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In the search of new compounds useful for the treatment of neurological disorders, medicinal plant research has progressed widely in the last decade, particularly because of the hypothesis that natural extracts possess a low or absent toxicity. Aloysia polystachia (Griseb.) Moldenke belonging to the family of Verbenaceae has been used in the traditional medicine for a variety of indications and as a sedative. Recently, some evidence reported that it exhibits some antidepressant- and anxiolytic-like effects involving the modulation of GABA system in preclinical studies in mice (1). However no investigations have been performed on its (geno)toxicity in long-term studies as it would be required for its use in neurological pathologies. In this study, we investigated the genotoxic potential of a water soluble extract of Aloysia polystachia leaves, administered orally every day for 90 days to 20 New Zealand white rabbits homogeneous for weight and age. Subjects were divided into four groups: the control group, and three experimental groups fed with a diet supplemented with 1g/kg; 1.5 g/kg and 2g/kg of Aloysia Polystachia, then peripheral blood was drawn from the vein auricularis marginalis before and after 45 and 90 days of treatment. Cytogenetic analysis was performed on each subject at each time point and chromosome aberrations (structural and numerical) and mitotic index were used as a measure of DNA damage and cytotoxicity (2). Results indicated that Aloysia polystachia extract induces a significant increase in the percentage of aberrant cells as well as in the aberration frequency (mainly chromatid breaks and fragments) associated with a decreasing trend of the mitotic index. The DNA damage was particularly higher after the first 45 days of treatment whereas it remains significantly high but almost unvaried until the end of the treatment. These data suggest that Aloysia polystachia extract has genotoxic and cytotoxic activity, even though further investigations are required to assess which compound of the extract could be responsible for the observed effects., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

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

Background. Myocardial perfusion imaging (MPI) with single photon emission tomography (SPET) is widely used in coronary artery disease evaluation. Recently major dosimetric concerns have arisen. The aim of this study was to evaluate if a pre-test scoring system could predict the results of stress SPET MPI, thus avoiding two radionuclide injections. Methods. All consecutive patients (n=309) undergoing SPET MPI during the first 6 months of 2014 constituted the study group. The scoring system is based on these characteristics: age >65 years (1 point), diabetes (2 points), typical chest pain (2 points), congestive heart failure (3 points), abnormal ECG (4 points), male gender (4 points), and documented previous CAD (5 points). The patients were divided on the basis of the prediction score into 3 classes of risk for an abnormal stress-first protocol. Results. An abnormal stress SPET MPI was present in 7/31 patients (23%) with a low risk score, in 24/90 (27%) with an intermediate score risk, and in 124/188 (66%) with an high score risk. ROC curve analysis showed good prediction of abnormal stress MPI. Conclusions. Our results suggest an appropriate use of a pre-test clinical prediction formula of abnormal stress MPI in a routine clinical setting.

Alzheimer’s disease (AD) is regarded as a multifactorial disease characterized by a complex pathogenesis including a cholinergic deficit - due to degeneration of cholinergic projections from the basal forebrain - and the extracellular accumulation of amyloid beta (Aβ) peptide. Aβ containing 39 to 42 amino acids is the predominant component of the senile plaques that, together with neurofibrillary tangles, are regarded as the neuropathological hallmarks of AD (Sorrentino et al. 2014). Aβ may assume different conformations changing from random coil or α-helical monomers to β-sheet structures forming toxic oligomers and/or β-sheet mature fibrils. In this framework, we studied the effect of acetylcholine (ACh) on the conformation of Aβ by circular dichroism analysis. Moreover we investigated the ability of ACh to protect neuronal cells from the toxic action of amyloid peptide and to modulate the neuroinflammatory response occurring via the phospholipase A2 (PLA2). Results show that the amount of Aβ(25-35) β-strand raised linearly in absence of ACh, whereas it remained almost constant in presence of ACh. In addition, in a micelle solution mimicking the membrane environment ACh was found effective in increasing and stabilizing the soluble and not toxic helical content of Aβ(25-35) suggesting that ACh is capable to preserve the soluble form of Aβ(25-35), reducing the incipit of Aβ aggregation. In order to assess the neuro-protective ability of ACh against toxic Aβ(25-35) accumulation, we used neural cell (NCC) cultures containing both astrocytes and glial cells prepared from brains embryos from timed pregnant Wistar rats and infused ACh for 48h. By immunostaining, we observed that ACh reduced Aβ(25-35)-induced cell death. Then, we tested the protective effect of ACh on inflammation induced by Aβ administration. NCC were challenged with Aβ(25-35) in the presence and absence of ACh and immunostained for astroglial and neuronal markers: results showed a reduction of the morphological features of astrogliosys in ACh treated cells. PLA2 expression analysis corroborated these data also underlying that ACh can negatively regulate inflammation pathways in glial cells., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

