pterostilbene and Inflammation

Resveratrol is a naturally occurring stilbene which has shown promising results as treatment for several neurodegenerative diseases. However, its application is limited due to its low efficacy and bioavailability. Here, we have designed and synthesized alkylated resveratrol prodrugs combining structural modification to improve antioxidant and anti-inflammatory properties and the preparation of prodrugs to extend drug bioavailability. For comparison we also studied resveratrol prodrugs and alkylated resveratrol derivatives. Methylated and butylated resveratrol derivatives showed the best in vitro neuroprotective and anti-inflammatory activity. The glucosyl- and glucosyl-acyl- prodrugs of these derivatives showed lower toxicity on zebra fish embryo. When neuroprotection was examined on pentylenetetrazole challenged zebra fish, they were capable of reverting neuronal damage but to a lower extent than resveratrol. Nevertheless, 3-O-(6'-O-octanoyl)-β-d-glucopyranoside resveratrol (compound 8) recovered AChE activity over 100% whereas resveratrol only up to 92%. In a 3-nitropropionic acid mice model of Huntington's disease, resveratrol derivative 8 delayed the onset and reduced the severity of HD-like symptoms, by improving locomotor activity and protecting against weight loss. Its effects involved an equal antioxidant but better anti-inflammatory profile than resveratrol as shown by SOD2 expression in brain tissue and circulating levels of IL-6 (11 vs 18 pg/mL), respectively. Finally, the octanoyl chain in compound 8 could be playing a role in inflammation and neuronal development indicating it could be acting as a double-drug, instead of as a prodrug.

Topics: Alkylation; Animals; Cell Survival; Cytokines; Dose-Response Relationship, Drug; Humans; Inflammation; Mice; Mice, Inbred C57BL; Molecular Structure; Neurodegenerative Diseases; Neurons; Nitro Compounds; Prodrugs; Propionates; RAW 264.7 Cells; Structure-Activity Relationship; Tumor Cells, Cultured; Zebrafish

Stilbenoids are a group of polyphenolic compounds found in plants, trees, berries, and nuts. Stilbenoids have been shown to serve an antimicrobial and antifungal function in plants. There is also evidence that as a part of the human diet, stilbenoids play an important role as antioxidants and may have anti-inflammatory effects. The PI3K/Akt pathway is a well-characterized signaling pathway controlling cellular functions involved in growth and cell cycle and in metabolism. There is also increasing evidence to show the involvement of this pathway in the regulation of inflammatory responses. In the present study, an attempt was made to investigate the anti-inflammatory properties of the naturally occurring stilbenoids pinosylvin (1), monomethylpinosylvin (2), resveratrol (3), pterostilbene (4), piceatannol (5), and rhapontigenin (6). Glycosylated derivatives of piceatannol and rhapontigenin, namely, astringin (7) and rhaponticin (8), respectively, were also investigated. In addition to the natural stilbenoids, pinosylvin derivatives (9-13) were synthesized and subjected to the testing of their effects on the PI3K/Akt pathway in inflammatory conditions. The investigated natural stilbenoids (except the glycosylated derivatives) were found to down-regulate Akt phosphorylation, which is a well-acknowledged marker for PI3K activity. It was also found that all of the studied natural stilbenoids had anti-inflammatory effects in vitro. The three most potent stilbenoids, piceatannol, pinosylvin, and pterostilbene, were selected for in vivo testing and were found to suppress inflammatory edema and to down-regulate the production of inflammatory mediators IL6 and MCP1 in carrageenan-induced paw inflammation in mice. When compared to the commercial PI3K inhibitor LY294002, the anti-inflammatory effects appeared to be quite similar. The results reveal hitherto unknown anti-inflammatory effects of natural stilbenoids and suggest that those effects may be mediated via inhibition of the PI3K/Akt pathway.

Topics: Animals; Anti-Inflammatory Agents; Biological Products; Cell Line; Chemokine CCL2; Down-Regulation; Inflammation; Inflammation Mediators; Interleukin-6; Macrophages; Male; Mice; Mice, Inbred C57BL; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature