Autoimmunity. 2015 Feb;48(1):46-56.

Markers of inflammation and oxidative stress studied in adjuvant-induced arthritis in the rat on systemic and local level affected by pinosylvin and methotrexate and their combination.

Bauerovaa* K, Acquavivab A, Ponista S, Gardib C, Vecchiob D, Drafia F, Arezzinib B, Bezakovac L, Kuncirovaa V, Mihalovaa D, Nosala R

* Corresponding author – address

PharmDr. Katarina Bauerova, Dr.Sc., Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Dubravska cesta 9, SK-841 04 Bratislava, Slovak Republic, e-mail:

aInstitute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Dubravska cesta 9, SK-841 04 Bratislava, Slovak Republic

cDepartment of Cellular and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University, Kalinčiakova 8, SK-832 32 Bratislava, Slovak Republic

bDepartment of Molecular and Developmental Medicine, University of Siena, via Aldo Moro 2, Siena, Italy



Oxidative stress (OS) is important in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA) and its experimental model-adjuvant arthritis (AA). Antioxidants are scarcely studied in autoimmunity, and future analyses are needed to assess its effects in ameliorating these diseases. Although there are studies about antioxidants effects on the course of RA, their role in combination therapy has not yet been studied in detail, especially on extra-articular manifestations of AA. During the 28-d administration of pinosylvin (PIN) in monotherapy and in combination with methotrexate (MTX) to AA rats, we evaluated the impact of the treatment on selected parameters. The experiment included: healthy controls, untreated AA, AA administered 50 mg/kg b.w. of PIN daily p.o., AA administered 0.4 mg/kg b.w. of MTX twice weekly p.o. and AA treated with a combination of PIN+MTX. AA was monitored using: hind paw volume, C-reactive protein, monocyte chemotactic protein-1 (MCP-1), thiobarbituric acid reactive substances (TBARS) and F2-isoprostanes in plasma, γ-glutamyltransferase activity in spleen, activity of lipoxygenase (LOX) in lung, heme oxygenase-1 (HO-1) and nuclear factor kappa B (NF-κB) in liver and lung. PIN monotherapy significantly improved the activation of NF-κB in liver and lung, HO-1 expression and activity of LOX in the lung, MCP-1 levels in plasma (on 14th d) and plasmatic levels of F2-isoprostanes. An important contribution of PIN to MTX effect was the reduction of OS (an increase of HO-1 expression in lung and reduction of plasmatic TBARS) and decrease of LOX activity in the lung.

PMID: 25046647



Rheumatoid arthritis (RA) is an autoimmune disease occurring in 0.5–1.0% of the adult population worldwide and it is associated with an increased mortality rate, mainly due to cardiovascular complications caused by inflammatory processes. RA is characterized by chronic and systemic inflammation that affects mainly the synovial membranes, articular cartilages, and bones [1]. Signs of systemic inflammation in patients with RA include constitutional features and localized extraarticular organ involvement (subcutaneous rheumatoid nodules, rheumatoid lung disease, vasculitis, pericarditis and pleuritic) [2].

During the development of adjuvant arthritis (AA) in Lewis rats we measured the inflammation and oxidative stress markers on systemic and local level. In this supplement we focus on the importance of extra-articular manifestation (EAM) of arthritis in lung and liver and the effect of PIN and its ability to improve methotrexate treatment particularly in suppressing the EAM of arthritis. We assessed antioxidant enzyme heme oxygenase-1 (HO-1) expression in lung and liver. The expression of HO-1

in AA was more decreased in lung than in liver, which is a novel finding in EAM of arthritis. Reduced expression of HO-1 in lung suggest an impaired antioxidant defense in this tissue. In a clinical study, severe pathologic changes in lung tissue were reported and revealed interstitial lung disease in about 50% of RA patients, of whom only 10% had clinically significant manifestation [3].