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

Mitophagy is a novel identified autophagic process through which defective and malfunctioning mitochondria are degraded or recycled. The balance between this process and mitochondrial biogenesis is crucial for post-mitotic neurons’ survival, as mitochondria are the primary energetic source required for the formation and function of axon terminals and dendritic spines (Amadoro et al 2014). As other cell types, neuronal populations also continually modulate size and number of these organelles, according to the variable energy demands and metabolic states throughout the entire lifetime and/or different sub-cellular compartments. In polarized neurons removal of damaged mitochondria can be a bioenergetically demanding task because these organelles need to be actively transported from the axons and dendrites in order to fuse to cell body resident lysosomes (Wang et al 2006). Physiologically aging as well as numerous human neurodegenerative disease, including Parkison’s disease, Huntington’s disease, amyotrophic lateral sclerosis and Alzheimer’s disease (AD) has been associated with alterations in quality control of neuronal mitochondria (Santos et al 2010), but still now, mitophagy pathway and its dysfunction in neudegenerative disease remain undisclosed. Data from several neurotoxic models show that mitochondria undergo to selective morphological changes in distribution, shape and interconnectivity which are hallmarks of mitophagy. These changes are typically studied at the confocal and TEM microscopes and quantified through a morphometric approach. Due to their polarized nature, neurons present a loss in the neuritic domain more pronounced in respect to the cell body compartment. In addition, morphometric analysis show that the mitochondria interconnectivity, area/perimeter ratio and elongation, major/minor axis ratio, are reduced in neurotoxic models and that these changes parallel in vivo findings from tissue derived from several neurodegenerative diseases. Thus, neurotoxic models, which are acute insults, are able to trigger mitophagy in similar way to human neurodegenerative diseases which, instead, develop the pathology in the course of several years., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Polycystic Ovary Syndrome (PCOS) is an endocrine disease. PCOS afflicts 5 to 10 % of women of reproductive age. The symptoms are: amenorrhea, oligomenorrhea, hirsutism, obesity, infertility, chronic hyperandrogenic anovulation and acne. Other risk factors aggravate this condition: insulin resistance, obesity, hypertension, dyslipidemia, inflammation and subclinical cardiovascular disease. Anxiety, depression and reduced quality of life are also common. This review highlights the mechanisms and the beneficial effects of acupuncture, exercise and resveratrol on animal models and on humans affected by PCOS.

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.

Macroautophagy is a highly regulated cellular process that serves to remove damaged proteins and organelles from the cell. Research during the last decade has made it increasingly clear that autophagy plays important roles in most of the major human diseases as well as in infection and immunity, with increasing evidence for selective autophagy of protein aggregates, organelles and pathogens (Levine et al., 2008). The purpose of autophagy is not the simple elimination of materials, but instead, autophagy serves as a dynamic recycling system that produces new building blocks and energy for cellular renovation and homeostasis. Here we provide a multidisciplinary sight of our current understanding of autophagy’s role in metabolic adaptation, intracellular quality control, and renovation during development and differentiation., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

Diabetic polyneuropathy (DPN), characterized by early hyperalgesia and increased nerve growth factor (NGF) availability, evolves in late irreversible neuropathic symptoms with reduced NGF support to sensory neurons. Electroacupuncture (EA) modulates NGF presence and activity in the peripheral nervous system, being effective for the treatment of DPN symptoms Our working hypotheses are that NGF plays an important pathogenic role in DPN development while EA could be useful in the therapy of DPN by modulating NGF expression and activity. Diabetes was induced in rats by streptozotocin (STZ) injection. One week after STZ, EA treatments were started and continued for three weeks. NGF system and hyperalgesia-related mediators were analyzed in the dorsal root ganglia (DRG), and in their spinal and skin innervation territories. Four weeks diabetes inceased NGF and NGF receptors and deregulated intracellular signaling mediators of DRG neurons hyper-sensitizations. EA in diabetic rats decreased both NGF and NGF receptors, normalized c-Jun N-terminal and p38 kinases activation, decreased transient receptor potential vanilloid-1 ion channel and possibly activated Nf-κB. Increase of NGF signaling might play an important role in the development of DPN. EA represent a supportive tools aimed at controlling DPN by modulating NGF signaling in diabetestargeted neurons., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

The bone resorption activity of osteoclasts is regulated at many levels, including differentiation of their monocytes precursors, fusion into multi-nucleated cells, migration to the resorption site, polarization of the mature osteoclasts and assembly of a podosome-based sealing zone. Another function of osteoclasts is relative to the integrity of the actin cytoskeleton, depending on the substratum upon which the osteoclasts are spread. There are two different structures of actin known as podosomes and sealing zone, actived in specialized matrix contacts and delimiting the membrane domain, where the ruffled border is formed. When a dual coculture of murine osteoblasts and murine mononuclear monocytes, in absolute absence of exogenous cytokines and other growth factors, was cultured on glass, the basic architecture of podosomes units and ruffled border was maintained regularly (1). We studied the osteoclast morphology and its behaviour in adhesion and in vesicle traffic by combination of light microscopy immunohistochemistry and transmission electron microscopy (TEM) immunolabeling. The adhesion and the fusion of preosteoclasts were observed by scanner electron microscopy (SEM). The osteoclasts produced by our physiological dual co-culture (without interaction of specific cytokines) are functionally and biologically active TRAP + and multinucleated cells. In fact the role of RANK, expressed by osteoblasts, controls the modulation of OPG bioavailability in the extracellular compartment. The fusion of monocytes is influenced by the presence of osteoblasts, that is based on RANK-RANKL interaction and communication between osteoblasts and preosteoclasts mediated by several molecules (2). Osteoclasts and osteoblasts can make direct contact, allowing membrane-bound ligands and receptors to interact and initiate intracellular signalling. RANKL-RANK complexes are likely internalized via rafts and then degraded in lysosomes. A recent study has shown that membrane-bound RANKL complexes to OPG is internalized by endocytosis process. A potential interaction of RANKL with clathrin components prior to the OPG binding, as shown by the kinetic results, OPG is intracellularly degraded after being internalized: our observation of osteoclast membrane at TEM has shown that immunogold labelled RANKL colocalizes with immunoglod labelled clathrin via the clathrin-coated-pit-mediated (3) pathway and both proteins are degraded by lysosome and proteasome pathway. 1) Nicolin V. et al.,. 2006 J.Mol.Histol.37 :171-177 2) Narducci P., et al.,. Acta Histochem.113(2):73-81. 3) Narducci P., et al., 2010, Eur J Histochem 54(1):e6., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011