Life Sciences 149 (2016) 65–71

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Gastroprotective effects of several H2RAs on ibuprofen-induced gastric ulcer in rats Jing Liu 1, Dan Sun 1, Jinfeng He 1, Chengli Yang, Tingting Hu, Lijing Zhang, Hua Cao, Ai-ping Tong, Xiangrong Song, Yongmei Xie, Gu He, Gang Guo, Youfu Luo, Ping Cheng ⁎, Yu Zheng ⁎ State Key Laboratory of Biotherapy, Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, PR China

a r t i c l e

i n f o

Article history: Received 29 July 2015 Received in revised form 29 January 2016 Accepted 10 February 2016 Available online 13 February 2016 Keywords: Ibuprofen Histamine-2 receptor antagonists Gastric ulcer index Lipid peroxidation Antioxidant enzymes

a b s t r a c t Ibuprofen is the first line of treatment for osteoarthritis and arthritis. The main side effects of ibuprofen especially in long-term treatment include gastric ulcer, duodenal ulcer and indigestion etc. Therefore, screening drugs with effective gastric protective effects and low toxicity for combination therapy with ibuprofen is necessary. The mechanism of gastric damage induced by ibuprofen is still unclear, however, cell damage caused by reactive oxygen species (ROS) is considered as the main reason. Preliminary screening of literature with the criteria of low toxicity led to four histamine-2 receptor antagonists (H2RAs): nizatidine, famotidine, lafutidine, and roxatidine acetate, which were selected for further investigation. These drugs were evaluated systemically by examining the gastric ulcer index, lipid peroxidation (LPO), membrane permeability, toxicity to main organs, and the influence on the activity of antioxidant enzymes, and myeloperoxidase (MPO). Nizatidine was found to be the best gastric protective agent. It exhibited excellent protective effect by increasing antioxidant enzyme activity, decreasing MPO activity, reducing LPO, and membrane permeability. Combination treatment with nizatidine and ibuprofen did not show any significant toxicity. Nizatidine was considered as a good option for combination therapy with ibuprofen especially for diseases that require long-term treatment such as arthritis and osteoarthritis. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Ibuprofen, a strong analgesic and anti-inflammatory drug, is used extensively in the treatment of rheumatoid arthritis, osteoarthritis, and fever. Similar to other non-steroidal anti-inflammatory drugs (NSAIDs), the long-term treatment of ibuprofen can lead to severe gastrointestinal side effects [22]. Since ibuprofen does not undergo biliary elimination [25], it often causes gastric-irritant effects, especially ulcer. A gastrointestinal-protective drug is recommended in conjunction with ibuprofen treatment, since the incidence of ulcer associated with ibuprofen is high. However, the mechanism of ibuprofen-induced gastric injury is still not clear, thus it is often co-administered with potent antacid preparations. Ibuprofen is the first line of treatment for conditions such as rheumatoid arthritis and osteoarthritis, which require long-term treatment. Therefore, it is very important to screen a lowtoxicity and effective drug with gastroprotective effects. The widely used protective agents in clinics are antacids, such as H2 receptor antagonists (H2RAs) and proton pump inhibitors (PPIs). However, several studies have reported that PPIs are associated with the risk of osteoporosis [34]. Osteoporosis often affects older people, and can lead to very ⁎ Corresponding authors. E-mail addresses: [email protected] (P. Cheng), [email protected], [email protected] (Y. Zheng). 1 The first three authors contributed equally to the work.

http://dx.doi.org/10.1016/j.lfs.2016.02.045 0024-3205/© 2016 Elsevier Inc. All rights reserved.

