Nuova ricerca

Manuela ZAVATTI

Personale tecnico amministrativo
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

Home | Curriculum(pdf) |


Pubblicazioni

2023 - Human Neuromuscular Junction on a Chip: Impact of Amniotic Fluid Stem Cell Extracellular Vesicles on Muscle Atrophy and NMJ Integrity [Articolo su rivista]
Gatti, Martina; Dittlau, Katarina Stoklund; Beretti, Francesca; Yedigaryan, Laura; Zavatti, Manuela; Cortelli, Pietro; Palumbo, Carla; Bertucci, Emma; Van Den Bosch, Ludo; Sampaolesi, Maurilio; Maraldi, Tullia
abstract

: Neuromuscular junctions (NMJs) are specialized synapses, crucial for the communication between spinal motor neurons (MNs) and skeletal muscle. NMJs become vulnerable in degenerative diseases, such as muscle atrophy, where the crosstalk between the different cell populations fails, and the regenerative ability of the entire tissue is hampered. How skeletal muscle sends retrograde signals to MNs through NMJs represents an intriguing field of research, and the role of oxidative stress and its sources remain poorly understood. Recent works demonstrate the myofiber regeneration potential of stem cells, including amniotic fluid stem cells (AFSC), and secreted extracellular vesicles (EVs) as cell-free therapy. To study NMJ perturbations during muscle atrophy, we generated an MN/myotube co-culture system through XonaTM microfluidic devices, and muscle atrophy was induced in vitro by Dexamethasone (Dexa). After atrophy induction, we treated muscle and MN compartments with AFSC-derived EVs (AFSC-EVs) to investigate their regenerative and anti-oxidative potential in counteracting NMJ alterations. We found that the presence of EVs reduced morphological and functional in vitro defects induced by Dexa. Interestingly, oxidative stress, occurring in atrophic myotubes and thus involving neurites as well, was prevented by EV treatment. Here, we provided and validated a fluidically isolated system represented by microfluidic devices for studying human MN and myotube interactions in healthy and Dexa-induced atrophic conditions-allowing the isolation of subcellular compartments for region-specific analyses-and demonstrated the efficacy of AFSC-EVs in counteracting NMJ perturbations.

2023 - Improved efficacy of quizartinib in combination therapy with PI3K inhibition in primary FLT3-ITD AML cells [Articolo su rivista]
Darici, Salihanur; Jørgensen, Heather G; Huang, Xu; Serafin, Valentina; Antolini, Ludovica; Barozzi, Patrizia; Luppi, Mario; Forghieri, Fabio; Marmiroli, Sandra; Zavatti, Manuela
abstract

: Acute myeloid leukemia is a heterogeneous hematopoietic malignancy, characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells, with poor outcomes. The internal tandem duplication (ITD) mutation of the Fms-like receptor tyrosine kinase 3 (FLT3) (FLT3-ITD) represents the most common genetic alteration in AML, detected in approximately 30% of AML patients, and is associated with high leukemic burden and poor prognosis. Therefore, this kinase has been regarded as an attractive druggable target for the treatment of FLT3-ITD AML, and selective small molecule inhibitors, such as quizartinib, have been identified and trialled. However, clinical outcomes have been disappointing so far due to poor remission rates, also because of acquired resistance. A strategy to overcome resistance is to combine FLT3 inhibitors with other targeted therapies. In this study, we investigated the preclinical efficacy of the combination of quizartinib with the pan PI3K inhibitor BAY-806946 in FLT3-ITD cell lines and primary cells from AML patients. We show here that BAY-806946 enhanced quizartinib cytotoxicity and, most importantly, that this combination increases the ability of quizartinib to kill CD34+ CD38-leukemia stem cells, whilst sparing normal hematopoietic stem cells. Because constitutively active FLT3 receptor tyrosine kinase is known to boost aberrant PI3K signaling, the increased sensitivity of primary cells to the above combination can be the mechanistic results of the disruption of signaling by vertical inhibition.

2022 - Exosomes Derived from Human Amniotic Fluid Mesenchymal Stem Cells Preserve Microglia and Neuron Cells from Aβ [Articolo su rivista]
Zavatti, M.; Gatti, M.; Beretti, F.; Palumbo, C.; Maraldi, T.
abstract

Background: Neuroinflammation is involved in neuronal cell death that occurs in neurodegenerative diseases such as Alzheimer’s disease (AD). Microglia play important roles in regulating the brain amyloid beta (Aβ) levels, so immunomodulatory properties exerted by mesenchymal stem cells may be exploited to treat this pathology. The evidence suggests that the mechanism of action of human amniotic fluid stem cells (hAFSCs) is through their secretome, which includes exosomes (exo). Methods: We examined the effect of exosomes derived from human amniotic fluid stem cells (hAFSCs-exo) on activated BV-2 microglia cells by lipopolysaccharide (LPS) as a neuroinflammation model. To investigate the exo effect on the interplay between AD neurons and microglia, SH-SY5Y neuroblastoma cells treated with Aβ were exposed to a conditioned medium (CM) obtained from activated BV-2 or co-culture systems. Results: We found that the upregulation of the markers of pro-inflammatory microglia was prevented when exposed to hAFSC-exo whereas the markers of the anti-inflammatory macrophage phenotype were not affected. Interestingly, the hAFSC-exo pretreatment significantly inhibited the oxidative stress rise and apoptosis occurring in the neurons in presence of both microglia and Aβ. Conclusion: We demonstrated that hAFSC-exo mitigated an inflammatory injury caused by microglia and significantly recovered the neurotoxicity, suggesting that hAFSC-exo may be a potential therapeutic agent for inflammation-related neurological conditions, including AD.

2021 - Amniotic fluid stem cell-derived extracellular vesicles counteract steroid-induced osteoporosis in vitro [Articolo su rivista]
Gatti, M.; Beretti, F.; Zavatti, M.; Bertucci, E.; Luz, S. R.; Palumbo, C.; Maraldi, T.
abstract

Background—Osteoporosis is characterized by defects in both quality and quantity of bone tissue, which imply high susceptibility to fractures with limitations of autonomy. Current therapies for osteoporosis are mostly concentrated on how to inhibit bone resorption but give serious adverse effects. Therefore, more effective and safer therapies are needed that even encourage bone formation. Here we examined the effect of extracellular vesicles secreted by human amniotic fluid stem cells (AFSC) (AFSC-EV) on a model of osteoporosis in vitro. Methods—human AFSC-EV were added to the culture medium of a human pre-osteoblast cell line (HOB) induced to differentiate, and then treated with dexamethasone as osteoporosis inducer. Aspects of differentiation and viability were assessed by immunofluorescence, Western blot, mass spectrometry, and histological as-says. Since steroids induce oxidative stress, the levels of reactive oxygen species and of redox related proteins were evaluated. Results—AFSC-EV were able to ameliorate the differentiation ability of HOB both in the case of pre-osteoblasts and when the differentiation process was affected by dexa-methasone. Moreover, the viability was increased and parallelly apoptotic markers were reduced. The presence of EV positively modulated the redox unbalance due to dexamethasone. Conclusion— these findings demonstrated that EV from hAFSC have the ability to recover precursor cell potential and delay local bone loss in steroid-related osteoporosis.

2021 - Identification of Sclerostin as a Putative New Myokine Involved in the Muscle-to-Bone Crosstalk [Articolo su rivista]
Magarò, Maria Sara; Bertacchini, Jessika; Florio, Francesca; Zavatti, Manuela; Potì, Francesco; Cavani, Francesco; Amore, Emanuela; De Santis, Ilaria; Bevilacqua, Alessandro; Reggiani Bonetti, Luca; Torricelli, Pietro; Maurel, Delphine B.; Biressi, Stefano; Palumbo, Carla
abstract

Bone and muscle have been recognized as endocrine organs since they produce and secrete “hormone-like factors” that can mutually influence each other and other tissues, giving rise to a “bone–muscle crosstalk”. In our study, we made use of myogenic (C2C12 cells) and osteogenic (2T3 cells) cell lines to investigate the effects of muscle cell-produced factors on the maturation process of osteoblasts. We found that the myogenic medium has inhibitory effects on bone cell differentiation and we identified sclerostin as one of the myokines produced by muscle cells. Sclerostin is a secreted glycoprotein reportedly expressed by bone/cartilage cells and is considered a negative regulator of bone growth due to its role as an antagonist of the Wnt/β-catenin pathway. Given the inhibitory role of sclerostin in bone, we analyzed its expression by muscle cells and how it affects bone formation and homeostasis. Firstly, we characterized and quantified sclerostin synthesis by a myoblast cell line (C2C12) and by murine primary muscle cells by Western blotting, real-time PCR, immunofluorescence, and ELISA assay. Next, we investigated in vivo production of sclerostin in distinct muscle groups with different metabolic and mechanical loading characteristics. This analysis was done in mice of different ages (6 weeks, 5 and 18 months after birth) and revealed that sclerostin expression is dynamically modulated in a muscle-specific way during the lifespan. Finally, we transiently expressed sclerostin in the hind limb muscles of young mice (2 weeks of age) via in vivo electro-transfer of a plasmid containing the SOST gene in order to investigate the effects of muscle-specific overproduction of the protein. Our data disclosed an inhibitory role of the muscular sclerostin on the bones adjacent to the electroporated muscles. This observation suggests that sclerostin released by skeletal muscle might synergistically interact with osseous sclerostin and potentiate negative regulation of osteogenesis possibly by acting in a paracrine/local fashion. Our data point out a role for muscle as a new source of sclerostin.

2021 - Possible Association Between DHEA and PKCε in Hepatic Encephalopathy Amelioration: A Pilot Study [Articolo su rivista]
Di Cerbo, A.; Roncati, L.; Marini, C.; Carnevale, G.; Zavatti, M.; Avallone, R.; Corsi, L.
abstract

Objective: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome caused by liver failure and by an impaired neurotransmission and neurological function caused by hyperammonemia (HA). HE, in turn, decreases the phosphorylation of protein kinase C epsilon (PKCε), contributing to the impairment of neuronal functions. Dehydroepiandrosterone (DHEA) exerts a neuroprotective effect by increasing the GABAergic tone through GABAA receptor stimulation. Therefore, we investigated the protective effect of DHEA in an animal model of HE, and the possible modulation of PKCε expression in different brain area. Methods: Fulminant hepatic failure was induced in 18 male, Sprague–Dawley rats by i.p. administration of 3 g/kg D-galactosamine, and after 30 min, a group of animals received a subcutaneous injection of 25 mg/kg (DHEA) repeated twice a day (3 days). Exploratory behavior and general activity were evaluated 24 h and 48 h after the treatments by the open field test. Then, brain cortex and cerebellum were used for immunoblotting analysis of PKCε level. Results: DHEA administration showed a significant improvement of locomotor activity both 24 and 48 h after D-galactosamine treatment (****p < 0.0001) but did not ameliorate liver parenchymal degeneration. Western blot analysis revealed a reduced immunoreactivity of PKCε (*p < 0.05) following D-galactosamine treatment in rat cortex and cerebellum. After the addition of DHEA, PKCε increased in the cortex in comparison with the D-galactosamine-treated (***p < 0.001) and control group (*p < 0.05), but decreased in the cerebellum (*p < 0.05) with respect to the control group. PKCε decreased after treatment with NH4Cl alone and in combination with DHEA in both cerebellum and cortex (****p < 0.0001). MTS assay demonstrated the synergistic neurotoxic action of NH4Cl and glutamate pretreatment in cerebellum and cortex along with an increased cell survival after DHEA pretreatment, which was significant only in the cerebellum (*p < 0.05). Conclusion: An association between the DHEA-mediated increase of PKCε expression and the improvement of comatose symptoms was observed. PKCε activation and expression in the brain could inhibit GABA-ergic tone counteracting HE symptoms. In addition, DHEA seemed to ameliorate the symptoms of HE and to increase the expression of PKCε in cortex and cerebellum.

