mg/kg of PMKS005 (c, g) or rebamipide (50 mg/kg) (d, h) 1 h before intragastric
aspirin administration. Macroscopic damage indices and histological damage indices are quantified (i, j). Scale bar = 200 lm. Results are expressed as mean ± SEM from 5 to 7 animals per group. P \ 0.05 compared with the control group. *P \ 0.05 compared with the aspirin group
Fig. 2 Cytoprotective activities of PMK-S005 in aspirin-induced gastric damage. Hexosamine concentrations (a) and adherent mucus (b) are significantly decreased after the intragastric administration of aspirin (200 mg/kg), and these reductions are significantly prevented
by pretreatment with 5 mg/kg of PMK-S005. Results are expressed as mean ± SEM from 5 to 7 animals per group. P \ 0.05 compared with the control group. *P \ 0.05 compared with the aspirin group
Effect of PMK-S005 on MPO, TNF-a, and IL-1b Levels in Aspirin-Induced Gastric Damage
various experimental gastric damages. Increased mucosal levels of MPO after aspirin administration were suppressed by pretreatment with 5 mg/kg of PMK-S005 or rebamipide (Fig. 3a). Pro-inflammatory cytokine, TNF-a, and IL-1b were also increased after aspirin administration, and these
Myeloperoxidase level, as an index of neutrophil infiltration into gastric mucosal tissue, is widely used as a marker in
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Fig. 3 Mucosal concentration of MPO (a), TNF-a (b), and IL-1b (c). The aspirin-administered rats showed marked increases in the MPO (a), TNF-a (b), and IL-1b levels (c). The increased MPO and TNF-a levels are significantly inhibited by pretreatment with 5 mg/kg of
PMK-S005 or rebamipide (50 mg/kg). Results are expressed as mean ± SEM from 5 to 7 animals per group. P \ 0.05 compared with the control group. *P \ 0.05 compared with the aspirin group
Fig. 4 Western blot analysis of cPLA2 (a), COX-1 (b), and COX-2 (c) expression. The aspirin-administered rats showed marked increases in cPLA2 (a) and COX-2 (c) levels in the gastric mucosa (representative blot is shown). These increases are prevented by
pretreatment with PMK-S005 (5 mg/kg) or rebamipide (50 mg/kg). COX-1 expression (b) is not changed. Results are expressed as mean ± SEM from 5 to 7 animals per group. P \ 0.05 compared with the control group. *P \ 0.05 compared with the aspirin group
increases were significantly suppressed by pretreatment with 5 mg/kg of PMK-S005 or rebamipide (Fig. 3b, c). Similar results were observed in the diclofenac and indomethacininduced gastric damage model (Supplementary Fig. S2, S5).
production through COX inhibition [24]. We observed that the mucosal level of PGE2 was significantly reduced after aspirin administration compared to that in the control rat (293.8–109.3; P = 0.042). Under this condition, pretreatment with PMK-S005 failed to inhibit the aspirin-induced reduction in PGE2 content (Fig. 5a). In contrast to PGE2, the increased LTB4 level after aspirin administration was significantly reduced by pretreatment with PMK-S005 (5 mg/kg; P = 0.005, 10 mg/kg; P = 0.009) or rebamipide (Fig. 5b). Similar results were observed in the diclofenac and indomethacin-induced gastric damage model (Supplementary Fig. S3, S6).
