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INTRODUCTION: Cannabis sativa L. (C. sativa) is used since ancient times to produce fabrics, baskets, and cords. Later, different ethnic groups used to burn the leaves and flowers of psychotropic cultivars with high Δ9-tetrahydrocannabinol (D9-THC) levels, during the religious or propitiatory rites to alter the state of consciousness. To date, it is not known whether also nonpsychotropic cultivars of C. sativa were used during these rites, and whether these varieties could have an effect on human behavior. This study aimed to evaluate the behavioral effects of an extract of nonpsychotropic C. sativa (NP-CS) in mice. MATERIALS AND METHODS: An extract of a nonpsychotropic cultivar of C. sativa dissolved in medium-chain triglyceride oil was used and the different phytochemical components were evaluated. The relative composition in terms of phytocannabinoid content was assessed by reverse phase high-performance liquid chromatography coupled to UV detection (RP-HPLC-UV), and the volatile components were analyzed by gas chromatography-mass spectrometry (GC-MS). In addition, the behavioral effect of NP-CS was assessed on a wild-type mouse model. The animals were treated for 14 days (oral gavage) and motility, anxiety, and social effects were assessed. RESULTS: RP-HPLC-UV analysis demonstrated that D9-THC was present in lower concentration with respect to other cannabinoids, like cannabidiol. Furthermore, the GC-MS analysis revealed the presence of several terpenoids. Concerning in vivo studies, chronic treatment with NP-CS did not alter body weight, motility, and anxiety and increased social interaction. CONCLUSIONS: This study highlighted the prosocial effects of NP-CS.

Autism spectrum disorders (ASDs) are a group of clinically heterogeneous neurodevelopmental disorders sharing common features related to impaired social and communication abilities in addition to stereotyped behaviors. ASD patients present encephalic morphological, physiological, and biomolecular alterations with low levels of melatonin due to alterations in its pathways. Therefore, even if ASDs have traditionally been framed as behavioral disorders, several lines of evidence are accumulating that ASDs are characterized by certain anatomical and physiological abnormalities, including oxidative stress and inflammation in peripheral biomarkers, but likewise present in human brain tissue also characterized by alterations in synaptic remodeling and neuromodulation. Melatonin has also protective and antioxidant properties, so we can therefore hypothesize that alterations in melatonin's pathways may be one of the causes of the symptomatology of autism. The aim of the present study was to analyze the beneficial effect induced by melatonin administration and its possible mechanism of action in a transgenic mouse model of autism, immediately after weaning. The male mice were daily treated per os with melatonin (10 mg/Kg/day) or vehicle for 8 weeks starting from the sixth week of life. The antioxidant modulation, the GABAergic/glutamatergic impairment, and the synaptic remodeling in the prefrontal cortex have been evaluated. Social and repetitive behaviors were also evaluated. The behavioral results showed no statistical evidences, instead the immunohistochemical results indicated the ability of melatonin to promote the activity of antioxidant system, the GABAergic/glutamatergic equilibrium, and the synaptic remodeling. The results show that melatonin may be a possible adjuvant therapeutic strategy in ASDs.

Alterations in KRAS have been identified as the most recurring somatic variants in the multiple myeloma (MM) mutational landscape. Combining DNA and RNA sequencing, we studied 756 patients and observed KRAS as the most frequently mutated gene in patients at diagnosis; in addition, we demonstrated the persistence or de novo occurrence of the KRAS aberration at disease relapse. Small-molecule inhibitors targeting KRAS have been developed; however, they are selective for tumors carrying the KRASG12C mutation. Therefore, there is still a need to develop novel therapeutic approaches to target the KRAS mutational events found in other tumor types, including MM. We used AZD4785, a potent and selective antisense oligonucleotide that selectively targets and downregulates all KRAS isoforms, as a tool to dissect the functional sequelae secondary to KRAS silencing in MM within the context of the bone marrow niche and demonstrated its ability to significantly silence KRAS, leading to inhibition of MM tumor growth, both in vitro and in vivo, and confirming KRAS as a driver and therapeutic target in MM.

Enzalutamide (MDV3100) is a potent second-generation androgen receptor antagonist approved for the treatment of castration-resistant prostate cancer (CRPC) in chemotherapy-naïve as well as in patients previously exposed to chemotherapy. However, resistance to enzalutamide and enzalutamide withdrawal syndrome have been reported. Thus, reliable and integrated preclinical models are required to elucidate the mechanisms of resistance and to assess therapeutic settings that may delay or prevent the onset of resistance. In this study, the prostate cancer multistage murine model TRAMP and TRAMP-derived cells have been used to extensively characterize in vitro and in vivo the response and resistance to enzalutamide. The therapeutic profile as well as the resistance onset were characterized and a multiscale stochastic mathematical model was proposed to link the in vitro and in vivo evolution of prostate cancer. The model showed that all therapeutic strategies that use enzalutamide result in the onset of resistance. The model also showed that combination therapies can delay the onset of resistance to enzalutamide, and in the best scenario, can eliminate the disease. These results set the basis for the exploitation of this “TRAMP-based platform” to test novel therapeutic approaches and build further mathematical models of combination therapies to treat prostate cancer and CRPC. Significance: Merging mathematical modeling with experimental data, this study presents the “TRAMP-based platform” as a novel experimental tool to study the in vitro and in vivo evolution of prostate cancer resistance to enzalutamide.

