Fundam Clin Pharmacol. 2014 Dec;28(6):616-26.
N-feruloylserotonin in preventive combination therapy with methotrexate reduced inflammation in adjuvant arthritis.
Viera Kuncirova1, Silvester Ponist1, Danica Mihalova1, Frantisek Drafi1, Radomir Nosal1, Alessandra Acquaviva2, Concetta Gardi2, Juraj Harmatha3, Iveta Hradkova4 and Katarina Bauerova1*
1Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava, Slovak Republic
2Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy
3Institute of Organic Chemistry and Biochemistry v.v.i., AS CR, Prague 6, Czech Republic
4Department of Diary and Fat Technology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology, Prague, Czech Republic
*Correspondence and reprints: firstname.lastname@example.org
Many of disease-modifying anti-rheumatic drugs often have side effects at high doses and/or during long-term administration. Increased efficacy without increased toxicity is expected for combination therapy of rheumatoid arthritis (RA). The aim of the study was to examine the effect of N-feruloylserotonin (N-f-5HT) and methotrexate (MTX) in monotherapy and in combination therapy on disease progression and inflammation in arthritic rats. Adjuvant arthritis was induced by intradermal injection of Mycobacterium butyricum in incomplete Freund’s adjuvant in Lewis rats. The experiment included healthy animals, arthritic animals without any drug administration, arthritic animals with administration of N-f-5HT in the oral daily dose of 15 mg/kg b.w., arthritic animals with administration of MTX in the oral dose of 0.3 mg/kg b.w. twice a week and arthritic animals treated with the combination of N-f-5HT and MTX. N-f-5HT in monotherapy reduced only activation of NF-κB and did not have any significant effect on other parameters monitored. Low-dose treatment of MTX decreased the level of IL-1β and MCP-1 on day 14 and activation of NF-κB in liver without significant effect on other parameters. N-f-5HT and MTX combination showed both the anti-arthritic (hind paw volume and arthritic score) and anti-inflammatory effect (plasmatic levels of IL-1β, IL-17, MCP-1, CRP, and activation of NF-κB in liver). In combination with MTX, N-f-5HT markedly potentiated the therapeutic effect of MTX low dose, which resulted in significant improvement of all parameters measured. The findings showed that the combination therapy simultaneously decreased multiple markers of inflammation, a result crucial for future therapy of RA.
Rheumatoid arthritis (RA) is characterized by chronic and systemic inflammation that affects mainly the synovial membranes, articular cartilages, and bones. RA also evokes marked inflammatory responses and immunological alterations in other organs, such as lungs, vascular tissue, liver and muscles. Although the pathogenesis of RA has not been completely elucidated, it is evident that inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-17 as well as inflammatory mediators such as cyclooxygenases (COX) and lipoxygenases (LOX) play an important role in RA . Adjuvant arthritis (AA) exhibits clinical and pathological features similar to human RA. AA is not only a model of polyarthritis but also induces pathologic changes in a variety of other tissue such as liver and spleen. AA is an experimental immunopathology in rats that is often used as a model for studying autoimmune chronic inflammation and new antiarthritic substances.
Methotrexate (MTX) in small doses has become the most frequently used DMARD in the therapy of RA. Despite the proven effectiveness of MTX in arthritis, it could also trigger side effects depending on the dosage: hepatotoxicity, nausea, mucous ulceration, hematotoxicity, teratogenicity. Combinations of MTX with other drugs has been suggested not only to improve the therapeutic efficacy but also to minimize toxic side effects in treating RA . One of the therapeutic strategies is to decrease the dose of MTX. Therefore in this study we used a low dose of MTX (0.3 mg/kg) twice a week alone and in combination with N-f-5HT (15 mg/kg b.w.), a substance with antioxidative properties and low toxicity to improve the efficacy of this traditional DMARD. N-feruloylserotonin (N-f-5HT; N-feruloyl-5-hydroxy-tryptamine) is a conjugated serotonin. It is a member of the indole hydrocinamic acid amides, with serotonin (5-HT) and ferulic acid (fa) as representative components of its structure. In our study we analyzed the effect of N-f-5HT, which were isolated from the seeds of Leuzea Carthamoides  (Scheme 1). Based on its proved antioxidative activity, we supposed that it would have a beneficial effect in the model of RA in the combination therapy with MTX. The mechanism of action and the effect of N-f-5HT in animals and humans have still not been thoroughly investigated and understood, calling for a specific follow-up studies. This study is the first to evaluate N-f-5HT in adjuvant arthritis and also the first to demonstrate its effect in combination with another drug.
