The results of the present study showed that serosal application of acetic acid to induce gastric ulcer has resulted in gastric damage with elevated levels of pro-inflammatory cytokines along with increased neutrophil infiltration, lipid peroxidation, cyclooxygenases and prostaglandins, in conjunction with depleted antioxidant GSH in the gastric tissue. On the other hand, NPW treatment, similar to that occurred with omeprazole treatment, facilitated the healing of gastric injury by suppressing oxidative damage and inflammation along with the replenishment of antioxidant capacity. Moreover, the upregulation of gastric COX-1 protein and NF-κB gene due to ulcer induction were abolished by both omeprazole and NPW treatments. On the other hand, COX-1 selective inhibitor ketorolac and the non-selective COX inhibitor indomethacin, but not the COX-2 inhibitor, partially reversed the NPW-induced alleviation in gastric oxidative injury, indicating that the anti-oxidant properties of NPW involve a COX-1-dependent mechanism.
Gastric ulcer is a chronic disease characterized by the episodes of relapses that may exist in the same area or recur at another location throughout the patient's life (Kangwan et al. 2014). Similar to human gastric ulcers that become chronic, ulcer induced by acetic acid (Okabe and Amagase 2005; TAKAGI et al. 1969) occurs by changes in multiple factors including prostaglandins, growth factors, nitric oxide, and cytokines, as well as of mucus adhesion pattern and microcirculation (Kobayashi et al. 2001). Ulcer healing consists of various physiological and structural mechanisms, such as angiogenesis and re-epithelialization, as well as oxidative stress and inflammation that include the upregulated activities of cyclooxygenase enzymes and prostaglandins (Kangwan et al. 2014; Yamane et al. 2022). Acetic acid-induced gastric injury is characterized by elevated pro-inflammatory cytokines like TNF-α, IL-1β and IL-6 in the gastric tissue accompanied by an increase of lipid peroxides and a decrease of antioxidant capacity (Ajeigbe et al. 2022; Eamlamnam et al. 2006; Xue et al. 2019), making the suppression of oxidative stress and inflammatory process a main target for the healing process of gastric ulcer. In accordance with the literature, the current results also demonstrated that neutrophil infiltration, lipid peroxidation, proinflammatory cytokine release, and the upregulation of COX enzymes and prostaglandins are evident in the acetic acid-induced gastric ulcer. Our results also showed that NPW treatment inhibited lipid peroxidation and MPO activity, reduced the release of proinflammatory cytokines and replenished the antioxidant GSH content, showing that the anti-ulcer effect of NPW occurs by inhibiting neutrophil recruitment and upregulating the antioxidant capacity during the chronic oxidative progression of gastric ulcer. Stimulation of NF-κB, which plays an important role in the development and progression of gastric ulcer, controls the magnitude of the inflammatory response in the gastric mucosa by modulating the production of a variety of cytokines and chemokines (Marta et al. 2020; Yeo et al. 2018). Accordingly, the agents that exert anti-inflammatory effects in acetic acid-induced gastric ulcer act by the inhibition of NF-κB (Konturek et al. 2008). We have previously reported that NPW improved sepsis-induced multiple organ injury and stress-induced gastric injury by suppressing oxidative stress via the inhibition of NF-κB signaling (Atici et al. 2022; Tamer et al. 2022). In the present study, the serum or gastric proinflammatory cytokines TNF-α and IL-1β as well as the mRNA expression of NF-κB were reduced by NPW treatment in the rat gastric tissue, indicating the anti-inflammatory action of NPW against acetic acid-induced gastric injury also occurs through the suppression of NF-κB pathway.
