Immunomodulating action of the 3-phenylcoumarin derivative 6,7-dihydroxy-3-[3′,4′-methylenedioxyphenyl]-coumarin in neutrophils from patients with rheumatoid arthritis and in rats with acute joint inflammation
To examine whether free (3-PD-5free) and/or liposomal (3-PD-5lipo) 6,7-dihydroxy-3-[3′,4′-methylenedioxyphenyl]-coumarin (3-PD-5) (1) modulate the effector functions of neutrophils from patients with rheumatoid arthritis under remission (i-RA) and with active disease (a-RA), in vitro; and (2) exert anti-inflammatory effect in a rat model of zymosan-induced acute joint inflammation.
Methods and results
Incorporation of 3-PD-5 into unilamellar liposomes of soya phosphatidylcholine and cholesterol was efficient (57.5 ± 7.9%) and yielded vesicles with low diameter (133.7 ± 18.4 nm), polydispersity index (0.39 ± 0.06), and zeta potential (− 1.22 ± 0.34 mV). 3-PD-5free (1 µM) and 3-PD-5lipo (3 µM) equally suppressed elastase release and reactive oxygen species generation in neutrophils from healthy subjects and i-RA and a-RA patients, stimulated with immune complexes. 3-PD-5free (20 µM) suppressed the release of neutrophil extracellular traps and chemotaxis in vitro, without clear signs of cytotoxicity. 3-PD-5lipo (1.5 mg/kg, i.p.) diminished joint edema and synovial infiltration of total leukocytes and neutrophils, without changing the synovial levels of TNF-α, IL-1β, and IL-6.
Altogether, the results reported herein indicate that 3-PD-5 is a promising modulator of the early stages of acute joint inflammation that can help to diminish not only excessive neutrophil infiltration in the synovia but also neutrophil activation and its outcomes in RA patients.
KeywordsNeutrophil Rheumatoid arthritis 3-Phenylcoumarin Chemotaxis Inflammation Liposome
Active rheumatoid arthritis
Leukotriene B4 receptor
CXCL8 receptor type 1
CXCL8 receptor type 2
Hank’s balanced saline solution
Hank’s balanced saline solution supplemented with gelatin
Inactive rheumatoid arthritis
Immobilized immune complexes
Median fluorescence intensity
Neutrophil extracellular traps
Free form of 3-PD-5
Liposomal form of 3-PD-5
Precipitated immune complexes
Precipitated immune complexes opsonized with human serum
Reactive oxygen species
Tumor necrosis factor α
The authors thank Dr. Adriana Balbina Paoliello-Paschoalato, Dr. Marcelo Dias Baruffi and Dr. Alexandre Kanashiro for scientific discussions, and Mr. Alcides Silva Pereira and Mrs. Nadir Mazzucato from the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil, for their helpful technical assistance.
LRA, MFA, LFM, and YMLV conceived the study; LRA, MFA, LFM, and AECS designed the experiments; RDRO selected the patients and determined their clinical parameters; LRA and LFM selected the healthy volunteers; LRA, MFA, CAC, and AECS performed the in vitro experiments; MFA, APLL, ASGFR, and AECS performed the in vivo experiments; LRA, MFA, LFM, LMK, AECS, and YMLV analyzed the data and discussed the results; MTP and FSE synthesized the 3-phenylcoumarin derivative and discussed the results; LRA, MFA, and LMK wrote the manuscript; and YMLV searched for funding and supervised the study. All authors read and approved the final manuscript.
This study was supported by the Brazilian funding agencies São Paulo Research Foundation (FAPESP, grants 2010/19504-0, 2012/23541-4, 2013/20810-7, and 2013/21331-5) and National Council for Scientific and Technological Development (CNPq, grants 482015/2012-8, 308111/2013-3, and 130209/2015-5).
Compliance with ethical standards
Conflict of interest
The authors declare that there are no conflicts of interest.
Ethics approval and consent to participate
All procedures performed in studies involving human participants complied with the ethical standards established by the Resolution 466/2012 of the Brazilian National Health Council, and with the 1964 Helsinki declaration and its later amendments. The Human Research Ethics Committee from the Ribeirão Preto Medical School Hospital and from the School of Pharmaceutical Sciences of Ribeirão Preto, both at the University of São Paulo, Ribeirão Preto, SP, Brazil, approved the study protocol (CEP/HCRP n. 15038/2015). All the participants signed an informed consent form to participate in this study. All procedures performed in studies involving animals complied with the ethical standards of The Animal Care Committee from the University of São Paulo, Brazil, which approved the study protocol (CEUA n. 15.1.1163.60.0).
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