2021 - Synergistic cytotoxicity of dual PI3K/mTOR and FLT3 inhibition in FLT3-ITD AML cells [Articolo su rivista]
Darici, S.; Zavatti, M.; Braglia, L.; Accordi, B.; Serafin, V.; Horne, G. A.; Manzoli, L.; Palumbo, C.; Huang, X.; Jorgensen, H. G.; Marmiroli, S.
abstract

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy, characterized by a heterogeneous genetic landscape and complex clonal evolution, with poor outcomes. Mutation at the internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic alterations in AML, associated with high relapse rates and poor survival due to the constitutive activation of the FLT3 receptor tyrosine kinase and its downstream effectors, such as PI3K signaling. Thus, aberrantly activated FLT3-kinase is regarded as an attractive target for therapy for this AML subtype, and a number of small molecule inhibitors of this kinase have been identified, some of which are approved for clinical practice. Nevertheless, acquired resistance to these molecules is often observed, leading to severe clinical outcomes. Therapeutic strategies to tackle resistance include combining FLT3 inhibitors with other antileukemic agents. Here, we report on the preclinical activity of the combination of the FLT3 inhibitor quizartinib with the dual PI3K/mTOR inhibitor PF-04691502 in FLT3-ITD cells. Briefly, we show that the association of these two molecules displays synergistic cytotoxicity in vitro in FLT3-ITD AML cells, triggering 90% cell death at nanomolar concentrations after 48 h.

2021 - The Interplay between HGF/c-met Axis and Nox4 in BRAF Mutated Melanoma [Articolo su rivista]
Beretti, Francesca; Farnetani, Francesca; Reggiani Bonetti, Luca; Fabbiani, Luca; Zavatti, Manuela; Maiorana, Antonino; Pellacani, Giovanni; Maraldi, Tullia
abstract

Melanoma is the leading cause of death due to cutaneous malignancy and its incidence is on the rise. Several signaling pathways, including receptor tyrosine kinases, have a role in the development and progression of melanocytic lesions and malignant melanoma. Among those, the hepatocyte growth factor (HGF)/c-met axis is emerging as a critical player because it can play a role in drug resistance. Indeed, 50% of melanoma patients present BRAF mutations, however, all responders develop resistance to the inhibitors typically within one year of treatment. Interestingly, BRAF inhibitors induce reactive oxygen species (ROS) in melanoma cells, therefore, the aim of this study was to investigate a possible interplay between HGF/c-met and ROS sources, such as NADPH oxidases (Nox).

2021 - Unravelling Heterogeneity of Amplified Human Amniotic Fluid Stem Cells Sub-Populations [Articolo su rivista]
Casciaro, Francesca; Zia, Silvia; Forcato, Mattia; Zavatti, Manuela; Beretti, Francesca; Bertucci, Emma; Zattoni, Andrea; Reschiglian, Pierluigi; Alviano, Francesco; Bonsi, Laura; Follo, Matilde Yung; Demaria, Marco; Roda, Barbara; Maraldi, Tullia
abstract

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector® (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector® profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine.

2020 - Comparison of the therapeutic effect of amniotic fluid stem cells and their exosomes on monoiodoacetate-induced animal model of osteoarthritis [Articolo su rivista]
Zavatti, M.; Beretti, F.; Casciaro, F.; Bertucci, E.; Maraldi, T.
abstract

The cartilage tissue engineering associated with stem cell-related therapies is becoming very interesting since adult articular cartilage has limited intrinsic capacity for regeneration upon injury. Amniotic fluid stem cells (AFSC) have been shown to produce exosomes with growth factors and immunomodulating molecules that could stop tissue degradation and induce cartilage repair. Based on this state of the art, the main aim of this study was to explore the efficacy of the secreted exosomes, compared to their AFSC source, in MIA-induced animal model of osteoarthritis mimicking a chronic and degenerative process, where inflammation is also involved and lead to irreversible joint damage. Exosomes, obtained by the use of a commercial kit, prior to the injection in animal knee joints, were characterized for the presence of typical markers and HGF, TGFβ, and IDO. Then, analyses were performed by histology, immunohistochemistry, and behavioral scoring up to 3 weeks after the treatment. Exosome-treated defects showed enhanced pain tolerance level and improved histological scores than the AFSC-treated defects. Indeed by 3 weeks, TGFβ-rich exosome samples induced an almost complete restoration of cartilage with good surface regularity and with the characteristic of hyaline cartilage. Moreover, cells positive for resolving macrophage marker were more easily detectable into exosome-treated joints. Therefore, a modulating role for exosomes on macrophage polarization is conceivable, as demonstrated also by experiments performed on THP1 macrophages. In conclusion, this study demonstrates for the first time the efficacy of human AFSC exosomes in counteract cartilage damage, showing a positive correlation with their TGFβ content.

2020 - Deregulated PTEN/PI3K/AKT/mTOR signaling in prostate cancer: Still a potential druggable target? [Articolo su rivista]
Braglia, L.; Zavatti, M.; Vinceti, M.; Martelli, A. M.; Marmiroli, S.
abstract

Although the prognosis of patients with localized prostate cancer is good after surgery, with a favorable response to androgen deprivation therapy, about one third of them invariably relapse, and progress to castration-resistant prostate cancer. Overall, prostate cancer therapies remain scarcely effective, thus it is mandatory to devise alternative treatments enhancing the efficacy of surgical castration and hormone administration. Dysregulation of the phosphoinositide 3-kinase pathway has attracted growing attention in prostate cancer due to the highly frequent association of epigenetic and post-translational modifications as well as to genetic alterations of both phosphoinositide 3-kinase and PTEN to onset and/or progression of this malignancy, and to resistance to canonical androgen-deprivation therapy. Here we provide a summary of the biological functions of the major players of this cascade and their deregulation in prostate cancer, summarizing the results of preclinical and clinical studies with PI3K signaling inhibitors and the reasons of failure independent from genomic changes.

2020 - Oxidative stress in Alzheimer's disease: In vitro therapeutic effect of amniotic fluid stem cells extracellular vesicles [Articolo su rivista]
Gatti, M.; Zavatti, M.; Beretti, F.; Giuliani, D.; Vandini, E.; Ottani, A.; Bertucci, E.; Maraldi, T.
abstract

Alzheimer's disease (AD) is characterized by abnormal protein aggregation, deposition of extracellular β-amyloid proteins (Aβ), besides an increase of oxidative stress. Amniotic fluid stem cells (AFSCs) should have a therapeutic potential for neurodegenerative disorders, mainly through a paracrine effect mediated by extracellular vesicles (EV). Here, we examined the effect of EV derived from human AFSCs (AFSC-EV) on the disease phenotypes in an AD neuron primary culture. We observed a positive effect of AFSC-EV on neuron morphology, viability, and Aβ and phospho-Tau levels. This could be due to the apoptotic and autophagic pathway modulation derived from the decrease in oxidative stress. Indeed, reactive oxygen species (ROS) were reduced, while GSH levels were enhanced. This modulation could be ascribed to the presence of ROS regulating enzymes, such as SOD1 present into the AFSC-EV themselves. This study describes the ROS-modulating effects of extracellular vesicles alone, apart from their deriving stem cell, in an AD in vitro model, proposing AFSC-EV as a therapeutic tool to stop the progression of AD.

2020 - Prolonged hypoxia delays aging and preserves functionality of human amniotic fluid stem cells [Articolo su rivista]
Casciaro, F.; Borghesan, M.; Beretti, F.; Zavatti, M.; Bertucci, E.; Follo, M. Y.; Maraldi, T.; Demaria, M.
abstract

Human amniotic fluid stem cells (hAFSCs) are an emerging tool in regenerative medicine because they have the ability to differentiate into various lineages and efficiently improve tissue regeneration with no risk of tumorigenesis. Although hAFSCs are easily isolated from the amniotic fluid, their expansion ex vivo is limited by a quick exhaustion which impairs replicative potential and differentiation capacity. In this study, we evaluate various aging features of hAFSCs cultured at different oxygen concentrations. We show that low oxygen (1% O2) extends stemness and proliferative features, and delays induction of senescence-associated markers. Hypoxic hAFSCs activate a metabolic shift and increase resistance to pro-apoptotic stimuli. Moreover, we observe that cells at low oxygen remain capable of osteogenesis for prolonged periods of time, suggesting a more youthful phenotype. Together, these data demonstrate that low oxygen concentrations might improve the generation of functional hAFSCs for therapeutic use by delaying the onset of cellular aging.

2020 - Protective Effects of Borago officinalis (Borago) on Cold Restraint Stress-Induced Gastric Ulcers in Rats: A Pilot Study [Articolo su rivista]
Di Cerbo, Alessandro; Carnevale, Gianluca; Avallone, Rossella; Zavatti, Manuela; Corsi, Lorenzo
abstract

Stress is a typical body's natural defense to a generic physical or psychic change. A specific linking mechanism between ulcer onset and psycho-physical stress prolonged exposure has been reported. We decided to investigate the possible effects of Borago officinalis L. (Borago) in preventing physical (stress)-induced gastric ulcers in a rat model. Eighty male Sprague-Dawley rats were randomly divided into 16 groups, pretreated with a control solution, omeprazole (20 mg/kg), Borago methanolic extract (25, 50, 100, 250, and 500 mg/kg), Borago organic extract (50, 100, 250, and 500 mg/kg), Borago aqueous extract (5, 10, 20, 30, and 40 mg/kg), and D(-)-2-Amino-5-phosphonovaleric acid (AP5) (25 mg/kg) and kept in stressful conditions such as water immersion and restraint-induced stress ulcers. The animals were sacrificed and their stomach scored for the severity and the number of gastric ulcers. Methanolic extract (500 mg/kg) significantly reduced both ulcer parameters (***p < 0.001 and **p < 0.01, respectively). Aqueous and organic extract significantly decreased severity score at 5 and 10 mg/kg (**p < 0.01 and ***p < 0.001, respectively), and at 250 and 500 mg/kg (***p < 0.001), respectively, while gastric ulcers' resulted number significantly reduced only at 10 mg/kg (*p < 0.05) and at 500 mg/kg (**p < 0.01), respectively. On the other hand, aqueous extract significantly increased the mucosal gastric content of cAMP (*p < 0.05) and NR2A and NR2B subunits (*p < 0.05 and **p < 0.01, respectively) at 5 mg/kg. Organic extract showed also a significant cytotoxic effect at 500 and 1,000 mg/kg with a 3T3 cell viability reduction of 43.6% (**p < 0.01) and 92.1% (***p < 0.001), respectively. Borago aqueous extract at 10 mg/kg could be considered as a potential protective agent against stress-induced ulcers, and it is reasonable to possibly ascribe such protective activity to a modulation of the NR2A and NR2B subunit expression.