Western Blotting of cPLA2, COX-1, and COX-2 The increased expression of cPLA2 4 h after aspirin administration (Fig. 4a) was significantly reduced by PMKS005 pretreatment (5 mg/kg; P = 0.007). COX-2 expression also showed a similar trend (Fig. 4c). However, COX-1 expression was not changed after aspirin administration, and PMK-S005 pretreatment did not cause any changes compared to that in the aspirin-treated rat (Fig. 4b). Similar results were observed in the diclofenac and indomethacininduced gastric damage model (Supplementary Fig. S3, S6). Effect of PMK-S005 on PGE2 and LTB4 Levels in Aspirin-Induced Gastric Damage The pathophysiology of NSAID-induced gastric injury depends, in part, on their ability to decrease prostaglandin
Discussion Garlic (Allium sativum) is a commonly used plant for seasoning in many different cultures of the world, especially in Asian countries, and its importance as a functional food against various pathologies has been explored [25–27]. However, the use of fresh raw garlic has been associated with stomach and digestion problems [28]; thus, increasing
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Fig. 5 Effects of PMK-S005 on the PGE2 (a) and LTB4 (b) level of the gastric mucosal tissue. Aspirin administration reduced the mucosal concentrations of PGE2 level and PMK-S005 pretreatment failed to inhibit the reduction in the PGE2 level (a). The increased LTB4 level in the gastric mucosa after aspirin administration is
significantly reduced by pretreatment with PMK-S005 or rebamipide (b). Results are expressed as mean ± SEM from 5 to 7 animals per group. P \ 0.05 compared with the control group. *P \ 0.05 compared with the aspirin group
the need to isolate a useful therapeutic agent with few harmful effects of garlic. SAC, the most abundant sulfurcontaining amino acid derived from garlic, has been reported to have multiple biological properties such as antioxidant [18], anti-inflammatory [19], anticancer [25], and anti-hepatotoxic activities [29]. In addition, SAC has already been shown to be absorbed in the gastrointestinal tract after oral administration and has been used for the treatment of hypertension without obvious signs of toxicity [30]. However, the mechanism underlying the protective effects of SAC against gastric damage is unclear. The present study demonstrated that PMK-S005 pretreatment exerts protective action against NSAIDs-induced gastric damage via (1) attenuating the lesions produced in three types of NSAIDs, (2) inducing mucin synthesis and secretion related to endogenous cytoprotection, (3) decreasing generation of inflammatory cytokines, and (4) reducing the induction of cPLA2, COX-2, and LTB4 after NSAIDs administration. All effects of PMK-S005 were consistently maximized at a concentration of 5 mg/kg, and it was more potent than rebamipide (50 mg/kg) in some model. To analyze the protective effect of PMK-S005 more clearly, rebamipide was adopted for comparison and the dosage used here was selected considering previous animal study [31, 32]. Rebamipide, a gastroprotective drug, was developed in Japan for the treatment of peptic ulcers [33]. The mechanisms of action include the stimulation of prostaglandin biosynthesis [34], free radical scavenging activity [35], anti-inflammatory activity [36], inhibition of the level of H. pylori colonization [37], restoration of Sonic hedgehog expression after H. pylori eradication [38], and induction of gastric ulcer healing [39]. Recently, rebamipide has even been shown to impede gastric cancer growth [40].
Although it was suggested that the extent of ulcer damage and related mechanisms might be different according to the type of NSAIDs [41], 5 mg/kg of PMKS005 consistently showed promising gastroprotective effects against three different types of NSAIDs. NSAIDsinduced gastropathy is associated with a decrease in gastric mucus production [42]. Our results showed that the gross and microscopic mucosal damage induced by NSAID was significantly attenuated by pretreatment with PMK-S005. In addition, this cytoprotective effect of PMK-S005 was closely related to the activation of gastric mucus synthesis and secretion, indicating that PMK-S005 acts through the defensive mechanism. Arachidonic acid, which is released from membrane phospholipids during the inflammatory process, is metabolized by two pathways. One is prostaglandin synthesis through COX and the other one is leukotriene synthesis through 5-LOX. LTB4, the by-product of 5-LOX and cPLA2, has been reported to play an important pathophysiological role in the development of gastroduodenal ulcer [43]. In the present study, the increase in cPLA2 expression after NSAIDs administration was attenuated by PMK-S005 pretreatment. In addition, PMK-S005 pretreatment inhibited NSAIDs-induced increase in MPO, TNF-a, and IL-1b levels in the gastric mucosa. These results suggest that PMK-S005 exerts anti-inflammatory activity by inhibiting cPLA2 signaling. In regards to NSAIDs-induced gastric damages, ROS produced by recruited leukocytes are also major factor [44]. In the present study, the gastroprotective effects of PMK-S005, a synthetic SAC, was comprehensively evaluated in the aspect of not only anti-inflammation but also antioxidant using NSAIDs-induced gastric damage model. Unlike our expectation, antioxidant enzyme HO-1 and
