Pharmacological influence on processes of adjuvant arthritis: Effect of the combination of an antioxidant active substance with methotrexate

Abstract

Oxygen metabolism has an important role in the pathogenesis of rheumatoid arthritis. A certain correlation was observed between oxidative stress, arthritis and the immune system. Reactive oxygen species produced in the course of cellular oxidative phosphorylation and by activated phagocytic cells during oxidative burst, exceed the physiological buffering capacity and result in oxidative stress. The excessive production of ROS can damage protein, lipids, nucleic acids, and matrix components. Patients with rheumatoid arthritis have an altered antioxidant defense capacity barrier. In the present study the effect of substances with antioxidative properties, i.e. pinosylvin and carnosine, was determined in monotherapy for the treatment of adjuvant arthritis (AA). Moreover carnosine was evaluated in combination therapy with methotrexate. Rats with AA were administered first pinosylvin (30 mg/kg body mass daily per os), second carnosine (150 mg/kg body mass daily per os) in monotherapy for a period of 28 days. Further, rats with AA were administered methotrexate (0.3 mg/kg body mass 2-times weekly per os), and a combination of methotrexate+carnosine, with the carnosine dose being the same as in the previous experiment. Parameters, i.e. changes in hind paw volume and arthritic score were determined in rats as indicators of destructive arthritis-associated clinical changes. Plasmatic levels of TBARS and lag time of Fe(2+)-induced lipid peroxidation (tau-FeLP) in plasma and brain were specified as markers of oxidation. Plasmatic level of CRP and activity of γ-glutamyltransferase (GGT) in spleen and joint were used as inflammation markers. In comparison to pinosylvin, administration of carnosine monotherapy led to a significant decrease in the majority of the parameters studied. In the combination treatment with methotrexate+carnosine most parameters monitored were improved more remarkably than by methotrexate alone. Carnosine can increase the disease-modifying effect of methotrexate treatment in rat AA.

Keywords: arthritis; carnosine; combination therapy; methotrexate; oxidative stress; pinosylvin.

Figure 1

Change of HPV measured on day 28 in the model of adjuvant arthritis treated with pinosylvin and carnosine in monotherapy. CO – control group, AA – Adjuvant arthritis group, AA-PIN – Adjuvant arthritis group administered pinosylvin, AA-CARN – Adjuvant arthritis group administered carnosine. Hind paw volume (HPV) is expressed in percentages [%]. Results are mean ± S.E.M., n=8-10. The symbol (*) shows significant difference ***p<0.001 versus CO, +p<0.05 versus AA.

Figure 2

Arthritic score in the model of adjuvant arthritis treated with pinosylvin and carnosine in monotherapy. CO – control group, AA – Adjuvant arthritis group, AA-PIN – Adjuvant arthritis group administered with pinosylvin, AA-CARN – Adjuvant arthritis group administered carnosine. Arthrogram is expressed in points [pts]. Results are mean ± S.E.M., n=8-10. The symbol (+) shows significant difference +p<0.05 vs AA.

Figure 3

GGT activity in spleen and joint on day 28 in the model of adjuvant arthritis treated with pinosylvin in monotherapy. CO – control group, AA – Adjuvant arthritis group, AA-PIN – Adjuvant arthritis group administered with pinosylvin. The activity of cellular γ-glutamyltransferase (GGT) in hind paw joint and spleen tissue homogenates is expressed in [nmoln p-nitroaniline/min/g tissue]. Results are mean ± S.E.M., n=8-10. The symbol (*) shows significant difference ***p<0.001 vs CO, +p<0.05 vs AA.

Figure 4

GGT activity in spleen and joint on day 28 in the model of adjuvant arthritis treated with carnosine in monotherapy. CO – control group, AA – Adjuvant arthritis group, AA-CARN – Adjuvant arthritis group administered carnosine. The activity of cellular γ-glutamyltransferase (GGT) in hind paw joint and spleen tissue homogenates is expressed in [nmoln p-nitroaniline / min / g tissue]. Results are mean ± S.E.M., n=8-10. The symbol (*) shows significant difference ***p<0.001 vs CO, +p<0.05 vs AA, +++p<0.001 vs AA.

Figure 5

TBARS measured on day 28 in the model of adjuvant arthritis treated with pinosylvin and carnosine in monotherapy. CO – control group, AA – Adjuvant arthritis group, AA-PIN – Adjuvant arthritis group administered with pinosylvin, AA-CARN – Adjuvant arthritis group administered with carnosine. Levels of TBARS in plasma are expressed in [nmoln/ml]. Results are mean ± S.E.M., n=8–10. The symbol (*) shows significant difference ***p<0.001 vs CO, +p<0.05 vs AA.

Figure 6

Effect of methotrexate and the combination of carnosine + methotrexate on adjuvant arthritis on the parameters of hind paw volume (HPV), activity of cellular γ-glutamyltransferase (GGT) in hind paw joint and spleen tissue homogenates and C-reactive protein (CRP) in plasma measured on day 28, expressed in percentage compared to the arthritis group considered as 100%. AA – Adjuvant arthritis group (100%), AA- MTX – Adjuvant arthritis group administered methotrexate and AA-CARN+MTX – Adjuvant arthritis group administered with carnosine+methotrexate. Results are mean ± S.E.M., n=8–10. The symbol (+) shows significant difference +p<0.05 vs AA, ++p<0.01 vs AA, +++p<0.001 vs AA, ##p<0.01 vs AA-MTX.

Figure 7

Effect of methotrexate and the combination of carnosine + methotrexate on adjuvant arthritis on the parameters of TBARS, tau-FeLP in plasma and brain on day 28, expressed in percentages in comparison to the arthritis group considered as 100%. AA – Adjuvant arthritis group (100%), AA- MTX – Adjuvant arthritis group administered methotrexate and AA-CARN+MTX – Adjuvant arthritis group administered carnosine+methotrexate. Results are mean ± S.E.M., n=8–10. The symbol (+) shows significant difference ++p<0.01 vs AA, #p<0.05 vs AA-MTX, ###p<0.001 vs AA-MTX.