Vitamin D3 mitigates type 2 diabetes induced by a high carbohydrate-high fat diet in rats: Role of the purinergic system.

This study evaluated the effect of vitamin D3 (VIT D3) supplementation on the enzymatic activities and density of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5-nucleotidase (E-5'-NT), adenosine deaminase (ADA), as well as the density of P2 × 7R, P2Y12R, A1R, A2AR receptors, IL-1ß, and oxidative parameters in type 2 diabetic rats. Forty male Wistar rats were fed a high carbohydrate-high fat diet (HCHFD) and received an intraperitoneal injection containing a single dose of streptozotocin (STZ, 35 mg/kg). Animals were divided into four groups: 1) control; 2) control/VIT D3 12 µg/kg; 3) diabetic; and 4) diabetic/VIT D3 12 µg/kg. Results show that VIT D3 reduced blood glucose, ATP hydrolysis, ADA activity, P2Y12R density (platelets), as well as ATP, ADP, and AMP hydrolysis and ADA activity (synaptosomes). Moreover, VIT D3 increased insulin levels and AMP hydrolysis (platelets) and improved antioxidant defense. Therefore, we suggest that VIT D3 treatment modulates hyperglycemia-induced changes via purinergic enzymes and receptor expression, consequently attenuating insulin homeostasis dysregulation in the diabetic state.

Purinergic signaling influences the neuroinflammatory outcomes of a testosterone-derived synthetic in female rats: Resistance training protective effects on brain health.

Physical exercise is recognized as a non-pharmacological approach to treat and protect against several neuroinflammatory conditions and thus to prevent brain disorders. However, the interest in ergogenic resources by athletes and bodybuilding practitioners is widespread and on the rise. These substances shorten the process of performance gain and improve aesthetics, having led to the prominent use and abuse of hormones in the past years. Recent evidence has shown that the purinergic system, composed of adenine nucleotides, nucleosides, enzymes, and receptors, participates in a wide range of processes within the brain, such as neuroinflammation, neuromodulation, and cellular communication. Here, we investigated the effects of the anabolic androgenic steroid (AAS) testosterone (TES) at a dose of 70 mg/kg/week in female rats and the neuroprotective effect of resistance exercise related to the purinergic system and oxidative stress parameters. Our findings showed a decrease in ATP and ADO hydrolysis in treated and trained animals. Furthermore, there was an increase in the density of purinoceptors (P2X7 and A2A) and inflammatory markers (IBA-1, NRLP3, CASP-1, IL-1ß, and IL-6) in the cerebral cortex of animals that received AAS. On the other hand, exercise reversed neuroinflammatory parameters such as IBA-1, NLRP3, CASP-1, and IL-1ß and improved antioxidant response and anti-inflammatory IL-10 cytokine levels. Overall, this study shows that the use of TES without indication or prescription disrupts brain homeostasis, as demonstrated by the increase in neuroinflammation, and that the practice of exercise can protect brain health.

Caffeic acid attenuates neuroinflammation and cognitive impairment in streptozotocin-induced diabetic rats: Pivotal role of the cholinergic and purinergic signaling pathways.

The present study evaluated the effect of caffeic acid (CA) on behavioral learning and memory tasks in the diabetic state. We also evaluated the effect of this phenolic acid on the enzymatic activities of acetylcholinesterase, ecto-nucleoside triphosphate diphosphohydrolase, ecto-5-nucleotidase and adenosine deaminase as well as on the density of M1R, α7nAChR, P2×7R, A1R, A2AR, and inflammatory parameters in the cortex and hippocampus of diabetic rats. Diabetes was induced by a single intraperitoneal dose of streptozotocin (55 mg/kg). The animals were divided into six groups: control/vehicle; control/CA 10 and 50 mg/kg; diabetic/vehicle; diabetic/CA 10 and 50 mg/kg, treated by gavage. The results showed that CA improved learning and memory deficits in diabetic rats. Also, CA reversed the increase in acetylcholinesterase and adenosine deaminase activities and reduced ATP and ADP hydrolysis. Moreover, CA increased the density of M1R, α7nAChR, and A1R receptors and reversed the increase in P2×7R and A2AR density in both evaluated structures. In addition, CA treatment attenuated the increase in NLRP3, caspase 1, and interleukin 1ß density in the diabetic state; moreover, it increased the density of interleukin-10 in the diabetic/CA 10 mg/kg group. The results indicated that CA treatment positively modified the activities of cholinergic and purinergic enzymes and the density of receptors, and improved the inflammatory parameters of diabetic animals. Thus, the outcomes suggest that this phenolic acid could improve the cognitive deficit linked to cholinergic and purinergic signaling in the diabetic state.

Neuromodulatory effect of the combination of metformin and vitamin D3 triggered by purinergic signaling in type 1 diabetes induced-rats.

