Despite recent advancements in pharmaceutical technology, the gastrotoxicity of non-steroidal anti-inflammatory drugs (NSAIDs), which frequently result in stomach ulcers and delayed healing, continues to be a significant issue. ^{1, 2} Gastric ulcers caused by the use of NSAIDs are caused by several factors, including the inhibition of prostaglandin-E2 (PGE2) or angiogenesis, the enhancement of the generation of free radicals, the induction of cyclooxygenase-2 (COX-2) expression, and the production of cytokines that are responsible for pro-inflammatory effects. ^{3– 5} In addition, it has been demonstrated that NSAIDs decrease the rate at which ulcers heal by inhibiting the development of pro-angiogenic factors. ⁶ These pro-angiogenic factors include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and essential fibroblast growth factor (bFGF). ^{7– 12} Indomethacin (IND) was found to have a more significant propensity to induce damage to the stomach than traditional NSAIDs. ¹³

Omeprazole, which is regarded as an important drug by the World Health Organization (WHO), is utilized extensively throughout the world to treat a variety of gastrointestinal conditions. Omeprazole inhibits the production of stomach acid by inhibiting a proton pump inside the stomach. ¹⁴ Furthermore, it lowers the levels of endogenous oxidative stress as well as cytokines that promote inflammation. ¹⁵ In recent years, several natural products have been introduced that, due to their anti-oxidative qualities, make it possible to ease gastrointestinal illnesses. ¹⁶ In this regards, several illnesses were effectively managed with botanical medicine, which entails the administration of plant-derived products. ^{17– 20} The natural therapies are safer and display minimal undesirable effects than manufactured drugs. ^{21– 25}

Natural substances and their medically active elements are extensively researched and evaluated as possibly successful therapies for a variety of diseases. ^{26– 31} These substances are being used to offer gastro-protection in a variety of contexts, notably the avoidance of chronic progressive gastric disease and acute stomach ulcers. ³²

Cranberries, also known as Vaccinium macrocarpon, are an important source of phytochemicals, particularly polyphenols, citric and malic acids, vitamin C, and triterpenoids, which are required for the antioxidant activity of the fruit. ^{4, 33} In addition to phenolic acids and benzoates, the red cranberry contains a high concentration of flavonoids, specifically proanthocyanins, anthocyanidins, and flavanols. ^{34, 35} There are also phenolic acids and benzoates present. Cranberry can treat a variety of other problems, including gingivitis, diarrhoea, and cardiovascular health. ³⁶ To the best of our knowledge, no previous studies were carried out to investigate the impact of cranberry extract on NSAID-induced gastric ulceration. This study aimed to evaluate the efficacy of cranberry extract in treating indomethacin-induced gastric ulcers in albino rats by exploring its effects on antioxidant enzymes, inflammatory markers, and histological findings. Furthermore, the possible mechanisms underlying its therapeutic effects are being investigated.

The results revealed an apparent induction of peptic ulcer with considerable macroscopic alterations in gastric tissue sections of the indomethacin group (G2) compared to the control group (G1). However, both the omeprazole (G3) and cranberry extract (G4) groups exhibited fewer macroscopic stomach modifications than the indomethacin group, culminating in a much lower severity and extent of the experimentally generated ulcer, as depicted in Figure 1.

The indomethacin group (G2) disclosed significantly reduced serum levels of antioxidant enzymes SOD and GPX in comparison with the normal control group (G1) (p < 0.05). Nonetheless, omeprazole (G3) and cranberry (G4) treatment groups demonstrated significantly higher serum levels of antioxidant enzymes SOD and GPX in comparison with the indomethacin induction (G2) group (p < 0.05). However, there were no substantial differences in SOD or GPX levels between the omeprazole (G3) and cranberry extract (G4) groups (P > 0.05) as seen in Figure 2.

Furthermore, serum levels of the inflammatory cytokine indicators TNF-α and IL-1β were substantially elevated in the indomethacin group (G2) when compared with the normal control group (G1) (p < 0.05). The omeprazole (G3) and cranberry (G4) treatment groups, however, presented a substantial reduction in serum levels of inflammatory markers TNF-α and IL-1β when compared with the indomethacin induction (G2) group (p < 0.05). On the other hand, it was demonstrated that the group treated with cranberry extract (G4) had substantially diminished serum levels of inflammatory mediators, including TNF-α and IL-1β, than the group treated with omeprazole (G3) (p < 0.05) as illustrated in Figure 3.