serious health consequence. The present study compared the protective effects of a few H2RAs and conducted a preliminary study of the protection mechanism. There is evidence supporting the fact that inflammatory cell infiltration can cause normal cell damage and play an important role in the formation of gastrointestinal ulcers [15,24]. The degree of injury of normal cells depends on two factors, the amount of reactive oxygen species (ROS) and the ability of normal cells to scavenge it. ROS is generated by phagocytes including but not limited to superoxide radical (O–2•), hydrogen peroxide (H2O2), hydroxyl radical (OH•), and hypochlorous acid (HOCl) which is generated by the myeloperoxidase (MPO) system [14, 21]. ROS is indicated as an important cause of lipid oxidation, which leads to changes in membrane fluidity and permeability ([7,38]; and [3]). Cells have different systems to scavenge ROS, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) etc. They protect the gastric mucosa against the toxic effects of oxygen-derived free radicals [29]. The imbalance between the generation and the scavenging of ROS, which may be triggered by several endogenous factors and aggressive exogenous factors, is the main cause of gastric mucosa injury [2,16,27]. H2RAs remove ROS through improving antioxidant enzyme activity. SOD catalyzes O–2• into less noxious H2O2, which is further degraded by CAT or GSH-Px [4]. CAT is an enzyme, which accelerates the degradation of H2O2 into water and oxygen [37]. The second pathway of H2O2 metabolism depends on the activity of GSH-Px [11] (Fig. 1).

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J. Liu et al. / Life Sciences 149 (2016) 65–71

Co., Ltd. (Anhui, China). All other chemicals used were of analytical grade. 2.2. Animal study

Fig. 1. Free radical scavenging by antioxidant enzymes. O–2•: superoxide radical; SOD: superoxide dismutase; H2O2: hydrogen peroxide; CAT: catalase; GSH-Px: glutathione peroxidase; GSH: reduced glutathione; GSSG: oxidized glutathione.

Antioxidant enzyme activities (SOD, CAT, and GSH-Px) of H2RA treated samples were investigated to evaluate the antioxidant capacity [17,23]. H2RAs also demonstrated to be hydroxyl radical scavengers, with powerful scavenging effect on HOCl [20,33,36]. HOCl is a strong oxidation agent produced by the MPO–H2O2–halide system, which is one of the strongest cytotoxicity in the ROS [31]. H2RAs can protect tissues by reducing the amount of MPO. Quantification of MPO was performed to evaluate the antioxidant capacity H2RAs [26,36]. Unsaturated fatty acids of the cellular membranes can be peroxided by ROS, causing disruption of membrane integrity [9,38,39]. When the main phospholipids of membrane are peroxided, the integrity of the membrane structure will be undermined and the membrane fluidity and permeability will be increased, resulting in the disruption of the balance of the internal and external membranes, leading to cell damage and thus ulcer development [26]. Malondialdehyde (MDA), the most well known aldehyde end product of lipid peroxidation (LPO), is an index of LPO [19]. To evaluate the development of gastric ulcers in H2RA-treated rats, a direct evaluation method was performed, through observing the gastric mucosal lesions under the microscope. Increased permeability of the gastrointestinal tract was observed prior to the formation of ulcers. In the present study, a preliminary screening was performed by retrieving literature with the screening criteria of low toxicity. Ranitidine demonstrated main side effects such as liver damage and anti-androgenic effects. Cimetidine inhibited hepatic drug metabolizing enzyme. Four H2RAs (famotidine (FAM), nizatidine (NIZ), roxatidine acetate (ROX), and lafutidine (LAF)) were studied to compare their protective effects on ibuprofen-induced ulcers. These four H2RAs were chosen because they have been widely used and have shown good clinical curative effect. In summary, the present study provided an assessment of the gastroprotective efficacy of the four H2RAs against ibuprofen-induced gastric ulcer, from the analysis of lesion index, histological studies, LPO, enzymic antioxidants SOD, CAT, GPx, MPO, and mucosal permeability of the gastric mucosa.

