According to the “Third International Consensus Definitions for Sepsis and Septic Shock” (Sepsis-3), sepsis is a clinical syndrome associated with life-threatening organ dysfunction caused by a dysregulated host response to infection ^[1]. In recent years, sepsis has presented an increase in its incidence, probably due to the increase in life expectancy, a higher prevalence of comorbidity, and due to the changes in definition that have existed as this pathology is more studied ^[2]. It affects between 19 and 48.9 million people a year and continues to be one of the main causes of morbidity and mortality in the world ^[2]. A subset of sepsis patients will develop or initially present with septic shock (SS), in which there are deep circulatory, cellular, or metabolic abnormalities profound enough to increase the risk of mortality ^[3]. Sepsis survivors have a twofold increased cardiovascular risk compared to the normal population, which is reflected in a higher incidence of major cardiovascular events, including myocardial infarction and acute heart failure ^[4]. The overall deterioration in health that occurs in post-sepsis patients might be due to progression of chronic disorders, residual organ insufficiency, and inadequate immune system functioning, and presumably a proper management of the acute phase of sepsis may result in less long-term complications ^[5].

Oxidative stress and inflammation are two central mechanisms that play a key role in the pathophysiology of sepsis. Therefore, attempts based on antioxidants and anti-inflammatory drugs have been tested in the therapeutic focus of this pathology. Thus, recent studies have been developed using antioxidant drugs, such as selenium ^[6], vitamin C ^[7], vitamin E ^[8], and N-acetylcysteine (NAC) ^[9], among other agents, for the treatment of sepsis. In addition, since uncontrolled inflammation would be exclusively responsible for the complications in patients with sepsis, several clinical trials aimed in the management of sepsis have been performed with drugs blocking the inflammatory cascade, including corticosteroids, anti-endotoxin antibodies, tumor necrosis factor and interleukin-1-receptor antagonists ^[10].

2. Oxidative Stress and Sepsis

2.1. Normal Response to Infection and Development of Sepsis

The normal host response to an infection begins with the recognition of microbial components by innate immune cells, leading to the release of proinflammatory cytokines such as interleukins, chemokines, and adhesion molecules, with the objective of recruiting additional inflammatory cells to eliminate the invading agents ^[11]. Under normal circumstances there is a balance between proinflammatory and anti-inflammatory mediators, resulting in a controlled inflammatory process, which enables the host to overcome the infection, leading to subsequent tissue repair. In contrast, when there is a dysregulation of inflammation, the phenomenon known as sepsis occurs ^[11]. The pathophysiological characteristics of sepsis involve a circle around a dysregulated response to infection, with an excessive release of proinflammatory cytokines ^[12], which are the mediators responsible for the systemic damage occurring under this condition ^[10].

2.2. Endothelial Dysfunction

The endothelium plays a fundamental role in vascular homeostasis as one of the main factors regulating the vasomotor tone ^[13], but it also constitutes a selective barrier to maintain tissue fluid homeostasis ^[14]. Moreover, in the presence of pathogens, the endothelium participates in preventing the spread of infection by releasing cytokines in order to recruit leukocytes and activate clot formation ^[13]. One of the main pathophysiological events in sepsis is endothelial dysfunction, which leads to a dysregulation of the vasomotor tone, a local imbalance between proinflammatory and anti-inflammatory species ^[12], and an increased permeability or loss of barrier functioning, with the latter resulting in a shift of circulating elements and tissue edema ^[14]. When there is a deep endothelial dysfunction a state of vasoplegia occurs, in which the endothelium loses the ability to regulate the vascular tone according to the metabolic demands of the tissues, thereby contributing to impaired microcirculatory blood flow and tissue hypoperfusion ^[15]. Accordingly, a state of hypoxia develops at the cellular level, leading to the activation of anaerobic metabolism with increased lactic acid production as a by-product ^[16], which is an independent mortality predictor in septic patients ^[17]. On the other hand, as part of the endothelial dysfunction a procoagulant state occurs, that causes development of intravascular fibrin deposition and clot formation, this phenomenon is called disseminated intravascular coagulation (DIC) ^[18]. It has been identified that the mechanism causing this event results from the inactivation of the metalloproteinase ADAMTS-13, which is the main inhibitor of the microthrombogenesis process ^[14]. Inflammatory mediators such as IL-6 and neutrophil-derived reactive oxygen species (ROS) are within the main agents responsible for the inactivation of this metalloproteinase ^[14]. In a later stage of DIC, bleeding may occur in parallel because of consumption of clotting factors and inhibitors ^[18].

