Evaluation of Quercetin and Green Tea in combination with Methotrexate for arthritis therapy (Acronym: PhytoArt 2.0)

Project leaders

Gianluigi Russo, Katarina Bauerova

Agreement

REPUBBLICA SLOVACCA - SAS - Slovak Academy of Sciences

Call

CNR-SAS 2016-2017

Department

Biology, agriculture and food sciences

Thematic area

Biology, agriculture and food sciences

Status of the project

Extended

Report for renewal

joint-report-2013-15-final-pdf.pdf

Research proposal

Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting approximately 1% of the population worldwide. Patients with RA have a significantly reduced life quality (degeneration of muscles and joints, muscle weakness, persistent pain) and they need a lifelong therapy. Resistance and adverse effects frequently occur during antiarthritic therapy. There is thus an urgent need for introduction of new substances into medical practice.
Reactive oxygen and nitrogen species can contribute to the pathogenesis of RA in a variety of ways, including induction of membrane oxidation and instability, irreversible damage to proteins and DNA, cartilage damage and induction of bone resorption. In addition, it has recently been appreciated that ROS/RNS can also modulate a variety of signaling events that control gene expression and affect cellular processes that participate in chronic inflammation.
In the past, our team in the frame of a CNR-SAV bilateral project (2010-12) and its renewal (2013-15) monitored oxidative stress (OS) and inflammation in an animal model of arthritis induced by adjuvant (AA), by means of different clinical and biochemical/immunological markers. This model was used to assess the efficacy of some natural drugs, such as pinosylvin (PIN), N-feruloylserotonin (N-f-5HT), quercetin (QUE) and standardized green tea extract (GS). In particular, we monitored the ability of these compounds to reduce the swelling of hind paws, and their effect on some markers of OS and pro-inflammatory cytokines.
Among the studied compounds, PIN and N-f-5HT were selected to test their possible effectiveness in the combination therapy with methotrexate (MTX), a basic antirheumatic drug very often used worldwide in rheumatology practice. In combination with MTX, PIN and N-f-5HT markedly potentiated the therapeutic effect of MTX low dose, which resulted in significant improvement of all parameters measured (Bauerova et al Autoimmunity, 2015; Bauerova et al World Biomedical Frontiers, 2015; Kuncirova et al Fundam Clin Pharmacol, 2014). Our findings showed that the combination therapy increased efficacy without increased toxicity, a result crucial for future therapy of RA.
Also QUE (at the daily dose of 150 mg/kg b.w. for 28 days) was able to ameliorate all markers of inflammation and improve anti-oxidant defences (Gardi et al Arch Biochem Biophys, 2015). Our data suggest that the effect of QUE on the inflammation in AA rats could occur through the blocking of the ERK signalling pathway and regulation of NF-kB activation. However, no beneficial effect was observed on clinical parameters, such as arthritic score. The lacking effect of QUE on clinical parameters raised conflictual data in the literature. We hypothesize that the described protective effects of QUE in ameliorating inflammatory and oxidative status of AA rats are not sufficient to overcome the imaginary threshold that separates pro-inflammatory processes induced by RA from the anti-inflammatory cellular responses triggered by QUE. This may be a consequence of the relatively low dose employed in the previous study, designed to mimic dietary supplementation of QUE. However, the effect of QUE on healthy rats observed in the present study suggests a potential preventive use of the molecule.
In the new project, we would like to verify this hypothesis, studying the effect of a chronic administration of QUE (at a dose superimposable to that applied in the previous study) "before" the induction of AA. This may allow target tissues to react more efficiently to the pro-inflammatory insult since their anti-inflammatory and antioxidant defences have been already potentiated by QUE, resulting in a better response to inflammation, and hopefully in absence of disease or a mild form of arthritis. An alternative possibility is to associate QUE with MTX in combination therapy to follow disease progression and inflammation in arthritic rats. As we previously observed with other natural compounds (e.g. PIN and N-f-SER), in AA rats, the combined administration of MTX
resulted in a potentiation of the therapeutic effect of MTX at low dose with a significant improvement of inflammation and oxidative status.
The aim of the previous bilateral project was to study the effect of GS on clinical and selected biochemical parameters in monotherapy as well as with combination therapy with MTX. GS was purchased from Indena (Italy) and orally administered at the daily dose of 200mg/kg b.w. for 28 days. At the end of the experiment, biological samples were taken. Plasma, serum and the organs (heart, lungs, spleen, liver, aorta, and brain) were frozen at -80°C. Part of them was analyzed in Bratislava, while others were shipped in dry ice to Italy. Unfortunately, these latter samples were delivered by Fedex thawed. Therefore, it has been possible to analyze only a limited amount of them. Despite this inconvenient, preliminary data indicate that: the main clinical parameters as arthritic score and hind paw volume were not therapeutically influenced; moreover, the effect of MTX was diminished. In agreement with the preliminary data obtained by SAV unit on the lack of clinical efficacy of GS in RA therapy, the CNR unit assayed the antioxidant power in plasma of treated rats showing that this parameter was unchanged or even reduced in the groups of AA animals treated with GS, suggesting its pro-oxidant effect. According to these data, also lung HO-1 (assayed by Siena unit) did not increase after GS treatment.
Concerning all scientific facts given above we would like in the proposed project repeat the experiment in the exactly same design with the aim to obtain the answer to many questions which are resulted from the first experiment, mainly: could GS act as a pro-oxidant? could GS worse the effect of MTX? If yes, which mechanism is responsible for this phenomenon? Finally, could be GS beneficial in lowering and/or inhibiting the pathological processes in arthritis?

Research goals

The aims of the present proposal will take advantage of the previous results obtained in the past bilateral program ending in 2015. We are going to use, for both aims, the well-established model of adjuvant arthritis represented by Lewis rats, in which the disease is induced by intradermal injection of heat-killed Mycobacterium butyricum suspended in incomplete Freund adjuvant into the base of the tail. The IEPT-SAV unit in Bratislava possesses a long lasting experience on this animal model. The effects of the two different treatments (WP1 and WP2) performed on AA rats will be assessed by a series of clinical and biochemical analyses performed by the IEPT-SAV and the Italian units (ISA-CNR and Siena University), respectively.

The work will be distributed within the two years foreseen by the project and will essentially include two workpackages (WPs):
WP-1: Study on the therapeutic effect of green tea against RA. In the case, the possibility of a combination therapy between MTX and a standardized preparation of green tea will be assayed in a rat model of AA to improve the efficacy of the canonical therapeutic protocol based on the administration of MTX;
WP-2: Study on the preventive effect of quercetin in a rat model of AA in association with MTX. QUE will be administered before the induction of experimental arthritis to verify the ability of the molecule to improve the therapeutic efficacy of MTX.

Last update: 18/04/2024