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NQO-1 levels were not increased after PMK-S005 administration and even decreased compared with those of NSAID-treated rats (data not shown). However, PMKS005 treatment increased antioxidant enzyme, HO-1, and superoxide dismutase-1 (SOD-1) expression as well as decreased the inflammatory genes iNOS and COX-2 expression in vitro system (unpublished data). We speculate that 1-h incubation after PMK-S005 may not be enough to exert its antioxidant effect, and frequency and duration of PMK-S005 administration could affect its antioxidant effects. This could be evaluated in the chronic NSAID model, which was not well established so far [45]. As the elderly patients require long-term NSAIDs therapy, several prevention methods including PPI have been used, but these methods have limited potency because of their adverse effects [13, 46]. Consequently, the gastroprotective properties of PMK-S005, a synthetic SAC, make it a plausible candidate for further studies. Moreover, since SAC is easily absorbed in the gastrointestinal tract and rapidly detected in several tissues (kidney, liver, lung, and brain), it has been suggested that the effects of PMK-S005 might not only be limited to the stomach but may also be applicable in inflammation-related diseases of other organs. Especially, several reports indicate that SAC is a potent antioxidant agent and prevents the oxidative damage observed in chronic degenerative diseases [47]. Therefore, we are planning to investigate the anti-aging property of PMK-S005 in a further study. In conclusion, the protective effect of PMK-S005, a synthetic SAC, against NSAIDs-induced gastric damage was demonstrated for the first time. This effect was attributable to its anti-inflammatory and cytoprotective activities through mucus synthesis. Acknowledgments This work was supported by Chungcheong leading industry promotion project of the Korean Ministry of Knowledge Economy. Conflict of interest None of the authors have any conflict of interest or financial arrangements that could potentially influence the described research.
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ORIGINAL ARTICLE
Protective Effects of Garlic Extract, PMK-S005, Against Nonsteroidal Anti-inflammatory Drugs–Induced Acute Gastric Damage in Rats Yoon Jeong Choi • Nayoung Kim • Ju Yup Lee • Ryoung Hee Nam Hyun Chang • Ji Hyung Seo • Kyu Keun Kang • Hee Jin Kim • Yun Jin Choi • Hye Seung Lee • Dong Ho Lee
•
Received: 29 June 2014 / Accepted: 15 September 2014 / Published online: 5 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Background PMK-S005 is synthetic s-allyl-L-cysteine (SAC), a sulfur-containing amino acid, which was initially isolated from garlic. The antioxidant and anti-inflammation activities of SAC have been demonstrated in diverse experimental animal models. Aims The purpose of this study was to investigate the gastroprotective effects of PMK-S005 against NSAIDsinduced acute gastric damage in rats. Methods Eight-week SD rats were pretreated with PMKS005 (1, 5, or 10 mg/kg) or rebamipide (50 mg/kg) 1 h before administration of NSAIDs including aspirin (200 mg/kg), diclofenac (80 mg/kg), and indomethacin
Electronic supplementary material The online version of this article (doi:10.1007/s10620-014-3370-5) contains supplementary material, which is available to authorized users. Y. J. Choi N. Kim (&) J. Y. Lee R. H. Nam H. Chang J. H. Seo K. K. Kang H. J. Kim Y. J. Choi D. H. Lee Department of Internal Medicine, Seoul National University Bundang Hospital, 82, Gumi-ro, 173 Beon-gil, Bundang-gu, Seongnam, Gyeonggi-do 463-707, Korea e-mail: [email protected] Y. J. Choi e-mail: [email protected] J. Y. Lee e-mail: [email protected] R. H. Nam e-mail: [email protected] H. Chang e-mail: [email protected] J. H. Seo e-mail: [email protected]