Prostate cancer (PCa) is a leading cause of cancer mortality in the male population commonly treated with androgen deprivation therapy (ADT) and relapsing as aggressive and androgen-independent castration-resistant prostate cancer (CRPC). In PCa the FGF/FGFR family of growth factors and receptors represents a relevant mediator of cancer growth, tumor-stroma interaction, and a driver of resistance and relapse to ADT. In the present work, we validate the therapeutic efficacy the FDA-approved FGFR inhibitor pemigatinib, in an integrated platform consisting of human and murine PCa cells, and the transgenic multistage TRAMP model of PCa that recapitulates both androgen-dependent and CRPC settings. Our results show for the first time that pemigatinib causes intracellular stress and cell death in PCa cells and prevents tumor growth in vivo and in the multistage model. In addition, the combination of pemigatinib with enzalutamide resulted in long-lasting tumor inhibition and prevention of CRPC relapse in TRAMP mice. These data are confirmed by the implementation of a stochastic mathematical model and in silico simulation. Pemigatinib represents a promising FDA-approved FGFR inhibitor for the treatment of PCa and CRPC alone and in combination with enzalutamide.

Methylglyoxal (MG), a potent glycotoxin that can be found in the diet, is one of the main precursors of Advanced glycation end products (AGEs). It is well known that modifications in lifestyle such as nutritional interventions can be of great value for preventing brain deterioration. This study aimed to evaluate in vivo how an oral MG treatment, that mimics a high MG dietary intake, could affect brain health. From our results, we demonstrated that MG administration affected working memory, and induced neuroinflammation and oxidative stress by modulating the Receptor for Advanced glycation end products (RAGE). The gene and protein expressions of RAGE were increased in the hippocampus of MG mice, an area where the activity of glyoxalase 1, one of the main enzymes involved in MG detoxification, was found reduced. Furthermore, at hippocampus level, MG mice showed increased expression of proinflammatory cytokines and increased activities of NADPH oxidase and catalase. MG administration also increased the gene and protein expressions of Presenilin-1, a subunit of the gamma-secretase protein complex linked to Alzheimer's disease. These findings suggest that high MG oral intake induces alteration directly in the brain and might establish an environment predisposing to AD-like pathological conditions.

The pharmacological research on the Cannabis sativa-derived compounds has never terminated. Among the phytocannabinoids without psychotropic effects, the prevalent one in Cannabis is cannabidiol (CBD). Although CBD was initially considered a type 2 cannabinoid receptor (CB2R) antagonist, it did not show a good cannabinoidergic activity. Furthermore, heterogeneous results were obtained in experimental animal models of anxiety disorders, psychotic stages and neurodegenerative diseases. Recently, CBD has been authorized by the FDA to treat some rare forms of epilepsy and many trials have begun for the treatment of autism spectrum disorders. This review aims to clarify the pharmacological activity of CBD and its multiple therapeutic applications. Furthermore, critical and conflicting results of the research on CBD are discussed with a focus on promising future prospects.

Phytochemicals and secondary metabolites able to interact with the endocannabinoid system (Cannabimimetics) have been recently described in a broad range of plants and fruits. These findings can open new alternative avenues to explore for the development of novel therapeutic compounds. The cannabinoids regulate many physiological and pathological functions in both animals and plants. Cannabis sativa is the main plant that produces phytocannabinoids inside resins capable to defend the plant from the aggression of parasites and herbivores. Animals produce anandamide and 2-arachidonoyl glycerol, which thanks to binding with main receptors such as type-1 cannabinoid receptor (CB1R) and the type-2 cannabinoid receptor (CB2R) are involved in inflammation processes and several brain functions. Endogenous cannabinoids, enzymes for synthesis and degradation of cannabinoids, and CB1R and CB2R constitute the endocannabinoid system (ECS). Other plants can produce cannabinoid-like molecules such as perrottetinene extracted from Radula perrottetii, or anandamide and 2-arachidonoyl glycerol extracted from some bryophytes. Moreover, several other secondary metabolites can also interact with the ECS of animals and take the name of cannabimimetics. These phytoextracts not derived from Cannabis sativa can act as receptor agonists or antagonist, or enzyme inhibitors of ECS and can be involved in the inflammation, oxidative stress, cancer, and neuroprotection. Finally, given the evolutionary heterogeneity of the cannabimimetic plants, some authors speculated on the fascinating thesis of the evolutionary convergence between plants and animals regarding biological functions of ECS. The review aims to provide a critical and complete assessment of the botanical, chemical and therapeutic aspects of cannabimimetic plants to evaluate their spread in the world and medicinal potentiality.

Pulmonary fibrosis is a progressive scarring disease of the lungs, characterized by inflammation, fibroblast activation, and deposition of extracellular matrix. The long pentraxin 3 (PTX3) is a member of the pentraxin family with non-redundant functions in innate immune responses, tissue repair, and haemostasis. The role played in the lungs by PTX3 during the fibrotic process has not been elucidated. In this study, the impact of PTX3 expression on lung fibrosis was assessed in an intratracheal bleomycin (BLM)-induced murine model of the disease applied to wild type animals, transgenic mice characterized by endothelial overexpression and stromal accumulation of PTX3 (Tie2-PTX3 mice), and genetically deficient Ptx3−/− animals. Our data demonstrate that PTX3 is produced during BLM-induced fibrosis in wild type mice, and that PTX3 accumulation in the stroma compartment of Tie2-PTX3 mice limits the formation of fibrotic tissue in the lungs, with reduced fibroblast activation and collagen deposition, and a decrease in the recruitment of the immune infiltrate. Conversely, Ptx3-null mice showed an exacerbated fibrotic response and decreased survival in response to BLM treatment. These results underline the protective role of endogenous PTX3 during lung fibrosis and pave the way for the study of novel PTX3-derived therapeutic approaches to the disease.