In addition, the histological outcomes of the present investigation demonstrated that gastric specimens from normal rats had normal appearance of the stomach mucosa, submucosa, and muscularis, as clarified in Figure 4A and Table 1.

In contrast, the rat stomach tittue section of the indomethacin group (G2) enjoyed substantial histopathological abnormalities characterised by extensive mucosal surface necrosis, profound congestion with mononuclear leukocyte infiltration, and marked degeneration of digestive glands as compared to the control group (G1), as shown in Figure 4B and Table 1.

The omeprazole treatment group (G3), on the other hand, displayed a major reductionS in indomethacin-induced histopathological irregularities as evidenced by mild congestion, mild edema, minor gastric bleeding, moderate inflammatory cell infiltration, and slight necrotic changes, as seen in Figure 4C and Table 1.

The rat gastric section of the cranberry extract group (G4) indicated dramatically diminished histopathological modifications, including mild sloughing of parietal cells, minor cellular swelling of chief cells, little gastric haemorrhage with minimal leukocyte infiltration, congestion, and oedema. Yet, the cranberry extract group’s stomach mucosa seemed to be comparable to that of the ontrol group, exhibiting normal cytoarchitecture as illustrated in Figure 4D and Table 1.

It has been demonstrated beyond a reasonable doubt that oxidative stress and inflammation play a significant part in the etiology of indomethacin-induced injury to the stomach. ^{86, 87} In the current study, the level of antioxidant enzymes (SOD and GPX) was found to be considerably lower in the group that was subjected to ulcer induction (G2) compared to the group that served as the control (G1) and the other treatment groups (G3, G4). Under typical circumstances, the oxidant and antioxidant defense mechanisms of the organism are in equilibrium with one another for the group that serves as the control. ⁸⁸ Diminished activities of SOD and GPx indicate impaired antioxidant pathways, which are frequently seen in many inflammatory diseases. ^{89– 91} These enzymes are necessary for neutralizing deleterious reactive oxygen species (ROS), and their absence can lead to further oxidative stress and inflammation. ^{92– 95}

The injection of an induction dosage of indomethacin results in the production of ROS and produces oxidative stress. ⁹⁶ According to research, indomethacin can bind to a region in the mitochondrial electron transport chain that is close to the complex and ubiquinone. This results in the uncoupling of the oxidative phosphorylation process and the formation of reactive oxygen species. ⁹⁷ Consequently, reactive oxygen species (ROS) are responsible for the inactivation of mitochondrial aconitase, which leads to the generation of free iron, which in turn generates more mitochondrial •OH. ⁹⁸ There is a correlation between oxidative stress and mitochondrial malfunction, the creation of mitochondrial permeability transition pores, and the generation of mitochondrial oxidative stress (MOS). ^{99– 102}

Regarding the omeprazole treatment group (G3), there was a significant increase in the level of antioxidant enzymes (SOD and GPX) as compared to the ulcer induction group (G2). These findings are consistent with the findings of another research investigation, which demonstrated that omeprazole possesses antioxidant activity in addition to its antisecretory characteristics. Omeprazole was found to be a powerful scavenger of hypochlorous acid (HOCl) even at a drug concentration, and it also showed significant inhibition of iron- and copper-driven oxidant damage at pH 5.3 and 3.5, respectively, according to research that was carried out to investigate the in-vitro antioxidant effects of omeprazole at specific pH levels. ¹⁰³ Another study discovered that OMP, because of its antioxidant activity, is characterized by the overexpression of superoxide dismutase in gastric mucosal cells. ¹⁰⁴

The cranberry treatment group (G4) showed enhanced levels of antioxidant enzymes, SOD and GPX; as compared to the Indomethacin ulcer induction group (G2). Cranberry extract contains several bioactive compounds, such as anthocyanin and flavonoids, which are well known for their antioxidant activity. ¹⁰⁵ Anthocyanins were found to induce the expression of several antioxidant enzymes, often mediated by Nrf2-dependent pathways responsible for inducing cytoprotective responses. In an earlier investigation to test the anti-oxidant function of Syzygium cumin (L.) Skeels on indomethacin-induced acute stomach ulceration, results indicated that the anthocyanin content of this plant dramatically upregulates SOD and GPx levels as compared to untreated rats. ¹⁰⁶ Another study conducted to explore this finding found that the mRNA and protein levels of Nrf2 in anthocyanins treatment groups were near to the control group and higher than the NSAIDs- ulcer induction group. This is related to the ability of these phytochemicals to induce Nrf2 expression or inhibit its proteasomal degradation by modifying the Nrf2–Keap1 complex. ¹⁰⁷

