ROS and RNS formation begins in the intestinal ischemia phase with adenosine triphosphate (ATP) accumulation generated in the anaerobic metabolism. There is degradation until the accumulation of hypoxanthine that at the beginning of the reperfusion phase, when there is reintroduction of oxygen to the intestinal tissue, interact with xanthine oxidase forming the superoxide anion (O₂⁻), the first ROS formed. From there, the organism launches defenses such as superoxide dismutase (SOD), attenuating and forming ROS such as hydrogen peroxide (H₂O₂). However, if the response to reperfusion injury continues, the hydrogen peroxide is transformed into hydroxyl from the metal Iron (Fe⁺) into hydroxyl (OH⁻), in the so-called Fenton reaction. And in parallel, there may be the activation of RNS with the formation of nitric oxide (NO) from L-arginine, mediated by inducible nitric oxide synthase (iNOS). The combination of NO and superoxide anion forms the highly reactive species called nitrite peroxide (ONOO) which will further damage the intestinal cell’s epithelium.

During the ischemic phase, mitochondrial oxidative phosphorylation is inhibited rendering a drop in the production and storage of adenosine triphosphate (ATP). ATP is successively degraded to adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine, inosine, and finally hypoxanthine. Lack of cellular energy causes sodium-potassium (Na⁺/K⁺) pump failure resulting in intracellular Na⁺ accumulation and K⁺ out of cells, ultimately leading to cellular edema and organelle dysfunction. In addition, an influx of calcium (Ca²⁺) and chloride (Cl⁻) ions into the intracellular environment occurs and triggers the activation of calpain protease, which in turn promotes the breakdown of a peptide bridge of the enzyme xanthine dehydrogenase (XDH) and subsequent formation of XO.

Although essential for the rescue of morphofunctional integrity of the affected tissues, restoration of mesenteric blood flow and consequent ischemic tissue reoxygenation has a deleterious effect because, paradoxically, reperfusion itself aggravates the damage [26,27]. Oxygen together with hypoxanthine and XO, synthesized during ischemia, catalyze the formation of ROS [28,29]. Re-introducing oxygen into the visceral circulation via reperfusion leads to the formation of O₂⁻ and hydrogen peroxide (H₂O₂) after successive monovalent reductions. In the presence of iron, copper, cobalt, chromium, or vanadium, the production of highly reactive hydroxyl radical (OH^.) is promoted via the Haber-Weiss and Fenton reactions [30]. There is an activity burst of the oxidative process characterized by the abundant production of multiple ROS and RNS within a few minutes after the restoration of blood flow [27]. The events underlying the damage caused by ischemia/reperfusion produce an uncontrolled and excessive release of ROS and RNS that overcome the organic line of defense represented by free radical scavengers [31].

The mitochondrial respiratory electron transport chain is the main intracellular site of ROS production and polymorphonuclear leukocytes play an important role in several pathological conditions also generating free radicals and nitric oxide (NO) synthesis. Different forms of mitochondrial dysfunction and tissue inflammation can affect the organ undergoing ischemia and reperfusion and may even compromise other organs and systems with a paracrine or and endocrine effect. This phase can lead to the failure of multiple organs and systems [23,32].