It is possible, that the decreased expression of HO-1 in lung tissue might be also involved in interstitial lung disease of patients with RA. Pinosylvin (PIN) in monotherapy was more effective in increasing HO-1 expression than MTX. The combination of PIN+MTX remained the most efficient in increasing this parameter and this effect was also significant in comparison to MTX monotherapy (Fig 1).


sp fig1

Figure 1. Effect of PIN and MTX in monotherapy and in combination therapy on the level of HO-1 expression measured on day 28 in the liver and lung of rats. CO – Control group, AA – Adjuvant arthritis group, AA-PIN – Adjuvant arthritis group administered pinosylvin, AA-MTX – Adjuvant arthritis group administered methotrexate, AA-PIN+MTX – Adjuvant arthritis group administered pinosylvin and methotrexate. Statistical significance was calculated compared to CO (*), AA (+) and MTX (#)


Involvement of NF-kB in the development of arthritis was demonstrated before [4]. Stilbens, such as PIN, are inhibitors of NF-kB [5], what may be important for the future therapy of RA. NF-κB was significantly increased in liver and lung tissues of AA animals’, and PIN significantly reduced this increase, both in monotherapy and in combined therapy with MTX (Fig. 2). NF-kB has been shown to be induced by stimuli that also up-regulate HO-1 gene expression (Scheme 1.). Li et al. have described a functional kB element in the mouse HO-1 promoter. These authors have shown that the NF-kB subunits p50 and p65 and the inducible NO-synthase mediated HO-1 up-regulation in vivo [6].


sp fig2

Figure 2. Effect of PIN and MTX in monotherapy and in combination therapy on NF-κB activation measured on day 28 in the liver and lung of rats. Abbreviations of experimental groups are the same as in Fig. 1. Statistical significance was calculated compared to CO (*) and to AA (+)


Interestingly, despite the activation of NF-kB in AA lung tissue, the expression of HO-1 was significantly reduced. Probably, in AA lung tissue, some signaling pathways which blocks HO-1 upregulation mediated by NF-kB are activated. Our results reflect the involvement of extra-articular manifestation in pathogenesis of AA, in which more attention should be paid in animal models of RA.

In this experiment we proved our hypothesis that one of the prospective possibilities of RA therapy would be the combination of immunosuppressive drugs (e.g. methotrexate) with molecules able of correcting the redox homeostasis of the organism. It can be concluded that PIN combination with MTX was beneficial in rat AA improving the redox homeostasis: in lung – expression of HO-1 and in monotherapy reduced the NF-κB activation.

sp fig3

Scheme 1. Activation of NF-κB and HO-1 by oxidative stress


Acknowledgments: The study was supported by grants: APVV-031507, APVV-0052-10, VEGA 2/0045/11 and performed in the frame of two SAS-CNR bilateral projects coordinated by Dr. Bauerova (Slovakia) and Dr. Russo (Italy): “Phytochemicals in ameliorating rheumatoid arthritis therapy: from preclinical studies to clinical applications“and „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”.



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[2] Turesson C. 2013.Extra-articular rheumatoid arthritis. Curr Opin Rheumatol. 25(3): 360-366.

[3] Liote, H. 2008. Pulmonary manifestation of rheumatoid arthritis. Rev Mal Respir. 25: 973–988.

[4] Alghasham, A., Z., Rasheed, 2014. Therapeutic targets for rheumatoid arthritis: Progress and promises. Autoimmunity 47(2): 77-94.

[5] van Loo, G., and R. Beyaert. 2011. Negative regulation of NF-κB and its involvement in rheumatoid arthritis. Arthritis Res Ther. 13: 221.

[6] Li Q., Guo Y., Ou Q., Cui C., Wu W., J., Tan W., et al. 2009. Gene transfer of inducible nitric oxide synthase affords cardioprotection by upregulating heme oxygenase-1 via a nuclear factor-{kappa}B-dependent pathway. Circulation 120: 1222–30.




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