2. Materials and methods 2.1. Materials Ibuprofen was obtained from Juhua Group Pharmaceutical Factory (Zhejiang, China), nizatidine was obtained from Macheng shi tianan Nano chemical material Co., Ltd. (Hubei, China), famotidine was purchased from Changzhou Longcheng Pharmaceutical Co., Ltd. (Jiangsu, China), lafutidine was purchased from Sichuan Kelun Pharmaceutical Co., Ltd. (Sichuan, China), rosatidine was obtained from Beijing Sihuan Pharmaceutical Co., Ltd. (Beijing, China), sucrose and sodium carboxymethyl cellulose were obtained from Shan He Excipents

Male Wistar rats (supplied by Vital River, China) weighing 200–250 g were used. All animal procedures were carried out according to the guidelines of the Animal Ethics Committee of Sichuan University. The animals were divided into six groups, with six rats in each group. The treatments of each were as follows: (1) Control group received CMC-Na (1%); (2) IBU (ibuprofen) group was administered with ibuprofen (200 mg/kg); (3) NIZ group was treated with nizatidine (31.25 mg/kg) following ibuprofen (200 mg/kg) administration; (4) FAM group was treated with famotidine (4.19 mg/kg) following ibuprofen (200 mg/kg) administration; (5) LAF group was treated with lafutidine (2.06 mg/kg) following ibuprofen (200 mg/kg) administration; (6) ROX group was treated with roxatidine (15.62 mg/kg) following ibuprofen (200 mg/kg) administration. All drugs given orally were suspended in CMC-Na (1%) suspension. After 6 consecutive days of treatment, the animals were sacrificed and the heart, liver, spleen, lung, kidney, and stomach were removed. The stomach was homogenized for determination of the activity of SOD, CAT, GSH-Px, MPO, and LPO. Sections of the major organs were processed for H&E histochemistry to assess toxicity. Another batch of animals was used for permeability study with the exact treatments as before. Each rat was administered sucrose solution (1 mL) and rat urine was collected for 8 h with metabolic cage. The level of sucrose in urine was measured. 2.3. Mucosal lesion The length and width of gastric ulcers were measured with a vernier caliper. The severity of the gastric lesions was evaluated with the method described by Guth et al. and expressed in terms of the ulcer index (UI) [12]. Briefly, the scoring of the ulcer was performed with a dissecting microscope (Carl Zeiss Microimaging GmbH37081 Göttingen, German). The size of the lesion (mm) was determined by measuring each lesion, and its greatest diameter was recorded as the length. The scores were relative value and were determined by the lesion size as follows: no lesion (score = 0), spot lesion (score = 1), lesion b 1 mm (score = 2), 1 b lesion b 2 mm (score = 3), 2 b lesion b3 mm (score = 4), lesion N3 mm (score = 5), and when the width N 1 mm, the score was double counted. The mean UI of each group was obtained by calculating the total scores divided by the number of animals. 2.4. Measurement of the membrane LPO LPO in gastric biopsies was assayed by estimating MDA using the thiobarbituric acid test [28]. In conditions with high temperatures and acidic environment, MDA reacts with thiobarbituric acid (TBA) to produce 3,5,5′-three methyloxazole-2.4-dione (trimethine), which is a stable, brown–red color that absorbs maximally at 532 nm. Therefore the content of MDA can be calculated according to the absorbance at 532 nm. Homogenate (0.5 mL of 10% (w/v)) was added to a solution containing 0.2 mL of 80 mg/mL sodium laurylsulfate (SDS), 1.5 mL acetic acid (20%), 1.5 mL 2-thiobarbituriate (80%) and 0.3 mL distilled water. The mixture was incubated at 98 °C for 60 min. After cooling, 5 mL of n-butanol:pyridine (15:1) was added and shaken vigorously. The mixture was then centrifuged at 4000 rpm for 10 min, and the organic layer was obtained and measured at 532 nm using an ultraviolet spectrophotometer (Lambda35 UV/VIS Spectrometer, PerkinElmer, USA). The standard curve was constructed with 1,1.3.3-tetramethoxypropane (TMP). The results were expressed as nmol MDA/g wet weight. The test recovery was over 90%.