2018 - Amniotic fluid stem cell exosomes: Therapeutic perspective [Articolo su rivista]
Beretti, Francesca; Zavatti, Manuela; Casciaro, Francesca; Comitini, Giuseppina; Franchi, Fabrizia; Barbieri, Veronica; La Sala, Giovanni B.; Maraldi, Tullia
abstract

It is widely accepted that the therapeutic potential of stem cells can be largely mediated by paracrine factors, also included into exosomes. Thus, stem cell-derived exosomes represent a major therapeutic option in regenerative medicine avoiding, if compared to stem cells graft, abnormal differentiation and tumor formation. Exosomes derived from mesenchymal stem cells (MSC) induce damaged tissue repair, and can also exert immunomodulatory effects on the differentiation, activation and function of different lymphocytes. Therefore, MSC exosomes can be considered as a potential treatment for inflammatory diseases and also an ideal candidate for allogeneic therapy due to their low immunogenicity. Amniotic fluid stem cells (AFSCs) are broadly multipotent, can be expanded in culture, and can be easily cryopreserved in cellular banks. In this study, morphology, phenotype, and protein content of exosomes released into amniotic fluid in vivo and from AFSC during in vitro culture (conditioned medium) were examined. We found that AFSC-derived exosomes present different molecules than amniotic fluid ones, some of them involved in immunomodulation, such transforming growth factor beta and hepatic growth factors. The immunomodulatory effect of AFSC's exosomes on peripheral blood mononuclear cells stimulated with phytohemagglutinin was compared to that of the supernatant produced by such conditioned media deprived of exosomes. We present evidence that the principal effect of AFSC conditioned media (without exosomes) is the induction of apoptosis in lymphocytes, whereas exposure to AFSC-derived exosomes decreases the lymphocyte's proliferation, supporting the hypothesis that the entire secretome of stem cells differently affects immune-response. © 2017 BioFactors, 44(2):158–167, 2018.

2018 - Nuclear Nox4 interaction with prelamin A is associated with nuclear redox control of stem cell aging [Articolo su rivista]
Casciaro, Francesca; Beretti, Francesca; Zavatti, Manuela; McCubrey, James A.; Ratti, Stefano; Marmiroli, Sandra; Follo, Matilde Y.; Maraldi, Tullia
abstract

Mesenchymal stem cells have emerged as an important tool that can be used for tissue regeneration thanks to their easy preparation, differentiation potential and immunomodulatory activity. However, an extensive culture of stem cells in vitro prior to clinical use can lead to oxidative stress that can modulate different stem cells properties, such as self-renewal, proliferation, differentiation and senescence. The aim of this study was to investigate the aging process occurring during in vitro expansion of stem cells, obtained from amniotic fluids (AFSC) at similar gestational age. The analysis of 21 AFSC samples allowed to classify them in groups with different levels of stemness properties. In summary, the expression of pluripotency genes and the proliferation rate were inversely correlated with the content of reactive oxygen species (ROS), DNA damage signs and the onset premature aging markers, including accumulation of prelamin A, the lamin A immature form. Interestingly, a specific source of ROS, the NADPH oxidase isoform 4 (Nox4), can localize into PML nuclear bodies (PML-NB), where it associates to prelamin A. Besides, Nox4 post translational modification, involved in PML-NB localization, is linked to its degradation pathway, as it is also for prelamin A, thus possibly modulating the premature aging phenotype occurrence.

2017 - DEVELOPMENT OF A NOVEL METHOD FOR AMNIOTIC FLUID STEM CELL STORAGE [Articolo su rivista]
Zavatti, Manuela; Beretti, Francesca; Casciaro, Francesca; Comitini, Giuseppina; Franchi, Fabrizia; Barbieri, Veronica; Bertoni, Laura; DE POL, Anto; LA SALA, Giovanni Battista; Maraldi, Tullia
abstract

Background - Current procedures for collection of human Amniotic Fluid Stem Cells (hAFSCs) imply that amniotic fluid cells were cultured in flask for two weeks, than can be devoted to research purpose. However, hAFSCs could be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of the study was to verify if a direct freezing of amniotic fluid cells is able to maintain and / or improve the potential of the sub-population of stem cells. Methods - We compared the potential of the hAFSCs depending on the moment in which they are frozen, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis, and differentiation potential of C and D samples were compared. Results - hAFSCs isolated from D samples expressed MSC markers until later passages, had a good proliferation rate, and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, the direct freezing induce a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. Conclusions - This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration affecting stem cell properties. This technique might be a reasonable approach in terms of costs and for the process of accreditation in GMP for a stem cell bank.

2016 - Estrogen receptor signaling in the ferutinin-induced osteoblastic differentiation of human amniotic fluid stem cells [Articolo su rivista]
Zavatti, Manuela; Guida, M; Maraldi, Tullia; Beretti, Francesca; Bertoni, Laura; LA SALA, Giovanni Battista; DE POL, Anto
abstract

Ferutinin is a diaucane sesquiterpene with a high estrogenic activity. Since ferutinin is able to enhance osteoblastic differentiation of human amniotic fluid stem cells (hAFSCs), the aim of this study was to evaluate the role of the estrogen receptors α (ERα) and G-protein coupled receptor 30 (GPR30) in ferutinin-mediated osteoblastic differentiation. Moreover, it was investigated if MEK/ERK and PI3K/Akt signaling pathways are involved in ferutinin-induced effects.

2016 - NADPH oxidase-4 and MATER expressions in granulosa cells: Relationships with ovarian aging [Articolo su rivista]
Maraldi, Tullia; Resca, Elisa; Nicoli, Alessia; Beretti, Francesca; Zavatti, Manuela; Capodanno, Francesco; Morini, Daria; Palomba, Stefano; LA SALA, Giovanni Battista; DE POL, Anto
abstract

Aims Relevant roles in follicular development and ovulation are played by maternal antigen that embryos require (MATER), product of a maternal effect gene, and by reactive oxygen species (ROS), indispensable for the induction of ovulatory genes. At the moment, the relationship between these two biological systems and their involvement in the ovarian aging have not been still clarified. The aim of the current experimental study was to analyse the age-related changes of the MATER and NOX proteins. Materials and methods MATER and ROS homeostasis was studied in granulosa cells (GCs) and cumulus cells (CCs) of infertile patients who undergone oocyte retrieval for in vitro fertilization cycles using Western blot and confocal immunofluorescence analysis. Samples were obtained from subjects with age ≥ 40 years (cases) and with age ≤ 37 years (controls). Key findings The expression pattern of MATER and NOX observed in GCs was not different from that observed in CCs. High levels of both proteins were detected in the control samples. A significant lower expression of both MATER and NOX4 was observed in the case versus control samples. Significance The expression of MATER and NOX4 proteins are closely related to the follicular development and ovulation with particular regard for ovarian aging.

2015 - Critical-size bone defect repair using amniotic fluid stem cell/collagen constructs: Effect of oral ferutinin treatment in rats [Articolo su rivista]
Zavatti, Manuela; Bertoni, Laura; Maraldi, Tullia; Resca, Elisa; Beretti, Francesca; Guida, Marianna; La Sala, Giovanni Battista; De Pol, Anto
abstract

Aims: This study aims to evaluate the bone regeneration in a rat calvarias critical size bone defect treated with a construct consisting of collagen type I and human amniotic fluid stem cells (AFSCs) after oral administration of phytoestrogen ferutinin. Main methods: In 12 week old male rats (n = 10), we performed two symmetric full-thickness cranial defects on each parietal region, and a scaffold was implanted into each cranial defect. The rats were divided into four groups: 1) collagen scaffold, 2) collagen scaffold + ferutinin at a dose of 2 mg/kg/5 mL, 3) collagen scaffold + AFSCs, and 4) collagen scaffold + AFSCs + ferutinin. The rats were sacrificed after 4 weeks, and the calvariae were removed, fixed, embedded in paraffin and cut into 7 pm thick sections. Histomorphometric measures, immunohistochemical and immunofluorescence analyses were performed on the paraffin sections. Key findings: The histomorphometric analysis on H&E stained sections showed a significant increase in the regenerated area of the 4th group compared with the other groups. Immunohistochemistly performed with a human anti-mitochondrial antibody showed the presence of AFSCs 4 weeks after the transplant. Immunofluorescence analysis revealed the presence of osteocalcin and estrogen receptors (ER alpha. and GPR30) in all-groups, with a greater expression of all markers in samples where the scaffold was treated with AFSCs and the rats were orally administered ferutinin. Significance: Our results demonstrated that the oral administration of ferutinin is able to improve the bone regeneration of critical-size bone defects in vivo that is obtained with collagen-AFSCs constructs.

2015 - Enrichment in c-Kit improved differentiation potential of amniotic membrane progenitor/stem cells [Articolo su rivista]
Resca, Elisa; Zavatti, Manuela; Maraldi, Tullia; Bertoni, Laura; Beretti, Francesca; Guida, Marianna; La Sala, Giovanni Battista; Guillot, P. V; David, A. L; Sebire, N. J; De Pol, Anto; De Coppi, P.
abstract

Introduction Human term placenta has attracted increasing attention as an alternative source of stem cells for regenerative medicine since it is accessible without ethical objections. The amniotic membrane (AM) contains at least two stem cell types from different embryological origins: ectodermal amniotic epithelial stem cells, and mesodermal mesenchymal stromal cells. Among the second group we studied the characteristics of amniotic mesenchymal cells (AMC) versus the ones enriched for the commonly used surface marker c-Kit (amniotic progenitor/stem cells-ASC), a stem cell factor receptor with crucial functions in a variety of biological systems and presents in early progenitors of different origin, as been already demonstrated in the enriched chorionic stem cells. Methods After isolation, cells from the amniotic membranes (amniotic cells-AC) were selected for c-Kit (ASC) and compared these cells with c-Kit unselected (AMC), evaluating the expression of other stem cell markers (Oct-4, Tra-1-81, SSEA-4), CD271 and Slug. Results Immunofluorescence analysis showed that ASC cells exhibited greater stem cell marker expression and included more CD271 and Slug positive cells. This was consistent with the interpretation that c-Kit enriched AC show greater stemness capacity compared to c-Kit unselected AMC. Discussion AMC and ASC can both differentiate into various cell types including adipogenic, osteogenic, chondrogenic, neurogenic and hepatic lineages, but the enrichment in c-Kit improved stemness and differentiation potential of ASC.

2015 - Nuclear Nox4 role in stemness power of human amniotic fluid stem cells [Articolo su rivista]
Maraldi, Tullia; Guida, Marianna; Zavatti, Manuela; Resca, Elisa; Bertoni, Laura; La Sala, Giovanni Battista; De Pol, Anto
abstract

Human amniotic fluid stem cells (AFSC) are an attractive source for cell therapy due to their multilineage differentiation potential and accessibility advantages. However the clinical application of human stem cells largely depends on their capacity to expand in vitro, since there is an extensive donor-to-donor heterogeneity. Reactive oxygen species (ROS) and cellular oxidative stress are involved in many physiological and pathophysiological processes of stem cells, including pluripotency, proliferation, differentiation, and stress resistance. The mode of action of ROS is also dependent on the localization of their target molecules. Thus, the modifications induced by ROS can be separated depending on the cellular compartments they affect. NAD(P)H oxidase family, particularly Nox4, has been known to produce ROS in the nucleus. In the present study we show that Nox4 nuclear expression (nNox4) depends on the donor and it correlates with the expression of transcription factors involved in stemness regulation, such as Oct4, SSEA-4, and Sox2. Moreover nNox4 is linked with the nuclear localization of redox sensitive transcription factors, as Nrf2 and NF-κB, and with the differentiation potential. Taken together, these results suggest that nNox4 regulation may have important effects in stem cell capability through modulation of transcription factors and DNA damage.

