**3.2 Natural substances isolated from yeast and mushrooms**

The control of inflammation in RA patients by natural and synthetic substances with antiinflammatory and/or antioxidant and immunomodulatory effects, which are safe also during long-term administration, could become a relevant part of RA therapy. Modulation of OS accompanying RA can offer a new approach and crucially modify treatment of this disease. The key goal of this proposal will be the investigation of the combination of immunosuppressive therapy of MTX with immunomodulators-antioxidants with the aim to achieve enhancement of its efficacy in RA treatment, which would enable dosage reduction in clinical conditions and, consequently, decrease the frequency of occurrence of its dosedependent side effects. Thus new ways of supplementary or combinatory RA therapy are of great importance. The aim is to find an alternative or additive to classical RA therapy with natural molecules without side effects possessing immunomodulatory, antiinflammatory, and antioxidative properties.

In recent decades, polysaccharides isolated from botanical sources (mushrooms, algae, lichens, and higher plants) have attracted a great deal of attention in the biomedical arena because of their broad spectrum of therapeutic properties and relatively low toxicity (Tzianabos, 2000). Plant and mushroom polysaccharides reveal immunomodulatory effects that depend on polysaccharide structure and molecular weight (low molecular weight – inhibition, high molecular weight – activation) (Schepetkin & Quinn, 2006). Prokopova (Prokopova et al., 1993) were the first to describe a therapeutic effect of simple carbohydrates on AA. We were first to report on the protective antioxidant and antiinflammatory activities of carboxylated (1-3)-beta-D-glucan isolated from *Saccharomyces cerevisiae* in Lewis rats with AA (Kogan et al., 2005). Glucomannans (GM) from *Candida utilis* were evaluated in the same model. The antiarthritic activity for cell-wall GM was associated with antioxidant activity *in vivo* (Bauerova et al., 2006; Mihalova et al., 2007). In the following experiment, the beneficial action of GM was revealed mainly in HPV decrease. Further a decrease of the activity of GGT in the spleen, hind paw joint and muscle tissue homogenates, decrease of the plasmatic activity of N-acetyl-beta-D-glucosaminidase (NAGA), and finally suppression of lysozyme and peroxidase activity assessed in peritoneal macrophages were observed in arthritic animals treated with GM. All these findings speak in favor of the antiinflammatory activity of GM. Moreover, a significant improvement of the arthritis induced suppression of total antioxidant status and decrease of the level of the arthritis-associated protein carbonyls in plasma were detected. In this experiment two doses of GM – 5 and 7.5 mg/kg b.w. were evaluated successfully. Peroral and intraperitoneal ways of administration were also compared (Bauerova et al., 2008b). In the following study, we tested the effect of GM in a higher dose of 15 mg/kg b.w. administered per os. On day 28 after *Mycobacterium butyricum* induced AA, GM was found to reduce HPV. Neutrophil count in whole blood was significantly increased on day 28 after induction of AA, yet GM in the dose of 15 mg/kg b.w. did not change it significantly. The spontaneous and PMA-induced CL was significantly increased in whole blood of rats with AA in comparison with healthy

Modern Pharmacological Approaches to Therapies:

**HPV (%)** 17.20±1.81 91.45±10.61

**TAS (mmol/l)** 1.02±0.21 0.52±0.20

**(nmol/ml)** 3.50±0.20 5.90±0.20

**AA** 732.90±128.56

+

++

Table 6. Plasma level of TNF- (tumor necrosis factor-) in an experiment with

Imunoglukan® (IMG) analyzed in time profile. For statistical analysis of data see table 1.

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

**AA-IMG** 339.80±117.40

**AA** 202.10±26.23

**AA-IMG** 98.58±27.43

28, IMG was more effective than GM.

**TBARS** 

statistical analysis of data see table 1.