(40 mg/kg). After 4 h, the gross ulcer index, histological index, and gastric mucus level were determined. Myeloperoxidase (MPO), TNF-a, IL-1b, PGE2, and LTB4 levels were estimated in the gastric mucosal tissue by ELISA. Protein expressions of cPLA2, COX-1, and COX-2 were assessed by Western blot analysis. Results Pretreatment with PMK-S005 significantly attenuated the NSAIDs-induced gastric damage and increased the gastric mucus level. In addition, PMK-S005 attenuated increases in MPO, TNF-a, and IL-1b production. The expressions of cPLA2 and COX-2 induced by NSAIDs were decreased by PMK-S005 pretreatment. PMK-S005 did not cause suppression of PGE2 synthesis induced by NSAIDs, but LTB4 production was significantly suppressed by PMK-S005. The effects of PMK-S005 were consistently maximized at a concentration of 5 mg/kg, which were frequently superior to those of rebamipide. H. J. Kim e-mail: [email protected] Y. J. Choi e-mail: [email protected] D. H. Lee e-mail: [email protected] N. Kim D. H. Lee Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea H. S. Lee Department of Pathology, Seoul National University Bundang Hospital, 82, Gumi-ro, 173 Beon-gil, Bundang-gu, Seongnam, Gyeonggi-do 463-707, Korea e-mail: [email protected]
K. K. Kang e-mail: [email protected]
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Conclusions These results strongly suggest that PMKS005 can be a useful gastroprotective agent against acute gastric mucosal damage by suppressing proinflammatory cytokines, down-regulating cPLA2, COX-2 and LTB4 expression, and increasing the synthesis of mucus. Keywords s-allyl-L-cysteine (SAC) Gastroprotection Anti-inflammatory agent Rat NSAID
Introduction Gastric ulcer is the most common disorder of the gastrointestinal tract, with increasing incidence and prevalence. Gastric ulcer is predominantly caused by infection with Helicobacter pylori (H. pylori) or other environmental factors such as stress, smoking, alcohol consumption, and the ingestion of nonsteroidal anti-inflammatory drugs (NSAIDs) [1, 2]. NSAIDs including aspirin are widely used as anti-inflammatory and analgesic agents. However, gastrointestinal toxicity associated with NSAIDs has emerged as an important medical problem [3]. Currently, the expanding use of NSAIDs in the elderly and its complications like acute gastric erosion, ulceration, bleeding, and perforation have become more frequent in old patients [4–7], and the mortality rate in the elderly due to ulcer bleeding and perforation has rapidly increased [8, 9]. Imbalances between the aggressive (acid and pepsin secretion, NSAIDs, or H. pylori) and defensive factors (mucus secretion, blood flow, nitric oxide, and prostaglandins) have been implicated in the etiology of ulcer disease. One of the main therapeutic approaches for this pathological condition is the suppression of acid secretion by proton pump inhibitors (PPIs) or H2 antagonists as well as the eradication of H. pylori by antibiotics. However, ulcers frequently relapse in spite of maintenance therapy with this drug and long-term treatment with anti-secretory agents can cause serious side effects, such as osteoporosis and hypergastrinemia, resulting in hyperplasia of enterochromaffin-like cells and the development of carcinoids in the gastric mucosa [10–13]. Our group suggested that the lower part of rat gastric mucosa was found to be replaced by connective tissue with accumulation of oxidative products as the rats get old [14]. In addition, impairment of apoptosis, angiogenesis, and sensory neuron activity via the activation of Egr-1 and PTEN increased the susceptibility of gastric mucosa to injury during aging [14]. This explains the increase in incidence of benign gastric ulcers in spite of a significant decrease in H. pylori positivity with increased consumption of NSAIDs in the elderly population [15]. As the elderly patients usually take aspirin or NSAIDs continuously in spite of its side effects, there is a growing need for gastroprotective agents, especially phytochemicals,
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which might inhibit gastric acid secretion, with antiinflammatory or antioxidant action without causing any side effect [16, 17]. One of the well-known sources of phytochemicals that have been studied is garlic and its derivative. PMK-S005 is a synthetic s-allyl-L-cysteine (SAC), which is a sulfur-containing amino acid, naturally found in garlic. SAC has been suggested to possess strong antioxidant and antiinflammatory activities in several studies [18, 19], but its gastroprotective effect has not been investigated. Therefore, in this study, the gastroprotective effects of a synthetic SAC, named PMK-S005, were evaluated in a rat model of gastric mucosal damage induced by three types of NSAIDs. In addition, the molecular mechanisms underlying its gastroprotective effect with respect to pro-inflammatory cytokines, mucin synthesis, as well as the arachidonic acid pathway were comprehensively examined.
