http://informahealthcare.com/txm ISSN: 1537-6516 (print), 1537-6524 (electronic) Toxicol Mech Methods, 2014; 24(8): 560–566 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2014.951815

RESEARCH ARTICLE

Protective effects of escin against indomethacin-induced gastric ulcer in mice Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Gazi Univ. on 02/04/15 For personal use only.

Tian Wang1*, Shanshan Zhao1*, Yucun Wang2, Yujiao Yang1, Le Yao1, Liuxiang Chu1, Hanhan Du1, and Fenghua Fu1 1

Department of Pharmacology, School of Pharmacy, Yantai University, Yantai, PR China and 2Division of Gastroenterology, Yantaishan Hospital, Yantai, PR China Abstract

Keywords

Escin, a natural mixture of triterpenoid saponin isolated from the seed of the horse chestnut, is reported to have a potent antiulcer activity against ethanol-induced gastric mucosal lesions. This study investigated the possible mechanisms underlying the gastroprotective effect of escin against indomethacin-induced gastric ulcer in mice. Gastric ulceration was induced by a single intragastric administration of indomethacin (18 mg/kg). The mice underwent intragastric treatment with escin at doses of 0.45, 0.9 or 1.8 mg/kg. Gastric lesion was estimated morphometrically and histopathologically 6 h after the indomethacin administration. The antioxidative parameters in gastric mucosa were measured. Moreover, the activity of myeloperoxidase and the contents of TNF-a, P-selectin and VCAM-1 in gastric tissues were determined. The results showed that escin protected gastric tissues against indomethacininduced gastropathy as demonstrated from a reduction in the ulcer index and an attenuation of histopathologic changes. Escin caused significant reductions of the contents of malondialdehyde, TNF-a, P-selectin, VCAM-1 and myeloperoxidase activity. The altered activities of superoxide dismutase, catalase and glutathione peroxidase in the stomach tissues were also ameliorated by escin treatment. The present study demonstrated that escin had a protective effect against indomethacin-induced gastric ulcer in mice, not only by virtue of its antioxidant potential, but also due to its anti-inflammatory effect.

Anti-inflammatory, antioxidant, escin, gastric ulcer, indomethacin

Introduction Peptic ulcer exists in a gastric and duodenal mucosa disorder with chronic and recurring character, caused by the acidity of gastric secretion. It is a recurrent chronic illness affecting 10% of the world population (Zapata-Colindres et al., 2006) and still a common global problem with increasing morbidity and mortality rates. An imbalance between protective factors (mucus and bicarbonate secretion, increased levels of antioxidants and production of prostaglandins) and mucosal aggressors (stress, ethanol exposure, smoking and frequent ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) has been associated with the etiology of peptic ulcer (Tarnawski et al., 2012). NSAIDs are one of the most commonly used medications throughout the world. Indomethacin, a representative of this group of drugs, is frequently used and is a clinically relevant

*These authors contributed equally to this work. Address for correspondence: Prof. Fenghua Fu, Department of Pharmacology, School of Pharmacy, Yantai University, 32 Qingquan Road, Yantai, Shandong 264005, PR China. Tel: +86-535-6706060. Fax: +86-535-6706066. E-mail: [email protected]

History Received 14 June 2014 Revised 26 July 2014 Accepted 27 July 2014 Published online 20 October 2014

experimental model for induction of acute gastric ulcer. Indomethacin causes gastric ulcers through various processes, including inhibition of prostaglandin synthesis, generation of reactive oxygen species (ROS), initiation of lipid peroxidation and infiltration of neutrophils (Suleyman et al., 2010). Although the inhibition of cyclooxygenase and deficiency of endogenous prostaglandin is accepted as a main mechanism implicated in indomethacin-induced gastropathy, there is increasing body of evidence suggesting the involvement of oxidative stress in this pathology (Naito et al., 1998). Indomethacin is a known inducer of ROS in animal models, which may contribute to mucosal injury (Utsumi et al., 2006). Furthermore, activated neutrophils produce many prooxidative and proinflammatory enzymes such as myeloperoxidase (MPO) and free radicals leading to oxidative burst, which may inflict gastric mucosa (Chatterjee et al., 2013). It is becoming increasingly apparent that leukocyte– endothelial cell interaction, caused by various adhesion molecules, is a critical and early event in the pathogenesis of NSAIDs-induced gastropathy (Panes et al., 1999). The NSAIDs-induced gastric mucosal injuries are significantly reduced in neutropenic rats and by immunoneutralization of vascular cell adhesion molecule-1 (VCAM-1), and P-selectin

