Figure 4. Effect of ACS14 on COX-2 expression (A) and PGE2 content (B) in gastric tissue. A. Representative Western blots and group data showing that ACS14 reversed Asp-upregulated COX-2 expression. n = 4. B. Both ACS14 and NaHS failed to change Asp- suppressed PGE2 level in gastric tissue. n = 7. Data are the means 6 SE.*P,0.05 compared with control; #P,0.05 compared with Asp; {P,0.05 compared with Asp+ACS14 1 mg/kg; 1P,0.05 compared with Asp+ACS14 5 mg/kg. doi:10.1371/journal.pone.0046301.g004 H2S is increasingly being recognized as a fundamental signaling molecule, and many H2S-releasing compounds were developed in recent years [15,32–34], such as H2S-releasing naproxen and H2S- releasing Asp. It has recently been found that NaHS significantly attenuated the gastric damage caused by Asp [31]. This prompted us to investigate whether H2S releasing Asp can still produce gastric injury. ACS14 is a developed H2S-releasing Asp. We found in the present study that ASC14 at the same dose as Asp failed to S-Aspirin Protects Aspirin-Induced Gastric Injury produce gastric injury, suggesting that H2S released from ACS14 may protect stomach against Asp-induced injury. We then moved on to study whether ACS14 at low doses can prevent harmful effect of Asp in stomach. We found that ACS14 at 1–10 mg/kg reversed Asp induced gastric damage in a concen- tration-dependent manner. ACS14 is known to release H2S in vitro and in vivo [15,35,36]. We also found in the present study that ACS14 (at 5 and 10 mg/kg) significantly increased the H2S concentrations in plasma. These data confirmed that the beneficial effect was from the released H2S from ACS14. COX is the rate-limiting enzyme to regulate the synthesis of prostaglandins by conversion of arachidonic acid to PGH2, the common precursor of bioactive prostaglandins. Two distinct COX isoforms were reported. COX-1 is responsible for constitutive prostaglandin formation, whereas COX-2 is usually induced in response to stress [37]. It was reported that Asp can rapidly up- regulate COX-2 expression in the stomach [38–40]. We found in the present study that ACS14 at 1–10 mg/kg reversed the up- regulated expression of COX-2, in a dose-dependent manner. This is consistent with a previous study that H2S significantly attenuated Asp-induced upregulation of COX-2 mRNA level [39]. Endogenous PGE2 derived from COX-2 is closely related to the recovery of gastric mucosal injury [41,42] and plays an important role for the maintenance of gastric mucosal integrity by preventing exogenous injury to the stomach and accelerating gastric mucosal healing [43]. It was found in the present study that Asp markedly decreased PGE2 production. We therefore proposed that the upregulated COX-2 produce level was secondary to a compen- satory response to inhibition of COX-2 activity and gastrin PG synthesis [38]. However, we found that neither ACS14 nor NaHS reversed Asp-impaired PGE2 production. Our data suggest that the protective effects of ACS14 and NaHS were not mediated by PGE2. Oxidative stress is associated with increased production of oxidizing species or a significant decrease in the capability of antioxidant defenses [44]. H2S scavenges oxygen-derived free radicals [9,45–49], which mediates the protective effects of NaHS against the toxicity of H2O2 in cells in vitro and also the ischemia- reperfusion-induced gastric mucosal damage in rats in vivo [47,50]. We found in the present study that ACS14 significantly reduced Asp-induced elevation of MDA, one of the markers of free radical species-related injury. Glutathione is the major cellular antioxidant and plays an important role in antioxidative stress by H2S [9,35,51–53]. H2S protects neurons from oxidative stress by increasing the levels of GSH [9,51,54]. We found in the present study that ACS14 significantly increased the gastric GSH level. In addition, ACS14 also reversed Asp-reduced protein expression of SOD, which is responsible for converting superoxide radicals to molecular oxygen and hydrogen peroxide within cytoplasm and mitochondria [55]. Our data suggest that ACS14 may protect the gastric mucosa against Asp-induced damage via upregulation of antioxidants level. We also examined the expressions of redox enzymes, NADPH oxidase. NADPH oxidase is a multicomponent enzyme that comprises p22phox, gp91phox, p40phox, p47phox, p67phox, and the small GTP-binding protein Rac [23–25,56]. We found in the present study that Asp significantly upregulated the protein expression levels of 22phox, p47phox and p67phox, but not that of gp91. These data suggest that Asp may activate NADPH oxidase by stimulating some subunits of the complex. ACS14 at 10 mg/kg obviously attenuated Asp-induced upregulation of p47phox and p67phox subunit expression and therefore protected gastric tissue. Although p22phox expression was further increased by ACS14 and PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e46301