Materials and Methods Animals Seven-week-old male Sprague–Dawley rats (Orient Co. Ltd., Seoul, Korea) were housed in a cage maintained at 23 °C, 12/12-h light/dark cycle under specific pathogenfree conditions. After 1 week of adaptation, 8-week-old rats weighing 250–300 g were used for the experiments. All of the experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University Bundang Hospital (IACUC number: BA1304-127-033-01). The rats were starved but allowed water for 12 h prior to the experiments. NSAIDs-Induced Gastric Mucosal Injury Model Either PMK-S005 (1, 5, or 10 mg/kg) or rebamipide (50 mg/kg) was administered orally to rats, which had been fasted for 12 h, 1 h prior to the induction of gastric damage. Acute gastric lesions were induced by intragastric administration of aspirin (200 mg/kg) suspended in 0.2 N HCl, diclofenac (80 mg/kg) suspended in 0.5 % carboxymethylcellulose (CMC) or indomethacin (40 mg/kg) suspended in 0.5 % CMC with 10 % ethanol. After 4 h, the animals were killed and the gastric lesions were scored. Gross Ulcer Index and Histological Index After killing the animals, the isolated stomachs were cut open along the greater curvature and washed in ice-cold saline. To investigate the degree of gross mucosal damage, the mucosal sides of the stomachs were photographed using a digital camera and part of the mucosa was immediately fixed with 10 % formalin solution. The gross damage of the
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gastric mucosa was assessed by three gastroenterologists, who were blinded to the treatments, using a gross ulcer index [20]. In addition, the mucosal damage was graded by assigning an index of histological injury [21]. Measurement of Adherent Mucus and Hexosamine The concentration of adherent gastric mucosal mucus was expressed as the concentration of Alcian blue adhering to the gastric mucosal surface (lg/g tissue) [22]. The excised stomach was extensively washed with ice-cold saline, and gastric mucosa was scraped with a slide glass and frozen at -70 °C until use. Finally, hexosamine appropriately obtained from hydrolyzed mucin was assayed using the method described by Neuhaus and Letzring [23].
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(cPLA2) antibody (1:1,000, Santa Cruz, CA, USA), cyclooxygenase (COX)-1 antibody (1:1,000; Santa Cruz, CA, USA), or cyclooxygenase (COX)-2 antibody (1:1,000; Cayman Chemical, Ann Arbor, MI, USA) at 4 °C overnight. The blots were incubated with secondary antibody (1:4,000; Santa Cruz, CA, USA), and an imaging analyzer was used to measure the band densities. Statistical Analysis Data are expressed as the mean ± standard errors (SEM). Statistical analyses were conducted with GraphPad Prism (GraphPad Software, La Jolla, CA, USA) and SPSS software (version 12.0; SPSS Inc., Chicago, IL, USA). Statistical significance was determined using the Mann–Whitney U test, and P \ 0.05 was considered to be statistically significant.
Measurement of Mucosal Myeloperoxidase (MPO), TNF-a, and IL-1b Results Ten milligrams of scraped mucosa was homogenized for 30 s with a polytron homogenizer in 200 ll of ice-cold lysis buffer (200 mM NaCl, 5 mM EDTA, 10 mM Tris (pH 7.4), 10 % glycerin, 1 mM PMSF, 1 lg/ml leupeptin, and 28 lg/ ml aprotinin). Suspensions were centrifuged at 13,000 rpm for 15 min, and the resulting supernatant was assayed using an MPO ELISA kit (HyCult Biotechnology, Uden, the Netherlands). For TNF-a and IL-1b, the appropriate kits from R&D systems (Minneapolis, MN, USA) were used by following the manufacturer’s instructions. Protein concentration was measured by using Bio-Rad protein assay kit (Bio-Rad laboratories, Hercules, MA, USA). Measurement of Mucosal PGE2 and LTB4 PGE2 and LTB4 levels were measured with a commercial kit (Cayman Chemical, Ann Arbor, MI, USA). Briefly, the scraped mucosa was weighed and homogenized with lysis buffer containing 0.1 M phosphate (pH 7.4), EDTA (1 mM), and indomethacin (10 lM). The samples were then centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant of each sample was used for the determination of PGE2 and LTB4 levels. Western Blotting for cPLA2, COX-1, and COX-2 The gastric mucosa was homogenized with lysis buffer containing 25 mM Tris–HCl (pH 7.4), EGTA (1 mM), DTT (1 mM), leupeptin (10 lg/ml), aprotinin (10 lg/ml), PMSF (1 mM), and Triton X-100 (0.1 %). Briefly, the proteins (each sample, 50 lg) were separated by SDS–PAGE (10 % wt/wt gel) and transferred to nitrocellulose membranes. The membranes were then blocked with dried milk (5 % wt/vol) and subsequently incubated with cytosolic phospholipase A2