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2.5. Measurement of gastric mucosa permeability The absorption of sucrose in the upper digestive tract is very limited, and it is readily hydrolyzed in the small intestine. Therefore, permeability of the stomach mucosa was assessed by determining the concentration of sucrose in urine [8]. When sucrose was subjected to heat under acidic condition, it was hydrolyzed into glucose and was able to be measured quantitatively. Sucrose was hydrolyzed into glucose and fructose using 225 μL H2SO4 (2 M) per 900 μL urine in a boiling water bath for 10 min. NaOH (360 μL, 2 M) was added, followed by 1.5 mL of PBS (pH = 7.0). After cooling, the mixture was centrifuged for 10 min at 13,000 rpm. The concentration of glucose was determined using a blood biochemical analyzer (Hitachi 7020, JP). Penetration rate is calculated according to the following equation: Penetration rateð%Þ ¼ V C

C  V  1000  100%: 0:66

total volume of urine (mL). glucose concentration (μg/mL).

2.6. Total protein content determination Antioxidant and MPO activity were expressed as U/mg protein, therefore the total protein content of the stomach tissue was determined and expressed as mg/g wet tissue. The total protein content was assayed according to the Coomassie Brilliant Blue G-250 Method (Bradford Method) [5]. Protein molecule contains NH3 +, which can bind to CBB dye molecules (negatively charged anionic form) to form a protein-dye complex, thus converting CBB from the red form to the blue form. Therefore, the absorbance of the solution was measured at a wavelength of 595 nm. The standard curve of total protein was formed using bovine serum albumin (BSA) standard solution. Each diluent (10 μL) was gently mixed with 200 μL of the protein assay solution, which contained 0.01% (w/v) Coomassie Brilliant Blue G-250, 4.7% (w/v) ethanol, and 8.5% (w/v) phosphoric acid. The absorbance was measured at 595 nm with an enzyme-labeled instrument (Multiscan MK3, ThermoLabsystems, USA). After, 10 μL of each sample protein solutions (10% tissue homogenate sample diluted 40 times with normal saline) was mixed with 200 μL of the protein assay solution, and the absorbance was measured at 595 nm. The protein concentrations were calculated according to the standard curve. 2.7. Antioxidation level of H2RAs 2.7.1. Measurement of superoxide dismutase (SOD) activity SOD activity was measured according to Alexander et al. [30]. The activity of SOD was evaluated by measuring its capacity to inhibit the production of a water-soluble formazan dye, which was produced by WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)2H-tetrazolium, monosodium salt). The rate of the reduction of WST-1 with O–2• was linearly related to xanthine oxidase (XO) activity, and this reduction was inhibited by SOD. One unit of SOD was defined as the amount of enzyme required to inhibit the rate of WST-1 reduction by 50% inhibition. The stomach homogenate (10%, w/v) was centrifuged for 10 min at 3000 rpm. Twenty microliter of supernatant (or water used as control) was mixed with 20 μL WST-1 (10 mM) solution, 20 μL xanthine (3 mM), 3 mM EDTA, and 200 μL xanthine oxidase solution (0.1 mM) to initiate the reaction. The mixture was incubated for 20 min at 37 °C and the changes in absorbance at 450 nm were recorded in an enzyme-labeled instrument (Multiscan MK3). A standard curve was constructed using the concentrations of SOD instead of stomach homogenate. The activity of SOD was expressed as U/mg protein.