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(Wallace et al., 1993). Increased plasma level of tumor necrosis factor-a (TNF-a) has been reported to induce leukocyte adherence after indomethacin administration, and its downregulation by pentoxifylline, dexamethasone or TNF-a antibody reduces gastric mucosal damage (Appleyard et al., 1996). Indomethacin-mediated gastric ulceration was reduced in TNF receptor-deficient mice (Souza et al., 2008). Therefore, TNF-a, as well as the TNF-a-dependent expression of adhesion molecules, can be considered a major player in the initiation, progression and persistence of indomethacininduced gastric mucosal inflammation and injury. Escin, the major active component of Aesculus hippocastanum, is a natural mixture of triterpene saponins. Accumulating experimental evidences suggest that escin has anti-inflammatory and anti-edematous effects. Escin prevents an edema in animal models of inflammation that produces the initial exudative phase, such as paw edema induced by irritative agents (Guillaume & Padioleau, 1994). Escin also inhibits the acetic acid-induced increase in capillary permeability and the adhesion formation in animal model (Fu et al., 2005). In another research, escin has been shown to downregulate adhesion molecules and selectin protein expression, and to reduce the adhesiveness and migration of neutrophils (Hu et al., 2004). Aesculus hippocastanum is traditionally used treat stomach ache, stomach problems in China. It was also reported that escin exerted a gastroprotective effect against ethanol-induced gastric mucosal lesions (Matsuda et al., 1999). It indicated that the gastroprotective effect of esicn could be partly explained through non-prostaglandindependent mechanisms involving its anti-inflammatory and vasoactive properties (Marhuenda et al., 1994). The aim of this study was to evaluate the gastroprotective effect of esicn on indomethacin-induced gastric lesions in mice.

Materials and methods Drug and chemicals Escin, a lyophilized powder used in the present experiment, was obtained from Luye Pharmaceutical Co., Ltd. (Yantai, PR China). Escin was approved by State Food and Drug Administration in China and launched in 1995. Indomethacin, horseradish peroxidase, nitroblue tetrazolium (NBT), GSH, trichloroacetic acid (TCA), H2O2, NADPH, adenosine triphosphate (ATP), Tris–HCl, ammonium molybdate, sodium laurylsulfate, acetic acid and 2-thiobarbiturate were purchased from Sigma (St. Louis, MO). KCl and MgCl2 (analytical reagent) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, PR China). The enzyme-linked immunosorbent assay (ELISA) kits of TNF-a, P-selectin and VCAM1 were obtained from Abcam (Cambridge, UK). Animals Swiss albino mice, weighing 20–22 g, were provided by the Experimental Animal Center of Shandong Luye Pharmaceutical Co., Ltd. The animals were housed in a climate-controlled room, maintained on a 12-h/12-h light/ dark cycle, and had ad libitum access to food and water. The experiments were performed according to the National Institute of Health Guidelines for the Care and Use of

