**2. Oxidative Stress Process**

An imbalance between the synthesis and accumulation of oxygen reactive species (ROS) in cells and tissues and the capacity of an organism to eliminate these reactive compounds results in oxidative stress [29]. Thus, organism homeostasis can be altered if higher accumulation of free radicals occurs. ROS are normally generated through different reactions like enzymatic and non-enzymatic, and they can have exogenous or endogenous sources. The enzymatic reactions responsible for ROS generation are characterized by phagocytosis, cytochrome P450 reactions, mitochondrial respiratory chain, and cyclooxygenase-synthesis of prostaglandin [30]. The non-enzymatic system involves the reaction of free oxygen with organic molecules or through tissue-radiation exposure [29]. The exogenous sources of free radicals production are represented by radiation [30], air pollution, and cigarette smoke while endogenous sources are represented by the enzymes systems: mitochondrial respiratory chain, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family, cyclooxygenase, and lipoxygenase [31–34].

ROS production direct measurement is a very complex and difficult process mostly because of ROS sources and species variety, low steady-state concentrations, high reactivity, and of the detection methods involved in the measurement analysis. Also, the oxidative stress assessment is performed by the indirect evaluation of ROS-induced damage on biological targets like DNA, proteins, membrane lipids, and others [35]. Additionally, ROS generation differs significantly in tissue localization, activation mechanisms, and functions in diseases. As a result, ROS levels must be kept within a range that enables organisms to

function normally. These concentration ranges may vary between tissues. However, the balance between physiological redox states and oxidative stress is fragile and is based on relative rates of ROS production and destruction. All of these make it difficult to establish an absolute scale that can offer the values of ROS concentrations in physiological and pathological conditions [36]. Usually, ROS in low and moderate concentrations doesn't possess a significant threat to homeostasis, their beneficial role being known in different physiological processes (e.g., the synthesis of different cellular structures, the help of defense system to neutralize pathogen agents) [29]. If the free radicals are present in large quantities, they cross the physiological barrier and can lead to several health issues, such as cardiovascular, neurological, kidney, respiratory, and rheumatoid diseases [32,34,37]. To counter the side effects of ROS, the organism possess antioxidant mechanisms represented by enzymatic systems (superoxide dismutase—SOD, catalase—CAT, glutathione peroxidase—GPx) and non-enzymatic ones (glutathione, vitamins A, C, and E) [32,33,37]. Superoxide dismutase (SOD) is responsible for O2 − reduction to H2O2. Also, another role of this enzyme is to prevent the formation of other free radicals (peroxynitrite—ONOO−). Further, the H2O2 molecule will be converted by catalase (CAT) in water and oxygen [33,38]. Glutathione peroxidase (GPx) is another enzyme with a role in degrading H2O2 and hydroperoxide molecules using glutathione (GSH) as a proton donor [32].

Another category of endogenous substances resulting from internal metabolism with consequences on human health due to an imbalance between synthesis and elimination is reactive nitrogen species (RNS). RNS are already known to take part in the pathophysiological process of different diseases (diabetes, Parkinson's disease, pulmonary, cardiovascular, rheumatological, liver, and neurological diseases) [39]. RNS are synthesized through the interaction of nitric oxide molecules with other reactive oxygen species. Nitric oxide (NO) is a versatile natural molecule found in organisms resulting from the breakdown of arginine to citrulline [40]. Nitric oxide possesses antimicrobial activity, promotes vascular relaxation with reducing blood pressure, and cell signaling. NO also acts as a scavenger molecule interacting with superoxide anion (O2 −) leading to peroxynitrite (ONOO−) formation. ONOO− leads to endothelial damage, DNA oxidation, and lipid peroxidation [40,41]. Nitrogen dioxide (NO2 −) is another RNS, that results from the interaction of NO with peroxyl radical, which produces lipid peroxidation, ascorbic acid oxidation, and alters tyrosine structure and function leading to the 3-nitrotyrosine formation (3NT) [40,42].

Even though the role of ROS and RNS in damaging the cells and signal transduction is well known, there are still several highly debated issues that need to be resolved. When present in low quantities, ROS and RNS operate as regulatory mediators in signaling processes; nevertheless, when present in high concentrations, they are toxic to living organisms by inactivating critical cellular components. This indicates that the concentrations of ROS and RNS control the change from their favorable to unfavorable effects, although the concentrations at which this change occurs are not well known [36].

The imbalance between the increased ROS and RNS production and decrease of antioxidant molecules will eventually lead to chronic inflammation [43]. During the oxidative stress process, the reactive oxygen/nitrogen species can initiate intracellular signaling and through which specific proinflammatory genes are expressed [43,44]. Generally, between oxidative stress and inflammation, there is a state of interdependency, with one potentiating the other via different mechanisms. For example, during oxidative stress at the brain level, lipids and proteins suffer alterations through oxidation which conduct to disruptions in neurons' communication with inflammation being stimulated [45]. During oxidative stress, DNA suffers damage resulting in different metabolites (8-oxo-7 8-dihydro-2 -deoxyguanosine, 8 oxo-guanine). It was demonstrated that 8-oxo-guanine presence stimulates the nuclear factor kappa B (Nf-*κ*B) [46]. The activation of Nf-*κ*B enhances the pro-inflammatory response through increased synthesis of inflammatory molecules (cytokines, chemokines) and also activation of immune cells [47–49]. Cytokines manage also to increase the ROS levels in non-immune cells. For example, it was shown that IL-6 stimulates the NADPH in lung cancer cell lines leading to an increase in ROS levels [47]. Oxidative stress leads to the release of arachidonic acid which under cyclooxygenase and lipoxygenase enzymes reactions, results in prostaglandins and leukotrienes synthesis [49]. Also, oxidative stress can be enhanced by inflammation. During inflammation, immune cells responsible for phagocytosis (neutrophils and macrophages) produce reactive oxygen and nitrogen species to dissolve the pathogen. In the case of an exaggerated inflammatory response, these reactive species exit from phagocytic cells resulting in tissue injury outside the inflammatory site [47].