Gynostemma pentaphyllum (var. Ginpent) (GP) is a variety of Cucurbit with anti-inflammatory and antioxidant effects in patients. In this manuscript, the main components present in the dry extract of GP have been identified using Ultra High Performance Liquid Chromatography quadrupole-time-of-flight mass spectrometry (UHPLC/Q-TOF-MS). In addition, the anti-inflammatory action of GP was evaluated in animal models with acute peripheral inflammation and motor alteration induced by lipopolysaccharide. The results showed that GP dry extract is rich in secondary metabolites with potential antioxidant and anti-inflammatory properties. We found that the treatment with GP induced a recovery of motor function measured with the rotarod test and pole test, and a reduction in inflammatory cytokines such as interleukin-1&beta, and interleukin-6 measured with the ELISA test. The data collected in this study on the effects of GP in in vivo models may help integrate the therapeutic strategies of inflammatory-based disorders.

Introduction: Cannabis sativa L. (C. sativa) is used since ancient times to produce fabrics, baskets, and cords. Later, different ethnic groups used to burn the leaves and flowers of psychotropic c...

Gynostemma pentaphyllum (var. Ginpent) (GP) is a variety of Cucurbit with anti-inflammatory and antioxidant effects in patients. In this manuscript, the main components present in the dry extract of GP have been identified using Ultra High Performance Liquid Chromatography quadrupole-time-of-flight mass spectrometry (UHPLC/Q-TOF-MS). In addition, the anti-inflammatory action of GP was evaluated in animal models with acute peripheral inflammation and motor alteration induced by lipopolysaccharide. The results showed that GP dry extract is rich in secondary metabolites with potential antioxidant and anti-inflammatory properties. We found that the treatment with GP induced a recovery of motor function measured with the rotarod test and pole test, and a reduction in inflammatory cytokines such as interleukin-1β and interleukin-6 measured with the ELISA test. The data collected in this study on the effects of GP in in vivo models may help integrate the therapeutic strategies of inflammatory-based disorders.

Nowadays, personalized cancer therapy relies on small molecules, monoclonal antibodies, or antibody–drug conjugates (ADC). Many nanoparticle (NP)-based drug delivery systems are also actively investigated, but their advantage over ADCs has not been demonstrated yet. Here, using the Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS), a class of polyavidins multifuctionalizable with stoichiometric control, we compare quantitatively anti-EGFR antibody(cetuximab)-targeted NPs to the corresponding ADC. We show that ANANAS tethering of cetuximab promotes a more efficient EGFR-dependent vesicle-mediated internalization. Cetuximab-guided ANANAS carrying doxorubicin are more cytotoxic in vitro and much more potent in vivo than the corresponding ADC, leading to 43% tumor reduction at low drug dosage (0.56 mg/kg). Advantage of cetuximab-guided ANANAS with respect to the ADC goes beyond the increase in drug-to-antibody ratio. Even if further studies are needed, we propose that NP tethering could expand application of the anti-EGFR antibody to a wider number of cancer patients including the KRAS-mutated ones, currently suffering from poor prognosis., The nature of the linker is known to affect the efficacy of antibody–drug conjugate (ADC). Here the authors show cetuximab-guided Avidin-Nucleic-Acid-Nanoassemblies to be superior to cetuximab-doxorubicin conjugate, and show its efficacy in KRAS mutant breast cancer, allowing for therapeutic expansion of anti-EGFR therapy.

Several studies on humans and mice support oxytocin's role in improving social behaviour, but its use in pharmacotherapy presents some important limiting factors. To date, it is emerging a pharmacological potential for melanocortin 4 receptor (MC4R) agonism in social deficits treatment. Recently, we demonstrated that the deletion of the NFKB1 gene, which encodes the p50 NF-κB subunit, causes impairment in social behaviours, with reductions in social interactions in mice. In this work, we tested the acute effects of THIQ, a selective melanocortin 4 receptor (MC4R) agonist. THIQ treatment increased social interactions both in wild type and p50−/− mice. In particular, after treatment with THIQ, p50−/− mice showed a prosocial behaviour analogous to that of basal WT mice. Moreover, intranasal treatment with an oxytocin antagonist blocked social interactions induced by THIQ, demonstrating that its prosocial effects are mediated by the oxytocin pathway. The data obtained reinforce using MC4R agonists to ameliorate social impairment in NDDs.

Multiple myeloma, the second most common hematologic malignancy, frequently relapses because of chemotherapeutic resistance. Fibroblast growth factors (FGF) act as proangiogenic and mitogenic cytokines in multiple myeloma. Here, we demonstrate that the autocrine FGF/FGFR axis is essential for multiple myeloma cell survival and progression by protecting multiple myeloma cells from oxidative stress–induced apoptosis. In keeping with the hypothesis that the intracellular redox status can be a target for cancer therapy, FGF/FGFR blockade by FGF trapping or tyrosine kinase inhibitor impaired the growth and dissemination of multiple myeloma cells by inducing mitochondrial oxidative stress, DNA damage, and apoptotic cell death that were prevented by the antioxidant vitamin E or mitochondrial catalase overexpression. In addition, mitochondrial oxidative stress occurred as a consequence of proteasomal degradation of the c-Myc oncoprotein that led to glutathione depletion. Accordingly, expression of a proteasome-nondegradable c-Myc protein mutant was sufficient to avoid glutathione depletion and rescue the proapoptotic effects due to FGF blockade. These findings were confirmed on bortezomib-resistant multiple myeloma cells as well as on bone marrow–derived primary multiple myeloma cells from newly diagnosed and relapsed/refractory patients, including plasma cells bearing the t(4;14) translocation obtained from patients with high-risk multiple myeloma. Altogether, these findings dissect the mechanism by which the FGF/FGFR system plays a nonredundant role in multiple myeloma cell survival and disease progression, and indicate that FGF targeting may represent a therapeutic approach for patients with multiple myeloma with poor prognosis and advanced disease stage. Significance: This study provides new insights into the mechanisms by which FGF antagonists promote multiple myeloma cell death.