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Laboratory Animals (NIH Publication No. 86-23, revised in 1986) and were approved by the Animal Ethics Committee of Yantai University. All efforts were made to minimize the number of animals used and their suffering. Experimental design Animals were randomly divided into the five groups each consisting of 14 mice: control group, indomethacin group, escin (0.45 mg/kg) group, escin (0.9 mg/kg) group and escin (1.8 mg/kg) group. The mice in escin groups were administered orally with escin at a dose of 0.45, 0.9 or 1.8 mg/kg. Two hours later, the mice in indomethacin group and escin groups were treated orally indomethacin at a dose of 18 mg/ kg, whereas the animals of control group were treated orally 1% carboxymethylcellulose in equivalent volumes. Histopathologic analysis of gastric mucosal lesions Six hours after indomethacin administration, animals were sacrificed through an overdose of chloral hydrate (600 mg/kg, intraperitoneal). The stomachs of eight mice in each group were removed and opened along the greater curvature. They were then washed with ice-cold saline and examined for macroscopical mucosal lesions. The ulcer index was assessed as previously described (Dokmeci et al., 2005). Two investigators blinded to the groups of animals performed the histologic experiments. Histologic sections were coded to eliminate an observer bias. The ulcerated portions of the stomach were fixed in 10% formaldehyde for 24 h, embedded in a paraffin block, and cut into 4-mm sections, deparaffinized in xylene and rehydrated through a series of decreasing concentrations of ethanol. The sections were stained with hematoxylin and eosin, and pathologic observation of the tissues was performed under a light microscope. Preparation of the homogenate of gastric tissues To prepare the homogenate, the six gastric tissues of each group were harvested and weighed. They were then homogenized in four volumes of 0.1 mol/L ice phosphate buffer (pH 7.4) and centrifuged at 5000 g for 30 min at 4  C. The supernatants were immediately stored at 80  C and used for the determination of the biochemical analysis and ELISA assay. The total protein was estimated by the previous method (Lowry et al., 1951). Assays of superoxide dismutase, catalase, glutathione peroxidase and malondialdehyde in the gastric tissues Superoxide dismutase (SOD) activity was determined by the NBT reduction method (McCord & Fridovich, 1969). The method was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, which reacts with NBT to form formazan dye. SOD activity was then measured at 560 nm by the degree of inhibition of this reaction. Catalase (CAT) activity in the gastric tissues was measured at 37  C by following the rate of disappearance of H2O2 at 240 nm (Aebi, 1984). One unit of CAT activity is defined as the amount of enzyme catalyzing the degradation of 1 mmol of H2O2 per min at 37  C. Glutathione peroxidase (GSH-Px) activity was measured by the spectrophotometric method developed by

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Paglia & Valentine (1967). One unit of GSH-Px activity was defined as the amount of the enzyme that converted 1 nmol NADPH to NADP+ per minute. The determination of malondialdehyde (MDA) content was performed according to the previous method (Ohkawa et al., 1979). The homogenate (200 mL) was added with a solution containing 8% sodium laurylsulfate (100 mL), 20% acetic acid (800 mL), 8% 2thiobarbiturate (800 mL) and distilled water (150 mL). The mixture was incubated at 98  C for 1 h. Upon cooling, 5 mL of n-butanol:pyridine (15:l) was added. The mixture was vortexed for 1 min and centrifuged for 30 min at 4000 g. The absorbance of the supernatant was measured at 532 nm.

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Assays of MPO, TNF-a, P-selectin and VCAM-1 in gastric tissues The MPO activity in gastric tissues was determined according to the previous method (Bradley et al., 1982). The MPO activity was calculated from the absorbance of the mixture at 450 nm, using horseradish peroxidase as the standard. The MPO activity is expressed as micromoles of H2O2 consumed per minute per milligram of protein at 25  C and pH 5.4. The levels of TNF-a, P-selectin and VCAM-1 in gastric tissues were estimated with an ELISA plate reader, using commercially available ELISA kits and following the manufacturer’s protocols. Duplicate samples were analyzed for each sample. Assay of H+/K+ ATPase activity in gastric tissues The H+/K+ ATPase activity in gastric tissues was assayed according to the method of Nagaya et al. (1987). The assay medium consisting of 70 mM Tris–Cl buffer, pH 6.8, 5 mM MgCl2 and the enzymes solution in the presence of 10 mM KCl in a total volume of 1 ml and was incubated for 1 h. The reaction was initiated by adding 2 mM ATP and further incubated at 37  C for 20 min. The reaction was stopped by 10% TCA. After centrifugation, 2.5 ml of ammonium molybdate were added to the supernatent, and the absorbance were read at 620 nm.

