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This review presents recent knowledge on the neuroprotective effects of vitamin D and their usefulness as oral supplementation when combined with other molecules, such as curcumin. A critical look at the effectiveness of vitamin D in this field is also provided. Vitamin D plays a crucial role in neuroprotection and in the cognitive decline associated with aging, where vitamin D's levels are related to the levels of several neurotrophic factors. An important role of vitamin D has also been observed in the mechanism of neuroinflammation, which is the basis of several aging conditions, including cognitive decline and neurodegeration; furthermore, the neuroprotective effect of vitamin D in the cognitive decline of aging has recently been reported. For this reason, many food supplements created for humans contain vitamin D alone or combined with other molecules with antioxidant properties. However, recent studies also explored negative consequences of the use at a high dosage of vitamin D. Vitamin D in tissues or brain cells can also modulate calbindin-D28K, parvalbumin, and calretinin, and is involved in immune function, thanks also to the combination with curcumin. Curcumin acts as a free radical scavenger and antioxidant, inhibiting lipid peroxidation and oxidative DNA damage. In particular, curcumin is a potent immune-regulatory agent and its administration has been reported to attenuate cognitive impairments. These effects could be exploited in the future to control the mechanisms that lead to the brain decay typical of neurodegenerative diseases.

Palmitoylethanolamide is a nutraceutical compound naturally produced in many plants and animal source foods, but the natural form is poorly water-soluble. It has demonstrated an anti-inflammatory role as a neuroprotective mediator, acting on several molecular targets of the central nervous system involved on brain aging process. In healthy adults, palmitoylethanolamide is an endogenous PPAR-α (peroxisome proliferator-activated receptor α) agonist through which it performs anti-inflammatory activity and provides its effects by activating the cannabinoid receptor. The different formulations of palmitoylethanolamide (micronized palmitoylethanolamide, FM-LipoMatrix ® palmitoylethanolamide and FM-LipoMatrix ® palmitoylethanolamide plus lipoic acid and vitamin D3) were analyzed starting from intestinal barrier, to verify their bioavailability, to in primary astrocytes in which cell viability, reactive oxygen species (ROS) and nitric oxide (NO) production, NFKB activity, MAPK, p53 and PPARα activities were investigated. Additionally, cannabinoid and estrogen receptors were analyzed using the western blot technique. The combination of palmitoylethanolamide in FM-LipoMatrix ® , lipoic acid and vitamin D3 shows better absorption predicting an improvement on plasma concentration; this formulation also shows a reduction in ROS and NO production and the data show the interaction of palmitoylethanolamide with cannabinoids and estrogen receptors inhibiting neuroinflammatory markers. All these data support the hypothesis of a new potential strategy to restore brain function and slow down brain aging in humans.

An important link between brain aging and a class of growth/survival factors called neurotrophins has recently been demonstrated. In particular, brain-derived neurotrophic factor (BDNF) plays a fundamental role during age-related synaptic loss, preventing cerebral atrophy and cognitive decline. The aim of the present study was to investigate whether the use of low dose BDNF sequentially kinetic activated (SKA) was able to counteract some mechanisms underlying the degeneration and aging of nervous tissue by increasing endogenous protection mechanisms. Both in vitro and in vivo experiments were performed to assess the ability of BDNF SKA to protect and regenerate survival-related molecular pathways, studying intestinal absorption in vitro and brain function in vivo. Our pioneering results show that BDNF SKA is able to induce the endogenous production of BDNF, using its receptor TrkB and influencing the apolipoprotein E expression. Moreover, BDNF SKA exerted effects on β-Amyloid and Sirtuin 1 proteins, confirming the hypothesis of a fine endogenous regulatory effect exerted by BDNF SKA in maintaining the health of both neurons and astrocytes. For this reason, a change in BDNF turnover is considered as a positive factor against brain aging.

