have been reviewed and approved by the Animal Ethics Committee at Xuzhou Medicine College. Asp-induced Gastric Mucosal Injury and ACS14 Treatment Gastric hemorrhagic lesions were induced by intragastric administration of Asp (200 mg/kg in 0.5% carboxymethyl cellulose solutions) in a volume of 1 ml/100 g body weight. To investigate the preventive effect of ACS14 on Asp-induced gastric mucosal injury, ACS14 synthesized as previously described [15] at doses of 1, 5 or 10 mg/kg (dissolved in DMSO) was injected intraperitoneally 30 min before the administration of Asp. Three hours after administration of Asp, the animals were killed by over- dose injection of pentobarbital sodium (60 mg/kg i.p.) and stomachs were harvested for other experiments. Gastric Damage: Macroscopic Analysis Both cardia and pylorus of stomach were ligated. 10 ml of 10% formaldehyde solution was injected into gastric cavity. The whole stomach was fixed in the same concentration of formaldehyde solution overnight. On the second day, the stomach was opened along the greater curvature, washed lightly and flattened on a piece of cardboard. The total number of gross mucosal lesions per stomach was counted and each lesion was scored according to the following scheme: grade1: petechial lesion, grade 2: lesion#2 mm, grade 3: 2, lesion#4 mm, grade 4: 4, lesion#6mm and grade 5: lesion greater than 6 mm. Measurement of H2S Concentration in Plasma and Gastric Tissue The method for measurement of H2S concentration was described in our previous publications [18,19]. Briefly, 75 ml plasma or gastric mucosal homogenates from each group were diluted in deionized water (final volume, 500 ml). H2S was trapped by addition of zinc acetate (1% w/v, 250 ml). Subsequently, N, N- dimethyl-p-phenylenediamine sulphate (20 mM; 133 ml) in 7.2 M HCl was added, followed by FeCl3 (30 mM; 133 ml) in 1.2 M HCl. Thereafter, trichloroacetic acid (10% w/v, 250 ml) was used to precipitate any protein that might be present in the culture media and upon centrifugation (10,000 g) absorbance (670 nm) of aliquots from the resulting supernatant (300 ml) was determined using a 96 well microplate reader [20]. Determination of PGE2 Levels Tissue from each rat stomach was removed, weighed (approximately 0.1 g), and placed in a test tube containing 1 ml of 0.1 M phosphate buffer, pH 7.4, 1 mM EDTA, and 10 mM indomethacin. The tissue was homogenized and centrifuged for 20 min at 1,000 g at 4uC. Prostaglandin E2 (PGE2) content in supernatant was determined by an enzyme immunoassay kit following the protocol described by the manufacturer (Biovol Technologies, China). Results are ex- pressed as picograms of PGE2 per milligram of protein. Proteins were determined by using the bicinchoninic acid (BCA) kit (Beyotime Institute of Biotechnology, China). Measurement of Malondialdehyde (MDA) Levels and Glutathione (GSH) Activity in Gastric Tissue Approximately 0.5 g of gastric tissue from individual rats was homogenized in 4.5 ml physiological saline and the supernatants were obtained by centrifugation at 2,000 g for 10 min. The protein concentration in the gastric mucosal homogenates was measured by using the bicinchoninic acid (BCA) kit (Beyotime S-Aspirin Protects Aspirin-Induced Gastric Injury Institute of Biotechnology, China). MDA levels and GSH activity in gastric tissue supernatants were measured using the enzyme-specific activity detection kits (Nanjing Jiancheng Bioengineering Co., China), according to the manufacturer’s instructions. Determination of Gastric Blood Flow Rats were anesthetized with pentobarbital sodium (60 mg/kg i.p.) and operated along the mid-line of abdomen to expose the stomach. Laser Doppler blood flow meter and miniature surface probes (moorVMS, UK) were used to record the blood flow. The acquired signal was converted to blood perfusion unit (BPU) and recorded with a computer. The curve was analyzed with moorVMS v1.0 software. Only stable signals were included and calculated. Blood flow was recorded for three times (15 s for each time) in each rat. Western Blot Analysis Gastric samples were lysed in buffer. The protein concentration of each lysate was determined using the BCA kit according to the manufacture’s protocol. 7.5%, 10% or 12.5% SDS-polyacrylamide gels were used depending on the molecular weight of the measured proteins. After electrophoresis, the polyvinylidene fluoride (PVDF) membranes were washed in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h, and then incubated with the relevant antibody at 4uC overnight. All antibodies (anti-SOD1 antibody, anti- XOD antibody, anti-COX2 antibody, anti-phosphorylated p22phox antibody, anti-p47phox antibody, anti-p67phox antibody and anti- gp91phox antibody) were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA. Membranes were washed three times in TBST buffer (10 mmol/l Tris, pH 7.5; 150 mmol/l NaCl, 0.05% Tween-20), followed by incubation with secondary antibody. The NBT/BCIP western blot analysis system according to the manufac- turer’s protocol was used for detection the protein signals. The results are the average of four independent experiments. Statistical Analysis All data were presented as mean 6 SEM. Statistical significance was assessed with one-way analysis of variance (ANOVA) followed by a post hoc (Bonferroni) test for multiple group comparison. Differences with p-value less than 0.05 were considered statistically significant. Results Effect of ACS14 and Asp on Gastric Mucosa We first compared the effect of ACS14 and Asp on gastric mucosa. We found that intragastric administration of ACS14 at 430 mg/kg (at equimolar doses to 200 mg/kg Asp) did not cause any damage in the gastric mucosa. However, Asp at 200 mg/kg induced severe mucosal damage. These data suggest that H2S released from ACS14 may protect against Asp-induced gastric damage (Fig. 1). Effect of ACS14 and NaHS on Asp-induced Gastric Mucosal Injury Treatment of rats with Asp (200 mg/kg) for 3 h significantly increased gastric mucosal injury. As shown in Fig. 2, Asp induced the appearance of multiple visible gastric petechial erosions. The size of erosions ranged from 2 to 10 mm in length and about 1 mm in width. This damage was reversed by ACS14 at 1– 10 mg/kg in a concentration-dependent manner. The significant effect was observed when ACS14 was at 5–10 mg/kg. NaHS (an PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e46301