2015 - Role of hepatocyte growth factor in the immunomodulation potential of amniotic fluid stem cells [Articolo su rivista]
Maraldi, Tullia; Beretti, Francesca; Guida, Marianna; Zavatti, Manuela; De Pol, Anto
abstract

Human amniotic fluid stem cells (hAFSCs) may be useful for regenerative medicine because of their potential to differentiate into all three germ layers and to modulate immune response with different types of secretion molecules. This last issue has not been completely elucidated. The aim of this study was to investigate the secretome profile of the hAFSC, focusing on the role of hepatocyte growth factor (HGF) in immunoregulation through short and long cocultures with human peripheral blood mononuclear cells. We found that HGF produced by hAFSCs exerts a cytoprotective role, inducing an increase in caspase-dependent apoptosis in human immune cells. This study provides evidence supporting the hypothesis that amniotic fluid is an ideal source of stem cells for expansion and banking properties for therapeutic use. hAFSCs not only are less immunogenic but also can secrete immunoregulatory factors that may be useful in autoimmune diseases or allogenic implants. SIGNIFICANCE: New information about the secretome pattern is reported in this paper. Human amniotic fluid stem cells (hAFSCs) possess immunomodulatory properties involving hepatocyte growth factor production. hAFSCs could be used in immunotherapies and might be able to avoid allogenic rejection

2014 - Ferutinin dose-dependent effects on uterus and mammary gland in ovariectomized rats [Articolo su rivista]
Ferretti, Marzia; Cavani, Francesco; Manni, Paola; Carnevale, Gianluca; Bertoni, Laura; Zavatti, Manuela; Palumbo, Carla
abstract

The present paper completes our recent study on the effects of phytoestrogen ferutinin in preventing osteoporosis and demonstrating the superior osteoprotective effect of a 2 mg/kg/day dose in ovariectomized (OVX) rats, compared to both estrogens and lower (0.5, 1 mg/kg/day) ferutinin doses. Morphological and morphometrical analyses were performed on the effects of different doses of ferutinin administrated for one month on uterus and on mammary gland of Sprague-Dawley OVX rats, evaluated in comparison with the results for estradiol benzoate. To verify whether ferutinin provides protection against uterine and breast cancer, estimations were made of both the amount of cell proliferation (by Ki-67), and the occurrence of apoptosis (by TUNEL), two processes that in unbalanced ratio form the basis for cancer onset. The results suggest that the effects of ferutinin are dose dependent and that a 2 mg/kg/day dose might offer a better protective action against the onset of both breast and uterine carcinoma compared to ferutinin in lower doses or estradiol benzoate, increasing cellular apoptosis in glandular epithelia.

2014 - Human amniotic fluid stem cells: neural differentiation in vitro and in vivo [Articolo su rivista]
Maraldi, Tullia; Bertoni, Laura; Riccio, Massimo; Zavatti, Manuela; Carnevale, Gianluca; Resca, Elisa; Guida, Marianna; Beretti, Francesca; LA SALA, Giovanni Battista; DE POL, Anto
abstract

The successful integration of stem cells after their implantation into the brain has become a central issue in modern neuroscience. In this study, we test the neural differentiation potential of c-Kit(+)/Oct-4(+) human amniotic fluid stem cells (hAFSCs) in vitro and their survival and integration in vivo. hAFSCs were induced towards neural differentiation and specific markers (GFAP, β-III tubulin, CNPase, MAP2, NeuN, synapsines, S100, PMP22) were detected by immunofluorescence and Western blot analysis. Glial proteins were expressed as early as 2 weeks after the initial differentiation stimulus, whereas neuronal markers started to appear from the third week of differentiation under culturing conditions of high cell density. This timeline suggested that glial cells possessed a promoting role in the differentiation of hAFSCs towards a neuronal fate. hAFSCs were then implanted into the lateral ventricle of the brain of 1-day-old rats, since neuronal development occurs up to 1 month after birth in this animal model. Our data showed that hAFSCs survived for up to 6 weeks post-implantation, were integrated into various areas of the central nervous system and migrated away from the graft giving rise to mature neurons and oligodendrocytes. We conclude that hAFSCs are able to differentiate and integrate into nervous tissue during development in vivo.

2013 - Effect of DHEA therapy on sexual behavior in female rats [Articolo su rivista]
Pluchino, N; Giannini, A; Cela, V; Santoro, An; Carnevale, Gianluca; Zavatti, Manuela; DI VIESTI, Vittoria; Benelli, Augusta; Genazzani, Andrea Riccardo; Zanoli, Paola
abstract

Delta-5 androgen therapies seem to enhance the sexual response in experimental animal models and in clinical trial. This study analyzed the influence of dehydroepiandrosterone (DHEA) administration on receptive and proceptive components of female rat sexual behavior. Ovariectomized (OVX) adult rats were divided in six groups submitted to the following treatments for 4 weeks: DHEA 0.5 and 5 mg/kg, by oral gavage, alone or in combination with estradiol benzoate 3 µg/rat; EB 3 and 10 µg/rat as control groups. All animals received progesterone (500 µg/rat) 4 h before the behavioral tests. All animals were tested for the following: receptivity and proceptivity weekly for 4 weeks; partner preference and paced mating behavior at the end of the treatments. Oral administration of DHEA at 5 mg/kg in EB primed rats was able to significantly increase proceptive behaviors, already after 1 week of treatment. The increase was more marked after 3 and 4 weeks of treatment. Behavioral changes were associated to modifications of circulating and brain level of allopregnanolone and beta-endorphin, although circulating hormonal levels were within a physiological range. Hormonal treatment using physiological doses of delta-5 androgens (DHEA) positively affects sexual motivation in OVX rats.

2013 - Enrichment in c-Kit(+) enhances mesodermal and neural differentiation of human chorionic placental cells [Articolo su rivista]
Resca, Elisa; Zavatti, Manuela; Bertoni, Laura; Maraldi, Tullia; DE BIASI, Sara; Pisciotta, Alessandra; A., Nicoli; LA SALA, Giovanni Battista; P. V., Guillot; A. L., David; N. J., Sebire; P. D., Coppi; DE POL, Anto
abstract

OBJECTIVE: Human term placenta (HTP) has attracted increasing attention as an alternative source of stem cells for regenerative medicine since the amniochorionic membrane harbors stem cells populations that are easily accessible, abundantly available without ethical objections. In the chorionic side of HTP we found a progenitor perivascular "niche" in which rare cells co-express Oct-4 and c-Kit. We investigated the stem cell characteristics and differentiation potential of a chorionic derived population enriched in c-Kit(+) cells and compared this to the unenriched population. STUDY DESIGN: Cells, isolated from the chorion of HTP, were expanded and enriched in c-Kit(+) cells (Chorionic Stem Cells-CSC). Histological staining, immunofluorescence, Western blot and flow cytometry were used to verify the stem cells characteristics of the populations and to compare the differentiation capability towards mesodermal and neural lineages in vitro. RESULTS: The expression of the pluripotent marker Oct-4 was greater in the CSCs compared to the unselected cells (Chorionic Cell-CC) but both Oct-4 and c-Kit expression decreased during passages. After differentiation, CSC displayed stronger chondrogenic and osteogenic potential and a greater adipogenic forming capacity compared to unselected ones. CSC differentiated better into immature oligodendrocytes while CC showed a neuronal progenitor differentiation potential. Moreover, both populations were able to differentiate in hepatogenic lineage. CONCLUSION: CSC display improved Oct-4 expression and a high differentiation potential into mesodermal lineages and oligodendrocytes.

2013 - Ferutinin promotes proliferation and osteoblastic differentiation in human amniotic fluid and dental pulp stem cells. [Articolo su rivista]
Zavatti, Manuela; Resca, Elisa; Bertoni, Laura; Maraldi, Tullia; Guida, Marianna; Carnevale, Gianluca; Ferrari, Adriano; DE POL, Anto
abstract

The phytoestrogen Ferutinin plays an important role in prevention of osteoporosis caused by ovariectomy-induced estrogen deficiency in rats, but there is no evidence of its effect on osteoblastic differentiation in vitro. In this study we investigated the effect of Ferutinin on proliferation and osteoblastic differentiation of two different human stem cells populations, one derived from the amniotic fluid (AFSCs) and the other from the dental pulp (DPSCs).AFSCs and DPSCs were cultured in a differentiation medium for 14 or 21days with or without the addition of Ferutinin at a concentration ranging from 10(-11) to 10(-4)M. 17β-Estradiol was used as a positive drug at 10(-8)M. Cell proliferation and expression of specific osteoblast phenotype markers were analyzed.MTT assay revealed that Ferutinin, at concentrations of 10(-8) and 10(-9)M, enhanced proliferation of both AFSCs and DPSCs after 72h of exposure. Moreover, in both stem cell populations, Ferutinin treatment induced greater expression of the osteoblast phenotype markers osteocalcin (OCN), osteopontin (OPN), collagen I, RUNX-2 and osterix (OSX), increased calcium deposition and osteocalcin secretion in the culture medium compared to controls. These effects were more pronounced after 14days of culture in both populations.The enhancing capabilities on proliferation and osteoblastic differentiation displayed by the phytoestrogen Ferutinin make this compound an interesting candidate to promote bone formation in vivo.

2013 - Human amniotic fluid-derived and dental pulp-derived stem cells seeded into collagen scaffold repair critical-size bone defects promoting vascularization. [Articolo su rivista]
Maraldi, Tullia; Riccio, Massimo; Pisciotta, Alessandra; Zavatti, Manuela; Carnevale, Gianluca; Beretti, Francesca; LA SALA, Giovanni Battista; A., Motta; DE POL, Anto
abstract

INTRODUCTION: The main aim of this study is to evaluate potential human stem cells, such as dental pulp stem cells (DPSC) and amniotic fluid stem cells (AFSC), combined with collagen scaffold, to reconstruct critical size cranial bone defects in animal model. METHODS: We performed two symmetric full-thickness cranial defects on each parietal region of rats and we replenished them with collagen scaffolds with or without stem cells already seeded into and addressed towards osteogenic lineage in vitro. After 4 and 8 weeks cranial tissue samples were taken for histological and immunofluorescence analysis. RESULTS: We observed a new bone formation in all the samples but the most relevant difference in defect correction were shown by stem cell-collagen samples at 4 weeks after implant, suggesting a faster regeneration ability of the combined constructs. The presence of human cells in the newly-formed bone was confirmed by confocal analysis with an antibody directed to a human mitochondrial protein. Furthermore, human cells were found to be an essential part of new vessel formation in the scaffold. CONCLUSIONS: All these data confirmed the strong potential of bioengineered constructs of stem cell-collagen scaffold for correcting large cranial defects in animal model and highlighting the role of stem cells in neo vascularization during skeletal defect reconstruction.