Background : Magnesium is involved in a wide variety of physiological processes including direct relaxation of smooth muscle. A magnesium imbalance can be considered the primary cause or consequence of many pathophysiological conditions. The smooth muscle tissue of the uterus, i.e., the myometrium, undergoes numerous physiological changes during life, fundamental for uterine activities, and it receives proven benefits from magnesium supplementation. However, magnesium supplements have poor absorption and bioavailability. Furthermore, no data are available on the direct interaction between intestinal absorption of magnesium and relaxation of the myometrium. Methods : Permeability in human intestinal cells (Caco-2 cells) and direct effects on myometrial cells (PHM1-41 cells) of two different forms of magnesium, i.e., sucrosomial and bisglycinate, were studied in order to verify the magnesium capacity of modulate contractility. Cell viability, reactive oxygen species (ROS) and nitric oxide (NO) production, magnesium concentration, contractility, and pathways involved were analyzed. Results : Data showed a better influence of buffered chelate bisglycinate on intestinal permeability and myometrial relaxation over time with a maximum effect at 3 h and greater availability compared to the sucrosomial form. Conclusions : Magnesium-buffered bisglycinate chelate showed better intestinal absorption and myometrial contraction, indicating a better chance of effectiveness in human applications.

Brain ageing is a complex multifactorial process characterized by gradual and continuous loss of neuronal functions. It is hypothesized that at the basis of brain ageing as well as age-related diseases, there is an impairment of the antioxidant defense system leading to an increase of oxidative stress. In this study, two different biological aspects involved in brain ageing and neurodegeneration have been investigated: oxidative stress and iron accumulation damage. In primary mouse astrocytes, the stimulation with 50  μ M lipoic acid (LA) and 100 nM vitamin D (vitD) was first investigated in a time-course study to determine the dosages to be used in combination and then in a permeability test using an in vitro blood-brain barrier. In a second set of experiments, the role of oxidative stress was investigated pretreating astrocytes with 200  μ M H 2 O 2 for 30 min. The ability of vitD and LA alone and combined together to prevent or repair the damage caused by oxidative stress was investigated after 24 h of stimulation by the MTT test, mitochondrial membrane potential measurement, and Western blot analysis. To induce neurodegeneration, cells were pretreated with 300  μ M catalytic iron for 6 days and then treated with vitD and LA alone and combined for additional 6 days to investigate the protection exerted by combination, analyzing viability, ROS production, iron concentration, and activation of intracellular pathways. In our study, the combination of LA and vitD showed beneficial effects on viability of astrocytes, since the substances are able to cross the brain barrier. In addition, combined LA and vitD attenuated the H 2 O 2 -induced apoptosis through the mitochondrial-mediated pathway. The combination was also able to counteract the adverse conditions caused by iron, preventing its accumulation. All these data support the hypothesis of the synergistic and cooperative activity exerted by LA and vitD in astrocytes indicating a possible new strategy to slow down ageing.

Objective: Wound healing is a dynamic, interactive, and complex process that involves a series of events, including inflammation, migration, proliferation, granulation tissue formation, and matrix remodeling. Despite the high frequency of serious slow-healing wounds, there is still no adequate therapy. The aim of this study is to evaluate a new highly diluted acetylcholine (Ach) formulation obtained through a sequential kinetic activation (SKA) method applied to a wound healing in vivo model to verify the hypothesis that a low dose of Ach could be a more physiological stimulus for healing, by stimulating muscarinic and nicotinic receptors and their related intracellular pathways. Approach: Two different concentrations (10 fg/mL and 1 pg/mL) and two formulations (either kinetically or nonkinetically activated) of Ach were used to verify the wound healing process. Area closure, histological aspect, and nicotinic and muscarinic receptors, matrix metalloproteinase 9 (MMP-9), Nestin, and von Willebrand's factor have been assessed by Western blot or ELISA and compared to 147 ng/mL Ach, used as positive control. Moreover, the systemic effect through plasmatic radical oxygen species (ROS) production and Ach concentration has been evaluated. Results: Ach SKA 1 pg/mL revealed a significant capacity to restore the integrity of tissue compared to other formulation and this effect was more evident after a single administration. Innovation: Topical application on skin of Ach SKA 1 pg/mL accelerates wound closure stimulating non-neuronal cholinergic system. Conclusion: Our results demonstrate for the first time the importance in an in vivo model of highly diluted SKA Ach during wound healing, suggesting a potential use in skin disease.