Rice (Oryza sativa L.) is the richest source of &gamma, oryzanol, a compound endowed with antioxidant and anti-inflammatory properties. &gamma, Oryzanol has been demonstrated to cross the blood-brain barrier in intact form and exert beneficial effects on brain function. This study aimed to clarify the effects of &gamma, oryzanol in the hippocampus in terms of cognitive function and protein expression. Adult mice were administered with &gamma, oryzanol 100 mg/kg or vehicle (control) once a day for 21 consecutive days following which cognitive behavior and hippocampal proteome were investigated. Cognitive tests using novel object recognition and Y-maze showed that long-term consumption of &gamma, oryzanol improves cognitive function in mice. To investigate the hippocampal proteome modulated by &gamma, oryzanol, 2D-difference gel electrophoresis (2D-DIGE) was performed. Interestingly, we found that &gamma, oryzanol modulates quantitative changes of proteins involved in synaptic plasticity and neuronal trafficking, neuroprotection and antioxidant activity, and mitochondria and energy metabolism. These findings suggested &gamma, oryzanol as a natural compound able to maintain and reinforce brain function. Although more intensive studies are needed, we propose &gamma, oryzanol as a putative dietary phytochemical for preserving brain reserve, the ability to tolerate age-related changes, thereby preventing clinical symptoms or signs of neurodegenerative diseases.

Background: Rice (Oryza sativa L.) is the main food source for more than half of humankind. Rice is rich in phytochemicals and antioxidants with several biological activities, among these compounds, the presence of &gamma, oryzanol is noteworthy. The present study aims to explore the effects of &gamma, oryzanol on cognitive performance in a mouse model of neuroinflammation and cognitive alterations. Methods: Mice received 100 mg/kg &gamma, oryzanol (ORY) or vehicle once daily for 21 consecutive days and were then exposed to an inflammatory stimulus elicited by lipopolysaccharide (LPS). A novel object recognition test and mRNA expression of antioxidant and neuroinflammatory markers in the hippocampus were evaluated. Results: ORY treatment was able to improve cognitive performance during the neuroinflammatory response. Furthermore, phase II antioxidant enzymes such as heme oxygenase-1 (HO-1) and NADPH-dehydrogenase-quinone-1 (NQO1) were upregulated in the hippocampi of ORY and ORY+LPS mice. Lastly, &gamma, oryzanol showed a strong anti-inflammatory action by downregulating inflammatory genes after LPS treatment. Conclusion: These results suggest that chronic consumption of &gamma, oryzanol can revert the LPS-induced cognitive and memory impairments by promoting hippocampal antioxidant and anti-inflammatory molecular responses.

Mice emit ultrasonic vocalizations (USVs) in different social conditions: pups maternal separation, juveniles play, adults mating and social investigation. The USVs measurement has become an important instrument for behavioural phenotyping in neurodevelopmental disorders (NDDs). Recently, we have demonstrated that the deletion of the NFκB1 gene, which encodes the p50 NF-κB subunit, causes NDDs phenotype in mice. In this study, we investigated the ultrasonic communication and the effects of an early social enrichment in mice lacking the NF-κB p50 subunit (p50 KO). In particular, USVs of wild-type (WT), p50 KO and KO exposed to early social enrichment (KO enriched) were recorded using an ultrasound sensitive microphone and analysed by Avisoft software. USVs analysis showed that p50 KO pups emit more and longer vocalizations compared to WT pups. On the contrary, in adulthood, p50 KO mice emit less USVs than WT mice. We also found significant qualitative differences in p50 KO mice USVs compared to WT mice; the changes specifically involved two USVs categories. Early social enrichment had no effect on USVs number, duration and type in p50 KO mice. Together, these data revealed social communication alterations in a mouse model of NDDs; these deficits were not recovered by early social enrichment, strengthening the fact that genetic background prevails on environmental enrichment.

Neuroferritinopathy is a rare genetic disease with a dominant autosomal transmission caused by mutations of the ferritin light chain gene (FTL). It belongs to Neurodegeneration with Brain Iron Accumulation, a group of disorders where iron dysregulation is tightly associated with neurodegeneration. We studied the 498–499InsTC mutation which causes the substitution of the last 9 amino acids and an elongation of extra 16 amino acids at the C-terminus of L-ferritin peptide. An analysis with cyclic voltammetry on the purified protein showed that this structural modification severely reduces the ability of the protein to store iron. In order to analyze the impact of the mutation in vivo, we generated mouse models for the some pathogenic human FTL gene in FVB and C57BL/6J strains. Transgenic mice in the FVB background showed high accumulation of the mutated ferritin in brain where it correlated with increased iron deposition with age, as scored by magnetic resonance imaging. Notably, the accumulation of iron–ferritin bodies was accompanied by signs of oxidative damage. In the C57BL/6 background, both the expression of the mutant ferritin and the iron levels were lower than in the FVB strain. Nevertheless, also these mice showed oxidative alterations in the brain. Furthermore, post-natal hippocampal neurons obtained from these mice experienced a marked increased cell death in response to chronic iron overload and/or acute oxidative stress, in comparison to wild-type neurons. Ultrastructural analyses revealed an accumulation of lipofuscin granules associated with iron deposits, particularly enriched in the cerebellum and striatum of our transgenic mice. Finally, experimental subjects were tested throughout development and aging at 2-, 8- and 18-months for behavioral phenotype. Rotarod test revealed a progressive impaired motor coordination building up with age, FTL mutant old mice showing a shorter latency to fall from the apparatus, according to higher accumulation of iron aggregates in the striatum. Our data show that our 498–499InsTC mouse models recapitulate early pathological and clinical traits of the human neuroferritinopathy, thus providing a valuable model for the study of the disease. Finally, we propose a mechanistic model of lipofuscine formation that can account for the etiopathogenesis of human neuroferritinopathy., Highlights • We developed two new neuroferritinopathy mice models (NF). • NF brains are characterized by iron/ferritin accumulation and oxidative damage. • NF brains show granules of lipofuscine associated with iron. • A mechanism of lipofuscine formation is proposed. • NF mice show impaired motor coordination increasing with age.