Several other flavonoids with antioxidant ability were reported in cranberries, such as rutin, apigenin, and quercetin. ¹⁰⁸ Rutin is among the flavonoids that exert antioxidant and free radical scavenging activities. ^{109, 110} In a study investigating the antioxidant activity in rats with indomethacin-induced ulcers, rutin could significantly lessen the oxidative stress biomarkers deteriorated by indomethacin treatment. ¹¹¹ Rutin has been proven in several laboratory investigations to boost glutathione levels and superoxide dismutase activity by capturing superoxide anions and scavenging free radicals, while also modulating TNF-α and IL-6 concentrations. ^{112– 114}

Regarding quercetin, the most potent antioxidant flavonoid, studies demonstrated that it has a beneficial effect in attenuating indomethacin-induced gastric ulcers in rats by increasing the antioxidants enzymes activity (Catalase, SOD, and GPX). ^{91, 94} The mechanisms behind the antioxidant activity are scavenging oxygen radicals, protecting against lipid peroxidation, and chelating metal ions. ^{115– 119}

In addition to oxidative stress, inflammation is also a crucial factor in the pathophysiology of gastropathy that is generated by nonsteroidal anti-inflammatory drugs. ^{120– 122} Based on the findings of the present study, it was observed that the level of inflammatory markers, specifically TNF-α and IL-1β, was considerably greater in the group that was subjected to ulcer induction (G2) compared to the group that served as the control (G1) and the other treatment groups (G3, G4). There is a direct connection between the formation of reactive oxygen species (ROS) and the oxidative stress that leads to a rise in the expression of TNF-α and IL-6 genes, which in turn leads to an increase in their levels through the nuclear factor kappa (NF-κB) dependent pathway. ^{39, 123, 124} More specifically, TNF-α acts to facilitate immune system responses and cellular proliferation. It encourages the translocation-related process of NF-κB, which aids communicating signals during inflammation. ^{20, 125– 128}

Regarding the Omeprazole treatment group, there was a significant decrease in TNF-α and IL-6 levels compared to the ulcer induction group (G2). This indicates that omeprazole has an anti-inflammatory effect independent of suppressing gastric secretion. The suggested mechanism behind these effects is postulated to be related to the down-regulation of nuclear factor kappa (NF-κB) with subsequent suppression of pro-inflammatory cytokines, as reported by other researchers. ^{129, 130}

Presently available therapeutic options for ulceration of the stomach feature a significant relapse probability. Natural remedies for ulceration management and therapy are not rare, since communities have historically used plant components. ¹³¹ A large fraction of the vegetable variety stays untapped for medicinal purposes. Antiulcer capabilities in botanical products exhibit potential, and many animal models are employed to assess their efficacy. ¹³² Competent experiments are required to evaluate the ulcer-preventing properties of botanicals and medicines. Such models are useful for understanding the pathological causes of wounds, as well as the antioxidative capabilities of critical medications or compounds with antiulcer effects. ¹³³ There are several models for assessing anti-ulcer medications, rendering it difficult to pick an acceptable model.

The group that received cranberry treatment (G4) demonstrated a noteworthy reduction in the levels of TNF-α and IL-6 when compared to the levels that were observed in the group that was subjected to Indomethacin ulcer induction (G2). There is a connection between this discovery and the anti-inflammatory properties of cranberry extracts, which are primarily comprised of anthocyanins and proanthocyanins. ^{39, 134} The principal mechanisms by which anthocyanin compounds diminish inflammation involve hampering NF-κB, a transcriptional element that is vulnerable in terms of inflammatory and oxidative processes. ¹³⁵ NF-κB is a substance produced by cells and located in the innermost part of the cell. It is inert due to its strong-affinity suppressor, IκB, which retains it in the cytoplasm and prevents its liberation. ¹³⁶ Once triggering events, like as oxidative damage, occurs, a substantial signaling chain is initiated. The chain of reactions promotes IKK-a and IKK-b, two kinases that metabolize IκB. ¹²⁹ Phosphorylation of IκB causes its disassociation, allowing NF-κB to relocate to the nucleus and attach to κB activation regions. This drives gene expression of chemotactic cytokines involving TNF-α and IL-1β. ¹³⁷ Additionally, the phosphorylation of Mitogen-activated protein kinase (MAPKs) enzymes was suppressed by anthocyanin extract, and as a result, activation is necessary to mitigate the inflammatory response. ¹³⁸ MAPKs, which include ERKs, c-JNKs, and p38, are a family of enzymes that react to various stimuli, one of which is inflammation. These enzymes, in turn, govern a wide variety of cellular responses, such as cell differentiation, mitosis, and apoptosis. ¹³⁹ It is necessary to phosphorylate MAPKs for them to become active, as their base form is inactive from a catalytic perspective. ¹⁴⁰