2013 - In vitro differentiation into insulin-producing β-cells of stem cells isolated from human amniotic fluid and dental pulp. [Articolo su rivista]
Carnevale, Gianluca; Riccio, Massimo; Pisciotta, Alessandra; Beretti, Francesca; Maraldi, Tullia; Zavatti, Manuela; Cavallini, Gian Maria; LA SALA, Giovanni Battista; Ferrari, Adriano; DE POL, Anto
abstract

AIM: To investigate the ability of human amniotic fluid stem cells and human dental pulp stem cells to differentiate into insulin-producing cells. METHODS: Human amniotic fluid stem cells and human dental pulp stem cells were induced to differentiate into pancreatic β-cells by a multistep protocol. Islet-like structures were assessed in differentiated human amniotic fluid stem cells and human dental pulp stem cells after 21 days of culture by dithizone staining. Pancreatic and duodenal homebox-1, insulin and Glut-2 expression were detected by immunofluorescence and confocal microscopy. Insulin secreted from differentiated cells was tested with SELDI-TOF MS and by enzyme-linked immunosorbent assay. RESULTS: Human amniotic fluid stem cells and human dental pulp stem cells, after 7 days of differentiation started to form islet-like structures that became evident after 14 days of induction. SELDI-TOF MS analysis, revealed the presence of insulin in the media of differentiated cells at day 14, further confirmed by enzyme-linked immunosorbent assay after 7, 14 and 21 days. Both stem cell types expressed, after differentiation, pancreatic and duodenal homebox-1, insulin and Glut-2 and were positively stained by dithizone. Either the cytosol to nucleus translocation of pancreatic and duodenal homebox-1, either the expression of insulin, are regulated by glucose concentration changes. Day 21 islet-like structures derived from both human amniotic fluid stem cells and human dental pulp stem cell release insulin in a glucose-dependent manner. CONCLUSION: The present study demonstrates the ability of human amniotic fluid stem cells and human dental pulp stem cell to differentiate into insulin-producing cells, offering a non-pancreatic, low-invasive source of cells for islet regeneration.

2013 - Inhibition of nuclear nox4 activity by plumbagin: effect on proliferative capacity in human amniotic stem cells. [Articolo su rivista]
Guida, Marianna; Maraldi, Tullia; Resca, Elisa; Beretti, Francesca; Zavatti, Manuela; Bertoni, Laura; LA SALA, Giovanni Battista; DE POL, Anto
abstract

Human amniotic fluid stem cells (AFSC) with multilineage differentiation potential are novel source for cell therapy. However, in vitro expansion leads to senescence affecting differentiation and proliferative capacities. Reactive oxygen species (ROS) have been involved in the regulation of stem cell pluripotency, proliferation, and differentiation. Redox-regulated signal transduction is coordinated by spatially controlled production of ROS within subcellular compartments. NAD(P)H oxidase family, in particular Nox4, has been known to produce ROS in the nucleus; however, the mechanisms and the meaning of this function remain largely unknown. In the present study, we show that Nox4 nuclear expression (nNox4) increases during culture passages up to cell cycle arrest and the serum starvation causes the same effect. With the decrease of Nox4 activity, obtained with plumbagin, a decline of nuclear ROS production and of DNA damage occurs. Moreover, plumbagin exposure reduces the binding between nNox4 and nucleoskeleton components, as Matrin 3. The same effect was observed also for the binding with phospho-ERK, although nuclear ERK and P-ERK are unchanged. Taken together, we suggest that nNox4 regulation may have important pathophysiologic effects in stem cell proliferation through modulation of nuclear signaling and DNA damage.

2012 - Effects of different doses of ferutinin on bone formation/resorption in ovariectomized rats. [Articolo su rivista]
Cavani, Francesco; Ferretti, Marzia; Carnevale, Gianluca; Bertoni, Laura; Zavatti, Manuela; Palumbo, Carla
abstract

This study analyzes the effects of different doses of ferutinin on bone loss caused by estrogen deficiency in ovariectomized rats, in comparison with estradiol benzoate. Thirty female Sprague-Dawley rats were ovariectomized and treated for 30 days from the day after ovariectomy. Static/dynamic histomorphometric analyses were performed on trabecular and cortical bone of lumbar vertebrae and femurs. Very low weight increments were recorded only in all F-OVX groups, with respect to the others. Although the great differences in weight, that could imply a decrease of bone mass in F-OVX groups compared to the control ovariectomized group (C-OVX), trabecular bone in lumbar vertebrae did not show significant differences, suggesting that ferutinin, opposing estrogen deficiency, inhibits bone resorption. Newly formed cortical bone was always low in all F-OVX groups and high in C-OVX, suggesting that it is mainly devoted in answering mechanical demands. In contrast, in distal femoral metaphyses, trabecular bone was reduced and the number of osteoclasts was increased in C-OVX with respect to all other groups, suggesting that it is mainly devoted in answering metabolic demands; moreover, ferutinin dose of 2 mg/kg seemed to be more effective than the lower doses used and estrogens, particularly in those skeletal regions with higher metabolic activity. Our results suggest that the role of ferutinin in preventing osteoporosis caused by estrogen deficiency is expressed in decreasing bone erosion; moreover, in all F-OVX groups bone turnover is very low and seems correlated to the trivial body weight increase, which, in turn, depends on ferutinin treatment.

2012 - Influence of Parmigiano Reggiano diet on male sexual behavior in rats: behavioral and neurochemical study [Articolo su rivista]
DI VIESTI, Vittoria; Carnevale, Gianluca; Carrozzo, Marina Maria; Zavatti, Manuela; Baraldi, Mario
abstract

Abstract: The influence of Parmigiano Reggiano (P.R.) cheese on copulatory behavior was studied in male rats. Sexually naїve, sluggish and potent rats were chronically fed with a diet of P.R. cheese for 10 d. Mount, intromission, ejaculation latencies and the percentage of mounting and ejaculating animals were recorded during the mating test. Microdialysis technique was used to detect the extracellular levels of dopamine (DA) and its metabolite dihydroxyphenylacetic acid (DOPAC) in rat brain following the P.R. diet. The P.R. diet was able to improve sexual behavior in naїve rats increasing the percentage of mounting and ejaculating animals. Moreover it was able to reduce latencies of mounts, intromissions and ejaculations and to increase the percentage of mounting and ejaculating animals in sluggish rats. Finally, in the microdialysis study an increase in DA and its metabolite DOPAC was found in P.R. fed naїve rats in comparison with control group.

2012 - Structural and histomorphometric evaluations of ferutinin effects on the uterus of ovariectomized rats during osteoporosis treatment [Articolo su rivista]
Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Benincasa, Marta; Sena, Paola; Carnevale, Gianluca; Zavatti, Manuela; Vittoria Di, Viesti; Zanoli, Paola; Palumbo, Carla
abstract

Aims: The effects of chronic administration of Ferutinin (phytoestrogen found in the plants of genus Ferula),compared with those elicited by estradiol benzoate, were evaluated, following ovariectomy, on the uterus ofovariectomized rats as regard weight, size, structure and histomorphometry.Main methods: The experimental study included 40 female Sprague–Dawley rats, assigned to two different protocols,i.e. preventive and recovering. In the preventive protocol, ferutinin (2 mg/kg/day)was orally administeredfor 30 days, starting from the day after ovariectomy; in the recovering protocol, ferutinin was administered, atthe same dosage, for 30 days starting fromthe 60th day after ovariectomy, when osteoporosiswas clearly established.Its effects were compared with those of estradiol benzoate (1.5 μg per rat twice a week, subcutaneouslyinjected) vs. vehicle-treated ovariectomized controls and vehicle-treated sham-operated controls. Uteri were removed,weighed and analysed under both the structural and histomorphometrical points of view.Key findings: Our data show that ferutinin acts, similarly to estradiol benzoate, on the uterus stimulating endometrialand myometrial hypertrophy; this notwithstanding, the phytoestrogen ferutinin, in contrast to estrogentreatment, appears to increase apoptosis in uterine luminal and glandular epithelia.Significance: Ferutinin, used in osteoporosis treatment primarily for bonemass recovering, seems in linewith aneventual protective function against uterine carcinoma, unlike estrogens so far employed in hormone replacementtherapy (HRT).

2011 - Anti-inflammatory activity of the non-peptidyl low molecular weight radical scavenger IAC in carrageenan-induced oedema in rats [Articolo su rivista]
Corsi, Lorenzo; Zavatti, Manuela; Geminiani, Elisa; Zanoli, Paola; Baraldi, Mario
abstract

Objective  In this research we investigated the anti-inflammatory activity of a non-peptidyl low molecular weight radical scavenger (IAC) in an acute and chronic animal model of inflammation.Methods  For this purpose the effect of IAC (10, 25, 50 mg/kg) was tested in rats on the associated behavioral responses to subsequent inflammatory and noxious challenges, such as hind paw oedema induced by intra-plantar injection of carrageenan and granuloma induced by subcutaneous implant of a cotton pellet, using indometacin (2.5 mg/kg) as reference drug. Moreover, the serum level of several cytokines was tested in the animal treated (or not) with IAC (50 mg/kg) both in the absence and presence of carrageenan-induced inflammation.Key findings  IAC showed a significant anti-inflammatory activity in both in acute and chronic models of inflammation. In addition IAC down regulated significantly the serum levels of interleukin (IL) 2 and IL6 whereas it increased the serum concentration of IL1α and glutathione.Conclusion  Although it remains to be elucidated whether or not the antioxidant property of IAC is directly responsible for the modulation of the tested cytokines, these results suggest IAC to be a possible candidate for a novel anti-inflammatory compound

2011 - Anxiolytic-like effect of Griffonia simplicifolia Baill. seed extract in rats [Articolo su rivista]
Carnevale, Gianluca; V. D., Viesti; Zavatti, Manuela; P., Zanoli
abstract

The seeds of Griffonia simplicifolia Barn.. a tropical shrub native to West Africa. are rich in 5-hydroxy-L-tryptophan (5-HTP), a direct precursor in the synthesis of serotonin (5-HT). In spite of the modern therapeutic application of Griffonia simplicifolia seed extract in mood disorders, no scientific evidence has been provided till now. For this reason the aim of our study was to investigate the effect of Griffonia simplicifolia seed extract on anxiety behavior. Griffonia simplicifolia seed extract, dosed at 1, 5, 10 and 25 mg/kg, was orally administered in rats which were submitted to the dark-light test and open field test. 60 min after the treatment. In the dark-light test, the administration of the extract at the doses of 10 and 25 mg/kg was able to significantly increase the time spent in the light compartment (P < 0.05). In the open field test, the extract dosed at 5, 10 and 25 mg/kg induced an anti-tigmotactic effect, as indicated by a significant increase of time spent in the central area of the open field (P < 0.01). In conclusion these findings indicate that Griffonia simplicifolia seed extract exerts anxiolytic-like effect in rats and suggest its potential usefulness for the treatment of anxiety in humans. (C) 2011 Elsevier GmbH. All rights reserved.

