**2.2 Experimental design of adjuvant arthritis with administration of coenzyme Q10**

Adjuvant arthritis (AA) was induced by intradermal injection of *Mycobacterium butyricum* in incomplete Freund's adjuvant to male Lewis rats [36, 37]. The experiment included healthy control animals (HC), arthritic animals (AA), and arthritic animals with administration of CoQ10 (liquid liposomal CoQ10—LiQSorb®) in the oral daily dose of 100 mg/kg b.w. (AA-CoQ ) by the use of gavage. The analyses were performed 28 days after the arthritis induction and in the beginning of CoQ10 supplementation. Concentrations of CoQ9, CoQ10, αT, and γT were determined by HPLC method with spectrophotometric detection at 275 nm (CoQ ) and 295 nm (tocopherols), using external standards [38, 39]. Total CoQ9 and CoQ10 (oxidized and reduced forms) in plasma was measured after oxidation with 1,4-benzoquinone [40]. Mitochondria from hind paw skeletal muscle tissue were isolated by means of differential centrifugation according to slightly modified methods [41, 42]. Mitochondrial proteins were estimated spectrophotometrically [43]. Data were collected and processed using CSW 32 chromatographic station (DataApex Ltd). Concentrations were calculated: in the plasma in μmol/l, in the tissue in nmol/g of wet weight, and in the mitochondria in nmol/mg of proteins. Total antioxidant status (TAS) in plasma was determined using the Randox Total Antioxidant Status kit with colorimetric detection at 600 nm. Markers of inflammation, C-reactive protein (CRP), and monocyte chemotactic protein-1 (MCP-1) were measured by ELISA. Data are expressed as mean ± SEM. Statistical significance between experimental groups was evaluated using Student's t-test, p < 0.05, which was considered as a significant result.

## **2.3 Evaluation of results of administration of coenzyme Q10 in experimental arthritis**

AA for a period of 28 days significantly increased markers of inflammation—CRP and MCP-1—and decreased TAS (**Table 1**). Concentrations of total CoQ9 (oxidized and reduced) and γT in plasma of AA rats increased significantly (**Table 2**).

In skeletal muscle tissue and mitochondria of AA rats, concentrations of oxidized form of coenzyme Q9 (CoQ9-OX) and αT decreased significantly and CoQ10-OX only slightly. Tissue γT increased compared to controls; in mitochondria the increase was marginally significant (p = 0.077), (**Tables 3** and **4**). Treatment of arthritic rats with CoQ10 (AA-CoQ) for 28 days partially suppressed inflammatory markers and increased TAS, but not statistically significant (**Table 1**). Elevated concentrations of total CoQ9 and γT in plasma were corrected to control values. Concentration of CoQ10 in plasma increased extremely, demonstrating a good bioavailability of CoQ10 administered (**Table 2**). In tissue and mitochondria, concentrations of CoQ9 and CoQ10 increased in comparison with AA rats and were comparable to controls. Concentrations of αT in tissue and mitochondria also increased, in the tissue at the limit of significance (p = 0.071) and in mitochondria without statistical significance (**Tables 3** and **4**).

Bioenergetic and antioxidant properties of CoQ10 are sufficiently described [44]. However, new research findings suggest that CoQ10 supplementation has also lowering effects on circulating inflammatory mediators, including CRP,

**155**

number of studies [45].

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

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

*Markers of inflammation: C-reactive protein (CRP), monocyte chemotactic protein (MCP-1),* 

**Plasma CoQ9-TOT CoQ10-TOT αT γT**

*Concentrations of total coenzyme Q9 (CoQ9-TOT), total coenzyme Q10 (CoQ10-TOT), α-tocopherol (αT),* 

**Tissue CoQ9-ox CoQ 10-ox αT γT**

HC 43.1 ± 3.01 1.90 ± 0.160 23.0 ± 1.23 0.98 ± 0.042 AA 32.7 ± 2.49\* 1.63 ± 0.187 18.7 ± 0.829\* 1.39 ± 0.155\* AA-CoQ 40.9 ± 4.07 2.43 ± 2.52+ 22.2 ± 1.42 1.07 ± 0.084

