**3.3 Endogenous antioxidants**

248 Rheumatoid Arthritis – Treatment

inhibitory effect of IMG became stronger with time. After IMG treatment, the MCP-1 level was also decreasing significantly on days 14 and 21 (Table 7). The time course of the MCP-1 level was found to be comparable for treated and untreated arthritis animals. The level of cytokine IL-4 was increasing with time – the maximum was observed on day 28 in AA animals. IMG exerted probably an indirect time dependent inhibitory effect on this cytokine

**(pg/ml) Day 14 Day 21 Day 28** 

**CO** 229.44±4.87 N.M. N.M.

Table 7. Plasma level of MCP-1 (monocyte chemotactic protein-1) in an experiment with Imunoglukan® (IMG) analyzed in time profile. For statistical analysis of data see table 1.

**(pg/ml) Day 14 Day 21 Day 28** 

**CO** 10.50±4.90 N.M. N.M.

Table 8. Plasma level of IL-4 (interleukin-4) in an experiment with Imunoglukan® (IMG)

 **and methotrexate combination in AA** 

On the basis of all the obtained results with GM and IMG, we finally chose IMG for

The study of Rovensky et al (Rovensky et al., 2011) was focused on the effect of IMG on inflammatory and arthritic markers in rats with AA during basal treatment with MTX. The treatment was prophylactic, which means that the animals were treated immediately after administration of the adjuvant, with the same design as used in our previous experiments. The results of our investigation confirmed the already reported effect of MTX treatment in rats with AA (Connolly et al., 1988; Welles et al., 1985). MTX at a dose of 1 mg/kg/week (0.5 mg/kg twice a week) suppressed but did not prevent arthritis development. In our study, MTX significantly suppressed the hind paw swelling and decreased arthrogram scores. IMG alone decreased both the hind paw swelling and the arthrogram on days 21 and 28. The remarkable finding was that IMG potentiated the beneficial effect of MTX; reduction of hind paw swelling and arthrogram scores on days 21 and 28 were more significant compared to

559.52±98.22 \*\*\*

363.28±89.57

\*

+

363.28±89.57 \*

40.43±4.58 \*\*\*

7.70±3.85 +++

<sup>+</sup> 287.42±108.02

(Table 8).

N.M. - not measured

N.M. - not measured

**3.2.1 Imunoglukan®**

**MCP-1** 

**IL-4** 

combinatory treatment of AA with MTX.

**AA** 360.62±51.26

**AA-IMG** 187.42±28.06

\*\*

++

**AA** 25.00±10.75 31.78±11.26

**AA-IMG** 23.05±4.50 10.15±4.15

analyzed in time profile. For statistical analysis of data see table 1.

Inflammation is one of the leading causes of mortality in the western world. Much evidence suggests a major role for dysregulation of the immune response to toxic stress (Itoh et al., 2003; Lynn & Golenbock, 1992). The intensive production of ROS associated with inflammation generally results in OS (Macdonald et al., 2003). Under conditions of high OS, the abilities of cells to eliminate ROS become exhausted, and dietary sources of antioxidants are requirered (Novoselova et al., 2009). We studied two important endogenous antioxidants – coenzyme Q10 (CoQ) and carnosine (CARN) as supplementary therapy in AA with the aim to contribute to the alternatives for dietetary complementary healing of RA. In Figure 3, two dose of CoQ - 20 (CoQ1) and 200 mg/kg b.w. (CoQ2) are compared with one dose of CARN – 150 mg/kg b.w. The experimental and statistical design was the same as described in section 3.1.

Fig. 3. Comparison of the effect of carnosine (CARN) and two different doses of coenzyme Q10 (CoQ) used as monotherapy in adjuvant arthritis (AA) on hind paw volume (HPV), GGT (-glutamyltransferase) activity in spleen and joint and level of TBARS (thiobarbituric acid reactive substances) in plasma measured on experimental day 28. Changes in parameters are illustrated in relation to untreated arthritic rats (100% represented by dotand-dash line). For statistical analysis of data see Fig. 1.

Modern Pharmacological Approaches to Therapies:

et al., 2010).

**Activity of GGT in joint (†)** 

**Protein carbonyls in plasma (nmol/mg protein)** 

**Protein carbonyls in brain tissue (nmol/mg protein)** 

**TBARS** 

† - nmol 4-nitroaniline /min /g tissue

For statistical analysis of data see table 1.

where IL-1 and MCP-1 are involved.

Substances Tested in Animal Models of Rheumatoid Arthritis 251

In the present experiment, the GGT activity was elevated in peripheral joint tissue. This finding is in good agreement with clinical studies of patients with RA who had increased levels of GGT not only in serum and urine but also in synovial fluid (Rambabu et al., 1990). CARN effectively reduced the activity of GGT in joint. Administration of CARN lowered the level of secondary products of lipid peroxidation in plasma measured as TBARS. Cheng (Cheng et al., 2011) showed that CARN, but not other conventional antioxidants, could protect neurons against MDA-induced injury through decomposition of protein crosslinking and may serve as a novel agent in the treatment of neurodegenerative diseases. The "anti-carbonyl" effect of CARN administration was also evidenced by other authors (Aldini

**Parameters CO AA AA-CARN** 

11.03±1.81 26.30±2.04

0.15±0.01 0.20±0.01

10.68±0.78 17.57±0.96

Table 10. Activity of GGT(-glutamyltransferase) in joint, plasmatic TBARS (thiobarbituric

experimental day 28 in an experiment with carnosine (CARN) evaluated as monotherapy.

We found that CARN was effective in decreasing protein carbonyls in plasma as well as in brain tissue homogenate of arthritic rats. These findings might provide, at least partially, an explanation for the antiinflammatory activity of CARN in chronic autoimmune disease, such as RA. The action of CARN resulted in decreased systemic inflammation in AA, monitored by plasmatic level of proinflammatory cytokine IL-1α and chemokine MCP-1. CARN was also effective in reducing the MCP-1 level in plasma in our experiment, suggesting that it may have a good potential in the treatment of chronic inflammatory diseases including RA

acid reactive substances), and protein carbonyls in plasma and brain measured on

**(nmol/ml)** 1.60±0.12 2.38±0.10

\*\*\*

\*\*

\*\*\*

\*\*

21.22±1.28 +

0.14±0.02 +

12.98±0.53 +

1.44±0.12 ++

Both antioxidants tended to improve HPV (not significantly) and significantly corrected the parameters of OS. No dose dependency was shown with exception of CoQ influence on GGT in spleen. In the next experiments we evaluated CARN in monotherapy of AA and CoQ in the lower dose for combinatory therapy with MTX in AA. The obtained results are described below.