Effect of PMK-S005 on Acute Gastric Damage Induced by Aspirin To investigate the gastroprotective effect of PMK-S005, we established an acute gastric damage model using NSAIDs including aspirin, diclofenac, and indomethacin. Administration of aspirin (200 mg/kg) induced multiple hemorrhagic erosions and ulcers, which were prevented by pretreatment with either PMK-S005 or rebamipide (Fig. 1a–h). Quantitatively, the gross ulcer indices were reduced from 10.3 to 0.2 after pretreatment with PMK-S005 (5 mg/kg; P = 0.026), but rebamipide pretreatment did not cause a statistically significant difference (Fig. 1i). In addition, the histological indices were dramatically reduced after pretreatment with 5 mg/kg of PMK-S005 (Fig. 1j). In accordance with that in the aspirin-induced gastric damage model, pretreatment with PMK-S005 ameliorated gastric ulceration in the diclofenac(80 mg/kg) and indomethacin (40 mg/kg)-induced gastric damage model (Supplementary Fig. S1, S4). Effect of PMK-S005 on Hexosamine and Adherent Mucus in Aspirin-Induced Gastric Damage Intragastric administration of aspirin significantly decreased gastric mucosal hexosamine (2.6 mg/g tissues) and adherent mucus concentration (352.1 lg/g tissues) compared to that in the control rat (Fig. 2a, b). However, pretreatment with 5 mg/kg of PMK-S005, before aspirin administration, significantly increased gastric mucosal hexosamine (4.6 mg/g tissues; P = 0.004) and adherent mucus concentration (490.1 lg/g tissues; P = 0.038; Fig. 2a, b). Similar results were observed in the diclofenac and indomethacin-induced gastric damage model (Supplementary Fig. S2, S5).
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Fig. 1 Gross (a–d) and histological findings (e–h) in the aspirininduced gastric damage of the rat stomach. Normal rat stomachs (a, e) are damaged by aspirin administration (200 mg/kg) (b, f), and most of the damage is prevented by pretreatment with 5 mg/kg of PMKS005 (c, g) or rebamipide (50 mg/kg) (d, h) 1 h before intragastric
aspirin administration. Macroscopic damage indices and histological damage indices are quantified (i, j). Scale bar = 200 lm. Results are expressed as mean ± SEM from 5 to 7 animals per group. P \ 0.05 compared with the control group. *P \ 0.05 compared with the aspirin group
Fig. 2 Cytoprotective activities of PMK-S005 in aspirin-induced gastric damage. Hexosamine concentrations (a) and adherent mucus (b) are significantly decreased after the intragastric administration of aspirin (200 mg/kg), and these reductions are significantly prevented
by pretreatment with 5 mg/kg of PMK-S005. Results are expressed as mean ± SEM from 5 to 7 animals per group. P \ 0.05 compared with the control group. *P \ 0.05 compared with the aspirin group
Effect of PMK-S005 on MPO, TNF-a, and IL-1b Levels in Aspirin-Induced Gastric Damage
various experimental gastric damages. Increased mucosal levels of MPO after aspirin administration were suppressed by pretreatment with 5 mg/kg of PMK-S005 or rebamipide (Fig. 3a). Pro-inflammatory cytokine, TNF-a, and IL-1b were also increased after aspirin administration, and these
Myeloperoxidase level, as an index of neutrophil infiltration into gastric mucosal tissue, is widely used as a marker in
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Fig. 3 Mucosal concentration of MPO (a), TNF-a (b), and IL-1b (c). The aspirin-administered rats showed marked increases in the MPO (a), TNF-a (b), and IL-1b levels (c). The increased MPO and TNF-a levels are significantly inhibited by pretreatment with 5 mg/kg of
PMK-S005 or rebamipide (50 mg/kg). Results are expressed as mean ± SEM from 5 to 7 animals per group. P \ 0.05 compared with the control group. *P \ 0.05 compared with the aspirin group
Fig. 4 Western blot analysis of cPLA2 (a), COX-1 (b), and COX-2 (c) expression. The aspirin-administered rats showed marked increases in cPLA2 (a) and COX-2 (c) levels in the gastric mucosa (representative blot is shown). These increases are prevented by
pretreatment with PMK-S005 (5 mg/kg) or rebamipide (50 mg/kg). COX-1 expression (b) is not changed. Results are expressed as mean ± SEM from 5 to 7 animals per group. P \ 0.05 compared with the control group. *P \ 0.05 compared with the aspirin group
increases were significantly suppressed by pretreatment with 5 mg/kg of PMK-S005 or rebamipide (Fig. 3b, c). Similar results were observed in the diclofenac and indomethacininduced gastric damage model (Supplementary Fig. S2, S5).