Figure 1. Effect of escin on ulcer index. Data were expressed as means ± SEM (n ¼ 8). Statistical significances were determined using ANOVA followed by the Student–Newman–Keuls test. ##p50.01 compared with control group; *p50.05, **p50.01 compared with indomethacin group.

of escin inhibited the gastric lesions. The histology of mice in control group showed intact mucosal epithelium, submucosa, and absence of any inflammatory cells infiltration. In the indomethacin group, superficial erosion in the mucosal epithelium with a presence of neutrophil infiltration in the serosal layer, and disruption of the mucosal layer were observed. On the other hand, stomach tissues of indomethacin-induced mice pretreated with escin demonstrated mild neutrophil infiltration in the serosal layer and slight disruption of the mucosal layer. (Figure 2). Effect of escin on the level of MDA in gastric tissues The lipidperoxidation end product MDA was quantitated in the gastric tissues in mice. Compared with the control group, the level of MDA was significantly increased following indomethacin treatment. Treatment with escin at a dose of 0.45, 0.9 or 1.8 mg/kg attenuated the increase of MDA content (Figure 3). Effects of escin on the activities of SOD, CAT and GSH-Px in gastric tissues

Data were expressed as means ± standard error of mean (SEM). Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Student– Newman–Keuls test. The p50.05 was considered as statistically significant.

A significant reduction in the activities of SOD, CAT and GSH-Px in the indomethacin-treated mice was observed as compared with the control group. In contrast, administration of escin at a dose of 0.45, 0.9 or 1.8 mg/kg to indomethacintreated animals partially prevented the reduction in the activities of SOD, CAT and GSH-Px in gastric tissues (Figure 4).

Results

Effect of escin on MPO activity in gastric tissues

Effect of escin on ulcer index

As shown in Figure 5, the MPO activity in the indomethacin group showed a significant increase compared to control group. Whereas escin significantly reduced the MPO activity in gastric tissues (Figure 5).

Statistical analysis

Administration of indomethacin induced a remarkably high ulcer index when compared to control group. Treatment of mice with escin in a dose of 0.45, 0.9 or 1.8 mg/kg significantly reduced the intensity of indomethacin-induced ulcers and decreased the ulcer index (Figure 1). Effect of escin on gastric mucosal lesion formation Gross pathologic studies showed that indomethacin caused multiple hemorrhagic erosions and bleeding in the stomach at 6 h after indomethacin administration. In contrast, treatment

Effects of escin on the levels of TNF-a, P-selectin and VCAM-1 in gastric tissues In this study, indomethacin caused a significant increase of the levels of TNF-a, P-selectin and VCAM-1 in gastric tissues. However, treatment with escin inhibited the increase of the levels of TNF-a, P-selectin and VCAM-1 significantly (Figure 6).

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Figure 2. Effect of escin on gastric mucosal lesion formation. Gross pathologic studies of effect of escin on gastric mucosal lesion formation. (A) Control group. (B) Indomethacin group. (C) Escin (0.45 mg/kg) group. (D) Escin (0.9 mg/kg) group. (E) Escin (1.8 mg/kg) group. Photomicrographs of escin on gastric mucosal lesion formation (H&E). (F) Control group. (G) Indomethacin group. (H) Escin (0.45 mg/kg) group. (I) Escin (0.9 mg/kg) group. (J) Escin (1.8 mg/kg) group. Bar ¼ 40 mm.

Figure 3. Effect of escin on MDA content in the gastric tissues. Data were expressed as means ± SEM (n ¼ 6). Statistical significances were determined using ANOVA followed by the Student–Newman–Keuls test. ##p50.01 compared with control group; *p50.05, **p50.01 compared with indomethacin group.