2.3. Systemic Complications in Sepsis

On a systemic level, the major events that can be identified in patients with sepsis are myocardial dysfunction, sepsis-induced acute kidney failure (S-AKI), and impaired immune system functioning, leading to a late stage of immunosuppression ^[19] with increased susceptibility to new infections. Myocardial dysfunction in septic patients is characterized by reversible biventricular dilation, decreased ejection fraction, and impaired response to fluid resuscitation and catecholamine stimulation ^[20]. Although mortality is substantially increased in patients with sepsis who develop cardiac dysfunction, these alterations appear to be reversible after 7–10 days in survivors ^[21]. This reversibility may be because myocardial damage in sepsis does not cause significant cell death in cardiac tissue, but a non-functioning hibernation-like state as part of an adaptive response to this phenomenon instead ^[10]. The foregoing is consistent with histological findings in patients who died of sepsis, where cell death in the heart was relatively minor and did not correlate with the profound level of organ dysfunction in these patients ^[10]. It is hypothesized that the depression of myocardial function occurs firstly due to mitochondrial dysfunction, which leads to excessive ROS production, and secondly because of a decreased nitric oxide (NO) availability, which is highly correlated with the contractile dysfunction observed in patients with sepsis ^[20]. Additionally, the gastrointestinal tract during sepsis is subjected to important changes. In particular, the most important ones being the intestinal barrier hyperpermeability, intestinal epithelial apoptosis and dysbiosis [26]^[22]. Indeed, during sepsis, the physiological integrity of the intestine, achieved through a balance of anti-inflammatory and inflammatory responses, is compromised [26]^[22]. This results in a disruption of the equilibrium between the host and its bacterial colonizers, leading to bacterial translocation, which, in addition to the hyperpermeability of the intestine, allows the passage of these pathogens into the systemic circulation [27]^[23]. Therefore, the intestine plays a very important role both in the worsening of intra-abdominal sepsis and in the spread of the infectious phenomenon to the rest of the body. Although the different complications in sepsis result from a deregulated pro-inflammatory response, after two hours of the sepsis onset an immunosuppressive phenomenon begins to be triggered [28]^[24] due to the depletion of CD4 and CD8 lymphocytes because of their splenic sequestration and also apoptosis of CD4 lymphocytes induced by the large number of cytokines in the early stage of sepsis development [28]^[24]. Therefore, the late phase of sepsis is characterized by an increased risk of secondary infections [28]^[24]. This correlates with postmortem findings in patients who died of sepsis, in whom a depletion of T cell populations was found compared to patients who died of non-infectious causes [29]^[25]. As identified in the previous sections, OS is transversally present in the pathophysiology of sepsis and its complications, so understanding this phenomenon is crucial for the identification of therapeutic agents able to target this phenomenon.

2.4. Oxidative Stress Definition and ROS Sources

Oxidative stress is a phenomenon that occurs because of an imbalance between oxidant potential and antioxidant defense system activity, in favor of the oxidants, leading to a disruption of the redox cell signaling system associated with molecular damage [30]^[26]. There are reactive species derived from oxygen, nitrogen, and sulfur, with the first two being particularly relevant in the pathophysiology of sepsis. Among the ROS, the superoxide radical anion (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (•OH), and oxygen singlet (¹O₂) are the most important. On the other hand, reactive nitrogen species (RNS) include NO˙, peroxynitrite anion (ONOO⁻), and nitrogen dioxide radical (NO₂) [31]^[27], among other species. It is important to emphasize that ROS and RNS are involved in a multitude of biological processes, but when found at specific concentrations and sites, over a specific time span, they are not associated with cellular damage [30]^[26]. Indeed, the first function of ROS that has been discovered is their role in neutrophils phagocytosis. What happens is that upon phagocytosis of pathogens, the ROS are produced by the NADPH oxidases (NOX) in the small volume of the phagosome [32]^[28].