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2.7.2. Measurement of CAT activity CAT activity was determined according to Korolyuk's method [18]. Decomposition of H2O2 in the presence of CAT was terminated by addition of ammonium molybdate and a pale yellow substance, which showed absorption at 410 nm. CAT activity was defined as the amount of enzyme required to decompose 1 μmol of H2O2 per s. For the measurement of CAT activity, 10% (w/v) homogenates were centrifuged for 10 min at 3000 rpm and supernatants were collected. The diluted supernatants (0.1 mL) were incubated with 2.0 mL of substrate (10 μmol/mL H2O2 in sodium–potassium phosphate buffer, pH 7.4) for 10 min at 37 °C. The enzymatic reaction was terminated by addition of 1.0 mL 32.4 mmol/L ammonium molybdate. The yellow complex of molybdate and H2O2 was measured at 410 nm using an ultraviolet spectrophotometer (Lambda35 UV/VIS Spectrometer, PerkinElmer, USA). Results were expressed as U/mg protein. 2.7.3. Evaluation of the involvement of GSH-Px GSH-Px was assayed with the method of Hafeman et al. [13]. Glutathione (GSH) and H2O2 were catalyzed into glutathione disulfide (GSSG) by GSH-Px. The surplus of GSH in the resulting supernatant was assayed by adding 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and the color (NTB) that developed was read at 421 nm immediately. The rate of TNB production was directly proportional to the concentration of GSH surpluses in the sample. GSH-Px activity was defined as the amount of enzyme required to reduce 1 μmol/L of GSH per min. Homogenates 10% (w/v) were centrifuged for 10 min at 3000 rpm. Supernatant (0.2 mL) was added into a pre-warmed (37 °C) reaction solution containing 1.0 mL of GSH (2 mmol/L), 1.0 mL of 0.40 M sodium phosphate buffer (pH 7.0) with 0.4 mM EDTA, 0.50 mL NaN3 (0.01 M), and water (final volume 4 mL). After incubation at 37 °C for 5 min, 1 mL H2O2 (1.25 mM) was added to the incubation medium and incubated for a further 3 min, then 1 mL of the mixture was removed and added to 4 mL of metaphosphoric acid. GSH in the protein free filtrates was determined by mixing 2.0 mL of the filtrate with 2.0 mL Na2HPO4 (0.4 M) and 1.0 mL DTNB reagent. Reactant was centrifuged and GSH surplus in the resulting supernatant was assayed by mixing 2.0 mL of supernatant with 2 mL of phosphate buffer (pH 7.0), and 1 mL DTNB. The absorbance was read at 421 nm immediately using an ultraviolet spectrophotometer (Lambda35 UV/VIS Spectrometer). A standard curve was constructed using GSH instead of stomach homogenate. The results of the GSH-Px activity in homogenate were expressed as U/mg protein. 2.8. MPO activity assay Neutrophil MPO was directly related to the neutrophil number, accounting for approximately 5% of the cell dry weight. Thus, the tissue neutrophil content was measured indirectly by determining the MPO activity according to the modified method of Bradley et al. [6]. The MPO activity was measured following the oxidation of o-dianisidine dihydrochloride by H2O2. The absorbance changes in the samples at 460 nm were recorded. In a microcuvette, 0.1 mL of supernatant was combined with 2.9 mL of 50 mM phosphate buffer (pH 6) containing 0.167 mg/mL o-dianisidine hydrochloride and 0.0005% H2O2. The change in absorbance at 460 nm was measured at 1 min intervals for 3 min by an ultraviolet spectrophotometer (Lambda35 UV/VIS Spectrometer). MPO activity was determined by the means of a standard curve constructed with serial dilutions of horseradish peroxidase. One unit of MPO activity was defined as that degrading 1 μmol of peroxide per min at 25 °C. 2.9. Histopathological assessment Histological evaluation was performed on the heart, liver, spleen, lung, and kidney. Tissue samples were preserved in 10% buffered formalin and processed for routine paraffin block preparation. Sections (approximately 4 μm) were cut and stained with hematoxylin and eosin.

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Table. 1 Ulcer index of gastric mucosal.

UI

Control

IBU

NIZ

FAM

LAF

ROX

0.00 ± 0.00

17.33 ± 13.56⁎

6.00 ± 4.69⁎⁎⁎

7.56 ± 7.78⁎⁎

20.56 ± 13.44

9.97 ± 14.69

Values are expressed as mean ± SD. ⁎ vs. control (p b 0.01). ⁎⁎ vs. IBU (p b 0.05). ⁎⁎⁎ vs. IBU (p b 0.01).