Blockade of COX-1 enzyme as well as treatment with the non-specific COX inhibitor indomethacin reversed the effects of NPW on neutrophil recruitment and antioxidant capacity, suggesting that COX-1 enzyme activity mediates the therapeutic effect of NPW on gastric oxidative damage. The primary functions of COX-1 are to maintain the integrity of the gastric mucosa and to control gastric blood flow and acid secretion, whereas COX-2 enzyme activated during an inflammatory response upregulates prostaglandin synthesis at the location of ulceration (Mizuno et al. 1997; Ricciotti and FitzGerald 2011). During both the acute inflammation phase and ulcer healing phase, COX-1 and COX-2 expressions are upregulated at the area of the gastric ulcer, particularly in areas of extensive tissue repair (Rodríguez et al. 2003). Previous studies showed that COX-1 and COX-2 was upregulated in H. pylori gastritis, especially the mid glandular zone and lamina propria inflammatory cells in the human gastric mucosa (Jackson et al. 2000), and also acetic acid-induced gastric ulcer model (Fagundes et al. 2020; Kolgazi et al. 2017). In agreement with these studies, our results confirmed that gastric COX-1 and COX-2 mRNA levels are elevated as a compensatory mechanism in response to a 3-day chronic gastric ulcer. On the other hand, NPW treatment depressed the degree of lipid peroxidation, neutrophil infiltration and pro-inflammatory cytokine levels, and elevated antioxidant capacity in conjunction with the downregulation of gastric COX-1 and COX-2 mRNA levels, suggesting that improved gastric damage due to NPW could be eliminating the need for compensatory upregulation of COX enzymes. Moreover, the suppressive effect of NPW on oxidative injury was reversed by selective blockade of COX-1, showing the specific role of the COX-1 enzyme activity in the protective effect of NPW, which was comparable to that of omeprazole treatment. Although we have previously reported that the protective effect of NPW against stress-induced gastric ulcer and acetic acid-induced colonic injury involves its modulatory effect on the COX enzyme system and cytokine production (Arabacı Tamer et al. 2023; Tamer et al. 2022), the involvement of COX enzymes in the healing-promoting effects of NPW in gastric ulcer was not identified before. Thus, the current study emphasizes that the protective effect of NPW against acute oxidative gastric injury as well as its facilitatory action in chronic healing of gastric ulcer involves its modulatory role in the activity of COX enzyme system.
Prostaglandins generated by particularly COX-2 enzyme, have a key role in ulcer healing process by increasing blood flow, stimulating mucosal bicarbonate secretion, preventing the disruption of mucosal barrier, accelerating cell proliferation and enhancing angiogenesis (Poonam et al. 2005; Takeuchi and Amagase 2018). Kolgazi et al. (2017) have demonstrated that blockade of selective COX-1 or COX-2 enzymes or non-selective COX-inhibitor indomethacin, reversed most of the therapeutic effects of nesfatin-1 peptide on acetic acid-induced gastric ulcer, while COX-2-blockade was consistently more effective. It has been reported that NSAIDs reduce PGE2 content in the gastric tissue and lead to significantly delayed chronic gastric ulcer healing in rats (Berenguer et al. 2002). Additionally, gastric PGE2 level was found to be elevated in parallel with the occurrence of oxidative stress and inflammation in acetic acid-induced gastric ulcer model (Ercan et al. 2020; Mohammadifard et al. 2021). Thus, PG levels, which are known to protect the gastric mucosa by increasing blood flow, were not altered by NPW treatment, while blockade of COX enzymes reduced the PG levels. Our previous results demonstrated that NPW treatment prevented reduction in blood flow during the chronic colitis and gastric ulcer by modulating COX enzyme system (Arabacı Tamer et al. 2023; Tamer et al. 2022). Since NPW receptors are widely expressed in the peripheral tissues that include blood vessels (Chottova Dvorakova 2018), NPW may have a regulatory function in the maintenance of gut microcirculation without affecting prostaglandins in the acetic acid-induced gastric ulcer, which may contribute to the ameliorative effect of NPW treatment in chronic gastric ulcer.
In conclusion, the present data revealed that the intraperitoneal administration of NPW improves gastric injury in rats via the inhibition of the pro-inflammatory cytokine production, oxidative stress and inflammation as well as the downregulation of COX-2 protein and NF-κB gene expressions. Our data indicate that additional experimental and clinical studies are necessary to explore the potential application of NPW for the enhancement of chronic gastric ulcer healing. It would be possible to learn more about the endogenous function of NPW and its interaction with the COX enzymes during the healing of gastric ulcers if specific NPW antagonists were to be developed.