2.5. Antioxidant Defense System

On the other hand, there is the antioxidant defense system, which has enzymatic and non-enzymatic mechanisms to counteract the production of ROS [31]^[27]. Enzymatic mechanisms include superoxide dismutase (SOD) that converts O₂⁻ to O₂ or the less reactive H₂O₂, glutathione peroxidase (GSH-Px) that catalyzes the conversion of H₂O₂, to water by converting reduced glutathione (GSH) to oxidized glutathione (GSSG) and catalase (CAT) that also catalyzes the breakdown of H₂O₂, [39]^[29], all of which constitute the first line of defense against the overproduction of ROS. Non-enzymatic mechanisms include a vast array of biomolecules such as vitamin C, α-tocopherol (vitamin E), GSH, carotenoids, flavonoids, polyphenols, and other exogenous antioxidants [40]^[30]. The role of SOD as part of the antioxidant defense system is reflected in a study in rats in which supplementation with artificial SOD (Glisodin^®) led to lower levels of the RNS nitrosamine and proinflammatory cytokines, which resulted in a lower incidence of S-AKI in the SOD-supplemented group [41]^[31]. GSH-Px is a selenoprotein, therefore adequate levels of selenium are required for the proper functioning of this enzyme [39]^[29]. Consequently, a correlation has been observed between low selenium levels and greater severity in patients hospitalized for sepsis [42]^[32]. Moreover, it has been observed that Glutathione peroxidase 3 (GSH-Px-3) activity is decreased in patients with sepsis, due to a lower expression of GSH-Px-3, as these patients usually have lower levels of selenium, which limits the synthesis of this enzyme [43]^[33]. The development of OS occurs in the context in which the production of ROS exceeds the capacity of the antioxidant defense system, resulting in damage to different biomolecules [30]^[26]. Protein carbonylation occurs by oxidative cleavage of protein backbones, resulting in structural modifications that affect the proper functioning of the protein [44]^[34]. At the membrane level, ROS generates lipid peroxidation (LPO), which alters the integrity of the cell membrane and membranous organelles [44]^[34]. Finally, DNA damage can also be found mainly in the guanine nucleotides, where oxidative modifications to the DNA result in mutations [44]^[34].

3. Antioxidant Treatments in Sepsis

Considering the role of OS in the pathophysiology of sepsis, the following section will explore a few antioxidant agents that have proven to have a beneficial effect against the deleterious effects of sepsis. The studied drugs are vitamin C, selenium, NAC, and vitamin E.

3.1. Vitamin C

Vitamin C, or ascorbate, is a water-soluble antioxidant compound that forms part of the antioxidant system in humans. Its antioxidant capacity comes from being an electron donor, reducing free radicals, and being oxidized to dehydroascorbate [31]^[27]. Normal levels of vitamin C vary between 50 to 70 µmol/L. The most described vitamin C transporters are Na⁺-dependent cotransporters SVCT1 and SVCT2 [31]^[27]. Many of the physiologic roles of vitamin C are important in patients with sepsis. These include the key antioxidant properties of vitamin C as ROS scavenging, stabilization of eNOS activity, repletion of other crucial body antioxidants such as vitamin E and GSH [31]^[27], and increase of norepinephrine and vasopressin endogenous synthesis. It acts as a cofactor of dopamine β-hydroxylase and tyrosine hydroxylase in the synthesis of norepinephrine [45]^[35]. Likewise, vitamin C acts as a cofactor in the synthesis of L-carnitine, which can reduce TNFα production, thus decreasing the severity of septic shock [46]^[36]. However, vitamin C not only acts as a direct antioxidant, but studies in rats have also shown that it has an inhibitory effect on NOX and iNOS, enzymes that are major sources of ROS [47]^[37]. In critically ill septic patients, plasma vitamin C levels greatly decrease and facilitate ROS and RNS generation [48]^[38], which is consistent with findings that correlate low vitamin C levels to a higher incidence of organic failure and worse outcomes on septic patients [49]^[39]. A clinical trial showed that 28-day mortality was significantly lower in septic patients receiving vitamin C, with a significant increase in intensive care units (ICU)-free days up to day 28, and hospital-free days up to day 60. However, these results were based on analyses that did not consider multiple comparisons and should therefore be considered exploratory ^[6]. It has been reported that vitamin C reduces 28-day mortality and dosage and duration of norepinephrine in intermittent intervals [50]^[40], also reducing the risk of pulmonary morbidity and organ failure [51]^[41]. Lamontagne et al. reported no statistical differences between placebo and supplemented group [50]^[40].

