*4.2.4 Levels of endogenous antioxidants*

The mammal organism has several mechanisms to counteract with OS by producing antioxidants, which are either produced in situ or externally supplied with foods or supplements. The nonenzymatic antioxidants are distinguished as metabolic antioxidants and nutrient antioxidants. Metabolic antioxidants referred also as endogenous antioxidants such as glutathione, lipoid acid, L-arginine, melatonin, coenzyme Q10, uric acid, bilirubin, metal-chelating proteins, and transferrin are produced by metabolic processes, while nutrient antioxidants are compounds that cannot be produced in the body and must be provided through foods or supplements, such as vitamin E, vitamin C, carotenoids, trace metals (selenium, manganese, zinc), flavonoids, and omega-3 and omega-6 fatty acids [75]. Decreased levels of nonenzymatic antioxidant glutathione and vitamin C were observed in the liver of AIA rats compared to the normal rats [76]. Antioxidant state showed that plasma vitamin E, vitamin C, vitamin A, and β-carotene were significantly lower in arthritic control rats than normal rats [77]. Reduction of plasmatic antioxidants is indicating reduced antioxidant capacity and elevation of oxidative stress during adjuvant arthritis which is similar to rheumatoid arthritis in human [78].


*RLU\*s, relative light units; PMA, phorbol-12-myristate-13-acetate; values are expressed as average standard error of mean, statistical significance (ANOVA-Tukey-Kramer post hoc test): \*\*\*p < 0.001 vs. CO.*

#### **Table 3.**

*Spontaneous and stimulated chemiluminescence and neutrophil count in whole blood of arthritic rats.*

## *Impact of Oxidative Stress on Inflammation in Rheumatoid and Adjuvant Arthritis: Damage… DOI: http://dx.doi.org/10.5772/intechopen.89480*

CoQ10 plays a central role in the electron transport chain and as a radicalscavenging antioxidant; therefore we studied its level in plasma during AA. In our experiments the arthritis process increased significantly the level of CoQ10 in comparison with healthy control rats. The arthritic processes also stimulated the synthesis of CoQ9 (dominant form of CoQ in rats) and its transport to plasma [79] (**Table 4**). In the skeletal muscle mitochondria, we have measured significant changes in levels of α- and γ-tocopherol (**Table 5**).

Similarly in AIA, also in patients with RA, a depletion of endogenous antioxidants was measured. The plasma concentration of beta-carotene and vitamin E, hemoglobin, and hematocrit were significantly lower in patients with RA than in controls. These results provide evidence for a potential role of raised lipid peroxidation and lowered enzymic and nonenzymic antioxidants in RA because of its inflammatory character. These results suggested that OS plays a very important role in the pathogenesis of RA [80, 78].

### *4.2.5 Changes in antioxidant enzymes*

In order to protect tissues from oxidative injuries, the body possesses enzymatic antioxidant enzymatic systems such as superoxide dismutases and catalase enzymes. It has been reported that AA decreases serum or synovial SOD and catalase activities together with other endogenous antioxidant systems [81]. Ramos-Romero et al. [82] showed a decrease in splenic catalase activity and, paradoxically, an increase in splenic total and mitochondrial SOD in AIA. The decreased catalase activity could be associated with the consumption of catalase in neutralizing the H2O2. On the other hand, increased splenic SOD activities could reflect the response of the body to increased ROS concentrations, and/or it could be due to the fact that arthritis was in its recovery phase 1 month after its induction. Moreover, SOD increase could also be explained by the increase in the oxidative stress found in arthritic rats and by the increased TNF-α secretion present in arthritis [82]. Both OS and TNF-α are shown to induce SOD synthesis [83]. It should be added that a similar increase in SOD activity was found in the plasma of RA patients [84] and in


*Values are expressed as average standard error of mean, statistical significance (ANOVA-Tukey-Kramer post hoc test): \*p < 0.05, \*\*p < 0.01 vs. CO.*

