**3. Hyaluronan in arthritis: regulation of inflammation through antioxidative effects**

### **3.1 The protective properties of hyaluronan**

*Antioxidants*

*\*p < 0.05. \*\*p < 0.01 vs. HC.*

**Table 4.**

*+ p < 0.05. ++p < 0.01 vs. AA.*

nonsignificantly [30].

*and γ-tocopherol (γT) in skeletal muscle mitochondria.*

metalloproteinases which may represent a new approach in the treatment of patients with osteoarthritis. Administration of CoQ10 in the dose 100 mg/kg for 28 days suppressed cartilage degeneration by inhibiting inflammatory mediators and OS in an experimental model of rat osteoarthritis [48]. Beneficial effects of CoQ10 supplementation on inflammatory cytokines and OS in RA patients were proved. In the double-blind, randomized controlled clinical trial in patients with RA, CoQ10 supplementation with 100 mg/day for 2 months led to a significant decrease of malondialdehyde (MDA) formation and a nonsignificant increase of total antioxidant capacity, indicating beneficial effects on OS. CoQ10 also suppressed overexpression of inflammatory cytokines TNF-α significantly and IL-6

*Concentrations of oxidized forms of coenzyme Q9 (CoQ9-OX), coenzyme Q10 (CoQ 10-OX), α-tocopherol (αT),* 

**Mitochondria CoQ 9-ox CoQ 10-ox αT γT**

HC 3.28 ± 0.14 0.144 ± 0.01 0.305 ± 0.02 0.042 ± 0.01 AA 2.67 ± 0.13\*\* 0.126 ± 0.01 0.216 ± 0.02\* 0.058 ± 0.01 AA-CoQ 3.16 ± 0.08++ 0.149 ± 0.01+ 0.289 ± 0.03 0.052 ± 0.01

**nmol/mg prt nmol/mg prt nmol/mg prt nmol/mg prt**

Our results show that administration of CoQ 10 to rats with induced adjuvant arthritis in the oral daily dose of 100 mg/kg b.w. for 28 days partially corrected inflammatory markers and TAS but without statistical significance (**Table 1**). CoQ 10 treatment corrected concentration of CoQ 9 in plasma to control value (**Table 2**). In the skeletal muscle tissue and isolated mitochondria, concentrations of CoQ 9 and CoQ 10 increased in comparison with AA rats and were comparable to controls. Concentrations of αT in tissue and mitochondria were also improved, in the tissue marginally significant and in mitochondria without statistical significance (**Tables 3** and **4**). Sufficient concentrations of CoQ together with αT, the main form of vitamin E, may be important in skeletal muscle function, in regulation of OS and inflammation. The role of vitamin E in regulation of diseases has been extensively studied in humans, animal models, and cell systems. It has been reported that isoforms of vitamin E may have opposing regulatory functions during inflammation, when supplementation with αT was anti-inflammatory and γT pro-inflammatory [33]. Different effects of vitamin E isoforms may result from differences in their metabolism, as αT is preferentially bound α-TTP (α-tocopherol transfer protein), while γT is metabolized mainly through cytochrome P450 and its concentrations in plasma and tissues are dependent on cytochrome P450 metabolism in the liver [49]. Our results show elevated concentrations of γT in plasma and skeletal muscle tissue of arthritic rats together with increased markers of inflammation and decreased TAS (**Tables 1–3**). This confirms the previous findings that inflammation and inhibition of the cytochrome P450 can increase γT concentration [50]. Treatment of arthritic animals with CoQ 10 corrected elevated levels of γT to control values and showed beneficial effect on concentrations of αT, CoQ 9, and CoQ 10 in the skeletal muscle tissue and mitochondria. This can help improve bioenergetic function of the skeletal muscle that is impaired by arthritic inflammatory

**156**

OS is important in the pathogenesis of autoimmune diseases such as RA and in its experimental model-adjuvant arthritis. The control of inflammation and OS in arthritic patients by hyaluronic acid (HA) is one of the approaches to the treatment of RA, concentration of which is reduced in the synovial fluid of patients suffering from arthritis. The most important aspect from a treatment perspective is the fact that HA has been found to be safe and well tolerable. The widespread use of HA also leads to lower use of nonsteroidal anti-inflammatory drugs, which may be an advantage for patients. HA is a high-molecular-weight, ubiquitous glycosaminoglycan (GAG) that naturally occurs within the cartilage and synovial fluid [51]. It is an anionic linear polysaccharide composed of alternating N-acetyl D-glucosamine and D-glucuronic acid residues attached by β(1-4) and β(1-3) glycosidic bonds (**Figure 5**) with molecular mass ranging from 6.5 to 10.9 MDa [52]. It is structurally the simplest compound among GAGs. HA has hydrophilic groups which not only form hydrogen bonds with each other but also interact with water molecules.