production through COX inhibition [24]. We observed that the mucosal level of PGE2 was significantly reduced after aspirin administration compared to that in the control rat (293.8–109.3; P = 0.042). Under this condition, pretreatment with PMK-S005 failed to inhibit the aspirin-induced reduction in PGE2 content (Fig. 5a). In contrast to PGE2, the increased LTB4 level after aspirin administration was significantly reduced by pretreatment with PMK-S005 (5 mg/kg; P = 0.005, 10 mg/kg; P = 0.009) or rebamipide (Fig. 5b). Similar results were observed in the diclofenac and indomethacin-induced gastric damage model (Supplementary Fig. S3, S6).
Western Blotting of cPLA2, COX-1, and COX-2 The increased expression of cPLA2 4 h after aspirin administration (Fig. 4a) was significantly reduced by PMKS005 pretreatment (5 mg/kg; P = 0.007). COX-2 expression also showed a similar trend (Fig. 4c). However, COX-1 expression was not changed after aspirin administration, and PMK-S005 pretreatment did not cause any changes compared to that in the aspirin-treated rat (Fig. 4b). Similar results were observed in the diclofenac and indomethacininduced gastric damage model (Supplementary Fig. S3, S6). Effect of PMK-S005 on PGE2 and LTB4 Levels in Aspirin-Induced Gastric Damage The pathophysiology of NSAID-induced gastric injury depends, in part, on their ability to decrease prostaglandin
Discussion Garlic (Allium sativum) is a commonly used plant for seasoning in many different cultures of the world, especially in Asian countries, and its importance as a functional food against various pathologies has been explored [25–27]. However, the use of fresh raw garlic has been associated with stomach and digestion problems [28]; thus, increasing
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Fig. 5 Effects of PMK-S005 on the PGE2 (a) and LTB4 (b) level of the gastric mucosal tissue. Aspirin administration reduced the mucosal concentrations of PGE2 level and PMK-S005 pretreatment failed to inhibit the reduction in the PGE2 level (a). The increased LTB4 level in the gastric mucosa after aspirin administration is
significantly reduced by pretreatment with PMK-S005 or rebamipide (b). Results are expressed as mean ± SEM from 5 to 7 animals per group. P \ 0.05 compared with the control group. *P \ 0.05 compared with the aspirin group
the need to isolate a useful therapeutic agent with few harmful effects of garlic. SAC, the most abundant sulfurcontaining amino acid derived from garlic, has been reported to have multiple biological properties such as antioxidant [18], anti-inflammatory [19], anticancer [25], and anti-hepatotoxic activities [29]. In addition, SAC has already been shown to be absorbed in the gastrointestinal tract after oral administration and has been used for the treatment of hypertension without obvious signs of toxicity [30]. However, the mechanism underlying the protective effects of SAC against gastric damage is unclear. The present study demonstrated that PMK-S005 pretreatment exerts protective action against NSAIDs-induced gastric damage via (1) attenuating the lesions produced in three types of NSAIDs, (2) inducing mucin synthesis and secretion related to endogenous cytoprotection, (3) decreasing generation of inflammatory cytokines, and (4) reducing the induction of cPLA2, COX-2, and LTB4 after NSAIDs administration. All effects of PMK-S005 were consistently maximized at a concentration of 5 mg/kg, and it was more potent than rebamipide (50 mg/kg) in some model. To analyze the protective effect of PMK-S005 more clearly, rebamipide was adopted for comparison and the dosage used here was selected considering previous animal study [31, 32]. Rebamipide, a gastroprotective drug, was developed in Japan for the treatment of peptic ulcers [33]. The mechanisms of action include the stimulation of prostaglandin biosynthesis [34], free radical scavenging activity [35], anti-inflammatory activity [36], inhibition of the level of H. pylori colonization [37], restoration of Sonic hedgehog expression after H. pylori eradication [38], and induction of gastric ulcer healing [39]. Recently, rebamipide has even been shown to impede gastric cancer growth [40].