Ethnopharmacological relevance Cannabis sativa L. (C. sativa) is an annual dioecious plant, which shares its origins with the inception of the first agricultural human societies in Asia. Over the course of time different parts of the plant have been utilized for therapeutic and recreational purposes, for instance, extraction of healing oils from seed, or the use of inflorescences for their psychoactive effects. The key psychoactive constituent in C. sativa is called Δ-9-tetrahydrocannabinol (D9-THC). The endocannabinoid system seems to be phylogenetically ancient, as it was present in the most primitive vertebrates with a neuronal network. N-arachidonoylethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG) are the main endocannabinoids ligands present in the animal kingdom, and the main endocannabinoid receptors are cannabinoid type-1 (CB1) receptor and cannabinoid type-2 (CB2) receptor. Aim of the study The review aims to provide a critical and comprehensive evaluation, from the ancient times to our days, of the ethnological, botanical, chemical and pharmacological aspects of C. sativa, with a vision for promoting further pharmaceutical research to explore its complete potential as a therapeutic agent. Materials and methods This study was performed by reviewing in extensive details the studies on historical significance and ethnopharmacological applications of C. sativa by using international scientific databases, books, Master’s and Ph.D. dissertations and government reports. In addition, we also try to gather relevant information from large regional as well as global unpublished resources. In addition, the plant taxonomy was validated using certified databases such as Medicinal Plant Names Services (MPNS) and The Plant List. Results and conclusions A detailed comparative analysis of the available resources for C. sativa confirmed its origin and traditional spiritual, household and therapeutic uses and most importantly its popularity as a recreational drug. The result of several studies suggested a deeper involvement of phytocannabinoids (the key compounds in C. sativa) in several others central and peripheral pathophysiological mechanisms such as food intake, inflammation, pain, colitis, sleep disorders, neurological and psychiatric illness. However, despite their numerous medicinal benefits, they are still considered as a menace to the society and banned throughout the world, except for few countries. We believe that this review will help lay the foundation for promoting exhaustive pharmacological and pharmaceutical studies in order to better understand the clinical relevance and applications of non-psychoactive cannabinoids in the prevention and treatment of life-threatening diseases and help to improve the legal status of C. sativa.

The use of different natural and/or synthetic preparations ofCannabis sativais associated with therapeutic strategies for many diseases. Indeed, thanks to the widespread diffusion of the cannabinoidergic system in the brain and in the peripheral districts, its stimulation, or inhibition, regulates many pathophysiological phenomena. In particular, central activation of the cannabinoidergic system modulates the limbic and mesolimbic response which leads to food craving. Moreover, cannabinoid agonists are able to reduce inflammatory response. In this review a brief history of cannabinoids and the protagonists of the endocannabinoidergic system, i.e. synthesis and degradation enzymes and main receptors, will be described. Furthermore, the pharmacological effects of cannabinoids will be outlined. An overview of the involvement of the endocannabinoidergic system in neuroinflammatory and metabolic pathologies will be made. Finally, particular attention will also be given to the new pharmacological entities acting on the two main receptors, cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2), with particular focus on the neuroinflammatory and metabolic mechanisms involved.

Ferritins, the main intracellular iron storage proteins, have been studied for over 60 years, mainly focusing on the mammalian ones. This allowed the elucidation of the structure of these proteins and the mechanisms regulating their iron incorporation and mineralization. However, ferritin is present in most, although not all, eukaryotic cells, comprising monocellular and multicellular invertebrates and vertebrates. The aim of this review is to provide an update on the general properties of ferritins that are common to various eukaryotic phyla (except plants), and to give an overview on the structure, function and regulation of ferritins. An update on the animal models that were used to characterize H, L and mitochondrial ferritins is also provided. The data show that ferritin structure is highly conserved among different phyla. It exerts an important cytoprotective function against oxidative damage and plays a role in innate immunity, where it also contributes to prevent parenchymal tissue from the cytotoxicity of pro-inflammatory agonists released by the activation of the immune response activation. Less clear are the properties of the secretory ferritins expressed by insects and molluscs, which may be important for understanding the role played by serum ferritin in mammals.