In physiological solutions hyaluronan manifests very unusual rheological properties and has exceedingly lubricious and very hydrophilic properties. This is the reason why HA occurs in the salt form, hyaluronate, and is present in every connective tissue and organ such as the skin, synovial fluid, blood vessels, serum, brain, cartilage, heart valves, and the umbilical cord. Synovial fluid in particular has the highest concentration of HA (3–4 mg/ml) compared to anywhere else in the body [53]. HA plays important physiological roles in living organisms which makes it an attractive biomaterial for various medical applications [54, 55]. HA has several diverse physiological functions. Because of its hygroscopic properties, hyaluronan significantly influences hydration and the physical properties of the extracellular matrix. In addition to its function as a passive structural molecule, hyaluronan also acts as a signaling molecule by interacting with cell surface receptors resulting in the activation and modulation of signaling cascades that influence inflammatory processes, including the antioxidant scavenging of the ROS and/or RNS arising from polymorphonuclear nucleosides' respiratory bursts as well as cell migration,

**Figure 5.** *Chemical structure of hyaluronan.*

#### **Figure 6.**

*Biological effects of hyaluronan oligosaccharides depend on their molecular weight.*

proliferation, and gene expression [55]. Moreover, there is a brisk metabolism of HA in humans, with approximately one-third (around 5 g) of total HA were degraded and replaced daily predominantly by the reticuloendothelial system [56]. Many physiological effects of HA may be functions of its molecular weight. Already in the year 2000, Camenisch and McDonald [57] published an overview of the effects of HA, dependent on its molecular weight. HA of an average mass of 0.2 MDa prolonged survival of peripheral blood eosinophils in vitro but HA of the mass of 3–6 MDa had a much lower effect. This observation follows from several previous reports suggesting distinct angiogenic and pro-inflammatory biological activities of lower molecular weight HA or HA oligomers. Lower molecular weight HA, but not high-molecular-weight HA, stimulates the production of metalloelastase and expression of inducible nitric oxide synthase in rat liver endothelial and Kupffer cells. In addition, it has been reported that low-molecular-weight degradation products of HA elicit pro-inflammatory responses by modulating the toll-like receptor-4 or by activating the nuclear factor kappa B (NF-kB). In contrast, high-molecular-weight HA manifests an anti-inflammatory effect via CD receptors and by inhibiting NF-kB activation [58] (**Figure 6**). During progression of inflammation and OS in the joints, HA depolymerizes into lower molecular weight compounds (2.7–4.5 MDa) which consequently diminish the mechanical and viscoelastic properties of the synovial fluid [51] as well as activate different signaling pathways. Randomized, doubleblinded, placebo-controlled trials have proven the effectiveness of HA (administered by the intra-articular injection or in the form of dietary supplements 48–240 mg/day) for the treatment of symptoms associated with synovitis [53].

Kogan et al. [59] suggest mechanisms, by which HA could exert its therapeutic effect: (i) restoration of elastic and viscous properties of the synovial fluid; (ii) induction of the endogenous synthesis of HA by synovial cells by the effect of exogenous HA, stimulation of chondrocyte proliferation, and inhibition of cartilage degradation; (iii) anti-inflammatory action of HA, since the therapy is associated with decreased inflammatory cell count in synovial fluid, modulation of cytokine expression, and reduction of ROS content; and (iv) analgesic effect. An important feature of HA is its antioxidant properties. The direct radical-scavenging properties of HA have been demonstrated in various experimental models. These results are in accord with the concept that hyaluronic acid mainly acts as a chemical ROS

**159**

they received:

**Figure 7.**

*The Role of Endogenous Antioxidants in the Treatment of Experimental Arthritis*

and/or RNS scavenger in extracellular space [55]. In favor of a direct antiradical activity, there is also the ability of hyaluronic acid (biopolymer) to form a viscous, pericellular meshwork that restricts ROS movement in close proximity to cells and thus interferes with the oxidative cascade [55]. The presence of CD44 hyaluronate receptors on the plasma membrane of granulocytes, which mediate the internalization of the biopolymer via endocytosis, offers another key to interpretation of the HA antioxidant mechanism of action, that is, the reduction in ROS and/or RNS is caused by hyaluronic acid internalization and the intracellular neutralization of the radicals [60]. One important pharmacological function of HA is the reduction of cellular superoxide generation and accumulation through nuclear factor erythroid 2-related factor 2 (Nrf2) regulation, which is a master transcription factor in cellular redox reactions. Antioxidants and phase II detoxifying enzymes such as catalase (CAT), superoxide dismutase (SOD), heme oxygenase-1, glutathione S-transferase, glutathione peroxidase (GPx), and thioredoxin are coordinated at transcription level by Nrf2, so the hyaluronic acid could affect the activity and quantity of these

*Hyaluronan increases enzymatic antioxidant defense through Nrf2 upregulation. HA, hyaluronan; AKT, serine/ threonine protein kinase; Nrf2, factor erythroid 2-related factor 2; CAT, catalase; SOD-1, superoxide dismutase-1; GPx-1, glutathione peroxidase; HO-1, hemeoxygenase-1; NQO-1, NAD(P)H (quinone)dehydrogenase 1.*

The aim of this study was to compare the effect of different molecular weights of hyaluronic acid (0.43, 0.99, and 1.73 MDa) applied in two different doses (0.5 and 5 mg/ kg b.w.), on the rat hind paw volume and parameters of OS: activity of antioxidant enzymes in erythrocytes (SOD, GPx, CAT), total antioxidant capacity, and concentration of lipid hydroperoxides (LPx, marker of oxidative damage to lipids) in plasma.