Effect of escin on H+/K+ ATPase activity in gastric tissues Compared with the H+/K+ ATPase activity in control group (1.25 ± 0.36 mmol Pi/mg protein/h), the H+/K+ ATPase activity in indomethacin group (1.90 ± 0.32 mmol Pi/mg protein/h) increased significantly. Treatment of escin at dose of 0.45, 0.9 or 1.8 mg/kg did not inhibit the increase of H+/K+ ATPase activity (1.75 ± 0.23, 1.84 ± 0.36 and 1.80 ± 0.38 mmol Pi/mg protein/h, respectively) induced by indomethacin.

Discussion The present study investigated the gastroprotective effects of escin on indomethacin-induced gastric damage in mice. Indomethacin caused a remarkable gastric damage in mice compared with untreated mice, as reflected by an increase in ulcer index and superficial erosion in the mucosal epithelium with presence of neutrophil infiltration. However, treatment with escin attenuated indomethacin-induced gastric ulcer

in mice. These results indicated that escin had a potential to prevent the gastric damages resulting from indomethacin administration. The H+/K+ ATPase was the dimeric enzyme responsible for H+ secretion by the gastric parietal cells. The inhibition of H+/K+ ATPase resulted in the reduction of gastric acid secretion and then showed protective effects against gastric ulcer. In the recent years, the drugs (e.g. omeprazole, pantoprazole) that inhibited H+/K+ ATPase had become preferred therapeutic choice due to their clinical efficacy. The findings in this study showed that escin had no effect on H+/K+ ATPase, which indicated the protective effects of escin against indomethacin-induced gastric ulcer was not associated with the inhibition of H+/K+ ATPase. It is generally accepted that the gastric ulcer production by indomethacin is related to its ability to inhibit the synthesis of prostaglandins, synthesized by cyclooxygenase-1 (COX-1) and COX-2 in the stomach tissue (De Souza et al., 2002). Prostaglandins form a vital component of gastric mucosal defense. However, previous studies demonstrated that the gastric mucosal protective action of escin was not mediated by endogenous prostaglandins. Therefore, the present study did not focus on exploring the COX-dependent mechanisms of the protective effects of escin on indomethacin-induced gastric ulcer. Recently, it has been shown that ROS possess an important role in the pathogenesis of mucosal damages caused by indomethacin in addition to the inhibition of COX enzymes (El-Abhar, 2010). ROS induced by indomethacin in the tissues might damage the membranes and stomach tissues by increasing the lipid peroxidation (Miura et al., 2002). In agree with previous report, our results showed that there was a significant increase in the level of MDA in the gastric tissues of mice administrated with indomethacin. However, the increase of MDA level in the gastric tissues induced by indomethacin was inhibited partially by the administration of escin. Organisms have enzymatic and non-enzymatic defense mechanisms against the toxicity and tissue damage of ROS (Mates et al., 1999). SOD is one of the antioxidant enzymes

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Figure 4. Effect of escin on the activities of SOD, CAT and GSH-Px in the gastric tissues. Data were expressed as means ± SEM (n ¼ 6). Statistical significances were determined using ANOVA followed by the Student–Newman–Keuls test. ##p50.01 compared with control group; *p50.05, **p50.01 compared with indomethacin group.

Figure 5. Effect of escin on the MPO activity in the gastric tissues. Data were expressed as means ± SEM (n ¼ 6). Statistical significances were determined using ANOVA followed by the Student–Newman–Keuls test. ##p50.01 compared with control group; *p50.05, **p50.01 compared with indomethacin group.

and plays an important role in ameliorating gastric damage by preventing oxidative damage. SOD destroys the highly reactive radical by converting it into the less reactive H2O2 that can be destroyed by the CAT reaction. The reduction in