Although it was suggested that the extent of ulcer damage and related mechanisms might be different according to the type of NSAIDs [41], 5 mg/kg of PMKS005 consistently showed promising gastroprotective effects against three different types of NSAIDs. NSAIDsinduced gastropathy is associated with a decrease in gastric mucus production [42]. Our results showed that the gross and microscopic mucosal damage induced by NSAID was significantly attenuated by pretreatment with PMK-S005. In addition, this cytoprotective effect of PMK-S005 was closely related to the activation of gastric mucus synthesis and secretion, indicating that PMK-S005 acts through the defensive mechanism. Arachidonic acid, which is released from membrane phospholipids during the inflammatory process, is metabolized by two pathways. One is prostaglandin synthesis through COX and the other one is leukotriene synthesis through 5-LOX. LTB4, the by-product of 5-LOX and cPLA2, has been reported to play an important pathophysiological role in the development of gastroduodenal ulcer [43]. In the present study, the increase in cPLA2 expression after NSAIDs administration was attenuated by PMK-S005 pretreatment. In addition, PMK-S005 pretreatment inhibited NSAIDs-induced increase in MPO, TNF-a, and IL-1b levels in the gastric mucosa. These results suggest that PMK-S005 exerts anti-inflammatory activity by inhibiting cPLA2 signaling. In regards to NSAIDs-induced gastric damages, ROS produced by recruited leukocytes are also major factor [44]. In the present study, the gastroprotective effects of PMK-S005, a synthetic SAC, was comprehensively evaluated in the aspect of not only anti-inflammation but also antioxidant using NSAIDs-induced gastric damage model. Unlike our expectation, antioxidant enzyme HO-1 and
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NQO-1 levels were not increased after PMK-S005 administration and even decreased compared with those of NSAID-treated rats (data not shown). However, PMKS005 treatment increased antioxidant enzyme, HO-1, and superoxide dismutase-1 (SOD-1) expression as well as decreased the inflammatory genes iNOS and COX-2 expression in vitro system (unpublished data). We speculate that 1-h incubation after PMK-S005 may not be enough to exert its antioxidant effect, and frequency and duration of PMK-S005 administration could affect its antioxidant effects. This could be evaluated in the chronic NSAID model, which was not well established so far [45]. As the elderly patients require long-term NSAIDs therapy, several prevention methods including PPI have been used, but these methods have limited potency because of their adverse effects [13, 46]. Consequently, the gastroprotective properties of PMK-S005, a synthetic SAC, make it a plausible candidate for further studies. Moreover, since SAC is easily absorbed in the gastrointestinal tract and rapidly detected in several tissues (kidney, liver, lung, and brain), it has been suggested that the effects of PMK-S005 might not only be limited to the stomach but may also be applicable in inflammation-related diseases of other organs. Especially, several reports indicate that SAC is a potent antioxidant agent and prevents the oxidative damage observed in chronic degenerative diseases [47]. Therefore, we are planning to investigate the anti-aging property of PMK-S005 in a further study. In conclusion, the protective effect of PMK-S005, a synthetic SAC, against NSAIDs-induced gastric damage was demonstrated for the first time. This effect was attributable to its anti-inflammatory and cytoprotective activities through mucus synthesis. Acknowledgments This work was supported by Chungcheong leading industry promotion project of the Korean Ministry of Knowledge Economy. Conflict of interest None of the authors have any conflict of interest or financial arrangements that could potentially influence the described research.
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