Nitric oxide (NO) dynamics underpin changes involving RNS. NO is produced from L-arginine by three main isoforms of nitric oxide synthase (NOS): epithelial NOS (eNOS), related to vasodilation and vascular regulation; neuronal NOS (nNOS), linked to various intracellular signaling pathways; and inducible NOS (iNOS), which has been reported to have beneficial microbicidal, antiviral, antiparasitic and antitumoral actions, but has also been implicated in the pathophysiology of colitis [33]. While the production of NO by nNOS and eNOS is regulated by a Ca²⁺/calmodulin-dependent mechanism, iNOS is activated in response to triggers such as endotoxins or cytokines, which can lead to rapid production of large amounts of NO. Several diseases have been associated with excessive levels of NO production, resulting in serious deleterious cell-physiological consequences [34,35,36,37,38]. All products formed by NO reactions are collectively called RNS. Despite the discovery of NO as an endothelium-derived relaxing factor, it plays a critical role in the pathophysiology of sepsis as an important mediator of endotoxin-induced arteriolar vasodilatation, hypotension, and shock [39]. At high concentrations, NO is importantly involved in inflammatory, infectious, and degenerative diseases [40]. Via reactions with other free radicals produced during oxidative stress, NO can be converted to nitrogen dioxide (NO₂), peroxynitrite (ONOO⁻), and dinitrogen trioxide (N₂O₃). NO₂ is formed from NO autoxidation (reaction of NO with oxygen). ONOO⁻ is a powerful electron oxidant and is formed through the diffusion-controlled reaction between O₂⁻ and NO; its most relevant targets are peroxiredoxins, glutathione peroxidase (GSH), CO₂, and metal centers. N₂O₃ can be formed from a reaction between NO₂ and NO and is considered an important intermediate in the autoxidation of NO. N₂O₃ is rapidly hydrolyzed to NO₂ [41]. All these compounds can subsequently react with various classes of biomolecules, including lipids, DNA, thiols, amino acids, and metals, leading to oxidation and nitration. If produced at high levels, RNS will detrimentally impact cell function, leading to changes in membrane integrity, loss of enzyme function, and DNA mutations [42].

It is noteworthy that, despite its typically beneficial antioxidant and vasodilatory functions, NO in high concentrations induces caspase-mediated apoptosis of epithelial cells in the intestinal tissue during ischemia and reperfusion. In addition, O₂⁻ rapidly reacts with NO to produce ONOO⁻, which is another potent oxidant [43]. In the vasculature, the reaction of NO with O₂⁻ leads to the formation of ONOO⁻ and decreases the vasorelaxant efficacy of NO. ONOO⁻ is a strong oxidant that can hydroxylate aromatic amino acids, oxidize thiols and lipids, and nitrate-free and protein-bound tyrosine residues. The number of possible reactions leading to secondary RNS formation illustrates the strong potential of NO to contribute to oxidative damage. High concentrations of NO, particularly in combination with increased oxidant production, cause tissue damage and inflammation through the production of NO₂, ONOO⁻ and other nitrating, nitrosating, and oxidizing intermediates, and via inhibition of metal-dependent enzymes [44,45].

Several enzymes, such as cytochrome P450, the enzyme complexes of the mitochondrial respiratory chain, XO [46], eNOS [47], heme oxygenase (HO) [48], myeloperoxidase (MPO) [49], lipoxygenase (LOX), cyclooxygenase (COX) [50], and NADPH oxidases (NOX) [51] generate ROS under pathological conditions leading to oxidative stress [52]. All these factors contribute to persistent oxidative stress in the cellular environment, which will result in progressive functional impairment of critical intracellular organelles and structures, including membranes, mitochondria, the endoplasmic reticulum, the cytoskeleton, and the nucleus. These deleterious effects occur mainly due to the oxidation of proteins, DNA, and lipids, ultimately culminating in cell death [53,54].

A balance between ROS levels and the activity of inactivating (antioxidant) enzymes is crucial for the maintenance of cellular homeostasis. Erythroid-related nuclear factor 2 (Nrf2) is a transcription factor that plays an important role in the response to oxidative stress to maintain redox balance. Under homeostatic conditions, Nrf2 is bound to its chaperone Keap1 (Kelch-like ECH association protein 1) in the cytoplasm. However, when oxidative stress occurs, Nrf2 dissociates from the inactive Keap1-Nrf2 complex and translocates to the nucleus, where it regulates specific gene expression to induce the synthesis of antioxidant enzymes [55]. O₂⁻ and H₂O₂ are inactivated by superoxide dismutase and catalase or the glutathione peroxidase system, respectively. OH^. is typically more harmful than these ROS, as this oxygen-derived free radical does not have an intracellular inactivator. Its production intensifies the severity of injuries to cell structures, causing DNA damage caused by adducts of lipid peroxidation, and the production of other free radicals (such as malondialdehyde, hydroperoxide, and ONOO⁻, among other substances capable of stimulating the adherence of granulocytes to the microvascular endothelium [55,56].