HC 0.328 ± 0.023 0.031 ± 0.004 19.9 ± 1.13 0.643 ± 0.051 AA 0.468 ± 0.044\*\* 0.027 ± 0.003 21.6 ± 0.72 0.834 ± 0.060\* AA-CoQ 0.237 ± 0.016++ 0.804 ± 0.069++ 19.6 ± 1.07 0.678 ± 0.043+

**Plasma CRP MCP-1 TAS**

HC 457.4 ± 21.3 1462 ± 159.2 0.673 ± 0.037 AA 602.2 ± 10.7\*\* 2925 ± 389.1\*\* 0.529 ± 0028\* AA-CoQ 563.3 ± 14.5 2539 ± 144.1 0.562 ± 0.033

**μg/ml pg/ml mmol/l**

**μmol/l μmol/l μmol/l μmol/l**

**nmol/g ww nmol/g ww nmol/g ww nmol/g ww**

interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α). Meta-analysis of clinical randomized controlled trials evaluated the effects of CoQ10 in some inflammatory diseases but with inconsistent results due to heterogeneity and limited

*Concentrations of oxidized forms of coenzyme Q9 (CoQ9-OX), coenzyme Q10 (CoQ10-OX), α-tocopherol (αT),* 

In an experimental study, an antiarthritic effect of CoQ10 against induced gouty

arthritis in rats was found [46]. CoQ10 treatment at the dosage 10 mg/kg/body weight for 3 days reduced paw edema, minimized lysosomal enzyme release, boosted antioxidant system, and suppressed lipid peroxidation. Protective mechanism of CoQ10 against cartilage degeneration induced by interleukin-1β was studied on isolated rat chondrocytes [47]. The study demonstrated the anticatabolic and cartilage protective potentials of CoQ10 by inhibition of overexpression of matrix

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

*and total antioxidant status (TAS) in plasma.*

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

**Table 1.**

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

**Table 2.**

*\*p < 0.05 vs. HC.*

*p < 0.05 vs. AA.*

*+*

**Table 3.**

*and γ-tocopherol (γT) in plasma.*

*+ p < 0.05. ++p < 0.01 vs. AA.* *The Role of Endogenous Antioxidants in the Treatment of Experimental Arthritis DOI: http://dx.doi.org/10.5772/intechopen.85568*


#### **Table 1.**

*Antioxidants*

**of coenzyme Q10**

as a significant result.

**arthritis**

(**Tables 3** and **4**).

other endogenous antioxidants have been considered as a risk factor for the development of RA [35]. We hypothesized that administration of CoQ10 could affect inflammation in arthritic rats by regulating the endogenous antioxidants and OS.

Adjuvant arthritis (AA) was induced by intradermal injection of *Mycobacterium butyricum* in incomplete Freund's adjuvant to male Lewis rats [36, 37]. The experiment included healthy control animals (HC), arthritic animals (AA), and arthritic animals with administration of CoQ10 (liquid liposomal CoQ10—LiQSorb®) in the oral daily dose of 100 mg/kg b.w. (AA-CoQ ) by the use of gavage. The analyses were performed 28 days after the arthritis induction and in the beginning of CoQ10 supplementation. Concentrations of CoQ9, CoQ10, αT, and γT were determined by HPLC method with spectrophotometric detection at 275 nm (CoQ ) and 295 nm (tocopherols), using external standards [38, 39]. Total CoQ9 and CoQ10 (oxidized and reduced forms) in plasma was measured after oxidation with 1,4-benzoquinone [40]. Mitochondria from hind paw skeletal muscle tissue were isolated by means of differential centrifugation according to slightly modified methods [41, 42]. Mitochondrial proteins were estimated spectrophotometrically [43]. Data were collected and processed using CSW 32 chromatographic station (DataApex Ltd). Concentrations were calculated: in the plasma in μmol/l, in the tissue in nmol/g of wet weight, and in the mitochondria in nmol/mg of proteins. Total antioxidant status (TAS) in plasma was determined using the Randox Total Antioxidant Status kit with colorimetric detection at 600 nm. Markers of inflammation, C-reactive protein (CRP), and monocyte chemotactic protein-1 (MCP-1) were measured by ELISA. Data are expressed as mean ± SEM. Statistical significance between experimental groups was evaluated using Student's t-test, p < 0.05, which was considered