2.10. Statistical analysis Results are expressed as mean ± SD. One-way analysis of variance (ANOVA) was performed, and the statistical comparisons among the groups were performed with Tukey's test using a statistical package program (SPSS 10.0 for Windows; SPSS, Chicago, IL, USA). 3. Results 3.1. Mucosa protection of nizatidine against ibuprofen-induced gastric damage Table 1 showed the ulcer index (UI) of gastric mucosa. Administration of 200 mg/kg ibuprofen for 6 consecutive days developed a significant (p b 0.01) gastric ulcer compared to that of the control group. In nizatidine and famotidine treated rats, UI decreased compared with those of ibuprofen-treated rats (p b 0.01,and p b 0.05, respectively). No changes were observed in the group treated with lafutidine combined with ibuprofen. UI also decreased in the roxatidine acetate group, however no significant difference was observed. Fig. 2 showed

the UI of each group. Combination treatment with nizatidine demonstrated the most effective protection against ibuprofen-induced gastric ulcer. Famotidine was also effective. However, the gastric ulcer prevention effects of lafutidine and roxatidine acetate were not ideal. 3.2. Decrease of LPO level in gastric tissue by nizatidine Ibuprofen induced a significant increase in LPO from 15.45 ± 2.64 nmol MDA/g wet weight of control group to 23.24 ± 4.40 nmol MDA/g wet weight (p b 0.01). This indicated that ibuprofen caused LPO in gastric cell membrane. The increase in MDA content caused by ibuprofen was reversed with H2RAs. However, only in the nizatidine group, a significant difference was observed compared to that of the ibuprofen group (p b 0.01). (Fig. 3(A)). 3.3. Improvement in gastric mucosa penetration by nizatidine Fig. 3(B) showed that ibuprofen caused significant increase in urinary excretion of sucrose versus control group (4.60 ± 0.86% vs. 0.16 ± 0.03%; p b 0.01), which indicated that mucosal permeability

Fig. 2. Gastric mucosa induced by ibuprofen, pretreated with or without H2RAs. Control: control group; IBU: ibuprofen treated group; NIZ: nizatidine treated group; FAM: famotidine treated group; LAF: lafutidine treated group; ROX: roxatidine treated group.

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Fig. 3. (A) Level of MDA in gastric mucosal; (B) permeability of gastric mucosal; (C) SOD activity of gastric tissue; (D) CAT activity of gastric tissue; (E) GSH-Px activity of gastric tissue; (F) effect of H2RAs on gastric MPO activity in ibuprofen-induced ulcer rat. Control: control group; IBU: ibuprofen treated group; NIZ: nizatidine treated group; FAM: famotidine treated group; LAF: lafutidine treated group; ROX: roxatidine treated group. ** vs. control (p b 0.01); * vs. control (p N 0.05); ## vs. IBU (p b 0.01).

