Fisetin: An Integrated Approach to Identify a Strategy Promoting Osteogenesis.

Flavonoids may modulate the bone formation process. Among flavonoids, fisetin is known to counteract tumor growth, osteoarthritis, and rheumatoid arthritis. In addition, fisetin prevents inflammation-induced bone loss. In order to evaluate its favorable use in osteogenesis, we assayed fisetin supplementation in both in vitro and in vivo models and gathered information on nanoparticle-mediated delivery of fisetin in vitro and in a microfluidic system. Real-time RT-PCR, Western blotting, and nanoparticle synthesis were performed to evaluate the effects of fisetin in vitro, in the zebrafish model, and in ex vivo samples. Our results demonstrated that fisetin at 2.5 µM concentration promotes bone formation in vitro and mineralization in the zebrafish model. In addition, we found that fisetin stimulates osteoblast maturation in cell cultures obtained from cleidocranial dysplasia patients. Remarkably, PLGA nanoparticles increased fisetin stability and, consequently, its stimulating effects on RUNX2 and its downstream gene SP7 expression. Therefore, our findings demonstrated the positive effects of fisetin on osteogenesis and suggest that patients affected by skeletal diseases, both of genetic and metabolic origins, may actually benefit from fisetin supplementation.

Characterization of Cytotoxic Lactose Binding Lectin from Sulphur Polypore, Laetiporus sulphureus (Agaricomycetes), from Algeria.

Mushroom lectins have important biological and biomedical applications. Most lectins purified from these organisms exhibit high toxicity in animal cells and toward microbial agents. They are able to induce cell growth inhibition and metabolism by their ability to interact with glyconjugate components (glycoproteins receptors, glycolipids) present in their membrane. After lectins bind to these membrane receptors, they induce cellular signalization chains in which gene expression is regulated and cell death programming (apoptosis) is activated. In this work, a new multimeric lectin was characterized from the rare saprobic edible mushroom, Laetiporus sulphureus strain TMES43, grown in the Algerian forest. Lectin was isolated with ammonium sulfate precipitation followed by affinity chromatography on a Sepharose 4B column, with specific activity of 1204.7 units of hemagglutination activity/mg and 35.55% yield. The protein has a tetrameric structure with a molecular weight of 36 kDa for each subunit, with a total molecular weight of approximately 140 kDa. In addition, a Mascot peptide fingerprint study on a matrix-assisted laser desorption ionization-time of flight tandem fragment showed identity with autophagy-related protein 16 from Meyerozyma guilliermondii (strain ATCC 6260/CBS 566/DSM 6381/JCM 1539/NBRC 10279/NRRL Y-324; Expasy ID: ATG16_PICGU) and no sequence similarity to known mushroom lectins. L. sulphureus hemagglutination activity was reduced by 5 mM of lactose and 10 mM of EDTA incubation and was recovered by metallic cations such as CaCl2, MgCl2, and ZnCl2. L. sulphureus purified lectin had no human ABO group specificity and showed low temperature and alkaline pH stabilities. The MTT preliminary assay showed that L. sulphureus purified lectin induced high cytotoxicity for tumor cells and normal cells.

Oxyresveratrol-Loaded PLGA Nanoparticles Inhibit Oxygen Free Radical Production by Human Monocytes: Role in Nanoparticle Biocompatibility.

Oxyresveratrol, a polyphenol extracted from the plant Artocarpus lakoocha Roxb, has been reported to be an antioxidant and an oxygen-free radical scavenger. We investigated whether oxyresveratrol affects the generation of superoxide anion (O2-) by human monocytes, which are powerful reactive oxygen species (ROS) producers. We found that oxyresveratrol inhibited the O2- production induced upon stimulation of monocytes with ß-glucan, a well known fungal immune cell activator. We then investigated whether the inclusion of oxyresveratrol into nanoparticles could modulate its effects on O2- release. We synthesized poly(lactic-co-glycolic acid) (PLGA) nanoparticles, and we assessed their effects on monocytes. We found that empty PLGA nanoparticles induced O2- production by resting monocytes and enhanced the formation of this radical in ß-glucan-stimulated monocytes. Interestingly, the insertion of oxyresveratrol into PLGA nanoparticles significantly inhibited the O2- production elicited by unloaded nanoparticles in resting monocytes as well as the synergistic effect of nanoparticles and ß-glucan. Our results indicate that oxyresveratrol is able to inhibit ROS production by activated monocytes, and its inclusion into PLGA nanoparticles mitigates the oxidative effects due to the interaction between these nanoparticles and resting monocytes. Moreover, oxyresveratrol can contrast the synergistic effects of nanoparticles with fungal agents that could be present in the patient tissues. Therefore, oxyresveratrol is a natural compound able to make PLGA nanoparticles more biocompatible.

Oxyresveratrol Inhibits R848-Induced Pro-Inflammatory Mediators Release by Human Dendritic Cells Even When Embedded in PLGA Nanoparticles.

Oxyresveratrol, a stilbene extracted from the plant Artocarpus lakoocha Roxb., has been reported to provide a considerable anti-inflammatory activity. Since the mechanisms of this therapeutic action have been poorly clarified, we investigated whether oxyresveratrol affects the release of the pro-inflammatory cytokines IL-12, IL-6, and TNF-α by human dendritic cells (DCs). We found that oxyresveratrol did not elicit per se the release of these cytokines, but inhibited their secretion induced upon DC stimulation with R848 (Resiquimod), a well-known immune cell activator engaging receptors recognizing RNA viruses. We then investigated whether the inclusion of oxyresveratrol into nanoparticles promoting its ingestion by DCs could favor its effects on cytokine release. For this purpose we synthesized and characterized poly(lactic-co-glycolic acid) (PLGA) nanoparticles, and we assessed their effects on DCs. We found that bare PLGA nanoparticles did not affect cytokine secretion by resting DCs, but increased IL-12, IL-6, and TNF-α secretion by R848-stimulated DCs, an event known as "priming effect". We then loaded PLGA nanoparticles with oxyresveratrol and we observed that oxyresveratrol-bearing particles did not stimulate the cytokine release by resting DCs and inhibited the PLGA-dependent enhancement of IL-12, IL-6, and TNF-α secretion by R848-stimulated DCs. The results herein reported indicate that oxyresveratrol suppresses the cytokine production by activated DCs, thus representing a good anti-inflammatory and immune-suppressive agent. Moreover, its inclusion into PLGA nanoparticles mitigates the pro-inflammatory effects due to cooperation between nanoparticles and R848 in cytokine release. Therefore, oxyresveratrol can be able to contrast the synergistic effects of nanoparticles with microorganisms that could be present in the patient tissues, therefore overcoming a condition unfavorable to the use of some nanoparticles in biological systems.