**3.2 Experimental design of adjuvant arthritis with administration of hyaluronan**

Male Lewis rats were randomly divided into groups according to the treatment

*DOI: http://dx.doi.org/10.5772/intechopen.85568*

antioxidant enzymes (**Figure 7**) [61].

(1) Not treated control groups (HC)

(2) Arthritic animals not treated with HA (AA)

*The Role of Endogenous Antioxidants in the Treatment of Experimental Arthritis DOI: http://dx.doi.org/10.5772/intechopen.85568*

#### **Figure 7.**

*Antioxidants*

**Figure 6.**

*Biological effects of hyaluronan oligosaccharides depend on their molecular weight.*

for the treatment of symptoms associated with synovitis [53].

proliferation, and gene expression [55]. Moreover, there is a brisk metabolism of HA in humans, with approximately one-third (around 5 g) of total HA were degraded

Many physiological effects of HA may be functions of its molecular weight. Already in the year 2000, Camenisch and McDonald [57] published an overview of the effects of HA, dependent on its molecular weight. HA of an average mass of 0.2 MDa prolonged survival of peripheral blood eosinophils in vitro but HA of the mass of 3–6 MDa had a much lower effect. This observation follows from several previous reports suggesting distinct angiogenic and pro-inflammatory biological activities of lower molecular weight HA or HA oligomers. Lower molecular weight HA, but not high-molecular-weight HA, stimulates the production of metalloelastase and expression of inducible nitric oxide synthase in rat liver endothelial and Kupffer cells. In addition, it has been reported that low-molecular-weight degradation products of HA elicit pro-inflammatory responses by modulating the toll-like receptor-4 or by activating the nuclear factor kappa B (NF-kB). In contrast, high-molecular-weight HA manifests an anti-inflammatory effect via CD receptors and by inhibiting NF-kB activation [58] (**Figure 6**). During progression of inflammation and OS in the joints, HA depolymerizes into lower molecular weight compounds (2.7–4.5 MDa) which consequently diminish the mechanical and viscoelastic properties of the synovial fluid [51] as well as activate different signaling pathways. Randomized, doubleblinded, placebo-controlled trials have proven the effectiveness of HA (administered by the intra-articular injection or in the form of dietary supplements 48–240 mg/day)

Kogan et al. [59] suggest mechanisms, by which HA could exert its therapeutic effect: (i) restoration of elastic and viscous properties of the synovial fluid; (ii) induction of the endogenous synthesis of HA by synovial cells by the effect of exogenous HA, stimulation of chondrocyte proliferation, and inhibition of cartilage degradation; (iii) anti-inflammatory action of HA, since the therapy is associated with decreased inflammatory cell count in synovial fluid, modulation of cytokine expression, and reduction of ROS content; and (iv) analgesic effect. An important feature of HA is its antioxidant properties. The direct radical-scavenging properties of HA have been demonstrated in various experimental models. These results are in accord with the concept that hyaluronic acid mainly acts as a chemical ROS

and replaced daily predominantly by the reticuloendothelial system [56].

**158**

*Hyaluronan increases enzymatic antioxidant defense through Nrf2 upregulation. HA, hyaluronan; AKT, serine/ threonine protein kinase; Nrf2, factor erythroid 2-related factor 2; CAT, catalase; SOD-1, superoxide dismutase-1; GPx-1, glutathione peroxidase; HO-1, hemeoxygenase-1; NQO-1, NAD(P)H (quinone)dehydrogenase 1.*

and/or RNS scavenger in extracellular space [55]. In favor of a direct antiradical activity, there is also the ability of hyaluronic acid (biopolymer) to form a viscous, pericellular meshwork that restricts ROS movement in close proximity to cells and thus interferes with the oxidative cascade [55]. The presence of CD44 hyaluronate receptors on the plasma membrane of granulocytes, which mediate the internalization of the biopolymer via endocytosis, offers another key to interpretation of the HA antioxidant mechanism of action, that is, the reduction in ROS and/or RNS is caused by hyaluronic acid internalization and the intracellular neutralization of the radicals [60]. One important pharmacological function of HA is the reduction of cellular superoxide generation and accumulation through nuclear factor erythroid 2-related factor 2 (Nrf2) regulation, which is a master transcription factor in cellular redox reactions. Antioxidants and phase II detoxifying enzymes such as catalase (CAT), superoxide dismutase (SOD), heme oxygenase-1, glutathione S-transferase, glutathione peroxidase (GPx), and thioredoxin are coordinated at transcription level by Nrf2, so the hyaluronic acid could affect the activity and quantity of these antioxidant enzymes (**Figure 7**) [61].