2011 - Effects of the cannabinoid antagonist SR 141716 on sexual and motor behaviour in receptive female rats [Articolo su rivista]
Zavatti, Manuela; Carnevale, Gianluca; A., Benelli; P., Zanoli
abstract

The aim of the present study was to investigate the effect of the cannabinoid antagonist/inverse agonist SR 141716 (SR) on the receptive behaviour and sexual motivation of female rats. Partner preference, receptivity and proceptivity were evaluated in ovariectomized female rats primed with oestrogen and progesterone and administered SR (1 or 2.5 mg/kg, i.p.) 20 min prior to testing. In the partner preference test, a reduced interest in both stimulus animals (a sexually active male and an ovariectomized hormone-primed female) was detected in rats treated with SR at both doses, but no effect on preference score was observed. In the receptivity test, pronounced reductions in lordosis quotient, lordosis rating and in the percentage of receptive females were found in SR-treated rats compared with control rats. Proceptive behaviours were not significantly affected by either dose of SR. In addition, we explored the behavioural effects induced by SR in female rats using the open field test. Only at the higher dose (i.e. 2.5 mg/kg) did SR markedly increased grooming and scratching behaviour. The results demonstrate the ability of SR to reduce female sexual receptivity, but not sexual motivation. The reduction does not seem strictly related to the motor alterations induced by the cannabinoid antagonist.

2011 - Experimental study on Satureja montana as a treatment for premature ejaculation [Articolo su rivista]
Zavatti, Manuela; Zanoli, Paola; Benelli, Augusta; Rivasi, Marianna; Baraldi, Claudia; Baraldi, Mario
abstract

AbstractAim of the studySatureja montana (winter savory) is a medicinal plant traditionally used to treat different disorders including male sexual dysfunction. In this study we evaluated the effect of Satureja montana hydroalcoholic extract on copulatory behavior of sexually potent male rats.Materials and methodsThe extract was orally administered acutely or repetitively for 8 consecutive days at the doses of 25 and 50 mg/kg. The main parameters of sexual behavior, mount (ML), intromission (IL), ejaculation (EL) latencies and post-ejaculatory interval (PEI), were evaluated in animals submitted to mating test and multiple ejaculations test. Testosterone serum levels were measured in rats acutely treated with Satureja montana extract dosed at 50 mg/kg. In addition the open field test was conducted to evaluate the locomotor behavior.ResultsWhen acutely administered at both dosages, the extract was able to significantly increase EL and decrease intromission frequency (IF) in comparison with controls. The significant increase in EL was found also when the extract was subacutely administered, daily for 8 consecutive days, at the dose of 25 mg/kg. In the multiple ejaculations test, EL values of treated rats were significantly increased during the 1st and 2nd sequence in comparison with controls; in addition only rats treated with the extract were able to reach the 4th ejaculation within 30 min. Testosterone serum level measured in rats acutely treated with Satureja montana at the dose of 50 mg/kg was significantly increased in rats in comparison with controls. Finally, the locomotor activity recorded in the open field test was not affected by the acute administration of the plant extract.ConclusionsThese data suggest that Satureja montana could be considered as a natural remedy for the treatment of premature ejaculation delaying ejaculation latency without exerting any negative effect on the other parameters of sexual behavior and without exerting a sedative effect. In addition the increased serum level of testosterone confirms the positive influence of Satureja montana on male sexual function.

2011 - Further Evidence of the Antiulcer Activity of IAC, a Novel Free Radical Scavenger [Articolo su rivista]
Carnevale, Gianluca; P., Zanoli; Zavatti, Manuela; M., Baraldi
abstract

It has been proposed that free radicals, reactive oxygen species (ROS) and reactive nitrogen species play a critical role in gastric mucosal damage. It is well known that the exposure of gastric mucosa to damaging factors such as stress and nonsteroidal anti-inflammatory drugs produces acute ulcers that are mainly mediated by ROS. The aim of the present study was to investigate the gastroprotective properties of bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate (IAC), a novel nonpeptidyl low-molecular-weight radical scavenger, in two different models of gastric ulcer in rats caused by ROS. IAC was orally administered at the doses of 50 and 100 mg/kg before gastric ulceration induced by indomethacin and water immersion and restraint stress. The number and severity of gastric lesions, following macroscopic inspection of the mucosa, were evaluated and expressed as an ulcer score. Oral administration of IAC dosed at 50 and 100 mg/kg was able to significantly prevent gastric ulceration induced by indomethacin and by stress. The gastroprotective effect of IAC on gastric mucosa could be attributed to its intrinsic antioxidant activity, suggesting it as a novel antiulcer agent. Copyright (C) 2011 S. Karger AG, Basel

2011 - Increased sexual motivation in female rats treated with Humulus lupulus L. extract [Articolo su rivista]
DI VIESTI, Vittoria; Carnevale, Gianluca; Zavatti, Manuela; Benelli, Augusta; Zanoli, Paola
abstract

Aim of the study: To evaluate the influence of Humulus lupulus extract on sexual behavior in female rats.Materials and Methods: Ovariectomized rats hormonally primed with estradiol benzoate (1.5 µg/rat) and progesterone (500 µg/rat) were acutely treated by oral gavage with Humulus lupulus extract dosed at 5, 10 and 25 mg/kg and then tested for partner preference and sexual receptivity.Results: The administration of Humulus lupulus extract at the highest dose significantly increased the preference for the stimulus male during the partner preference test and the number of proceptive behaviors during the receptivity test, without affecting the lordosis response. Conclusions: Humulus lupulus extract increased sexual motivation in hormone-primed female rats.

2011 - Influence of Griffonia simplicifolia on male sexual behavior in rats: Behavioral and neurochemical study [Articolo su rivista]
Carnevale, Gianluca; V. D., Viesti; Zavatti, Manuela; A., Benelli; P., Zanoli
abstract

The seeds of Griffonia simplicifolia Baill. are rich in 5-HIP (5-hydroxytryptophan), a direct precursor of the neurotransmitter serotonin. In the present study we investigated the influence of the plant extract on male sexual behavior. The seed extract was orally administered to Sprague-Dawley male rats at three dose levels (25, 50 and 100 mg/kg) both acutely and subchronically (daily for 9 days). Mating test with receptive female rats was performed 60 min after the acute treatment or the last dose when repetitively administered. Mount, intromission and ejaculation latencies and post-ejaculatory interval were recorded. Food intake and body weight were measured over the 9-day period of treatment. Microdialysis technique was used to detect the extracellular levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in rat brain following the acute administration of the extract dosed at 100 mg/kg. The acute treatment significantly increased mount latency (at any dosage), intromission and ejaculation latencies (at 100 mg/kg) and post-ejaculatory interval (at 50 and 100 mg/kg). On the contrary the subchronic treatment failed to exert a significant influence on copulatory behavior. The daily administration of the extract dosed at 50 and 100 mg/kg for 9 days significantly reduced food intake and body weight. Finally in the microdialysis experiments we found a dramatic increase in 5-HT and its metabolite 5-HIAA. (C) 2011 Elsevier GmbH. All rights reserved.

2010 - Evidence that isopropylthioxanthone (ITX) is devoid of anxiolytic and sedative effect [Articolo su rivista]
Campioli, Enrico; Zavatti, Manuela; Avallone, Rossella; Puja, Giulia; Losi, Gabriele; Baraldi, Mario
abstract

Isopropylthioxanthone (ITX) is a well-known photo-initiator in ultraviolet light-cured inks frequently used in milk packaging materials, yoghurt, ready-to-feed infant formula, and other drinks. Traces of ITX have beenfound in milk and, as a consequence, there was considerable interest in studying the biological activity of this molecule and its potential hazard for the human health. Although the ITX genotoxic effects have been excluded 10 by the European Food Safety Authority (EFSA), the US Environmental Protection Agency (USEPA) is still examining its possible toxic potential depending on a dose–effect ratio. Little is known about the ITX activity on the function of the central nervous system and cerebral neurotransmitters. Using behavioural, biochemical, andelectrophysiological tests, the authors have found that: (1) ITX did not exert an in vivo anxiolytic or sedativeeffect when administered orally to rats; (2) ITX did not affect the binding characteristics of central and peripheral15 benzodiazepine receptors studied in vitro; and (3) ITX did not influence the ability of GABA to increase thechloride channel permeability studied by patch clamp technique in a single neuron of cultured cerebellargranule cells.

2010 - Griffonia simplicifolia negatively affects sexual behavior in female rats [Articolo su rivista]
Carnevale, Gianluca; DI VIESTI, Vittoria; Zavatti, Manuela; Benelli, Augusta; Zanoli, Paola
abstract

At present Griffonia simplicifolia is used in food supplement aimed to treat mood disorders as well asto reduce food intake and body weight. The plant has gained increasing interest for its high content in5-hydroxy-l-tryptophan (5-HTP) particularly in the seed. The present study was designed to evaluatethe influence of a seed extract of the plant, dosed at 25, 50 and 100 mg/kg, on the sexual behavior ofovariectomized hormone-primed rats after acute and subchronic treatment. The single administrationof G. simplicifolia significantly reduced lordosis response and increased rejection behavior in female ratstreated with the highest dose while it did not influence proceptive behaviors. On the other hand the subchronicadministration of the extract significantly reduced proceptivity but not receptivity, and increasedrejection behavior. All the tested dosages were able to markedly decrease food intake and body weightafter a 9-day treatment. Taken together the present results, possibly ascribed to increased levels of 5-hydroxytryptamine (5-HT) in the brain, suggest a cautious administration of the plant extract owing toits negative influence on female sexual behavior.

2010 - Influence of ferutinin on bone metabolism in ovariectomized rats. II: Role in recovering osteoporosis. [Articolo su rivista]
Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Zavatti, Manuela; Resca, Elisa; Carnevale, Gianluca; Benelli, Augusta; Zanoli, Paola; Palumbo, Carla
abstract

The study investigates the influence of ferutinin (a phytoestrogen extracted from Ferula Hermanis root) on bone metabolism in ovariectomized rats. The study represent the complection of previous investigations (published in 2009, concerning the ferutinine role in preventing osteoporosis due to estrogen deficiency). The present investigation concerns the role of Ferutinine in recovering osteoporosis.

2010 - New aspects of Ferutinin effect in preventing osteoporosis [Abstract in Rivista]
Ferretti, Marzia; Cavani, Francesco; Bertoni, Laura; Zavatti, Manuela; Taronna, ANGELO PIO; Carnevale, Gianluca; Benelli, Augusta; Zanoli, Paola; Marotti, Gastone; Palumbo, Carla
abstract

The results of the study suggest that ferutinin role, in preventing osteoporosis due to estrogen deficiency, is expressed in inhibiting osteoclast erosion rather than in enhancing osteoblast deposition (as previously suggested); moreover, in all F-OVX groups the bone turnover is very low and seems correlated to the trivial body weight increase, which, in turn, depends on ferutinin treatment.

2009 - Anti-ulcer activity of IAC, a novel free-radical scavenger, in rats. [Articolo su rivista]
Zavatti, Manuela; Corsi, Lorenzo; Zanoli, Paola; Baraldi, Mario
abstract

Objectives We investigated the ability of a novel low-molecular-weight free-radical scavenger, bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate (IAC), to protect the gastric mucosa from indometacin-induced ulceration.Methods The pharmacological effects of IAC, administered orally or by intraperitoneal injection, on the gastric mucosa were assessed in a rat model of gastric ulceration induced by indometacin. The effect of IAC on the level of prostaglandin PGE2 in the gastric mucosa was also investigated. Results IAC, which has no ulcerative activity per se, had a preventive and protective activity against indometacin-induced gastric ulceration. This effect could be only partially attributed to a modulatory effect of IAC on PGE2 levels; it is more likely to be due to the antioxidant activity of the compound. Conclusions Taking into account the properties of IAC and the mechanisms underlying gastric inflammation elicited by non-steroidal anti-inflammatory drugs, IAC may represent a novel anti-ulcer agent.