**2.3 Evaluation of results of administration of coenzyme Q10 in experimental** 

and reduced) and γT in plasma of AA rats increased significantly (**Table 2**).

AA for a period of 28 days significantly increased markers of inflammation—CRP and MCP-1—and decreased TAS (**Table 1**). Concentrations of total CoQ9 (oxidized

In skeletal muscle tissue and mitochondria of AA rats, concentrations of oxidized form of coenzyme Q9 (CoQ9-OX) and αT decreased significantly and CoQ10-OX only slightly. Tissue γT increased compared to controls; in mitochondria the increase was marginally significant (p = 0.077), (**Tables 3** and **4**). Treatment of arthritic rats with CoQ10 (AA-CoQ) for 28 days partially suppressed inflammatory markers and increased TAS, but not statistically significant (**Table 1**). Elevated concentrations of total CoQ9 and γT in plasma were corrected to control values. Concentration of CoQ10 in plasma increased extremely, demonstrating a good bioavailability of CoQ10 administered (**Table 2**). In tissue and mitochondria, concentrations of CoQ9 and CoQ10 increased in comparison with AA rats and were comparable to controls. Concentrations of αT in tissue and mitochondria also increased, in the tissue at the limit of significance (p = 0.071) and in mitochondria without statistical significance

Bioenergetic and antioxidant properties of CoQ10 are sufficiently described [44]. However, new research findings suggest that CoQ10 supplementation has also lowering effects on circulating inflammatory mediators, including CRP,

**2.2 Experimental design of adjuvant arthritis with administration** 

**154**

*Markers of inflammation: C-reactive protein (CRP), monocyte chemotactic protein (MCP-1), and total antioxidant status (TAS) in plasma.*


#### **Table 2.**

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


#### **Table 3.**

*Concentrations of oxidized forms of coenzyme Q9 (CoQ9-OX), coenzyme Q10 (CoQ10-OX), α-tocopherol (αT), and γ-tocopherol (γT) in the skeletal muscle tissue.*

interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α). Meta-analysis of clinical randomized controlled trials evaluated the effects of CoQ10 in some inflammatory diseases but with inconsistent results due to heterogeneity and limited number of studies [45].

In an experimental study, an antiarthritic effect of CoQ10 against induced gouty arthritis in rats was found [46]. CoQ10 treatment at the dosage 10 mg/kg/body weight for 3 days reduced paw edema, minimized lysosomal enzyme release, boosted antioxidant system, and suppressed lipid peroxidation. Protective mechanism of CoQ10 against cartilage degeneration induced by interleukin-1β was studied on isolated rat chondrocytes [47]. The study demonstrated the anticatabolic and cartilage protective potentials of CoQ10 by inhibition of overexpression of matrix


**Table 4.**

*Concentrations of oxidized forms of coenzyme Q9 (CoQ9-OX), coenzyme Q10 (CoQ 10-OX), α-tocopherol (αT), 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 nonsignificantly [30].

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

**157**

**Figure 5.**

*Chemical structure of hyaluronan.*

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

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

process. Elevated concentrations of endogenous antioxidants can contribute to

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,

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

**3.1 The protective properties of hyaluronan**

regulation of oxidative stress.

**antioxidative effects**

process. Elevated concentrations of endogenous antioxidants can contribute to regulation of oxidative stress.