increased due to ibuprofen. The increase was reduced to a certain extent (p b 0.01) in the groups with combination treatment of nizatidine, famotidine, or lafutidine (0.87 ± 0.20%, 2.50 ± 0.61%, and 1.37 ± 0.44%, respectively). The efficacy of nizatidine was superior to that of famotidine and lafutidine (p b 0.01, p b 0.05, respectively). Lafutidine showed better efficacy than famotidine (p b 0.01). However, roxatidine acetate was not effective in preventing mucosal permeability. 3.4. Total protein content The total protein content of each rat was determined based on the CBB method. The standard curve: Y = 1.2941X + 0.3823. (r = 0.995, X: protein concentration; Y: absorption value). 3.5. Restoration of antioxidant enzyme levels by nizatidine The antioxidant activity was expressed as U/mg protein. Administration of ibuprofen produced a significant decrease in SOD activity compared to that of the control group (129.25 ± 13.47 U/mg protein vs. 189.02 ± 20.71 U/mg protein) (p b 0.01). Nizatidine, famotidine, and roxatidine acetate restored the activity of the enzyme (183.23 ± 31.95 U/mg protein, 178.78 ± 36.61 U/mg protein, 178.25 ± 43.17 U/mg protein; p b 0.01, p b 0.01, p b 0.05, respectively) compared to those of the ibuprofen group. Lafutidine failed to prevent decrease of SOD activity caused by ibuprofen (160.12 ± 36.42 U/mg protein) (Fig. 3(C)). Ibuprofen did not affect CAT activity compared to that of the control group (0.75 ± 0.28 U/mg protein vs. 0.82 ± 0.28 U/mg protein). Similarly, co-treatment with the four H2RAs showed no significant difference in CAT activity (nizatidine: 0.77 ± 0.33 U/mg protein; famotidine: 0.97 ± 0.20 U/mg protein; lafutidine: 0.80 ± 0.33 U/mg protein; roxatidine acetate: 0.88 ± 0.40 U/mg protein) (Fig. 3(D)). Fig. 3(E) showed the influence of administration with or without of H2RAs on the activity of GSH-Px in ibuprofen-induced ulcers in healthy rats. Rats treated with ibuprofen showed a significant decrease in the GSH-Px levels (211.89 ± 40.79 U/mg protein; p b 0.01) when compared to those of the control group (304.51 ± 43.03 U/mg protein). The reduction was prevented by nizatidine, famotidine, and roxatidine acetate (p b 0.01) relative to the ibuprofen group (294.25 ± 49.54 U/mg protein, 310.04 ± 77.50 U/mg protein, 289.76 ± 75.95 U/mg protein, respectively).

3.6. Decrease of gastric MPO activity by nizatidine MPO activity increased from a basal concentration 1.33 ± 0.77 U/g (control) wet weight to 1.90 ± 0.51 U/g wet weight after the administration of ibuprofen (p b 0.01). The increase in MPO activity was inhibited (p b 0.01) by nizatidine and roxatidine acetate (1.31 ± 0.15 and 1.19 ± 0.25 U/g wet weight, respectively). Famotidine and lafutidine showed no effect on the increased MPO activity caused by ibuprofen. The effect of nizatidine was significantly better than that of famotidine (p b 0.05) and lafutidine (p b 0.05) (Fig. 3(F)). 3.7. Low toxicity caused by combination treatment with ibuprofen and H2RAs Histopathological assessment (Fig.4) showed no obvious toxicity on the major organ systems including the heart, liver, spleen, lung, and kidney in all groups. This suggested that ibuprofen in combination with H2RA did not increased toxicity relative to ibuprofen alone. 4. Discussion The dosage used in this experiment when conversed to the clinical dosage for adults (presumed weight of 70 kg), was 1538 mg/day, which did not reach the maximum clinical dose. After administration of IBU 200 mg/kg for 6 consecutive days, serious ulcer developed on the gastric mucosal of rats with a high incidence (close to 100% in the ibuprofen group). It indicated that long-term administration of high dose of ibuprofen might cause high incidence of gastric ulcer. Thus, screening gastrointestinal protective agent for combination treatment with ibuprofen is necessary. Although, the mechanism of gastrointestinal side effect of ibuprofen is still unclear, it is well documented in the literature that the pathogenesis in animals is multifactorial: superficial aggressive cellular necrosis and the release of tissue-derived mediators. Therefore, studies were undertaken to select drugs that had protective effects against gastric mucosal damage induced by ibuprofen. In this study, ibuprofen inhibited the activity of SOD and GSH-Px, thus, decreased ROS scavenging activity, and had no significant effect on CAT. MPO that generates oxygen free radicals increased, leading to excessive accumulation of ROS in the gastric mucosa. The main phospholipids of the cell membrane were oxidized, the integrity of the membrane structure was undermined, and membrane fluidity and