#### **Table 4.**

inflammatory diseases such as AIA, not only in tissues directly affected by the

Recent evidence from animal models of RA emphasized the importance of neutrophils in the initiation and progression of AIA [70]. Progressive erosion of articular cartilage is a prominent feature of this disease. Not surprisingly, immunosuppressive approaches such as blockade of CD4+ lymphocytes effectively reduce the intensity of damage and the progression of AIA. The report of Santos et al. [71] convincingly demonstrates a requirement not only for CD4+ lymphocytes but also for neutrophils, the latter determined by the protective effects of neutrophil depletion. The sequence of events showed that CD4+ cells are necessary for the establishment of the immune response, which leads to the recruitment of neutrophils,

with the involvement of cytokines (TNF-α, IL-1) and the IL-8 family of

chemokines. The combination of products (oxidants, proteinases, and cytokines) from stimulated neutrophils, synovial macrophages, and lymphocytes is important to set the stage for acute and progressive polyarthritis [72]. We assessed ROS production in stimulated neutrophils of arthritic rats, and it was found to be increased, with a maximum on day 14 and 21 of AIA. Neutrophils in the whole blood of AIA animals reacted excessively to stimulation and produced 6–9 times more ROS [73]. We also demonstrated oxidative damage of tissues in AIA: ROS levels in the joint and the spleen were significantly elevated [74] (**Table 3**).

The mammal organism has several mechanisms to counteract with OS by producing antioxidants, which are either produced in situ or externally supplied with foods or supplements. The nonenzymatic antioxidants are distinguished as metabolic antioxidants and nutrient antioxidants. Metabolic antioxidants referred also as endogenous antioxidants such as glutathione, lipoid acid, L-arginine, melatonin, coenzyme Q10, uric acid, bilirubin, metal-chelating proteins, and transferrin are produced by metabolic processes, while nutrient antioxidants are compounds that cannot be produced in the body and must be provided through foods or supplements, such as vitamin E, vitamin C, carotenoids, trace metals (selenium, manganese, zinc), flavonoids, and omega-3 and omega-6 fatty acids [75]. Decreased levels of nonenzymatic antioxidant glutathione and vitamin C were observed in the liver of AIA rats compared to the normal rats [76]. Antioxidant state showed that plasma

vitamin E, vitamin C, vitamin A, and β-carotene were significantly lower in arthritic control rats than normal rats [77]. Reduction of plasmatic antioxidants is indicating reduced antioxidant capacity and elevation of oxidative stress during adjuvant arthritis which is similar to rheumatoid arthritis in human [78].

CO 41,802 2452 150,789 9159 12,174 747 AIA 168,203 12815\*\*\* 1,165,603 94470\*\*\* 40,260 3325\*\*\* *RLU\*s, relative light units; PMA, phorbol-12-myristate-13-acetate; values are expressed as average standard error*

*Spontaneous and stimulated chemiluminescence and neutrophil count in whole blood of arthritic rats.*

*of mean, statistical significance (ANOVA-Tukey-Kramer post hoc test): \*\*\*p < 0.001 vs. CO.*

**Spontaneous PMA stimulated Neutrophil count in 1 μL of**

**blood**

disease (cartilage, bone, and skeletal muscle) (**Table 2**).

*4.2.3 Production of reactive oxygen species by neutrophils*

*Animal Models in Medicine and Biology*

*4.2.4 Levels of endogenous antioxidants*

**Chemiluminescence**

**(RLU\*s)**

**Table 3.**

**204**

*Concentrations of total coenzyme Q9 (CoQ9-TOT), total coenzyme Q10 (CoQ10-TOT), α-tocopherol (αT), and γ-tocopherol (γT) in plasma.*


*Values are expressed as average standard error of mean, statistical significance (ANOVA-Tukey–Kramer post hoc test): \*p < 0.05 vs. CO.*

#### **Table 5.**

*Concentrations of total coenzyme Q9 (CoQ9-TOT), total coenzyme Q10 (CoQ10-TOT), α-tocopherol (αT), and γ-tocopherol (γT) in skeletal muscle mitochondria.*

the synovial membrane of mice with collagen-induced arthritis [85]. Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen, thus preventing the oxidation of biological structures by hydrogen peroxide. Authors demonstrated the elevated and LPO activity and NO level and decreased GSH, SOD, and catalase activities in AIA rats [86]. OS in AIA model is depleting antioxidant enzymes, which is in good agreement with human RA studies.

predominantly promoted by the NF-κB pathway via the activation of MuRF1 transcription factor which ultimately induces immoderate proteolysis of muscle proteins by activating the ubiquitin-proteasome system. Moreover, Castillero et al. [97] observed overexpression of MuRF1 as well as several other myogenic factors, such

*Impact of Oxidative Stress on Inflammation in Rheumatoid and Adjuvant Arthritis: Damage…*

Another important pathogenic factor of RC is reduced physical activity, which appears to be the result of either poor pain management of inflamed and swollen joints, metabolic changes, or merely general caution for physical activity. Lower physical activity leads to reduced muscle fiber stimulation, which significantly disrupts the cycle of muscle proteolysis and proteosynthesis in favor of proteolysis [98]. One of the possible triggers of RC could also be increased free radical concen-