Thus, inflammation and oxidative stress, are tightly interconnected [50] initiation and maintenance of many pathological conditions, their prevention and control could provide a safe alternative in chronic disease management.

#### **3. Inflammation**

Inflammation represents a pathophysiological mechanism of defense that acts in case of homeostasis perturbations provoked by infectious agents or trauma. Immune cells enact this mechanism which has a role in locating the pathological agent, digestion, and resolution of the inflammation with restoring homeostasis. Thus, inflammation could be considered a protective response, but an uncontrolled inflammation may be potentially harmful and may lead to many acute and chronic diseases [51].

Inflammation could be acute or chronic depending on the interval of time from the onset of homeostasis impairment until the development of the entire process and the appearance of clinical symptoms. Acute inflammatory diseases present a rapid and nonspecific immune response which usually lasts up to 2 weeks with the resolution of the inflammation process [52]. If the process is not healed, this can lead to a prolonged immune response called chronic inflammation, which lasts from months to years [53]. Thus, chronic inflammations may require a long-lasting management and they become a burden not only for individuals but also for society due to higher costs and health assistance. For all these reasons, there is a perpetual race to discover new molecules with pharmacological properties that may limit the chronic evolution of inflammation.

The etiology of it is also variable, different agents like infectious agents or trauma could trigger cells belonging to both innate and adaptive immunity. The most important aspect of acute inflammation is the recognition of the pathogen or damaged tissue through circulating molecules, which signals innate immune cells and leads to a cascade of biological reactions [54,55]. These reactions are mediated by several proinflammatory molecules, such as cytokines and chemokines, which act interconnected in a signaling network, leading to the recruitment of more immune cells and mediators at the site of inflammation [56,57]. Vasodilation also occurs at the site of inflammation to bring the immune cells there. This process is facilitated by local mediators (nitric oxide, prostaglandins) produced by endothelial and inflammatory cells, leading to the translocation of vascular fluid into interstitial space and enhancing the migration of immune cells [58,59].

Neutrophils represent the first and the most important type of cells belonging to innate immunity that act at the site of inflammation. Neutrophils create a toxic environment by releasing cytotoxic compounds from their vesicles, such as proteases and reactive oxygen and nitrogen species, that destroy the pathogen and the surrounding tissue [55,60]. After the pathogen is destroyed, the resolution process begins, and monocytes are recruited for wound healing. Monocytes block other new inflammatory processes with possible downside effects for the host [55].

Clinical signs and symptoms that characterize inflammation reflect these pathophysiological processes. Locally at the inflammation site, heat, edema, redness, pain, and impaired function could be observed [57]. Cellular injury inducing an immune response activates the intrinsic blood coagulation pathway [61]. Coagulation is activated to create the fibrin clot, which has a role in the isolation of the inflammation process, but also it enhances the pro-inflammatory response [62]. Another mechanism that intervenes in innate immunity is the complement system, which consists in several proteins that could be activated. These

proteins, through a series of chain reactions, create a so-called "membrane complex attack" that disrupts the microbe's cell membrane and induces death [58,59,62].

The acute inflammatory response may transit to a chronic response if the neutrophils fail to eliminate the pathogen agent in the first place. After this, the innate immune response is followed by an adaptive immune response characterized by the presence of macrophages and lymphocytes. Besides them, fibroblasts and plasma cells can be found at the site of chronic inflammation [63]. Monocytes are a group of cells that migrate from blood to different tissues and differentiate themselves into macrophages. Macrophages work in innate immune response alongside neutrophils and in the adaptive immune response through activating lymphocytes [59,63]. Lymphocytes T (Ly T) are activated by macrophages and will be divided into different populations with specific role in the adaptive immune response. However, their primary function is to enhance the immune system, stimulating all immune cells to give a specific defense response against the etiological agent [63]. Lymphocytes B (Ly B), activated by Ly T cells, differentiate in plasma cells which produce antibodies against different types of antigens [64]. The chronic process is associated with tissue destruction, and the repair process is maintained by fibroblasts that secrete collagen, representing the main component needed for wound healing [65]. Thus, acute inflammation is characterized by vasodilatation, and innate immune cells, while the chronic one is described by the involvement of adaptive immune cells, and fibroblast proliferation with significant changes in wound healing. Chronic inflammation is an important component of many diseases, such as atherosclerosis, diabetes, metabolic disorders, cancer, and autoimmune conditions, so the conversion of acute inflammation to a chronic one is a desirable outcome.