Furthermore, it has been proposed that the anti-inflammatory action that is brought about by cranberry extracts is due to the flavonoid component of the cranberry, which prevents neutrophils from initiating the infiltration process. ¹⁴¹ Several factors can lead to neutrophil infiltration, including the reduction in mucosal blood flow after IND administration. ¹⁴² According to the findings of a study, the flavonoid product known as rutin helped to boost the activity of cNOS, which in turn led to an increase in the levels of nitric oxide in the mucosal tissues of the stomach. Nitric oxide that is produced from cNOS can increase mucosal blood flow and tissue perfusion, which ultimately results in a significant reduction in neutrophil infiltration. ¹⁴³

According to the results obtained, the highest ulcer index score was reported for the Indomethacin ulcer induction group (G2), which was significantly higher than the control, Omeprazole, and cranberry treatment groups. This is due to gastrotoxicity of a high dose of indomethacin inducing an inflammatory response and oxidative stress damaging the gastric mucosa coupled with decreasing the mucus and bicarbonate layer as a result of NSAIDs use which makes gastric tissue more liable to acidic damage of the gastric secretions. ¹³ On the other hand, the Omeprazole treatment group (G3) showed less ulcer score than the indomethacin ulcer induction group (G2) since omeprazole is a PPI that inhibits Na+/K+ ATPase enzyme leading to suppression of acid secretion. ¹⁴ Furthermore, from the previous results, omeprazole displayed antioxidant and anti-inflammatory actions that collectively contributed to their anti-ulcer effects. ¹⁵ The cranberry extracts treatment group showed an ulcer score that is significantly lower than the Indomethacin ulcer induction group (G2) and Omeprazole treatment group (G3) because of their potent antioxidant and anti-inflammatory effects owing to their flavonoid content. ³⁶ The limitations of the current study could be summarized by the small number of animals used and the lack of estimation of the exact molecular mechanism through which cranberry extract exerts its gastro-protective effect. Estimation of Malondialdehyde (MDA) tissue levels, being the product of lipid peroxidation and the expression level of nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA is usually used to predict the pathways of enhancing antioxidant activity while the nuclear factor kappa (NF-κB) mRNA to estimate the anti-inflammatory activity of the extract. Moreover, it is advised that future studies on female rats be conducted to identify gender-related differences. Furthermore, there is an urgent need for future research to explore the composition of phytomolecules present in cranberry extract using various separation techniques such as HPTLC-MS, NMR, LC-MS, etc.

The current study’s findings revealed that cranberry extract proved its efficacy as a potential treatment for gastric ulcers caused by nonsteroidal anti-inflammatory drugs (NSAIDs) due to its anti-inflammatory and antioxidant properties, as confirmed by biochemical and histological analysis.

The research project has been approved by the Institutional Review Board (IRB) of the University of Baghdad’s College of Medicine. The Declaration of Helsinki’s requirements and guidelines were scrupulously adhered to in the course of conducting the current investigation. The ethical authority at University of Baghdad’s College of Medicine confirmed the required documentation and client data with approval number (UoB.Med.03-29) on December 31, 2023.

Zaid Mahmood Abdul Majeed conducted the investigation, wrote and refined the first draft of the document, participated in its design and provided financing and other forms of assistance, donated supplies, equipment, and lab animals, and finished the final copy of the research article.

Mohammed Qasim Yahya Malallah A. Al-atrakji created the theoretical framework for the researched project, specified the parameters of the exploratory analysis, and outlined the main goals through an in-depth assessment of the findings, supplemented with insightful criticism and supervision.