Effect of PPAR agonist LYSO-7 on installation and healing of gastric ulcers induced in mice.

Gastric ulcer is a chronic disease that presents high prevalence, and effectiveness of pharmacological treatments available is limited by several adverse effects. In this study is shown the mechanism of action and toxicological effects of the molecule indole-thiazolidine LYSO-7 in different models of gastric ulcer. Male Swiss mice were treated with vehicle LYSO-7 (5, 25, or 50 mg/kg, p.o.) or bezafibrate (25 or 50 mg/kg, p.o.) 1 hour before the oral administration of Et/HCl (60%/0.03 M) or indomethacin (100 mg/kg). In another set of assays, animals were pre-treated with GW9962, a PPARγ antagonist (2 mg/kg, i.p.), anti-granulocyte antibody (50 µL, i.p.) or L-NAME (70 mg/kg, i.p.) 1 hour before the treatment with vehicle or LYSO-7. One hour after administration of the Et/HCl solution, neutrophils were quantified in the blood and bone marrow, the gastric microcirculatory network was studied in situ by intravital microscopy, in the gastric tissue were quantified the percentage of injured area, MPO activity, PPARγ gene and protein expression, iNOS and eNOS protein expression, and catalase, SOD, GPx, GR and GST activity. One hour after indomethacin administration, gastric tissue was removed to verify the efficacy of LYSO-7 on inflammatory mediator secretion. Chronic ulcer assay induced by acetic acid was carried out in Balb/c WT or ANXA1-/-, applying 20µL of acetic acid in the subserosal layer of the stomach and 24 hours after induction, animals were treated during seven days, once a day, with LYSO-7 (50 mg/kg), bezafibrate (50 mg/kg) or vehicle. Assays were performed with macrophages recruited to the peritoneum by sodium thioglycollate (3%, i.p.) and neutrophils by oyster glycogen (1%, i.p.). Acute and chronic toxicological and mutagenicity assays were also conducted. The results obtained show that LYSO-7 treatment decrease the injured area, neutrophil influx and microcirculatory stasis evoked by Et/HCl administration. Protective effects were reversed in animals pretreated with GW9962, indicating the involvement of PPARγ. Neutrophil influx is a determinant of the gastric lesion, once the depletion of these cells decreased the gastric damage, indicating that in the neutrophil mobilization blockade from the bone marrow to blood and to injured tissue may be a gastroprotective mechanism of LYSO-7. The vascular stasis reversion in the microcirculation is mediated by NO, but not the neutrophil influx, since the pretreatment with L-NAME abolished the effects of LYSO-7 on blood flow. This effect was dependent on increase and decrease of eNOS and iNOS protein expression, respectively. LYSO-7 positively altered the activity of antioxidant enzymes in the gastric tissue. Furthermore, LYSO-7 reduced the injured area and the concentration of TNFα and increased IL-10 in the gastric tissue in the indomethacin-induced ulcer model. In the resolution of inflammation, LYSO-7 treatment decreased the percentage of the injured area, increased the neutrophils apoptosis and the efferocytosis of apoptotic neutrophils by peritoneal macrophages, inhibited the TNFα release and increased the secretion of IL-10, IL-1β and VEGF in the supernatant of phagocytosis assay. The resolution of gastric lesions, as well as, the induction of phagocytosis by LYSO-7 was reduced in animals ANXA1-/-. This data shown the relation of PPARγ and ANXA1, as PPARγ expression is reduced in macrophages obtained from ANXA1-/- animals. Toxicological studies showed that LYSO-7 has low acute and chronic toxicity in vivo, and did not cause mutagenicity in bone marrow erythrocytes. The data obtained show that LYSO-7 acts as PPARγ in the modulation of gastric ulcer and modulate neutrophil migration and blood flow in the microcirculation. The transactivation and transrepression of eNOS and iNOS, respectively, blocking the neutrophil influx into the injury, antioxidant enzymes activation in the gastric epithelium and inhibition of inflammatory mediators release seem to be the mechanisms action of LYSO-7 in gastric cytoprotection. Additionally, LYSO-7 operates in the resolution of inflammation promoting downregulation in the secretion of inflammatory mediators and increases the neutrophil apoptosis and efferocytosis of apoptotic neutrophils. (AU)