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Fig. 4. Representative images of the heart, liver, spleen, lung, and kidney tissues stained by hematoxylin–eosin with microscopic analysis (magnification of 200×) for each group. Control: control group; IBU: ibuprofen treated group; NIZ: nizatidine treated group; FAM: famotidine treated group; LAF: lafutidine treated group; ROX: roxatidine treated group.

permeability increased, resulting in disruption of the balance inside and outside of cells and ultimately cell damage. Therefore, it was inferred that ROS played an important role in gastric lesions induced by ibuprofen. The antiulcerogenic activity of some H2RAs has been reported [20, 26,36]. In the present study, H2RAs were used to protect the gastric mucosal against lesions induced by ibuprofen. As suggested in the penetration experiment described above, significant decrease was observed in mucosal permeability with combination treatment of nizatidine, famotidine, and lafutidine compared to those of the ibuprofen control group. Among them, the efficacy of nizatidine was superior to that of famotidine and lafutidine (p b 0.01 and p b 0.05, respectively), indicating that nizatidine was more effective in preventing early lesion of the gastric ulcers. UI significantly decreased in the nizatidine and famotidine group (p b 0.01 and p b 0.05, respectively). However, lafutidine and roxatidine acetate did not significantly reduced UI. These results strongly suggested that nizatidine protected the gastric mucosal better than the other H2RAs investigated. Famotidine was suggested as the most effective gastric acid suppressive agent among the existing H2RAs [35], but in the present study, results indicated that it was not the most effective for ibuprofen-induced gastric damage in rats. The mechanism of gastrointestinal damage induced by NSAIDs (such as ibuprofen) is still unclear. Gastric lesions characterized by hemorrhage, edema, inflammatory infiltration, and loss of epithelial cells [32], were observed through microscopic examination in ibuprofentreated gastric tissue (Fig.2) in the present study, which was consistent with previous report. It is possible for ibuprofen to induce apoptosis in gastric mucosal cells, since it appears to increase leukocyte infiltration into the gastric mucosa, which is followed by ROS production [10]. Recently, cell damage caused by ROS was considered as the main reason for NSAID induced gastric damage [1]. Our results indicated that the scavenging effects on active oxygen free radicals of H2RAs were important in protecting gastric ulcers induced by ibuprofen instead of their acid-suppressing effect. SOD, GSH-PX, and MPO were used as direct indicators of H2RAs to scavenge ROS. The oxidation of unsaturated lipids

was mainly due to the ROSs that were contained in tissue such as O–2•. H2RA antioxidant activity was related to the scavenging of ROS. It appeared that the stronger the scavenging effect, the better the protective effect of H2RA against gastric damage. Nizatidine demonstrated the strongest antioxidant activity out of the H2RAs studied, which supported its best protection against gastric damage. In summary, nizatidine demonstrated the best protective effect among the four selected H2RAs on ibuprofen-induced ulcers, and could be applied as a protective agent of ibuprofen in clinic or developed into compound preparation with ibuprofen. 5. Conclusion The present study demonstrated that ibuprofen caused lipid peroxidation and increased membrane permeability, and ultimately led to gastric ulceration, by reducing the activity of antioxidant enzymes and increasing MPO activity. Nizatidine was found to be the most effective gastric protective agent, since it exhibited excellent protective effects through increasing the activity of antioxidant enzymes, decreasing MPO activity, reducing LPO and membrane permeability. Nizatidine would be a good option for combination therapy with ibuprofen, especially concerning conditions that require long-term treatment such as arthritis and osteoarthritis. This study also provided the theoretical basis for developing a compound preparation containing ibuprofen and nizatidine. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This work was supported by China Postdoctoral Science Foundation (2013M542421, 2014T70877), Science and Technology Support Project

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Gastroprotective effects of several H2RAs on ibuprofen-induced gastric ulcer in rats.

Ibuprofen is the first line of treatment for osteoarthritis and arthritis. The main side effects of ibuprofen especially in long-term treatment includ...
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