The aim of this study was to compare the effect of different molecular weights of hyaluronic acid (0.43, 0.99, and 1.73 MDa) applied in two different doses (0.5 and 5 mg/ kg b.w.), on the rat hind paw volume and parameters of OS: activity of antioxidant enzymes in erythrocytes (SOD, GPx, CAT), total antioxidant capacity, and concentration of lipid hydroperoxides (LPx, marker of oxidative damage to lipids) in plasma.

#### **3.2 Experimental design of adjuvant arthritis with administration of hyaluronan**

Male Lewis rats were randomly divided into groups according to the treatment they received:

	- (a) Group NHA (Mw(HA) = 0.43 MDa, in an oral daily dose of 0.5 mg/kg b.w.)
	- (b) Group 5NHA (Mw(HA) = 0.43 MDa, in an oral daily dose of 5 mg/kg b.w.)
	- (c) Group SHA (Mw(HA) = 0.99 MDa, in an oral daily dose of 0.5 mg/kg b.w.)
	- (d) Group 5SHA (Mw(HA) = 0.99 MDa, in an oral daily dose of 5 mg/kg b.w.)
	- (e) Group VHA (Mw(HA) = 1.79 MDa, in an oral daily dose of 0.5 mg/kg b.w.)
	- (f) Group 5VHA (Mw(HA) = 1.79 MDa, in an oral daily dose of 5 mg/kg b.w.).

Adjuvant arthritis was induced by a single intradermal injection of heat-inactivated *Mycobacterium butyricum* [36, 37]. Blood was collected to obtain plasma and erythrocytes. Total antioxidant capacity and concentration of LPx were determined in plasma. Isolated erythrocytes were washed three times with 0.15 mol/l NaCl solution. After centrifugation (900 × g, 5 min, 4°C), erythrocytes were hemolyzed by adding a triple volume of cold distilled water and stored at −20°C until further analyses. Activities of Cu/Zn-superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), and the concentration of hemoglobin (Hb) were determined in the hemolysate of erythrocytes. From clinical parameters hind paw volume (HPV) was evaluated [62]. The activity of SOD was determined using a commercial kit. The results are expressed in U of SOD per mg Hb. The activity of GPx was also determined by a commercial kit. The results are expressed in μkat per g Hb. CAT activity was determined by a modified method according to [63], and the results are expressed in μkat per g Hb. The total antioxidant capacity of plasma was measured using the Trolox equivalent antioxidant capacity (TEAC) assay [64]. Quantification was performed using the dose-response curve for the reference of antioxidant Trolox, a water-soluble form of vitamin E. The results are presented as mmol of Trolox per ml of plasma. The level of LPx in plasma was measured using the method previously described by [64], and the results are presented in nmol per ml of plasma. The experimental data were expressed as the mean ± SEM. Statistical analysis was performed using Student's t-test. The limit for statistical significance was set at p < 0.05.

#### **3.3 Evaluation of results of administration of hyaluronans in experimental arthritis**

The onset of AA confirmed the increased hind paw volume in arthritic groups (data not shown). HA administration did not cause a significant reduction of HPV in any molecular weight and at any doses used. Parameters of OS are summarized in **Table 5**. Rats with AA had significantly higher activity of SOD and CAT in erythrocytes as well as higher concentration of LPx in comparison to HC group. Activity of GPx was marginally increased (p = 0.054) and TEAC was not changed.

The effect of hyaluronic acid on antioxidant enzyme activities, the total antioxidant capacity of plasma, and the effect on LPx concentration are summarized in **Table 6**. We have found significantly higher erythrocyte SOD activity after administration of HA in all molecular forms and doses, whereas GPx activity was significantly higher only after HA administration at the higher dose. At a lower

**161**

*The Role of Endogenous Antioxidants in the Treatment of Experimental Arthritis*

**Parameter HC AA P** SOD (U/mg Hb) 422.65 ± 15.93 546.48 ± 14.25 0.0001 GPx (μkat/g Hb) 51.10 ± 1.45 56.86 ± 2.26 0.054 CAT (μkat/g Hb) 2.61 ± 0.18 3.06 ± 0.09 0.044 TEAC (mmol/l) 4.01 ± 0.07 4.04 ± 0.09 n.s. LPx (nmol/ml) 20.76 ± 3.55 53.34 ± 5.83 0.003

*Activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in erythrocytes, total antioxidant capacity (TEAC), and concentration of lipoperoxides (LPx) in plasma were measured on the 28th day.*

dose, we observed a significantly elevated GPx activity only in the SHA group (Mw(HA) = 0.99 MDa). For both enzymes, we have also noticed a significant difference between the effects of the same molecular forms of HA but administered at different doses. The higher HA dose (5 mg/kg b.w.) significantly increased the GPx