Background Three functional ferritin genes have been identified so far in mammals, and they encode the cytosolic Heavy (FTH) and Light chain (FTL) and the mitochondrial ferritin. The expression of a transcript by a fourth ferritin-like gene (Ferritin-Heavy-Polypeptide-Like-17, FTHL17) on the X chromosome was reported in mouse spermatogonia and in early embryonic cells. Methods The intronless human FTHL17 gene encodes a protein with 64% identity to human FTH with substitution of key residues of the ferroxidase center. The gene was cloned into vectors for expression in Escherichia coli and mammalian cells, linked to a flag-tag. Results The recombinant FTHL17 from E. coli purified as an assembled 24-mer ferritin devoid of ferroxidase activity and with a reduced physical stability. When transiently expressed in mammalian cells the flag-FTHL17 assembled in ferritin shells that showed reduced stability to denaturants compared with flag H and L ferritins. Immunocytochemistry with anti-flag antibody decorated the nuclei of flag-FTHL17 transfected COS cells, but not those of the cells transfected with flag-FTH or flag-FTL. Conclusions We concluded that FTHL17 encodes a ferritin-like protein without ferroxidase activity. Its restricted embryonic expression and partial nuclear localization suggest that this novel ferritin type may have functions other than iron storage. General significance The work confirms the presence of a fourth functional human ferritin gene with properties distinct from the canonical cytosolic ones.

Mitochondrial ferritin (FtMt) is a functional ferritin targeted to mitochondria that is highly expressed in the testis. To investigate the role of FtMt in the testis we set up a series of controlled matings between FtMt gene-deletion mice (FtMt–/–) with FtMt+/+ mice. We found that the number of newborns per litter and the fertility rate were strongly reduced for the FtMt–/– males, but not for the females, indicating that FtMt has an important role for male fertility. The morphology of the testis and of the spermatozoa of FtMt–/– mice was normal and we did not detect alterations in sperm parameters or in oxidative stress indices. In contrast, we observed that the cauda epididymides of FtMt–/– mice were significantly lighter and contained a lower number of spermatozoa compared with the controls. Also, the ATP content of FtMt–/– spermatozoa was found to be lower than that of FtMt+/+ spermatozoa. These data show that FtMt contributes to sperm epididymis maturation and to male fertility., The work was partially supported by MIUR grant PRIN10–11 to P. A. and by Telethon grant GGP1099 to P. A.

Hepcidin is a peptide hormone that controls systemic iron availability and is upregulated by iron and inflammation. Heparins have been shown to be efficient hepcidin inhibitors both in vitro and in vivo, even when their anticoagulant activity has been abolished by chemical reactions of oxidation/reduction (glycol-split). We analyzed a modified heparin type, characterized by a high, almost saturated, sulfation degree and low molecular weight. It inhibited hepcidin expression in hepatic HepG2 cells, and when used in mice, it readily suppressed liver hepcidin mRNA and serum hepcidin, with a significant decrease of spleen iron. This occurred also in inflammation-model, LPS-treated animals, and after heparin chronic 10-day treatments. The heparin had low/absent anticoagulant activity, as tested for factor-Xa and -IIA, APTT and anti Xa. It reduced triglyceride levels in the mice. This heparin acts faster and is more potent than the glycol split-heparins, probably because of its smaller molecular weight and higher sulfation degree. This modified heparin has potential applications for the treatment of diseases with high hepcidin levels.

Aim Oxidative stress is considered one of the main events that lead to aging and neurodegeneration. Antioxidant treatments used to counteract oxidative damage have been associated with a wide variety of side effects or at the utmost to be ineffective. The aim of the present study was to investigate the antioxidant property of a natural mineral, the tribomechanically micronized zeolite (MZ). Main methods Cell death and oxidative stress were assessed in retinoic acid differentiated SH-SY5Y cells, a neuronal-like cell line, after a pro-oxidant stimulus. In vivo evaluation of antioxidant activity and amyloidogenic processing of beta amyloid have been evaluated in a transgenic model of aging related neurodegeneration, the APPswePS1dE9 transgenic mice (tg mice) after a five-month long period of water supplementation with MZ. Key findings The study showed that 24 h of cell pretreatment with MZ (1) protected the cells by radical oxygen species (ROS)-induced cell death and moreover (2) induced a reduction of the mitochondrial ROS production following a pro-oxidant stimulation. Looking for an antioxidant effect of MZ in vivo, we found (3) an increased activity of the endogenous antioxidant enzyme superoxide dismutase (SOD) in the hippocampus of tg mice and (4) a reduction in amyloid levels and plaque load in MZ treated tg mice compared to control tg mice. Significance Our results suggest MZ as a novel potential adjuvant in counteracting oxidative stress and plaque accumulation in the field of neurodegenerative diseases.

Alterations in genes that regulate neurodevelopment can lead to cortical malformations, resulting in malfunction during postnatal life. The NF-κB pathway has a key role during neurodevelopment by regulating the maintenance of the neural progenitor cell pool and inhibiting neuronal differentiation. In this study, we evaluated whether mice lacking the NF-κB p50 subunit (KO) present alterations in cortical structure and associated behavioral impairment. We found that, compared with wild type (WT), KO mice at postnatal day 2 present an increase in radial glial cells, an increase in Reelin protein expression levels, in addition to an increase of specific layer thickness. Moreover, adult KO mice display abnormal columnar organization in the somatosensory cortex, a specific decrease in somatostatin- and parvalbumin-expressing interneurons, altered neurite orientation, and a decrease in Synapsin I protein levels. Concerning behavior, KO mice, in addition to an increase in locomotor and exploratory activity, display impairment in social behaviors, with a reduction in social interaction. Finally, we found that risperidone treatment decreased hyperactivity of KO mice, but had no effect on defective social interaction. Altogether, these data add complexity to a growing body of data, suggesting a link between dysregulation of the NF-κB pathway and neurodevelopmental disorders pathogenesis.