2009 - Effect of leptin on the development of primary ossification centers in mouse fetuses. [Abstract in Rivista]
Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Zavatti, Manuela; Benelli, A.; Palumbo, Carla
abstract

Leptin may be considered a significat cartilage/bone growth factor.

2009 - Experimental evidence of the anaphrodisiac activity of Humulus lupulus L. in naïve male rats [Articolo su rivista]
Zanoli, Paola; Zavatti, Manuela; Rivasi, Marianna; Benelli, Augusta; Avallone, Rossella; Baraldi, Mario
abstract

Ethnopharmacological relevance: In the folk medicine Humulus lupulus L. (hops) is mainly recommendedas a mild sedative with antispasmodic and digestive properties. It is also reputed to exert an anaphrodisiaceffect but it is still lacking the experimental evidence of this activity.Aim of the study: To evaluate the influence of Humulus lupulus extract on sexual behavior of both naïveand sexually potent male rats; thereafter to investigate the role of 8-prenylnarigenin (8-PN) in the effectdisplayed by the hop extract.Materials and methods: Sprague–Dawley male rats both naïve and sexually potent were acutely administeredwith the hop extract dosed at 5, 10, 25 and 50 mg/kg. In addition the extractwas administered dailyfor 10 consecutive days at the dose of 0.25 mg/kg/day in sexually potent animals. The pure compound8-PN was acutely administered in naïve rats at the dosages of 5, 12.5 and 25g/kg. All the animals werescreened for their sexual behavior manifestation during the mating test.Results: In naïve rats the acute administration of Humulus lupulus extract at the doses of 25 and 50 mg/kgsignificantly reduced the percentage of mounting and ejaculating animals, in comparison to vehiclecontrols. The other parameters recorded during the mating test were not affected by the hop extract.In sexually potent rats nor the acute neither the repeated administration of the extract modified theircopulatory behavior.The pure compound 8-PN failed to influence male sexual behavior of naïve rats.Conclusion: Humulus lupulus extract exerted an anaphrodisiac effect only in naïve rats by inhibiting theirmounting and ejaculating behavior. The presence of 8-PN in the extract could be only partially involvedin the observed anaphrodisiac effect.

2009 - Influence of Eurycoma longifolia on the copulatory activity of sexually sluggish and impotent male rats [Articolo su rivista]
Zanoli, Paola; Zavatti, Manuela; C., Montanari; Baraldi, Mario
abstract

AIM OF THE STUDY: The root of Eurycoma longifolia Jack, native to South East Asia, has long been used as a male aphrodisiac remedy to treat sexual disorders. In the study we evaluated the influence of Eurycoma longifolia Jack on sexual behavior (including both motivation and copulatory performance) of sexually sluggish and impotent male rats. MATERIALS AND METHODS: The root powder of the plant was orally administered to adult Sprague-Dawley male rats, classified as sexually sluggish or impotent taking in account their behavior in pre-experimental tests. Groups of 8 animals each were submitted to three different types of treatment: (1) acute at 3 dose levels (250, 500 and 1000 mg/kg); (2) subacute (daily for 6 days) at the dose of 500 mg/kg and (3) subchronic (daily for 12 days) at the same dose (500 mg/kg). Mount, intromission and ejaculation latencies and post-ejaculatory interval were recorded during the mating test in order to evaluate sexual performance. In addition the partner preference test was used to assess sexual motivation. Testosterone serum levels were measured in subacutely treated rats and compared with the values of controls receiving vehicle. RESULTS: Concerning the copulatory activity of sexually sluggish rats, both acute (dosed at 500 and 1000 mg/kg) and subacute treatments with the root powder significantly reduced ejaculation latencies, increasing also the percentage of mounting and ejaculating animals; in addition the subacute administration reduced post-ejaculatory interval. In impotent rats both subacute and subchronic treatments increased the percentage of mounting and ejaculating rats. The motivational behavior of sluggish rats during the partner preference test was not affected by the treatments. Testosterone serum levels were increased in rats subacutely treated in comparison with controls. CONCLUSION: Eurycoma longifolia root improved sexual performance but not motivation in sluggish rats after acute or subacute administration. The effect could be mainly ascribed to increased testosterone levels.

2009 - Influence of ferutinin on bone metabolism in ovariectomized rats. I: role in preventing osteoporosis [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Resca, Elisa; Casolari, Barbara; Carnevale, Gianluca; Zavatti, Manuela; C., Montanari; Benelli, Augusta; Zanoli, Paola
abstract

Phytoestrogens play a role in maintaining bone mass in the post-menopausal period for their putative function as osteoprotective agents. The aim of the present study was to investigate the influence of Ferutinin, a phytoestrogen found in the plants of Ferula genus, on bone loss in ovariectomized rats. Such an animal model can simulate the various clinical syndromes deriving from osteoporosis. The effect of the daily oral administration of ferutinin to ovariectomized rats (dosed at 2 mg/kg per day for 30 and 60 days) was compared to that of estradiol benzoate (subcutaneously administered at the dose of 1.5 microg/rat twice a week). After the sacrifice, histomorphometrical analyses were performed on trabecular bone of L4-L5 vertebrae and distal femoral metaphysis, as well as on cortical bone of femoral diaphysis; biochemical parameters (bone mineral components and markers) were also evaluated from the rat serum. The histomorphometrical analyses of trabecular and cortical bone from lumbar vertebrae and femur showed that ferutinin has the same antiosteoporotic effect of estradiol benzoate on bone mass, and in some cases is even stronger. This fact suggests that it could prevent osteoporosis caused by severe estrogen deficiency in ovariectomized rats. The possibility of using ferutinin as an alternative to the commonly employed hormonal replacing therapy in post-menopausal women is discussed.

2009 - Leptin increases growth of primary ossification centers in fetal mice [Articolo su rivista]
Bertoni, Laura; Ferretti, Marzia; Cavani, Francesco; Zavatti, Manuela; Resca, Elisa; Benelli, Augusta; Palumbo, Carla
abstract

The effect of peripheral leptin on fetal primary ossification centers during the early phases of bone histogenesis was investigated by administration of leptin to pregnant mice. Fourteen pregnant mice were divided into two groups. The treated pregnant group was subcutaneously injected in the intrascapular region with supraphysiologic doses (2 mg kg(-1)) of leptin (Vinci Biochem, Firenze, Italy) in a volume of 0.1 mL per 10 g body weight, at the 7th, 9th and 11th day of gestation. The control group was treated with physiological solution in the same manner and same times as the treated group. The new-born mice were killed 1 day after birth and the primary ossification centers were stained with Alizarin Red S after diaphanizing the soft tissues in 1% potassium hydroxide. The development of both endochondral and intramembranous ossification centers was morphometrically analysed in long bones. The results showed that the ossification centers of mice born by mothers treated with leptin grow more rapidly in both length and cross-sectional area compared with mice born by the untreated mothers. As the development of long bones depends on endochondral ossification occurring at proximal and distal epiphyseal plates as well as on intramembranous ossification along the periosteal surface, it appears that leptin activates the differentiation and proliferation of both chondrocytes and osteoblasts. The role of leptin as a growth factor of cartilage and bone is discussed in the light of the data reported in the literature.

2009 - Neuropharmacological activity of Humulus lupulus L. [Capitolo/Saggio]
Zanoli, Paola; Zavatti, Manuela
abstract

The traditional use of hops in the treatment of sleep disturbances has been supported by experimental studies performed in rodents, as described in the present chapter. An inhibitory influence on the central nervous system was suggested by the finding of a reduction in locomotor behavior together with an enhancement of pentobarbital hypnotic activity, following the oral administration of hop extracts. A lipophilic hop extract showedan antidepressant-like activity in an animal model of behavioral despair, without a significant anxiolytic activity. Among hop components, the bitter acids have been carefully investigated for their role in the neuropharmacological activities of hops. In particular, the fraction containing alfa -acids has been recognizedto be mainly responsible for the sedative effects of hops. On the other hand the fraction containing beta -acids appears to exert a central stimulatory activity, probably due to a reduction in the GABAergic activity. This hypothesis is supported by its effects in animal models of sedation and convulsions as well as in electrophysiological studies on cerebellar granule cells in culture.Clinical trials specifically related to the sedative properties of hops are still inadequate since human studies were mainly performed using valerian–hop combination extracts.

2009 - The phytoestrogen ferutinin affects female sexual behavior modulating ERα expression in the hypothalamus [Articolo su rivista]
Zanoli, Paola; Zavatti, Manuela; Geminiani, Elisa; Corsi, Lorenzo; Baraldi, Mario
abstract

This study was designed to assess the effect of the phytoestrogenic compound ferutinin, chronically administered in ovariectomized progesterone primed rats, alone or in combination with estradiol benzoate. After 2, 3 and 4 weeks of treatments, female rats were tested for receptive (lordosis) and proceptive behaviors (hops, darts and ear wigglings). Ferutinin given alone markedly increased the intensity of the lordotic response in ovariectomized rats but failed to significantly affect proceptivity. On the other hand estradiol benzoate significantly increased both receptive and proceptive behaviors. When administered in combination with estradiol, ferutinin reduced the increase in receptivity and proceptivity due to estrogen effects, acting as an antiestrogen. At the end of the behavioral experiments, animals were sacrificed and Western blot analysis of estrogen receptor α (ERα) levels was performed in the dissected hypothalami. Ferutinin increased ERα expression when administered alone, as estradiol did, but decreased the response to estradiol when administered in combination. These results suggest that ferutinin displays estrogenic or antiestrogenic activity through ERα in the hypothalamus, depending on the absence or the presence of estrogen priming

2009 - The phytoestrogen ferutinin improves sexual behavior in ovariectomized rats [Articolo su rivista]
Zavatti, Manuela; Benelli, Augusta; C., Montanari; Zanoli, Paola
abstract

The present study was designed to examine the effect of ferutinin chronic administration on sexual behavior of ovariectomized non-estrogen-primed rats. Starting from 3 weeks after ovariectomy, female rats were orally treated with ferutinin at the doses of 0.2 and 0.5mg/kg, daily for 4 weeks. Ferutinin's effect was compared with that of estradiol benzoate, subcutaneously injected at the dose of 1.5mug/rat twice a week. Animals were tested for sexual motivation, receptivity and proceptivity after 1, 2 and 3 weeks of treatment and for paced mating behavior after 4 weeks of treatment. Before each experimental test, they received progesterone injection (500mug/rat). Both dosages of ferutinin significantly increased the receptive behavior in a time-dependent manner, as well as estradiol benzoate did. Also proceptive behaviors increased in ferutinin-treated animals in comparison with control ones. During the partner preference test ferutinin was able to induce a significant preference for a sexually active male over a sexually receptive female. Moreover, ferutinin restored a normal paced mating behavior, which had been suppressed by ovariectomy. These results show that ferutinin exerts an estrogenic activity in ovariectomized non-estrogen-primed female rats.

2008 - Effect of the phytoestrogen ferutinin in preventing and recovering osteoporosis: histomorphometric analysis of bone mass in ovariectomized rats. [Abstract in Rivista]
Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Resca, Elisa; Carnevale, Gianluca; Zavatti, Manuela; Benelli, A.; Zanoli, P.; Palumbo, Carla
abstract

Ferutinin seems to display the same effects on bone mass obtained with estradiol in OVX rats.

