Activity of pinosylvin administered in monotherapy and in combination with methotrexate on the development of rat adjuvant arthritis

Oxidative stress (OS) has been implicated in various pathological conditions involving several diseases and aging. Rheumatoid arthritis (RA) is a common severe joint disease that involves all age groups. The pathogenesis of RA is associated predominantly with the formation of free radicals at the site of inflammation. However, knowledge on the role of OS in the progression of RA is scarce and the link between OS and inflammation status in arthritis should be more precisely investigated. Pinosylvin (PIN), 3,5-dihydroxy-trans-stilbene, is mainly found in the heartwood of Pinus sylvestris. PIN used in this study was synthesized in the Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Prague, Czech Republic by Ing. Juraj Harmatha, PhD. The aim of the present study was to examine the effect of PIN on the progression of adjuvant-induced arthritis (AA) in rats in monotherapy and in combination with methotrexate (MTX), which is a classical immunosuppressant drug. AA was induced by a single intradermal injection of heat-inactivated Mycobacterium butyricum in incomplete Freund’s adjuvant. The experiments included healthy animals, arthritic animals not treated, arthritic animals treated with MTX, with PIN, and with a combination of PIN and MTX. The two latter groups received a daily oral dose of 50 mg/kg b.w. of PIN, either alone or with MTX in an oral dose of 0.4 mg/kg b.w. twice a week during 28 experimental days. We found that PIN potentiated both the antiarthritic (decrease of hind paw volume) and the antioxidant effect of MTX (reduction of plasmatic levels of TBARS). Activity of GGT in spleen, level of MCP-1 and CRP in plasma were not improved by addition of PIN to MTX due to the prominent effect of MTX alone on these parameters. Arthritic animals showed increased OS, evaluated as plasma levels of isoprostanes. PIN alone or in combination with MTX strongly reduced isoprostane levels (about 50%). On the contrary, a significant decline in Nrf2-regulated antioxidant defences, such as hemeoxygenase-1 (HO-1), was observed in the lung (about 40%) but not in the liver from AA rats. In the AA lung, PIN alone increased the levels of HO-1 by about 30% more than MTX. Moreover, the combination therapy was the most effective in increasing the levels of HO-1 (3-fold in respect to AA values). OS can also activate NF-B, which plays a critical role in the transcription of proinflammatory genes. Our data showed a marked increase in NF-B in the lung and liver from AA animals. This increase was strongly reduced by PIN alone as well as in combination with MTX. Our results suggest that the anti-inflammatory activity of PIN is mediated by suppression of NF-kB activation in the liver and lung of arthritic animals. In summary, combined administration of PIN and MTX suppressed arthritic progression in rats more effectively than did MTX alone. This natural compound is able to reduce OS in vivo and may help improve the treatment of rheumatoid arthritis. Acknowledgement: VEGA 2/0045/11, APVV-0315-07, CNR/SAV bilateral project 2010-2012: In vitro and in vivo models of arthritic processes for studying the mechanisms of inflammation and oxidative stress link-up. New perspectives for arthritis therapy.