**IL-1α**

**TNF-α**

N.M. - not measured

N.M. - not measured

Substances Tested in Animal Models of Rheumatoid Arthritis 247

TBARS accompanied with a more pronounced increase of TAS measured in plasma on day

**Parameters CO AA AA-GM AA-IMG** 

\*\*\*

\*

\*\*\*

Table 4. Changes in hind paw volume (HPV), plasmatic TAS (total antioxidant status) and

The good antioxidative and antiinflammatory effect of IMG stimulated us to study in a more complex way its effect on the course of the main cytokines/chemokines in AA. Daily administration of IMG suppressed significantly the levels of pro-inflammatory cytokines IL-1α (Table 5) and TNF-α (Table 6) on all days monitored. Moreover, the observed

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

**CO** 186.44±77.42 N.M. N.M.

Table 5. Plasma level of IL-1 (interleukin-1) in an experiment with Imunoglukan® (IMG)

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

**CO** 59.64±13.58 N.M. N.M.

\*\*\* 588.58±272.50 633.06±117.40

\*\*\* 100.15±28.35 100.65±42.85

49.50±4.30 +

168.88±38.98 +

plasmatic TBARS (thiobarbituric acid reactive substances) in an experiment with glucomannan (GM) and Imunoglukan® (IMG) measured on experimental day 28. For

66.57±3.84 +

54.50±7.77 +

+

++

\*

184.40±18.44 +

29.45±2.90 +

0.89±0.18 1.16±0.13

5.70±0.30 4.70±0.30

controls. GM 15 mg/kg b.w. decreased spontaneous as well as PMA-stimulated CL. CL of spleen and joint in rats with AA was significantly increased in comparison with controls (3.38±1.07 mV/1mg wet weight *vs.* 1.33±0.16 mV/1mg wet weight, and 6.63±1.34 mV/100mg wet weight *vs.* 1.11±0.11 mV/100mg wet weight). GM significantly decreased CL of joints, while CL of the spleen was not affected by GM. The obtained results showed that GM reduced ROS generation in arthritic rats. The predominant decrease of extracellular ROS production suggests a protective effect of GM against tissue damage, especially in the hind paw joint of arthritic rats (Drabikova et al., 2009).

Further we decided to compare the effect of GM to Imunoglukan®, a beta-(1,3/1,6)-D-glucan (IMG), which was isolated from *Pleurotus ostreatus.* Figure 2 shows a comparison of the effect of GM and IMG on HPV as well as on OS parameters. GM was tested in two doses: 7.5 (GM1) and 15 mg/kg b.w (GM2). IMG was evaluated also in two doses: 1 (IMG1) and 2 mg/kg b.w. (IMG2). The experimental and statistical design was the same as described in section 3.1. of this chapter. Both GM as IMG were effective in reducing HPV and improving the oxidative status. As no clear dose dependency was shown, we chose for the next experiment higher doses with the aim to detect the more effective immunomodulator for combinatory therapy with MTX. The experiment included healthy intact animals as reference controls (CO), arthritic animals without any drug administration (AA), and arthritic animals with the administration of GM (AA-GM) in the oral daily dose of 15 mg/kg b.w. and of IMG (AA-IMG) in the oral daily dose of 2 mg/kg b.w. Table 4 shows that as to the capability of lowering the HPV no differences between GM and IMG were found. However, as to the antioxidant potential expressed as a more prominent decrease of

Fig. 2. Comparison of the effect of different glucomannan (GM) and Imunoglukan® (IMG) doses 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 dot-and-dash line). For statistical analysis of data see Fig. 1.

controls. GM 15 mg/kg b.w. decreased spontaneous as well as PMA-stimulated CL. CL of spleen and joint in rats with AA was significantly increased in comparison with controls (3.38±1.07 mV/1mg wet weight *vs.* 1.33±0.16 mV/1mg wet weight, and 6.63±1.34 mV/100mg wet weight *vs.* 1.11±0.11 mV/100mg wet weight). GM significantly decreased CL of joints, while CL of the spleen was not affected by GM. The obtained results showed that GM reduced ROS generation in arthritic rats. The predominant decrease of extracellular ROS production suggests a protective effect of GM against tissue damage, especially in the