As mentioned in the previous text, ROS and RNS concentrations have been reported to be elevated in the joint area as well as plasma. This may indicate that an increase in free radicals levels could also be found in skeletal muscle tissue. There are several sites of free radical production in muscles including mitochondria, sarcoplasmic reticulum, and sarcolemma [99]. As metabolically highly active organs, muscles dramatically increase their oxygen consumption during physical activity in order to compensate various energy-dependent processes. Concurrently excessive amounts of oxidants are produced, which then serve as messenger molecules in multiple intracellular cascades. The main site of free radical generation is mitochondria during aerobic metabolism and oxidative phosphorylation. It has been shown that complexes I and III and more recently complex II of mitochondrial electron transport chain are key producers of ROS in muscle fibers [100]. Several authors suggest that the major ROS produced in muscle cells is superoxide anion (O2•), which is a very unstable radical and rapidly undergoes reduction resulting in dismutation into hydrogen peroxide (H2O2) [101]. Even though H2O2 is quite a stable nonradical molecule, excessive concentrations of H2O2 could ultimately result in increased generation of hydroxyl radical (•OH)–a highly reactive ROS which could potentially damage various cellular molecules and disrupt many intracellular mechanisms. Free radicals are also regularly produced by several enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) family as well as xanthine oxidase (XO) [99]. In skeletal muscles only Nox 2 and Nox 4 isoforms have been found, and it is believed that both of these isoforms are localized primarily in mitochondria [102]. However, the precise mechanism by which the increased activity of these enzymes is promoted is to date poorly understood. Under physiological conditions, excessive concentrations of free radicals are regularly scavenged and converted into non-radical molecules by antioxidant defense system molecules. However, several studies have observed low concentrations of some nonenzymatic antioxidants such as GSH [41] as well as low activity of enzymatic antioxidants such as SOD and glutathione peroxidase (GPX) in RA, which could potentially affect muscle tissue [103]. It has been proposed that decreased physical activity in RC patients could play a major role in oxidative damage of muscle cells since lower muscle stimulation reduces antioxidant capacity thus causing impaired

Several long-term studies have reported a number of negative effects of free radicals in muscles at the molecular level. Oxidative damage of lipids, particularly in cell membranes [105], as well as nucleic acids in the DNA [106] is of great importance to normal cellular functioning, lately there has been a great deal of emphasis

Proteins as functional units of the cell can cause great damage to the cell itself if its space conformation is disrupted. Perhaps the most common protein modification caused by imbalance of oxidative status is carbonylation of side chains of multiple

as atrogin-1/MAFbx ubiquitin ligases in adjuvant arthritis.

*DOI: http://dx.doi.org/10.5772/intechopen.89480*

tration and onset of OS.

balance in oxidant-antioxidant ratio [104].

**207**

on protein modifications caused by ROS in multiple diseases.

Activity of glutathione peroxidase (GPx) in blood serum and muscles of rats with AIA increased and activity of glutathione reductase (GR) in these tissues increased in comparison with the control. Probably, changes in enzyme activity are a defense response of the body to ROS generation in RA and can be a result of ROS activation or stimulation of their synthesis [87]. Similarly in the study of Sahu et al. [88], CIA increased antioxidant enzyme GPx and GR activities in joints, liver, kidney, and spleen tissues of rats.

Several pathologic factors have been suggested to be involved in the overexpression of heme oxygenase-1 HO-1 in RA lesions. In addition to superoxides and pro-inflammatory cytokines, hypoxia may play an important role in HO-1 expression in the lesions [89, 90]. AIA is an experimental model widely used to evaluate etiopathogenetic mechanisms in chronic inflammation. Devesa et al. [91] have examined the participation of HO-1 in AIA. They have found an increased nitric oxide (NO) production in the paw preceded the upregulation of HO-1, whereas selective inhibition of inducible NO synthase (iNOS) after the onset of arthritis lowered HO-1 expression, suggesting that this enzyme may depend on NO produced by iNOS. Administration of the HO-1 inhibitor protoporphyrin IX ameliorated the symptoms of arthritis. This compound significantly decreased leukocyte infiltration, erosion of articular cartilage, and osteolysis, as well as the production of inflammatory mediators. In this model, HO-1 can be involved in vascular endothelial growth factor production and angiogenesis. These results support a role for HO-1 in mediating the progression of the disease in this model of chronic arthritis [91]. Our research group showed that extra-articular manifestations of AIA are present also in lung, where the expression of heme oxygenase-1 was reduced during AIA [60].