Subanalysis based on the molecular weight of the administered HA revealed the higher SOD activity in the VHA group compared to NHA (p = 0.0013), in the 5SHA and 5VHA groups compared to 5NHA (p = 0.0025, respectively, p = 0.0308), and VHA compared to SHA (p = 0.0107). The higher GPx activity was found in SHA group in comparison to NHA (p = 0.0425), in 5SHA, respectively, 5VHA in comparison to 5NHA (p = 0.0217 respectively p = 0. 0058), and lower activity in VHA group in comparison to SHA (p = 0.0069). A similar trend was observed in the effect of HA on the total antioxidant capacity of plasma. The values of this parameter were increased in all groups but significantly only when HA was administered

Also, the higher HA dose significantly increased the total antioxidant capacity compared to the lower dose. Differences in the effect of HA with different molecular weights were seen only in the 5VHA group, whereas TEAC was significantly increased when compared to the 5NHA group (p = 0.0212). On the other hand, HA in all molecular weights and at both monitored doses significantly reduced CAT activity. The effect of different doses was found only in SHA and VHA groups, where the higher dose significantly reduced activity in SHA (p = 0.0004) and significantly increased activity in VHA (p = 0.0192). At higher doses, we found significant

reductions of CAT activity in VHA compared to both NHA and SHA (p = 0.0018 and p = 0.0001) and in 5SHA compared to 5NHA (p = 0.0194). Concentration of LPx was significantly reduced in all monitored groups, with no differences in the effect

Our study, for the first time, evaluated the ability of the HA to affect the activity of erythrocyte antioxidant enzymes, as well as total antioxidant capacity and LPx of rats with AA. We have found increased activities of antioxidant enzymes (SOD, GPx and CAT) in erythrocytes of AA rats with increased plasma LPx concentration. Administration of different molecular weights of HA (0.43, 0.99, and 1.73 MDa) applied in two different doses (0.5 and 5 mg/kg b.w.) resulted in a further increase in activities of these enzymes, but we observed a decreased concentration of plasma LPx. Inflammatory diseases, including RA, are characterized by sustained overproduction of ROS, accompanied by disruption of the antioxidant defense system resulting in local and systemic OS development in the affected joint-synovial fluid [65], and in addition to the joints, plasma and some organs are affected [66]. The results of the present work showed that in spite of the increased antioxidant enzyme

of different molecular weights of HA or in the effect of doses.

activity compared to the lower dose (0.5 mg/kg b.w.).

*Control group (HC), arthritis group (AA), statistical significance (P).*

*Oxidative stress markers in rats with adjuvant arthritis.*

at a higher concentration.

**Table 5.**

*DOI: http://dx.doi.org/10.5772/intechopen.85568*


*The Role of Endogenous Antioxidants in the Treatment of Experimental Arthritis DOI: http://dx.doi.org/10.5772/intechopen.85568*

*Control group (HC), arthritis group (AA), statistical significance (P).*

*Activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in erythrocytes, total antioxidant capacity (TEAC), and concentration of lipoperoxides (LPx) in plasma were measured on the 28th day.*

#### **Table 5.**

*Antioxidants*

0.5 mg/kg b.w.)

0.5 mg/kg b.w.)

5 mg/kg b.w.).

(3) Arthritic animals treated with hyaluronic acid (HA) during 28 days:

(a) Group NHA (Mw(HA) = 0.43 MDa, in an oral daily dose of

(c) Group SHA (Mw(HA) = 0.99 MDa, in an oral daily dose of

(f) Group 5VHA (Mw(HA) = 1.79 MDa, in an oral daily dose of

**3.3 Evaluation of results of administration of hyaluronans in experimental** 

GPx was marginally increased (p = 0.054) and TEAC was not changed.

The onset of AA confirmed the increased hind paw volume in arthritic groups (data not shown). HA administration did not cause a significant reduction of HPV in any molecular weight and at any doses used. Parameters of OS are summarized in **Table 5**. Rats with AA had significantly higher activity of SOD and CAT in erythrocytes as well as higher concentration of LPx in comparison to HC group. Activity of

The effect of hyaluronic acid on antioxidant enzyme activities, the total antioxidant capacity of plasma, and the effect on LPx concentration are summarized in **Table 6**. We have found significantly higher erythrocyte SOD activity after administration of HA in all molecular forms and doses, whereas GPx activity was significantly higher only after HA administration at the higher dose. At a lower

Adjuvant arthritis was induced by a single intradermal injection of heat-inactivated *Mycobacterium butyricum* [36, 37]. Blood was collected to obtain plasma and erythrocytes. Total antioxidant capacity and concentration of LPx were determined in plasma. Isolated erythrocytes were washed three times with 0.15 mol/l NaCl solution. After centrifugation (900 × g, 5 min, 4°C), erythrocytes were hemolyzed by adding a triple volume of cold distilled water and stored at −20°C until further analyses. Activities of Cu/Zn-superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), and the concentration of hemoglobin (Hb) were determined in the hemolysate of erythrocytes. From clinical parameters hind paw volume (HPV) was evaluated [62]. The activity of SOD was determined using a commercial kit. The results are expressed in U of SOD per mg Hb. The activity of GPx was also determined by a commercial kit. The results are expressed in μkat per g Hb. CAT activity was determined by a modified method according to [63], and the results are expressed in μkat per g Hb. The total antioxidant capacity of plasma was measured using the Trolox equivalent antioxidant capacity (TEAC) assay [64]. Quantification was performed using the dose-response curve for the reference of antioxidant Trolox, a water-soluble form of vitamin E. The results are presented as mmol of Trolox per ml of plasma. The level of LPx in plasma was measured using the method previously described by [64], and the results are presented in nmol per ml of plasma. The experimental data were expressed as the mean ± SEM. Statistical analysis was performed using Student's t-test. The limit for statistical significance was set at p < 0.05.