2008 - Improved sexual behavior in male rats treated with a Chinese herbal extract: hormonal and neuronal implications [Articolo su rivista]
Zanoli, Paola; Benelli, Augusta; Zavatti, Manuela; Rivasi, Marianna; Baraldi, Claudia; Baraldi, Mario
abstract

Aim: To investigate the influence of an extract obtained from five Chinese medicinal plants on sexual behavior ofadult male rats. Methods: The extract was administered at doses of 30, 60 and 120 mg/kg by oral gavage, acutely (onetime, 45 min before mating test) or subchronically (daily for 10 days) in sexually potent and sexually sluggish/impotentrats. Sexual behavior, serum levels of luteinizing hormone (LH) and testosterone (T) were evaluated in treatedrats and compared with controls receiving vehicle. The effect of the extract on central dopaminergic neurotransmissionwas assessed in the nucleus accumbens using a microdialysis technique. Results: In sexually potent rats, bothacute and subchronic treatment with the extract dosed at 30 and 60 mg/kg reduced mount latency and intromissionlatency. In sluggish/impotent rats, the acutely administered extract at the dose of 60 mg/kg shortened ejaculationlatency, whereas subchronically administered at the doses of 30 and 60 mg/kg, reduced mount, intromission andejaculation latencies, increasing also the percentage of mounting and ejaculating rats. The extract dosed at 60 mg/kgsignificantly increased LH and T following acute and subchronic administration and increased 3,4-dihydroxyphenylaceticacid levels in the nucleus accumbens, 30 min after the acute administration. Conclusion: The improvement in bothappetitive and consummatory components of sexual behavior observed in male rats treated with the extract could beascribed to increased serum T level in parallel with the activation of the central dopaminergic system.

2008 - Influence of ferutinin on bone mass and its side effects in ovariectomized rats. [Abstract in Rivista]
Ferretti, Marzia; Palumbo, Carla; Bretoni, L.; Cavani, Francesco; Resca, E.; Benincasa, Marta; Carnevale, Gianluca; Zavatti, Manuela; Montanari, C.; Benelli, A.; Zanoli, P.; Marotti, Gastone
abstract

Ferutinin seems to display the same effects on bone mass recorded with estradiol, but with respect to estrogens it seems to extert a protection against uterine carcinoma.

2008 - Pharmacognostic and pharmacological profile of Humulus Lupulus L. [Articolo su rivista]
Zanoli, Paola; Zavatti, Manuela
abstract

The present review describes the morphological, phytochemical and ethnopharmacological aspects of Humulus lupulus L. (Cannabinaceae) and summarizes the most interesting findings obtained in the preclinical and clinical research related to the plant. The female inflorescences of Humulus lupulus (hops), well-known as bittering agent in brewing industry, have long been used in traditional medicine mainly to treat sleep disturbances. However the sedative activity is still under investigation in order to recognize the active principles responsible for the neuropharmacological effects observed in laboratory animals, and their mechanism of action. Here we report the data from our experiments as well as those obtained from other researchers, focusing on the variability of the results. Other traditional applications of hops as stomachic, antibacterial and antifungal remedy have been supported by in vivo and/or in vitro investigations. In recent years some prenylated chalcones present in hops have received much attention for their biological effects: in particular, xanthohumol has been shown to exert cancer chemopreventive activity in in vitro experiments, while 8-prenylnaringenin has been characterized as one of the most potent phytoestrogens isolated until now. Nevertheless much additional work is needed to open up new biomedical application of these compounds.

2007 - Evidence that the β-acid fraction of hops reduces central GABAergic neurotransmission [Articolo su rivista]
Zanoli, Paola; Zavatti, Manuela; Rivasi, Marianna; F., Brusiani; Losi, Gabriele; Puja, Giulia; Avallone, Rossella; Baraldi, Mario
abstract

Humulus lupulus (hops) is traditionally used as a tranquilizing herbal remedy. Here, we investigated the in vivo and in vitro effect of hop β-acids on central nervous system function. Oral administration of β-acids (5–10 mg/kg) in rats produced an increased exploratory activity in the open field, a reduction in the pentobarbital hypnotic activity and a worsening of picrotoxin-induced seizures. When dosed at 10 mg/kg, β-acids increased, in the elevated plus maze, open arm entries reducing in parallel those in closed arms. In the forced swimming test, we observed a reduction in the immobility time that could suggest an antidepressant-like activity. Electrophysiological studies performed on cerebellar granule cells in culture showed that the β-acids fraction decreased GABA-evoked current in a dose-dependent way. The effect was not inhibited by the benzodiazepine antagonist Ro 15-1788. Benzodiazepine receptors involvement was also excluded by [3H]-Ro 15-1788 binding assay. In conclusion, the behavioral effects of β-acids fraction could be explained by a reduction in the GABAergic activity although we cannot rule out the involvement of other neurotransmitter systems

2007 - Phytoestrogen effects on bone mass in ovariectomized rats: preliminary histomorphometric analysis. [Abstract in Rivista]
Ferretti, Marzia; Palumbo, Carla; Cavani, Francesco; Bertoni, Laura; Resca, E.; Carnevale, Gianluca; Zavatti, Manuela; Montanari, C.; Benelli, A.; Zanoli, P.; Marotti, Gastone
abstract

Phytoestrogens ferutinine could prevent in rats the risk of osteoporosis in estrogen deficient conditions and it could enhance the recover of bone mass in osteoporotic OVX rats.

2006 - Role of ferutinin in the impairment of female sexual function induced by Ferula hermonis [Articolo su rivista]
Zavatti, Manuela; C., Montanari; Zanoli, Paola
abstract

In the present study, we evaluated the effects of single components of Ferula hermonis extract on female rat sexual behaviour. Ovariectomized rats hormonally primed with estradiol benzoate (1.5 or 10 mu g/rat s.c.) and progesterone (500 mu g/rat s.c.) were acutely treated by oral gavage with ferutinin, teferin and teferdin (2.5 mg/kg). Thereafter they were tested for: a) partner preference, b) receptivity, c) proceptivity, d) paced mating behaviour. In the partner preference test, the choice of the female for a sexually active male was not influenced by the different treatments. Similarly, during the paced mating test, the contact-return latencies as well as the percentage of exits from the male compartment were not different in control and treated rats. Therefore none of the three compounds showed the capacity to alter sexual motivation. On the other hand, ferutinin, but not teferin and teferdin, significantly inhibited female receptivity. These results suggest a primary role of ferutinin in the impairment of sexual behaviour elicited by F hermonis extract in hormone primed-female rats.

2005 - Activity of single components of Ferula hermonis on male rat sexual behavior [Articolo su rivista]
Zanoli, Paola; Rivasi, Marianna; Zavatti, Manuela; F., Brusiani; Vezzalini, Francesca; Baraldi, Mario
abstract

The influence of the single components of Ferula hermonis extract on sexual behavior was studied in male rats. Sexually potent and sluggish/impotent animals were orally treated acutely (2.5 mg/kg) and subchronically (0.25 mg/kg/day for 10 days) with ferutinin, teferdin and teferin. Ferutinin alone acutely administered in potent rats was able to reduce mount and intromission latencies, while in sluggish/impotent animals, it induced the same effects and additionally shortened the ejaculation latency, as teferdin did. Both substances increased testosterone levels in rats. Unlike teferdin, ferutinin subchronically administered in potent rats negatively affected appetitive and consummatory sexual behavior, reducing also testosterone serum levels. In conclusion, if repetitively administered, ferutinin was able to stimulate sexual behavior after acute ingestion, but exerted a negative influence on the sexual capacity of potent male rats, whereas teferdin only improved copulatory performance of sluggish/impotent animals.

2005 - Ferula hermonis impairs sexual behavior in hormone-primed female rats [Articolo su rivista]
Zanoli, Paola; Zavatti, Manuela; Rivasi, Marianna; Baraldi, Mario
abstract

The influence of Ferula hermonis root extract on sexual behavior was studied in female rats. Sexual receptivity, proceptivity and paced mating behavior were evaluated in ovariectomized females primed with estradiol benzoate (EB) and progesterone (P) and then treated with F hermonis extract acutely (30 and 60 mg/kg) or subehronically (1 and 10 mg/kg daily for 10 consecutive days). A significant reduction in lordosis responses was observed in rats after acute (60 mg/kg) or subehronic (1 and 10 mg/kg) administration of the plant extract. Similarly, a decrease in proceptive behaviors was exhibited by F hermonis treated rats in comparison with EB + P controls. No difference was found in the patterns of paced mating behavior between control and treated animals. The present results demonstrate that the acute or repeated ingestion of F hermonis specifically impairs the receptive and proceptive components of female sexual behavior. The effect could be the consequence of an antiestrogenic action of the extract in hormone-primed female rats.

2005 - New insight in the neuropharmacological activity of Humulus lupulus L [Articolo su rivista]
Zanoli, Paola; Rivasi, Marianna; Zavatti, Manuela; Brusiani, F.; Baraldi, Mario
abstract

The purpose of the present study was to investigate the effects of Humulus lupulus CO2 extract and its fraction containing α-acids on the central nervous system of rats. Both tested substances were able to prolong pentobarbital sleeping time, without affecting the latency to the loss of the righting reflex. This effect was dose-dependent, starting from a minimal dose of 10 mg/kg. Neither the extract nor its α-acid fraction affected the locomotor activity in the open field test or exerted an anxiolytic effect in rats submitted to the elevated plus-maze test. Interestingly both compounds reduced the immobility time during the behavioral despair test when administered three times (24, 5 and 1 h) before the test. In conclusion this report shows that Humulus lupulus CO2 extract exerts: (a) a pentobarbital sleep-enhancing property without influencing the motor behavior of rats; (b) an antidepressant activity. The same effects were elicited by the administration of the Humulus lupulus fraction containing α-acids, which can be considered as the major responsible for the enhanced pentobarbital effect and for the antidepressant property.

2005 - Pharmacological characterisation of beta-acids fraction of hops [Abstract in Atti di Convegno]
Zavatti, Manuela; Zanoli, Paola; Rivasi, Marianna; Avallone, Rossella; Baraldi, Mario
abstract

Humulus lupulus (hops) is traditionally used as a tranquilizing herbal remedy. Here, we investigated the in vivo and in vitro effect of hop -acids on central nervous system function. Oral administration of -acids (5–10 mg/kg) in rats produced an increased exploratory activity in the open field, a reduction in the pentobarbital hypnotic activity and a worsening of picrotoxin-induced seizures. When dosed at 10 mg/kg, -acids increased, in the elevated plus maze, open arm entries reducing in parallel those in closed arms. In the forced swimming test, we observed a reduction in the immobility time that could suggest an antidepressant-like activity. Electrophysiological studies performed on cerebellar granule cells in culture showed that the -acids fraction decreased GABA-evoked current in a dose-dependent way. The effect was not inhibited by the benzodiazepine antagonist Ro 15-1788. Benzodiazepine receptors involvement was also excluded by [3H]-Ro 15-1788 binding assay. In conclusion, the behavioraleffects of -acids fraction could be explained by a reduction in the GABAergic activity although we cannot rule out the involvement of other neurotransmitter systems.