Fig. 1 Change of HPV on experimental days 14 and 28 in adjuvant arthritis (AA) rats treated with N-f-5HT in combination with MTX. The figure shows the change of hind paw volume in time profile. AA-N-f-5HT – adjuvant arthritis group administered N-feruloylserotonin, AA-MTX – adjuvant arthritis group administered methotrexate, AA-N-f-5HT+MTX – adjuvant arthritis group administered N-feruloylserotonin and methotrexate. Hind paw volume (HPV) is expressed in percentages [%]. Results are mean ± S.E.M., n=8-10. Statistical significance was evaluated using unpaired Student´s t-test. The symbol (*; +; #) shows significant difference +++ p <0.001 vs AA, + p <0.05 vs AA, # p <0.05 vs AA-MTX.
Current evidence suggests that inflammation is primarily responsible for the development of arthritis. In this model, swelling of the rat hind paws and increase in the arthritis score are indicators of chronic systemic inflammation. N-f-5HT alone did not exert an effect on the parameters of inflammation measured (except activation of Nf-κB), though in combination with MTX it exerted a synergistic effect in decreasing the levels of inflammatory parameters. The remarkable finding was that N-f-5HT potentiated beneficial effect of MTX; reduction of hind paw volume on all experimental days was more significant compared to the rats treated with MTX alone (Fig.1). This study provides evidence that the combination therapy with two substances – low dose MTX (0.3 mg/kg b.w.) and N-f-5HT (15 mg/kg b.w.) resulted in synergistic beneficial effects on amelioration of the severity of arthritis (measured as change of hind paw volume and arthritic score) and on reduction of inflammation in AA rats evaluated by analysis of key inflammatory parameter IL-1β analyzed on day 14 and 28 (Fig. 2).
Fig. 2 Effect of N-f-5HT and MTX in monotherapy and in combination therapy on levels of IL-1β in plasma measured on days 14 and 28. The figures show the levels of IL-1β in plasma determined by an enzyme immunoassay kit in plasma samples of Lewis rats measured on day 14 and 28. CO – control group, AA – adjuvant arthritis group, AA-N-f-5HT – adjuvant arthritis group administered N-feruloylserotonin, AA-MTX – adjuvant arthritis group administered methotrexate, AA-N-f-5HT+MTX – adjuvant arthritis group administered N-feruloylserotonin and methotrexate. Levels of IL-1β in plasma are expressed in [pg/ml]. Results are mean ± S.E.M., n=8-10. Statistical significance was evaluated using unpaired Student´s t-test. The symbol (*; +, #) shows significant difference ***p <0.001 vs CO, * p <0.05 vs CO, +++ p <0.001 vs AA, ++ p <0.01 vs AA, + p <0.05 vs AA, ## p <0.05 vs AA-MTX.
Activation of NF-κB plays a central role in the regulation of inflammatory and oxidative processes and its involvement in the development of arthritis was demonstrated before . We assumed that the systemic inflammation accompanying the development of AA will have significant impact on liver. Liver plays a pivotal role in regulating homeostasis; therefore, damage to the liver can alter critical functions. In our study we demonstrated significantly increase activation of NF-κB in liver of adjuvant arthritis rats. Combination therapy data revealed that MTX together with N-f-5HT synergistically reduced the activation of Nf-κB in liver inflammatory tissue (Tab.1). This data suggests that the therapeutic effect of combination therapy on adjuvant arthritis in rats isediated via modulation the immune system and correction of cytokine imbalance. However, the reduction of activation of NF-κB is not necessarily the only mechanism of action.
Tab. 1 Effect of N-f-5HT and MTX in monotherapy and in combination therapy on NF-κB activation in rats liver. The table shows NF-κB activation assessed by p65-DNA binding in the liver of Lewis rats measured on day 28. CO – control group, AA – adjuvant arthritis group, AA-N-f-5HT – adjuvant arthritis group administered N-feruloylserotonin, AA-MTX – adjuvant arthritis group administered methotrexate, AA-N-f-5HT+MTX – adjuvant arthritis group administered N-feruloylserotonin and methotrexate. NF-κB activation was assessed by measuring p65-DNA binding in the liver. Activation of NF-κB in liver is expressed in [Abs/mg of total protein]. Results are mean ± S.E.M., n=8-10. Statistical significance was evaluated using unpaired Student´s t-test. The symbol (*; +; #) shows significant difference ** p <0.01 vs CO, + p <0.05 vs AA, ++ p <0.01 vs AA, p ## <0.01 vs AA-MTX.
It can be concluded that the combination of the standard antirheumatic drug with natural substances possessing anti-inflammatory and antioxidative activities as N-f-5HT may be a fruitful approach towards a new and safer therapy of RA.
We wish to thank Prof. Jan Šmidrkal (Institute of Chemical Technology, Prague, Czech Republic) and Ing. Juraj Harmatha, PhD (Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic) for the synthesis and analysis of N-f-5HT in this experiment. The study was supported by grants: 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):”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” and “Phytochemicals in ameliorating rheumatoid arthritis therapy: from preclinical studies to clinical applications“
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