Further we decided to compare the effect of GM to Imunoglukan®, a beta-(1,3/1,6)-D-glucan (IMG), which was isolated from *Pleurotus ostreatus.* Figure 2 shows a comparison of the effect of GM and IMG on HPV as well as on OS parameters. GM was tested in two doses: 7.5 (GM1) and 15 mg/kg b.w (GM2). IMG was evaluated also in two doses: 1 (IMG1) and 2 mg/kg b.w. (IMG2). The experimental and statistical design was the same as described in section 3.1. of this chapter. Both GM as IMG were effective in reducing HPV and improving the oxidative status. As no clear dose dependency was shown, we chose for the next experiment higher doses with the aim to detect the more effective immunomodulator for combinatory therapy with MTX. The experiment included healthy intact animals as reference controls (CO), arthritic animals without any drug administration (AA), and arthritic animals with the administration of GM (AA-GM) in the oral daily dose of 15 mg/kg b.w. and of IMG (AA-IMG) in the oral daily dose of 2 mg/kg b.w. Table 4 shows that as to the capability of lowering the HPV no differences between GM and IMG were found. However, as to the antioxidant potential expressed as a more prominent decrease of

Fig. 2. Comparison of the effect of different glucomannan (GM) and Imunoglukan® (IMG) doses 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 dot-and-dash line). For

statistical analysis of data see Fig. 1.

hind paw joint of arthritic rats (Drabikova et al., 2009).



Table 4. Changes in hind paw volume (HPV), plasmatic TAS (total antioxidant status) and plasmatic TBARS (thiobarbituric acid reactive substances) in an experiment with glucomannan (GM) and Imunoglukan® (IMG) measured on experimental day 28. For statistical analysis of data see table 1.

The good antioxidative and antiinflammatory effect of IMG stimulated us to study in a more complex way its effect on the course of the main cytokines/chemokines in AA. Daily administration of IMG suppressed significantly the levels of pro-inflammatory cytokines IL-1α (Table 5) and TNF-α (Table 6) on all days monitored. Moreover, the observed


N.M. - not measured

Table 5. Plasma level of IL-1 (interleukin-1) in an experiment with Imunoglukan® (IMG) analyzed in time profile. For statistical analysis of data see table 1.


#### N.M. - not measured

Table 6. Plasma level of TNF- (tumor necrosis factor-) in an experiment with Imunoglukan® (IMG) analyzed in time profile. For statistical analysis of data see table 1.

Modern Pharmacological Approaches to Therapies:

additional and beneficial effect to that of MTX treatment.

**3.3 Endogenous antioxidants** 

described in section 3.1.

Substances Tested in Animal Models of Rheumatoid Arthritis 249

the rats treated with MTX alone. Hetland et al. (1998) showed that β-glucan reduced growth of *Mycobacterium tuberculosis* in macrophage cultures and had a protective effect against *Mycobacterium bovis*, BCG infection in BALB/c mice (Hetland & Sandven, 2002). Certain microbes, fungi and viruses led to the generation and activation of autoimmune T cells resulting in the development of a particular autoimmune disease in genetically susceptible individuals. Thus IMG, an effective activator of the immune system may also be beneficial in humans in preventing or eliminating bacterial infections, which are known to induce reactive arthritis. In our studies, we tested the pure β-glucan - Imunoglukan® isolated from *Pleurotus ostreatus*. This β-glucan decreased arthritis development in rats and had an

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

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 dot-

and-dash line). For statistical analysis of data see Fig. 1.

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 (Table 8).


#### N.M. - not measured

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.


N.M. - not measured

Table 8. Plasma level of IL-4 (interleukin-4) in an experiment with Imunoglukan® (IMG) analyzed in time profile. For statistical analysis of data see table 1.

On the basis of all the obtained results with GM and IMG, we finally chose IMG for combinatory treatment of AA with MTX.

#### **3.2.1 Imunoglukan® and methotrexate combination in AA**

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 the rats treated with MTX alone. Hetland et al. (1998) showed that β-glucan reduced growth of *Mycobacterium tuberculosis* in macrophage cultures and had a protective effect against *Mycobacterium bovis*, BCG infection in BALB/c mice (Hetland & Sandven, 2002). Certain microbes, fungi and viruses led to the generation and activation of autoimmune T cells resulting in the development of a particular autoimmune disease in genetically susceptible individuals. Thus IMG, an effective activator of the immune system may also be beneficial in humans in preventing or eliminating bacterial infections, which are known to induce reactive arthritis. In our studies, we tested the pure β-glucan - Imunoglukan® isolated from *Pleurotus ostreatus*. This β-glucan decreased arthritis development in rats and had an additional and beneficial effect to that of MTX treatment.