This work demonstrates the cooperative effect of Q10, vitamin D3, and L-arginine on both cardiac and endothelial cells. The effects of Q10, L-arginine, and vitamin D3 alone or combined on cell viability, nitric oxide, and reactive oxygen species productions in endothelial and cardiac cells were studied. Moreover, the involvement of PI3K/Akt and ERK/MAPK pathways leading to eNOS activation as well as the involvement of vitamin D receptor were also investigated. The same agents were tested in an animal model to verify vasodilation, nitric oxide, and reactive oxygen species production. The data obtained in this work demonstrate for the first time the beneficial and cooperative effect of stimulation with Q10, L-arginine, and vitamin D3. Indeed, in cardiac and endothelial cells, Q10, L-arginine, and vitamin D3 combined were able to induce a nitric oxide production higher than the that induced by the 3 substances alone. The effects on vasodilation induced by cooperative stimulation have been confirmed in an in vivo model as well. The use of a combination of Q10, L-arginine, and vitamin D to counteract increased free radical production could be a potential method to reduce myocardial injury or the effects of aging on the heart., (© 2018 S. Karger AG, Basel.)

Background: Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural antioxidant polyphenol able to exert a wide range of biological effect on several tissues. Despite its important beneficial properties, it has a low water solubility, which limits its therapeutic applications in humans. Resveratrol also acts as a phytoestrogen that modulates estrogen receptor (ER)-mediated transcription. In addition, it has been shown that ovarian tissues benefit greatly from vitamin D3, which exerts its beneficial effects through VDR receptors. The aim was to evaluate the cooperative effects of resveratrol combined with vitamin D3 on ovarian cells and tissues and some other organs as well. Moreover, the modulation of specific intracellular pathways involving ER and VDR receptors has been studied., Methods: The experiments were performed both in vitro and in vivo, to analyze cell viability, radical oxygen species production, signal transductions through Western Blot, and resveratrol quantification by HPLC., Results: Cell viability, radical oxygen species production, and intracellular pathways have been studied on CHO-K1 cells. Also, the relative mechanism activated following oral intake in female Wistar rats as animal model was investigated, evaluating bioavailability, biodistribution and signal transduction in heart, kidney, liver and ovarian tissues. Both in in vitro and in vivo experiments, resveratrol exerts more evident effects when administered in combination with vitD in ovarian cells, showing a common biphasic cooperative effect: The role of vitamin D3 in maintaining and supporting the biological activity of resveratrol has been clearly observed. Moreover, resveratrol plus vitamin D3 blood concentrations showed a biphasic absorption rate., Conclusions: Such results could be used as a fundamental data for the development of new therapies for gynecological conditions, such as hot-flashes.

This study compares the absorption characteristics of two iron-based dietary supplements and their biocompatibility to bisglycinate iron, a common chelated iron form. The Caco-2 cell line-a model of human intestinal absorption-and GTL-16 cell line-a model of gastric epithelial cells-were used to perform the experiments; in the first experiments, the kinetics of absorption have been evaluated analyzing the divalent metal transporter 1 (DMT1) expression. Three different iron combinations containing 50 µM iron (named Fisioeme ® , Sideral ® and bisglycinate) were used for different stimulation times (1-24 h). After this, the effects of the three iron formulations were assessed in both a short and a long time, in order to understand the extrusion mechanisms. The effects of the three different formulations were also analyzed at the end of stimulation period immediately after iron removal, and after some time in order to clarify whether the mechanisms were irreversibly activated. Findings obtained in this study demonstrate that Fisioeme ® was able to maintain a significant beneficial effect on cell viability compared to control, to Sideral ® , and to iron bisglycinate. This observation indicates that Fisioeme ® formulation is the most suitable for gastric and intestinal epithelial cells., Competing Interests: The authors declare no conflict of interest.

