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N6-Isopentenyladenosine (iPA) is a cytokinin identified in plants but also present in a free form or as a modified nucleoside bound to selenocysteine tRNA in human cells. It is an adenosine modified by an isopentenyl chain which derives from dimethylallil pyrophosphate (DMAPP), an intermediate of the mevalonate pathway. iPA is required for efficient translational decoding of selenoproteins, and can modulate a variety of biological processes including cell cycle progression, DNA synthesis and apoptosis (1). Recently, it has been shown that iPA can exhibit immunomodulatory and anti-inflammatory properties by activating NK cells and modulating cytokine production in a way depending on the concentration used (2). In order to further investigate the anti-inflammatory properties of iPA and its possible mechanisms of action, we analyzed its ability to inhibit TNFα-induced inflammation, either in normal human bronchial epithelial cells or in a model of exacerbated inflammation represented by bronchial cells derived from a Cystic Fibrosis (CF) patient bearing the ΔF508 mutation. Results showed that iPA inhibited IL-8 and RANTES release in both type of cells in a different manner. The analysis of the key enzymes of the STAT3 and NF-κB signalling pathways showed that iPA decreased the phosphorylation of STAT3 enzyme and markedly increased the expression of the direct NF-κB inhibitor, IκBα. These results were corroborated analyzing directly the NF-κB activity in HEK 293/T cells transfected with a NF-κB reporter plasmid. In these cells, iPA was also able to decrease IκBα levels. Of interest, we found that iPA also increased the expression of the antioxidant selenoprotein glutathione peroxidase only in CF cells. Altogether these data suggest that iPA can negatively regulate inflammation with a general mechanism of action involving the inhibition of NF-κB pathway but also propose that, in the presence of an altered inflammatory response such as in CF disease, iPA might act by modulating expression and/or synthesis of glutathione peroxidase., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Cystic fibrosis (CF) is a genetic life-shortening condition in Caucasians. Despite being a multi-organ disease, CF is classically diagnosed by symptoms of acute/chronic respiratory disease, with persistent pulmonary infections (Amaral, 2015). CF is caused by mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) protein. Inheritance of mutant CFTR alleles results in surface liquid depletion and defective mucociliary clearance leading to pulmonary failure. Defects in CFTR perturb the regulation of many intracellular signaling pathways including the NF-kB pathway causing excessive production of pro-inflammatory mediators. Current CF therapies are directed to delay CF lung damage by restoring CFTR function and controlling abnormal inflammation. However, only few anti-inflammatory drugs are effective for CF treatments (mainly oral corticosteroids and ibuprofen), these drugs have limited beneficial effects in presence of considerable side effects. Flavonoids have been reported as promising anti-inflammatory drugs and some of them seem to act as CFTR direct activators (Amaral, 2015). From this respect, herbal remedies or plant bioactive molecules may be of great interest. To this aim, we tested the anti-inflammatory activity of apolar extracts from the roots of three Peonia species (Paeoniaceae family), namely P. rockii P. ostii and P. lactiflora, on CFTR ΔF508/ΔF508 CuFi1 cells and normal counterpart. The effects of the extracts on intrinsic as well as TNFα-induced inflammation were evaluated by determining IL-8, IL-6 and RANTES production. Further- more, to study the direct effect of the extracts on NF-kB activation, Human Embryonic Kidney cells were used in transient transfection of NF-kB reporter plasmid and NF-kB activity and cytokine productions were also evaluated. Results showed a significant anti-inflammatory activity of all three Peonia extracts with the P. lactiflora being the most effective. Furthermore, we also tested the anti-inflammatory potential of the pure flavonoid naringin in the same model systems. We found that naringin was able to reduce cytokine release through inhibiting the key enzymes of the NF-kB and MAPK/ ERK pathways. Interestingly, preliminary results on spray dried pharmaceutical formulations of this molecule, show that naringin co-sprayed with leucine improves pharmacological activity of the flavonoid neat raw drug., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Ethnopharmacological relevance: The roots of Paeonia species are widely used in Traditional Chinese Medicine for various diseases and, mainly, for their anti-inflammatory activity. Aim of the study: This research aimed to investigate the composition of extracts from three peonies, the herbaceous species, P.lactiflora, and the tree peonies of the section Moutan, P. rockii, and P. ostii,and their property to inhibit inflammation in bronchial epithelial and Human Embryonic Kidney (HEK 293/T) cells. Materials and methods: Paeonia apolar extracts were obtained by maceration from dried roots of three species using chloroform as extraction solvent. Composition and concentrations of the chemotaxonomic markers, benzoic acid and monoterpene derivatives, were established by a HPLC-DAD method. Total polyphenols content was determined by the Folin-Ciocalteau colorimetric assay. Anti-inflammatory activity was studied in bronchial epithelial cells affected by Cystic Fibrosis (CF), CuFi1, and the normal counterpart NuLi1. Cell proliferation and viability were evaluated by BrdU incorporation and MTT assays. The ability of the extracts to modulate cytokines (IL-6) and chemokines (IL-8 and RANTES) secretion was tested by ELISA specific immunoassays. Moreover, the anti-inflammatory activity was confirmed in HEK 293/T cells transfected with a NF- ?B reporter plasmid determining NF- ?B activity by luciferase assay. Results: The total polyphenol content, expressed as benzoic acid equivalents, ranged from 105.2-110.0 (P.rockii and P.ostii) to 347.0 (P. lactiflora) ?g/mg. HPLC analysis indicated that the amount of benzoic acid and monoterpenes, paeoniflorigenone and benzoylpaeoniflorin, was almost superimposable (10.4%, 15.1%, and 6.5% w/w) in P. rockii and P. ostii, whereas P. lactiflora was characterized by a very high concentration of benzoic acid (34.5% w/w). All Paeonia extracts at sub-toxic concentrations strongly reduced RANTES production in unstimulated as well as TN F?-stimulated CuFi1 cells. Moreover, P. lactiflora also reduced IL-8 secretion. In NuLi1 cells Paeonia extracts determined only a reduction of RANTES, even though with a lesser extent. This interesting effect on chemokine secretion seems to be correlated to a direct inhibition of NF- ?B activity, as revealed in HEK 293/T cells. Conclusion: Among the three peonies, P. lactiflora, possess anti-inflammatory activity due to inhibition of chemokines (IL-8 and RANTES) release, particularly in CF cells. Results confirmed the traditional anti-inflammatory use of peony and suggested a relevant potential application in the treatment of Cystic Fibrosis.