(b) Group 5NHA (Mw(HA) = 0.43 MDa, in an oral daily dose of 5 mg/kg b.w.)

(d) Group 5SHA (Mw(HA) = 0.99 MDa, in an oral daily dose of 5 mg/kg b.w.)

(e) Group VHA (Mw(HA) = 1.79 MDa, in an oral daily dose of 0.5 mg/kg b.w.)

**160**

**arthritis**

*Oxidative stress markers in rats with adjuvant arthritis.*

dose, we observed a significantly elevated GPx activity only in the SHA group (Mw(HA) = 0.99 MDa). For both enzymes, we have also noticed a significant difference between the effects of the same molecular forms of HA but administered at different doses. The higher HA dose (5 mg/kg b.w.) significantly increased the GPx activity compared to the lower dose (0.5 mg/kg b.w.).

Subanalysis based on the molecular weight of the administered HA revealed the higher SOD activity in the VHA group compared to NHA (p = 0.0013), in the 5SHA and 5VHA groups compared to 5NHA (p = 0.0025, respectively, p = 0.0308), and VHA compared to SHA (p = 0.0107). The higher GPx activity was found in SHA group in comparison to NHA (p = 0.0425), in 5SHA, respectively, 5VHA in comparison to 5NHA (p = 0.0217 respectively p = 0. 0058), and lower activity in VHA group in comparison to SHA (p = 0.0069). A similar trend was observed in the effect of HA on the total antioxidant capacity of plasma. The values of this parameter were increased in all groups but significantly only when HA was administered at a higher concentration.

Also, the higher HA dose significantly increased the total antioxidant capacity compared to the lower dose. Differences in the effect of HA with different molecular weights were seen only in the 5VHA group, whereas TEAC was significantly increased when compared to the 5NHA group (p = 0.0212). On the other hand, HA in all molecular weights and at both monitored doses significantly reduced CAT activity. The effect of different doses was found only in SHA and VHA groups, where the higher dose significantly reduced activity in SHA (p = 0.0004) and significantly increased activity in VHA (p = 0.0192). At higher doses, we found significant reductions of CAT activity in VHA compared to both NHA and SHA (p = 0.0018 and p = 0.0001) and in 5SHA compared to 5NHA (p = 0.0194). Concentration of LPx was significantly reduced in all monitored groups, with no differences in the effect of different molecular weights of HA or in the effect of doses.

Our study, for the first time, evaluated the ability of the HA to affect the activity of erythrocyte antioxidant enzymes, as well as total antioxidant capacity and LPx of rats with AA. We have found increased activities of antioxidant enzymes (SOD, GPx and CAT) in erythrocytes of AA rats with increased plasma LPx concentration. Administration of different molecular weights of HA (0.43, 0.99, and 1.73 MDa) applied in two different doses (0.5 and 5 mg/kg b.w.) resulted in a further increase in activities of these enzymes, but we observed a decreased concentration of plasma LPx.

Inflammatory diseases, including RA, are characterized by sustained overproduction of ROS, accompanied by disruption of the antioxidant defense system resulting in local and systemic OS development in the affected joint-synovial fluid [65], and in addition to the joints, plasma and some organs are affected [66]. The results of the present work showed that in spite of the increased antioxidant enzyme


*NHA (Mw(HA) = 0.43 MDa, 0.5 mg/kg b.w.); 5NHA (Mw(HA) = 0.43 MDa, 5 mg/kg b.w.); SHA (Mw(HA) = 0.99 MDa, 0.5 mg/kg b.w.); 5SHA (Mw(HA) = 0.99 MDa, 5 mg/kg b.w.); VHA (Mw(HA) = 1.79 MDa, 0.5 mg/kg b.w.); 5VHA (Mw(HA) = 1.79 MDa, 5 mg/kg b.w.).*

*Activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in erythrocytes, total antioxidant capacity (TEAC), and concentration of lipoperoxides (LPx) in plasma were measured on the 28th day.*

**Table 6.**

**163**

tion from early lineages [77].

*The Role of Endogenous Antioxidants in the Treatment of Experimental Arthritis*

activities found in erythrocytes of rats with AA, lipid peroxidation in plasma is increased in comparison to control group. Lipid peroxides are generated at the site of tissue injury due to increased ROS production during chronic inflammation and diffuse into blood where they can be estimated [67]. Studies have reported raised levels of MDA, 4-hydroxynonenal, and other markers of oxidative lipid damage in the serum, plasma, and erythrocytes of RA patients [65, 68]. Kumar et al. [68] also found increased plasma SOD activity in patients with RA, similarly to Mazzetti et al. [69]. Similar results were found in early type 2 diabetes patients [70], where increased antioxidant defense in plasma and erythrocytes is explained as a potential mechanism that can overcome oxidative damage induced by ROS overproduction. There are some reports on erythrocyte SOD, CAT, and GPx activities in patients with RA or in rats with AA, but the results are controversial [71, 72]. It is possible that differences between different investigators' results, regarding antioxidant status, are due to differences in the stage of the disease. Chronic inflammation may deplete antioxidant defenses, whereas acute inflammation can upgrade them [73]. In our study, we did not notice a change in total plasma antioxidant capacity in the group of arthritic rats, similarly to Bracht et al. [66] in the mono-arthritic rats. Vijayakumar et al. [74] confirmed susceptibility of erythrocytes to peroxide stress. They have found not only elevated plasma lipid peroxidation but also the excessive lipid peroxidation in erythrocytes and erythrocyte membranes. In addition, they found decreased glutathione levels and GPx activity in plasma but increase in eryth

rocytes of RA patients as compared to healthy subjects. Superoxide radicals play an important role as a chemical mediator on the inflammatory response to RA. The increased activity of plasma SOD observed in the abovementioned studies as well as increased activity of SOD in erythrocytes observed in our study could therefore be found due to their function in dismutation of superoxide radicals excess. Thus, the activities of antioxidant enzymes in blood cells including erythrocytes could reflect the rate of OS in the affected cells. This could be a suitable approach for assessing

Our study demonstrated that HA (in all molecular weights and in both doses),

ers [61]. Supplementary to its primary role in cytoprotection, Nrf2 is also linked to differentiation, proliferation, growth, and apoptosis, and it is thought that Nrf2 has evolved from an original role in hematopoiesis and the regulation of cell differentia

Based on this, we could assume that during proliferation and differentiation of hematopoietic stem cells, expression of antioxidant enzymes can be induced. In the induction of expression, the CD44 receptor, which binds HA and mediates its role as a signal molecule, could have importance. We could just speculate if the activities of antioxidant enzymes in erythrocytes reflect the effect of administered HA on the activities of these enzymes in other tissues as well. As we observed an increase in both SOD and GPx activities in erythrocytes of AA rats under HA supplementation, we anticipate a similar mechanism of HA action in other cells, e.g., in chondrocytes, where the increase in antioxidant potential could provide antioxidant protection

orally administrated in a rat model of AA, affected all measured markers of OS. Furthermore, erythrocyte antioxidant markers including SOD and GPx, and total antioxidant capacity of plasma, increased significantly during 28 days of supplementation. On the other hand, we have found decreased erythrocyte CAT activity and plasma concentration of LPx. Based on our results, we cannot give a clear explanation how the HA can affect all observed parameters. Numerous studies have confirmed the effect of HA on the activity of these enzymes but in other cells and tissues and not in erythrocytes [75, 76]. It was confirmed that HA can reduce cellular superoxide generation and its accumulation through Nrf2 regulation which can induce transcription of antioxidant enzymes such as SOD, GPx, CAT, and oth

the effect of therapy aimed to reduce inflammation and OS.




*DOI: http://dx.doi.org/10.5772/intechopen.85568*

*Effect of hyaluronic acid on oxidative stress markers.*

#### *The Role of Endogenous Antioxidants in the Treatment of Experimental Arthritis DOI: http://dx.doi.org/10.5772/intechopen.85568*

activities found in erythrocytes of rats with AA, lipid peroxidation in plasma is increased in comparison to control group. Lipid peroxides are generated at the site of tissue injury due to increased ROS production during chronic inflammation and diffuse into blood where they can be estimated [67]. Studies have reported raised levels of MDA, 4-hydroxynonenal, and other markers of oxidative lipid damage in the serum, plasma, and erythrocytes of RA patients [65, 68]. Kumar et al. [68] also found increased plasma SOD activity in patients with RA, similarly to Mazzetti et al. [69]. Similar results were found in early type 2 diabetes patients [70], where increased antioxidant defense in plasma and erythrocytes is explained as a potential mechanism that can overcome oxidative damage induced by ROS overproduction. There are some reports on erythrocyte SOD, CAT, and GPx activities in patients with RA or in rats with AA, but the results are controversial [71, 72]. It is possible that differences between different investigators' results, regarding antioxidant status, are due to differences in the stage of the disease. Chronic inflammation may deplete antioxidant defenses, whereas acute inflammation can upgrade them [73].