The physiological effects of acetylcholine on keratinocytes depend on the presence of nicotinic and muscarinic receptors. The role of nonneuronal acetylcholine in keratinocytes could have important clinical implications for patients with various skin disorders such as nonhealing wounds. In order to evaluate the efficacy of highly diluted acetylcholine solutions obtained by sequential kinetic activation, we aimed to investigate the effects of these solutions on normal human keratinocytes. Two different concentrations (10 fg/mL and 1 pg/mL) and formulations (kinetically activated and nonkinetically activated) of acetylcholine were used to verify keratinocyte viability, proliferation, and migration and the intracellular pathways involved using MTT, crystal violet, wound healing, and Western blot compared to 147 ng/mL acetylcholine. The activated formulations (1 pg/mL and 10 fg/mL) revealed a significant capacity to increase migration, cell viability, and cell proliferation compared to 147 ng/mL acetylcholine, and these effects were more evident after a single administration. Sequential kinetic activation resulted in a statistically significant decrease in reactive oxygen species production accompanied by an increase in mitochondrial membrane potential and a decrease in oxygen consumption compared to 147 ng/mL acetylcholine. The M1 muscarinic receptor was involved in these effects. Finally, the involvement of ERK/mitogen-activated protein kinases (MAPK) and KI67 confirmed the effectiveness of the single treatment on cell proliferation. The intracellular pathways of calcium were investigated as well. Our results indicate for the first time that highly diluted and kinetically activated acetylcholine seems to play an active role in an in vitro model of wound healing. Moreover, the administration of acetylcholine within the physiological range may not only be effective but is also likely to be safe., (© 2016 S. Karger AG, Basel.)

Background: Gastric diseases are a worldwide problem in modern society, as reported in the USA, in the range of 0.5-2 episodes/year/person and an incidence of 5-100 episodes/1000/week according to seasons and age. There is convincing evidence that oxidative stress is involved in the pathogenesis of acute gastric injury. Acid secreted from gastric parietal cells determines mucosal injuries which in turn cause inflammation and oxidative stress. Consequent inflammation produces free radicals by mitochondria thus causing lipid peroxidation, oxidative and acidic stress, which can lead to cell apoptosis. Vitamin D 3, the active form of vitamin D, may counteract intracellular cell death and improve epithelial regeneration., Methods: This study was planned to assess whether vitamin D 3 is a protective factor against acid injury and oxidative stress in gastric epithelial cells. Primary epithelial cells and GTL-16 cells have been used to test the effects of Grisù® alone or in combination with vitamin D 3 during oxidative stress or high acid exposition measuring cell viability, ROS production, cellular adhesion time along with apoptotic, autophagic and survival pathways. The combined effect of Grisù® and vitamin D 3 was found more effective in counteracting the negative consequences of oxidative stress and acidity conditions than some other gastroprotective agents, such as Maalox® or Gaviscon®., Results: In case of oxidative stress or acidity condition the stimulation with Grisù® alone caused an improvement of cell viability and a reduction of ROS production on epithelial gastric cells. In addition, the adhesion time of the cells was improved. All these effects were increased by the presence of vitamin D 3 . Similar data were also observed in primary gastric epithelial cells confirming the results obtained in GTL-16 cells., Conclusions: These results suggest that Grisù® in combination with vitamin D 3 may exert a gastroprotective effect to maintain or restore the integrity of gastric epithelium through an antioxidant pathway, inhibiting apoptosis and activating survival kinases. Moreover, the combination of Grisù® and vitamin D 3 improves cell viability and decreases ROS production compared to other gastroprotective agents combined with vitamin D 3 . All these data were validated using primary cells isolated from gastric tissue.

Background: Recently, vitamin D3 (1alpha, 25-dihydroxyvitamin D) has shown its capability to take part in many extraskeletal functions and its serum levels have been related to patient survival rate and malignancy of many types of neoplasms, including ovarian cancers. Catalytic iron is a free circulating form of iron that is able to generate reactive oxygen species and consequently to promote a number of cellular and tissutal dysfunctions including tumorigenesis. In fertile women an important source of catalytic iron is derived from retrograde menstruation. Epithelial secretory cells from fimbriae of fallopian tubes are greatly exposed to catalytic iron derived from menstrual reflux and so represent the site of origin for most serous ovarian cancers. The aim of this study was to assess whether vitamin D3 can play a role in counteracting catalytic iron-induced oxidative stress in cells from fimbriae of fallopian tubes., Methods: The cells, isolated from women undergoing isteroannessiectomy, were treated with catalytic iron 50-75-100 mM and vitamin D3 at a concentration ranging from 0.01 to 10 nM to study cell viability, radical oxygen species production, p53, pan-Ras, Ki67 and c-Myc protein expressions through Western Blot, and immunocytochemistry or immunofluorescence analysis., Results: The pre-treatment with vitamin D3 1 nM showed its beneficial effects that consists in a significant decrease in ROS production. In addition a novel finding is represented by the demonstration that pre-treatment with vitamin D3 is also able to significantly counteract tumoral biomarkers activation, such as p53, pan-Ras, Ki67 and c-Myc, and consequently the catalytic iron-induced cellular injury., Conclusions: This study demonstrates for the first time that vitamin D3 plays an important role in preventing catalytic iron-dependent oxidative stress in cultured fimbrial cells. These results support the hypothesis that vitamin D3 could counteract carcinogenic changes induced by catalytic iron.