Hepcidin controls systemic iron availability, and its excess contributes to the anemia of chronic diseases, the most prevalent anemia in hospitalized patients. We previously reported that heparins are efficient hepcidin inhibitors both in vitro and in vivo, but their anticoagulant activity limits therapeutic use. We studied nonanticoagulant heparins produced by N-acetylation and oxidation/reduction (glycol-split) that lost antithrombin-binding affinity. Four nonanticoagulant heparins inhibited hepcidin expression in hepatic HepG2 cells and primary hepatocytes. The 2 most potent ones used in mice suppressed liver hepcidin expression and serum hepcidin in 6 hours, with a significant decrease of spleen iron. This occurred also in lipopolysaccharide (LPS)-treated animals that mimic inflammation, as well as after chronic 1-week treatments, without evident adverse effects on coagulation. Heparin injections increased iron mobilization and facilitated the recovery from the anemia induced by heat-killed Brucella abortus, a model of inflammatory anemia. The heparins were used also in Bmp6(-/-) mice. A single dose of heparin reduced the already low level of hepcidin of these mice and prevented its induction by LPS. These nonanticoagulant compounds impair bone morphogenetic protein /sons of mothers against decapentaplegic signaling with no evident adverse effect in vivo, even when administered chronically. They may offer a strategy for the treatment of diseases with high hepcidin levels.

Mitochondrial ferritin is a functional ferritin that localizes in the mitochondria. It is expressed in the testis, heart, brain, and cells with active respiratory activity. Its overexpression in cultured cells protected against oxidative damage and reduced cytosolic iron availability. However, no overt phenotype was described in mice with inactivation of the FtMt gene. Here, we used the doxorubicin model of cardiac injury in a novel strain of FtMt-null mice to investigate the antioxidant role of FtMt. These mice did not show any evident phenotype, but after acute treatment to doxorubicin, they showed enhanced mortality and altered heart morphology with fibril disorganization and severe mitochondrial damage. Signs of mitochondrial damage were present also in mock-treated FtMt−/− mice. The hearts of saline- and doxorubicin-treated FtMt−/− mice had higher thiobarbituric acid reactive substance levels, heme oxygenase 1 expression, and protein oxidation, but did not differ from FtMt+/+ in the cardiac damage marker B-type natriuretic peptide (BNP), ATP levels, and apoptosis. However, the autophagy marker LC3 was activated. The results show that the absence of FtMt, which is highly expressed in the heart, increases the sensitivity of heart mitochondria to the toxicity of doxorubicin. This study represents the first in vivo evidence of the antioxidant role of FtMt. Key message Mitochondrial ferritin (FtMt) expressed in the heart has a protective antioxidant role. Acute treatment with doxorubicin caused the death of all FtMt−/− and only of 60 % FtMt+/+ mice. The hearts of FtMt−/− mice showed fibril disorganization and mitochondrial damage. Markers of oxidative damage and autophagy were increased in FtMt−/− hearts. This is the first in vivo evidence of the antioxidant role of FtMt. Electronic supplementary material The online version of this article (doi:10.1007/s00109-014-1147-0) contains supplementary material, which is available to authorized users.

Ferritin is the main intracellular protein of iron storage with a central role in the regulation of iron metabolism and detoxification. Nucleotide insertions in the last exon of the ferritin light chain cause a neurodegenerative disease known as Neuroferritinopathy, characterized by iron deposition in the brain, particularly in the cerebellum, basal ganglia and motor cortex. The disease progresses relentlessly, leading to dystonia, chorea, motor disability and neuropsychiatry features. The characterization of a good animal model is required to compare and contrast specific features with the human disease, in order to gain new insights on the consequences of chronic iron overload on brain function and behavior. To this aim we studied an animal model expressing the pathogenic human FTL mutant 498InsTC under the phosphoglycerate kinase (PGK) promoter. Transgenic (Tg) mice showed strong accumulation of the mutated protein in the brain, which increased with age, and this was accompanied by brain accumulation of ferritin/iron bodies, the main pathologic hallmark of human neuroferritinopathy. Tg-mice were tested throughout development and aging at 2-, 8- and 18-months for motor coordination and balance (Beam Walking and Footprint tests). The Tg-mice showed a significant decrease in motor coordination at 8 and 18 months of age, with a shorter latency to fall and abnormal gait. Furthermore, one group of aged naïve subjects was challenged with two herbicides (Paraquat and Maneb) known to cause oxidative damage. The treatment led to a paradoxical increase in behavioral activation in the transgenic mice, suggestive of altered functioning of the dopaminergic system. Overall, data indicate that mice carrying the pathogenic FTL498InsTC mutation show motor deficits with a developmental profile suggestive of a progressive pathology, as in the human disease. These mice could be a powerful tool to study the neurodegenerative mechanisms leading to the disease and help developing specific therapeutic targets.

It has previously been reported that growth hormone secretagogues (GHS) may have a role in the regulation of bone metabolism in animals and humans. In this study we evaluated the effect of ghrelin, the endogenous ligand of GHS receptors, on the proliferation rate and on osteoblast activity in primary cultures of rat calvaria osteoblasts. In the same experiments, we compared the effects of ghrelin with those of hexarelin (HEXA) and EP-40737, two synthetic GHS with different characteristics. Both ghrelin and HEXA (10−11−10−8 M) significantly stimulated osteoblast proliferation at low concentrations (10−10 M). Surprisingly, EP-40737 demonstrated an antiproliferative effect at 10−9−10−8 M, whereas lower concentrations had no effect on cell proliferation. Ghrelin and HEXA significantly increased alkaline phosphatase (ALP) and osteocalcin (OC) production. At variance with these peptides, EP-40737 did not significantly stimulate ALP and OC. The mRNA for GHS-R1a receptors and the corresponding protein were detected in calvarial osteoblasts by RT-PCR and Western blot respectively, indicating that ghrelin and GHS may bind and activate this specific receptor. We conclude that endogenous ghrelin and synthetic GHS modulate proliferation and differentiation of rat osteoblasts, probably by acting on their specific receptor.