Several studies have indicated the vitamin D deficiency in the development of macro- and microvascular complications of diabetes mellitus (DM) including DM-related cognitive dysfunction. The purinergic system plays an important role in the modulation of a variety of mechanisms, including neuroinflammation, plasticity, and cell-cell communication. In addition, purines, their receptors, and enzymes can regulate the purinergic axis at different levels in type 1 DM (T1DM). This study evaluated the effects of vitamin D3 alone or in combination with metformin in the behavioral performance of streptozotocin-induced T1DM rats. The effects of this combination on the metabolism of ATP and ADP were also studied by NTPDase (CD39), AMP by 5'-nucleotidase (CD73), and adenosine by adenosine deaminase (E-ADA) in the brain and peripheral lymphocytes of type 1 diabetic STZ-induced rats. The results showed that anxiety and memory loss from the DM condition reverted after 30 days of vitamin D3 treatment. Furthermore, the DM state affected systemic enzymes, with no effect on the central enzymes hydrolyzing extracellular nucleotides and nucleosides. Vitamin D3 treatment positively regulated ectonucleotidase (NTPDase and 5'-nucleotidase) activity, E-ADA, and the purinergic receptors as a mechanism to prevent oxidative damage in the cerebral cortex of T1DM rats. A neuroprotector effect of vitamin D3 through adenosine signaling was also observed, by regulating A1 and A2A receptors proteins levels. The present findings suggest that purinergic signaling through vitamin D3 modulation may be a novel alternative strategy for T1DM treatment, and may compensate for the negative changes in the central nervous system.

Impact of free curcumin and curcumin nanocapsules on viability and oxidative status of neural cell lines.

Curcumin is an active polyphenol substance found in the highest concentrations in the roots of Curcuma longa. Its health benefits have led to recent increases in the consumption of curcumin. It has anti-inflammatory and antioxidant activities and is a potent neuroprotective against diseases of the brain. Nevertheless, its low bioavailability and its relative difficulty crossing the blood-brain barrier limit curcumin's use for these purposes. Curcumin-loaded nanoparticles may be an effective treatment for several diseases although there is a paucity of studies reporting its safety in the central nervous system (CNS). Therefore, this study aimed to identify non-neurotoxic concentrations of free curcumin and two nanoformulations of curcumin. Cell lines BV-2 and SH-SY5Y, both originating from the CNS, were evaluated after 24, 48, and 72 h of treatment with free curcumin and nanocapsules We measured viability, proliferation, and dsDNA levels. We measured levels of reactive oxygen species and nitric oxide as proxies for oxidative stress in culture supernatants. We found that free curcumin was toxic at 10 and 20 µM, principally at 72 h. Nanoformulations were more neurotoxic than the free form. Safe concentrations of free curcumin are between 1-5 µM, and these concentrations were lower for nanoformulations. We determined the ideal concentrations of free curcumin and nanocapsules serving as a basis for studies of injuries that affect the CNS.

Curcumin modulates neurogliogenesis and purinergic receptor expression in neural precursor cells infected with Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis, and its congenital transmission is of paramount concern. During embryonic development, infection with the parasite causes irreversible damage to the still-forming fetus's central nervous system (CNS). In the pathogenesis of neurotoxoplasmosis, purinergic receptors prejudice neuroprotection, neuroinflammation, and activation of microbicide mechanisms against the parasitic vacuole. This study used curcumin as a treatment for neural precursor cells (NPCs) infected with T. gondii. The congenital toxoplasmosis induction consisted of maternal infection with the VEG strain, and NPCs were obtained from the telencephalon of mouse embryos. Curcumin at increasing concentrations was administered in vitro to analyze NPC metabolic activity, cell number, and size, as well as neurogliogenesis, proving to be effective in recovering the size of infected NPCs. Curcumin partially re-established impaired neurogenesis. Purinergic A1, A2A, and P2X7 receptors may be related to neuroprotection, neuroinflammatory control, and activation of mechanisms for inducing the parasite's death. ERK 1/2 was highly expressed in infected cells, while its expression rates decreased after the addition of the treatment, highlighting the possible anti-inflammatory action of curcumin. These findings suggest that curcumin treats neurological perturbations induced by toxoplasmosis.

Amazon-derived nutraceuticals: Promises to mitigate chronic inflammatory states and neuroinflammation.

Nutraceuticals have been the focus of numerous research in recent years and accumulating data support their use for promoting some health benefits. Several nutraceuticals have been widely studied as supplements due to their functional properties ameliorating symptoms associated with neurological disorders, such as oxidative stress and chronic inflammatory states. This seems to be the case of some fruits and seeds from the Amazon Biome consumed since the pre-Columbian period that could have potential beneficial impact on the human nervous system. The beneficial activities of these food sources are possibly related to a large number of bioactive molecules including polyphenols, carotenoids, unsaturated fatty acids, vitamins, and trace elements. In this context, this review compiled the research on six Amazonian fruits and seeds species and some of the major nutraceuticals found in their composition, presenting brief mechanisms related to their protagonist action in improving inflammatory responses and neuroinflammation.