Recent studies have postulated a role for vitamin D and its receptor on cerebral function, and anti-inflammatory, immunomodulatory and neuroprotective effects have been described; vitamin D can inhibit proinflammatory cytokines and nitric oxide synthesis during various neurodegenerative insults, and may be considered as a potential drug for the treatment of these disorders. In addition, iron is crucial for neuronal development and neurotransmitter production in the brain, but its accumulation as catalytic form (Fe(3+)) impairs brain function and causes the dysregulation of iron metabolism leading to tissue damage due to the formation of toxic free radicals (ROS). This research was planned to study the role of vitamin D to prevent iron damage in neuroblastoma BE(2)M17 cells. Mechanisms involved in neurodegeneration, including cell viability, ROS production, and the most common intracellular pathways were studied. Pretreatment with calcitriol (the active form of vitamin D) reduced cellular injury induced by exposure to catalytic iron., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Objective: Recent evidence strongly suggests that the fallopian tube is a site of origin of ovarian cancer. Although histological data show iron deposition in the fallopian tubes, its role remains unclear. To establish whether catalytic iron has a possible role in ovarian carcinogenesis, we isolated human fimbrial secretory epithelial cells (FSECs)., Methods: Fimbrial secretory epithelial cells, isolated from women undergoing isteroannessiectomy, were treated with different doses of catalytic iron (0.05-100 mM) to study cell viability; NO production; p53, Ras, ERK/MAPK, PI3K/Akt, Ki67, and c-Myc protein expressions through Western blot analysis; and immunocytochemistry or immunofluorescence., Results: In FSECs treated with catalytic iron for up to 6 days, we observed an increase in cell viability, NO production, and p53, pan-Ras, ERK/MAPK, PI3K/Akt, Ki67, and c-Myc activations (P < 0.05) in a dose-dependent and time-dependent manner. These same results were also observed in FSECs maintained for respectively 2 and 4 weeks in the absence of catalytic iron after 6 days of stimulation., Conclusions: Our model aimed at studying the main nongenetic risk factor for ovarian cancer, providing an alternative interpretation for the role of menstruation in increasing risk of this pathology. This in vitro model mimics several features of the precursor lesions and opens new scenarios for further investigations regarding the correlation between damages produced by repeated retrograde menstruation carcinogenic stimuli.

Secretin has been implicated in cardiovascular regulation through its specific receptors, as well as through β-adrenoceptors and nitric oxide, although data on its direct effect on coronary blood flow and cardiac function have remained scarce. The present study aimed to determine the primary in vivo effect of secretin on cardiac function and perfusion and the mechanisms related to the autonomic nervous system, secretin receptors and NO. In addition, in coronary endothelial cells the intracellular pathways involved in the effects of secretin on NO release were also examined. In 30 pigs, intracoronary secretin infusion at 2.97 pg for each millilitre per minute of coronary blood flow at constant heart rate and aortic blood pressure increased coronary blood flow, maximal rate of change of left ventricular pressure, segmental shortening, cardiac output and coronary NO release (P<0.05). These responses were graded in a further five pigs. Moreover, while blockade of muscarinic cholinoreceptors (n=5) and of α-adrenoceptors (n=5) did not abolish the observed responses to secretin, blockade of β1-adrenoceptors (n=5) prevented the effects of secretin on cardiac function. In addition, blockade of β2-adrenoceptors (n=5) and NO synthase inhibition (n=5) prevented the coronary response and the effect of secretin on NO release. All these effects were abolished by a secretin receptor inhibitor (n=5). In coronary endothelial cells, the increased NO production caused by secretin was found to be related to cAMP/protein kinase A signalling activated as downstream effectors of stimulation of secretin receptors and β2-adrenoceptors. In conclusion, in anaesthetized pigs secretin primarily increased cardiac function and perfusion through the involvement of specific receptors, β-adrenoceptors and NO release.