superoxide dismutase activity after indomethacin treatment has been previously recorded (Odabasoglu et al., 2008). GSHPx is proposed as potential chemopreventive agents for their antioxidant and detoxification properties (Abdallah, 2010). GSH-Px is important component of the detoxification system and present in high concentration in the gastric tissues. GSH-Px depletion in gastric mucosa after indomethacin administration results in lipid peroxidation, and gastric damages (Halici et al., 2005). Similarly, in the present study, the levels of SOD, CAT and GSH-Px in mice stomach tissues were significantly reduced by administration of indomethacin. On the other hand, treatment of escin at 0.45, 0.9 or 1.8 mg/kg resulted in a significant increase in the activities of SOD, CAT and GSH-Px. These findings indicated that escin possessed a protective effect against the indomethacin-caused oxidative damage in the gastric tissues of mice. The increase in the production of ROS does not fully account for the etiopathogenesis of indomethacin-induced gastric damage. It was demonstrated that indomethacin produced a significant increase in TNF-a level in gastric tissues, which is one of the aggressive factors in ulcerogenesis (Abbas & Sakr, 2013). Previous study has demonstrated that

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Figure 6. Effect of escin on the contents of TNF-a, P-selectin and VCAM-1 in the gastric tissues. Data were expressed as means ± SEM (n ¼ 6). Statistical significances were determined using ANOVA followed by the Student–Newman–Keuls test. ##p50.01 compared with control group; *p50.05, **p50.01 compared with indomethacin group.

NSAIDs administration in rats causes a dose-dependent increase in plasma TNF-a level (Santucci et al., 1994). In line with this hypothesis, pretreating rats with specific TNF-a synthesis inhibitors or selective anti-TNF-a receptor monoclonal antibodies prevents gastric mucosal damage in NSAIDs-treated rats without interfering with prostaglandin metabolism (Appleyard et al., 1996). The timecourse of TNFa release correlates with the timecourse of gastic injury and neutrophil margination, suggesting that this cytokine is responsible for gastric neutrophil activation and accumulation (Yadav et al., 2012). The expression of adhesion molecules involved in neutrophil recruitment and transmigration is regulated by TNF-a. TNF-a is able to stimulate the expression of P-selectin and VCAM-1 (Chandrasekharan et al., 2007). Neutrophils have been implicated in the damage associated with indomethacin-induced gastric damage. Activated neutrophils produce many pro-oxidative and proinflammatory enzymes such as MPO and oxygen free radicals leading to oxidative burst, which inflicted gastric mucosal damage (Karakus et al., 2009). MPO is abundantly expressed in neutrophils and secreted from activated neutrophils (Evans et al., 1996). Its over-expression is commonly associated with inflammatory diseases (Wu et al., 1999). Several studies have reported there is an increase in MPO

activity after indomethacin administration, indicating that neutrophils infiltration is involved in indomethacin-induced gastropathy (Motawi et al., 2008). This study showed a significantly high TNF-a level in indomethacin group. Furthermore, the augmented expression of P-selectin and VCAM-1 were also detected after indomethacin treatment. MPO activity is considered an index for the evaluation of neutrophils infiltration and inflammation (Nishida et al., 1997). The findings showed that indomethacin administration led to an increase of MPO activity in the gastric tissues in mice. Consistent with the fact, in this experiment the examination of stomach histology revealed the presence of neutrophil infiltration in the serosal layer. However, escin reduced the content of TNF-a, P-selectin and VCAM-1 in gastric tissues. Moreover, escin also ameliorated the gastric inflammation and neutrophil infiltration induced by indomethacin. Collectively, these results demonstrated that escin can inhibit neutrophils infiltration and subsequent inflammation to prevent indomethacininduced gastric ulcer in mice. In conclusion, this study demonstrated the protective effects of escin against indomethacin-induced gastric ulcer in mice, not only by virtue of its antioxidant potential, but also due to its anti-inflammatory effect.

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Acknowledgements The authors would like to thank Prof. Lon Clark for the English language revision.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The study was supported by the Project of Fundamental Study Funds of the Shandong Provincial Education Department (Grant No. J11LF56), the Doctoral Foundation of Yantai University (Grant No. YX10B18) and the Taishan Scholar Project.

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Protective effects of escin against indomethacin-induced gastric ulcer in mice.

Escin, a natural mixture of triterpenoid saponin isolated from the seed of the horse chestnut, is reported to have a potent antiulcer activity against...
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