Doxorubicin (DOX) is an anticancer drug with cardiotoxic side effects mostly caused by iron homeostasis dysregulation. Mitochondria are involved in iron trafficking and mitochondrial ferritin (FtMt) was shown to provide protection against cellular iron imbalance. Therefore, we hypothesized that FtMt overexpression could limit DOX effects on iron homeostasis. Heart's homogenates of DOX-treated C57BL/6 mice were analyzed for cytosolic and mitochondrial iron-related proteins' expression and activity, revealing high cytosolic ferritin and ferritin-bound iron, low transferrin-receptor 1 and a strong hepcidin upregulation. Mitochondrial iron-related proteins (aconitase, succinate-dehydrogenase, frataxin) seemed, however, unaffected, although a partial inactivation of superoxide dismutase 2 was detected. Importantly, the ectopic expression of FtMt in human HeLa cells partially reverted DOX-induced iron imbalance. Our results, while confirming DOX effects on iron homeostasis, demonstrate that DOX affects more cytosolic than mitochondrial iron metabolism both in murine hearts and human HeLa cells and that FtMt overexpression is able to prevent most of these effects in HeLa cells.

Maccarinelli, F.; et al. Photoacoustic molecular imaging for in vivo liver iron quantitation. Journal of Biomedical Optics 21(5): 056008 (2016). (Mar 1, 2016). DOI: http://dx.doi.org/10.1117/1.JBO.21.5.056008, Copyright 2017 Society of Photo Optical Instrumentation Engineers (SPIE). One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this publication for a fee or for commercial purposes, or modification of the contents of the publication are prohibited., A recent study showed that ferritin is a suitable endogenous contrast agent for photoacoustic molecular imaging in cultured mammalian cells. We have therefore tested whether this imaging technique can be used for in vivo quantification of iron in mouse livers. To verify this hypothesis, we used multispectral optoacoustic tomography (MSOT) to image albino CD1 mice before and after experimental iron loading. Postmortem assays showed that the iron treatment caused a 15-fold increase in liver iron and a 40-fold increase in liver ferritin levels, while in vivo longitudinal analysis using MSOT revealed just a 1.6-fold increase in the ferritin/iron photoacoustic signal in the same animals. We conclude that MSOT can monitor changes in ferritin/iron levels in vivo, but its sensitivity is much lower than that of ex vivo iron assays., The work was partially supported by PRIN10-11 grant to P.A. F.M. was partially supported by a fellowship by Consorzio Interuniversitario Biotecnologie.

Hepcidin is a major regulator of iron homeostasis, and its expression in liver is regulated by iron, inflammation, and erythropoietic activity with mechanisms that involve bone morphogenetic proteins (BMPs) binding their receptors and coreceptors. Here we show that exogenous heparin strongly inhibited hepcidin expression in hepatic HepG2 cells at pharmacologic concentrations, with a mechanism that probably involves bone morphogenetic protein 6 sequestering and the blocking of SMAD signaling. Treatment of mice with pharmacologic doses of heparin inhibited liver hepcidin mRNA expression and SMAD phosphorylation, reduced spleen iron concentration, and increased serum iron. Moreover, we observed a strong reduction of serum hepcidin in 5 patients treated with heparin to prevent deep vein thrombosis, which was accompanied by an increase of serum iron and a reduction of C-reactive protein levels. The data show an unrecognized role for heparin in regulating iron homeostasis and indicate novel approaches to the treatment of iron-restricted iron deficiency anemia.

Background Impaired regulation of hepcidin in response to iron is the cause of genetic hemochromatosis associated with defects of HFE and transferrin receptor 2. However, the role of these proteins in the regulation of hepcidin expression is unclear.Design and Methods Hepcidin expression, SMAD and extracellular signal-regulated kinase (Erk) phosphorylation and furin expression were analyzed in hepatic HepG2 cells in which HFE and transferrin receptor 2 were down-regulated or expressed, or furin activity specifically inhibited. Furin expression was also analyzed in the liver of transferrin receptor 2 null mice.Results We showed that the silencing of HFE and transferrin receptor 2 reduced both Erk phosphorylation and furin expression, that the exogenous expression of the two enhanced the induction of phosphoErk1/2 and furin by holotransferrin, but that this did not occur when the pathogenic HFE mutant C282Y was expressed. Furin, phosphoErk1/2 and phosphoSMAD1/5/8 were down-regulated also in transferrin receptor 2-null mice. Treatment of HepG2 cells with an inhibitor of furin activity caused a strong suppression of hepcidin mRNA, probably due to the inhibition of bone morphogenic protein maturation.Conclusions The data indicate that transferrin receptor 2 and HFE are involved in holotransferrin-dependent signaling for the regulation of furin which involved Erk phosphorylation. Furin in turn may control hepcidin expression.

Hamsters exposed for eight weeks to short photoperiod (LD 10:14) or treated with melatonin in the late afternoon under long photoperiod (LD 14:10) had significantly higher number of cytosolic androgen receptors in the pituitaries, hypothalami and harderian glands, as compared to the long photoperiod (LD 14:10) exposed controls. The numerical value of the apparent Kd was two to three times lower in the hypothalami and pituitaries, but not in the harderian glands of the animals from these groups. These results indicate that alterations in receptor numbers and affinity constants may be responsible for the dramatic changes in the sensitivity of the hypothalamo-pituitary axis to the negative feedback actions of the gonadal steroids, observed under inhibitory photoperiods and that this effect could be duplicated by late afternoon melatonin treatment.