In our study, we did not notice a change in total plasma antioxidant capacity in the group of arthritic rats, similarly to Bracht et al. [66] in the mono-arthritic rats. Vijayakumar et al. [74] confirmed susceptibility of erythrocytes to peroxide stress. They have found not only elevated plasma lipid peroxidation but also the excessive lipid peroxidation in erythrocytes and erythrocyte membranes. In addition, they found decreased glutathione levels and GPx activity in plasma but increase in erythrocytes of RA patients as compared to healthy subjects. Superoxide radicals play an important role as a chemical mediator on the inflammatory response to RA. The increased activity of plasma SOD observed in the abovementioned studies as well as increased activity of SOD in erythrocytes observed in our study could therefore be found due to their function in dismutation of superoxide radicals excess. Thus, the activities of antioxidant enzymes in blood cells including erythrocytes could reflect the rate of OS in the affected cells. This could be a suitable approach for assessing the effect of therapy aimed to reduce inflammation and OS.

Our study demonstrated that HA (in all molecular weights and in both doses), orally administrated in a rat model of AA, affected all measured markers of OS. Furthermore, erythrocyte antioxidant markers including SOD and GPx, and total antioxidant capacity of plasma, increased significantly during 28 days of supplementation. On the other hand, we have found decreased erythrocyte CAT activity and plasma concentration of LPx. Based on our results, we cannot give a clear explanation how the HA can affect all observed parameters. Numerous studies have confirmed the effect of HA on the activity of these enzymes but in other cells and tissues and not in erythrocytes [75, 76]. It was confirmed that HA can reduce cellular superoxide generation and its accumulation through Nrf2 regulation which can induce transcription of antioxidant enzymes such as SOD, GPx, CAT, and others [61]. Supplementary to its primary role in cytoprotection, Nrf2 is also linked to differentiation, proliferation, growth, and apoptosis, and it is thought that Nrf2 has evolved from an original role in hematopoiesis and the regulation of cell differentiation from early lineages [77].

Based on this, we could assume that during proliferation and differentiation of hematopoietic stem cells, expression of antioxidant enzymes can be induced. In the induction of expression, the CD44 receptor, which binds HA and mediates its role as a signal molecule, could have importance. We could just speculate if the activities of antioxidant enzymes in erythrocytes reflect the effect of administered HA on the activities of these enzymes in other tissues as well. As we observed an increase in both SOD and GPx activities in erythrocytes of AA rats under HA supplementation, we anticipate a similar mechanism of HA action in other cells, e.g., in chondrocytes, where the increase in antioxidant potential could provide antioxidant protection

*Antioxidants*

**162**

**Parameter** SOD (U/mg Hb)

GPx (μkat/g Hb)

CAT (μkat/g Hb)

TEAC (mmol/l)

LPx (nmol/ml)

*\*p < 0.05.*

*\*\*p < 0.01.*

*\*\*\*p < 0.001 vs. AA.*

*(Mw(HA) = 1.79 MDa, 0.5 mg/kg b.w.); 5VHA (Mw(HA) = 1.79 MDa, 5 mg/kg b.w.).*

*measured on the 28th day.*

**Table 6.**

*Effect of hyaluronic acid on oxidative stress markers.*

**AA** 546.48 ± 14.25

56.86 ± 2.26 3.06 ± 0.09 4.04 ± 0.09 53.34 ± 5.83

4.23 ± 0.09 24.35 ± 1.19\*\*

55.53 ± 3.297

2.61 ± 0.15\*

**NHA** 615.26 ± 23.00\*

**5NHA** 817.37 ± 28.45\*\*\*

71.17 ± 3.76\*\* 2.40 ± 0.08\*\*\*

4.57 ± 0.03\*\* 26.84 ± 1.70\* *NHA (Mw(HA) = 0.43 MDa, 0.5 mg/kg b.w.); 5NHA (Mw(HA) = 0.43 MDa, 5 mg/kg b.w.); SHA (Mw(HA) = 0.99 MDa, 0.5 mg/kg b.w.); 5SHA (Mw(HA) = 0.99 MDa, 5 mg/kg b.w.); VHA* 

*Activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in erythrocytes, total antioxidant capacity (TEAC), and concentration of lipoperoxides (LPx) in plasma were* 

661.41 ± 24.34\*\*

64.95 ± 2.65\*

2.81 ± 0.12 4.07 ± 0.10 25.3 ± 4.67\*\*

**SHA**

**5SHA** 944.73 ± 41.35\*\*\*

84.89 ± 4.20\*\*\*

2.06 ± 0.10\*\*\*

4.80 ± 0.24\*\* 27.98 ± 3.45\*

**VHA** 777.17 ± 30.91\*\*\*

55.44 ± 1.40 2.02 ± 0.05\*\*\*

4.19 ± 0.10 25.13 ± 5.53\*\*

**5VHA**

869.42 ± 34.96\*\*\*

93.52 ± 6.83\*\*\*

2.28 ± 0.09\*\*\*

4.73 ± 0.06\*\*

24.49 ± 2.67\*

of synovial fluid and reduction of lipoperoxidation not only in the synovium but also in the plasma as what we have found in our study. Also, the direct antioxidant ability of HA, which has been described, could contribute to the reduction of lipoperoxidation [55]. However, further studies need to be made to confirm these assumptions.
