S-Aspirin Protects Aspirin-Induced Gastric Injury Figure 3. Effect of ACS14 on H2S concentration and basal gastric mucosal blood flow in rats. A & B. ACS14 increased H2S concentrations in both blood plasma (A) and gastric tissues (B). n = 7. C. ACS14 increased basal gastric mucosal blood flow in rats. n = 8. Data are the means6SE. *P,0.05 compared with control; #P,0.05 compared with Asp; {P,0.05 compared with Asp+ACS14 1 mg/kg. doi:10.1371/journal.pone.0046301.g003 with Asp (200 mg/kg). This effect was not rescued by treatment with either ACS14 or NaHS (Fig. 4B). ACS14 and NaHS Decreased Asp-induced Gastric Oxidative Stress It is well known that Asp-induced gastric injury is caused by oxidative stress [3,21]. We therefore examined the levels of gastric MDA, one of the markers of free radical species-related injury. As expected, the levels of gastric MDA were significantly elevated in Asp group of rats, as compared with the control group (Fig. 5A, P,0.05). Pretreatment with ACS14 at 10 mg/kg obviously reduced the elevated MDA level. Treatment with NaHS (0.73 mg/kg) showed a trend of decreasing gastric MDA when compared with Asp alone group, but no significant difference was found. The results suggest that ACS14 appeared to be a potent antioxidant regulator to attenuate the Asp-induced gastric injury in rats. GSH is the most important antioxidant. As shown in Fig. 5B, Asp markedly suppressed intracellular GSH production from 10.2760.54 mmol/g protein to 7.5961.54 mmol/g protein. This is consistent with the previous findings [22]. Pretreatment with ACS14 at 5 and 10 mg/kg significantly increased the gastric GSH level. SOD-1 is one of three superoxide dismutases responsible for destroying free superoxide radicals in the body. As shown in Fig. 5C, Asp treatment significantly decreased the expression of SOD-1 in the gastric tissue. Similarly, pretreatment with ACS14 and NaHS reversed the down-regulation of SOD-1 expression induced by Asp. Xanthine oxidase (XOD) is an oxidase which produces reactive oxygen species. XOD expression in gastric tissue was significantly increased by Asp treatment (Fig. 5D). Treatment with ACS14 and NaHS suppressed Asp-induced upregulation of XOD expression. The NADPH oxidase is a membrane-bound enzyme complex. It generates superoxide by transferring electrons from NADPH to molecular oxygen to produce the superoxide. NADPH comprises p22phox, gp91phox, p40phox, p47phox, p67phox, and the small GTP- binding protein Rac [23–25]. As shown in Fig. 6, Asp significantly upregulated the protein expression of p22phox (Fig. 6A), p47phox (Fig. 6B) and p67phox (Fig. 6C), pretreatment with ACS14 and NaHS significantly attenuated the expression of p47phox and p67phox, but enhanced the expression of p22phox. However, all the three drugs, Asp, ACS14 and NaHS had no significant effect on gp91phox (Fig. 6D). Discussion Asp is widely used as an anti-inflammatory and analgesic drug. However, Asp at therapeutic dose for pain-relief often induces gastrointestinal adverse effects including gastric ulcer and bleed- ing. At low doses, Asp is also used to prevent cardiovascular and cerebrovascular disease [26,27]. The recent clinical studies showed that even at low dose to prevent cardiovascular disease. Asp also induces gastroduodenal complications [28–31]. The annoying adverse effect may largely limit the clinical uses of NSAIDs. Therefore, development of new salicylate drugs which may not produce gastrointestinal toxicity is necessary and urgent. PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e46301