3.2. Selenium

Selenium is an essential micronutrient that acts as an enzymatic cofactor of more than 30 selenoproteins [52]^[42]. This protein group has different biological functions, particularly related to redox cell signaling and antioxidant response, thyroid hormone metabolism, and humoral and cellular immune response [53]^[43]. Approximately 60% of serum selenium is incorporated to selenoprotein P (SePP), 30% to GSH-Px, and 5–10% to albumin [52]^[42]. The GSH-Px family catalyzes various hydroperoxides reductions and has synergy with vitamin E in antioxidant defense against LPO [54]^[44]. The thioredoxin reductase family catalyzes the conversion of H₂O₂ to H₂O. Endothelial dysfunction caused by sepsis leads to SePP adhesion to the endothelium, which is believed to be a protection mechanism against major damage caused by OS [52]^[42]. In catabolic states, selenium urinary excretion increases. It has been described that selenoproteins inhibit NF-κB by redox cell signaling and therefore reduce cytokine storm and ROS/RNS production [55]^[45]. Critically ill patients coursing sepsis have lower levels of selenium [52]^[42], and concentrations lower than 0.7 µmol/L have been associated with higher mortality and organ dysfunction in patients on ICU ^[6]. In a clinical trial developed by Angstwurm et al. [56]^[46], in patients with severe sepsis or septic shock and APACHE III score > 70 who were infused with 1 mg of sodium selenite followed by continuous 1 mg daily infusion for 14 days, a decrease was shown in 28-day mortality versus placebo group, and adequate selenium and GSH-Px-3 serum levels were found. Adjuvant therapy with a continuous dose of 750 μg/24 h of sodium selenite could be beneficial in septic patients with acute lung injury, as shown by Kočan et al. [57]^[47]. Evidence surrounding selenium treatment benefits for sepsis as a monotherapy is controversial. In a metanalysis made by Kong et al. [58]^[48], selenium administration as an adjuvant to standard therapy in severe sepsis of septic shock showed no difference in 28-day mortality but showed a decrease in all-cause mortality.

3.3. N-acetylcysteine

N-acetylcysteine is a thiol precursor of L-cysteine, which is the rate-limiting amino acid for GSH synthesis [59]^[49]. L-cysteine is transported to the intracellular space by the Na⁺-dependent alanine–serine–cysteine system. Nevertheless, because of NAC’s permeability through the membrane, it does not require active transport [60]^[50]. Between the three amino acids which GSH is composed of (glutamate, glycine, and cysteine), cysteine is the least concentrated in the cytosol [60]^[50], therefore in OS, its concentration determines the rate of GSH synthesis, and, in consequence, GSH-Px activity as part of the antioxidant response. Additionally, NAC acts as a direct ROS scavenger, as it can be oxidized by multiple free radicals, and it reacts with H₂O₂, resulting in H₂O as a byproduct [61]^[51]. Given NAC’s important role as GSH precursor and its direct action in ROS and RNS, multiple clinical trials have been done where NAC supplementation could be beneficial in pathologies where OS has a major role in their development. In a study developed by Spies et al. ^[9], 58 patients that required hemodynamic support who developed sepsis were given 150 mg/Kg of NAC in 15 min followed by lowering it to 12.5 mg/kg for 90 min. Forty-five percent of the patients responded by an increase of >10% of VO₂, and a decrease in PCO₂ levels, concluding an increase in tissue oxygenation and cardiac function.

3.4. Vitamin E

α-tocopherol, the main molecular form of vitamin E, is a lipid-soluble molecule capable of regulating the production of reactive species in mitochondria in a dose-dependent manner [62]^[52]. Vitamin E acts as a free-radical scavenging antioxidant, however it does not act against non-radical oxidant species [63]^[53]. A study made by Weber et al. [64]^[54] reported an association between low selenium levels and α-tocopherol independently with higher levels of apoptosis in patients with severe sepsis versus non sepsis ICU patients and healthy patients. In septic shock patients, vitamin E is associated with decreased levels of procalcitonin ^[7]. A study made with pig models showed an indirect association between vitamin E levels and LPO byproducts, and a rapid increase in oxidative stress biomarkers [65]^[55].