Chlorpyrifos pesticide promotes oxidative stress and increases inflammatory states in BV-2 microglial cells: A role in neuroinflammation.

The exposure to environmental stressors, such as organophosphate (OP) pesticides, has been associated with the development of neurodegenerative diseases. Chlorpyrifos (CPF) is the worldwide most used OP pesticide and one of the most hazardous pesticides as it can cross the blood-brain barrier. Since studies evaluating the effects of CPF on brain immune cells are scarce, this research investigated the oxidative and inflammatory responses of CPF exposure in murine microglial cells. BV-2 cells were exposed to different concentrations of CPF pesticide (0.3-300 µM). CPF induced activation of microglial cells, confirmed by Iba-1 and CD11b marking, and promoted microglial proliferation and cell cycle arrest at S phase. Moreover, CPF exposure increased oxidative stress production (NO, MDA, and O2∙), and upregulated pro-inflammatory cytokines (IL-1ß and NLRP3) genes expression in BV-2 cells. Overall, data showed that CPF exposure, at the lowest concentrations, acted by promoting pro-oxidative and pro-inflammatory states in microglial cells. These results provide important information on the potential role of microglial activation in CPF-induced neuroinflammation and add to the expanding knowledge on the neurotoxicity of OP.

Aluminum-Induced Alterations in Purinergic System Parameters of BV-2 Brain Microglial Cells.

Aluminum (Al) is ubiquitously present in the environment and known to be a neurotoxin for humans. The trivalent free Al anion (Al3+) can cross the blood-brain barrier (BBB), accumulate in the brain, and elicit harmful effects to the central nervous system (CNS) cells. Thus, evidence has suggested that Al increases the risk of developing neurodegenerative diseases, particularly Alzheimer's disease (AD). Purinergic signaling has been shown to play a role in several neurological conditions as it can modulate the functioning of several cell types, such as microglial cells, the main resident immune cells of the CNS. However, Al effects on microglial cells and the role of the purinergic system remain elusive. Based on this background, this study is aimed at assessing the modulation of Al on purinergic system parameters of microglial cells. An in vitro study was performed using brain microglial cells exposed to Al chloride (AlCl3) and lipopolysaccharide (LPS) for 96 h. The uptake of Al, metabolism of nucleotides (ATP, ADP, and AMP) and nucleoside (adenosine), and the gene expression and protein density of purinoceptors were investigated. The results showed that both Al and LPS increased the breakdown of adenosine, whereas they decreased nucleotide hydrolysis. Furthermore, the findings revealed that both Al and LPS triggered an increase in gene expression and protein density of P2X7R and A2AR receptors, whereas reduced the A1R receptor expression and density. Taken together, the results showed that Al and LPS altered the setup of the purinergic system of microglial cells. Thus, this study provides new insights into the involvement of the purinergic system in the mechanisms underlying Al toxicity in microglial cells.

Diabetes and hypertension: Pivotal involvement of purinergic signaling.

Diabetes mellitus (DM) and hypertension are highly prevalent worldwide health problems and frequently associated with severe clinical complications, such as diabetic cardiomyopathy, nephropathy, retinopathy, neuropathy, stroke, and cardiac arrhythmia, among others. Despite all existing research results and reasonable speculations, knowledge about the role of purinergic system in individuals with DM and hypertension remains restricted. Purinergic signaling accounts for a complex network of receptors and extracellular enzymes responsible for the recognition and degradation of extracellular nucleotides and adenosine. The main components of this system that will be presented in this review are: P1 and P2 receptors and the enzymatic cascade composed by CD39 (NTPDase; with ATP and ADP as a substrate), CD73 (5'-nucleotidase; with AMP as a substrate), and adenosine deaminase (ADA; with adenosine as a substrate). The purinergic system has recently emerged as a central player in several physiopathological conditions, particularly those linked to inflammatory responses such as diabetes and hypertension. Therefore, the present review focuses on changes in both purinergic P1 and P2 receptor expression as well as the activities of CD39, CD73, and ADA in diabetes and hypertension conditions. It can be postulated that the manipulation of the purinergic axis at different levels can prevent or exacerbate the insurgency and evolution of diabetes and hypertension working as a compensatory mechanism.

Superoxide-hydrogen peroxide imbalance differentially modulates the keratinocytes cell line (HaCaT) oxidative metabolism via Keap1-Nrf2 redox signaling pathway.

The purpose of this study was to investigate the effect of a superoxide-hydrogen peroxide (S-HP) imbalance of the superoxide dismutase manganese dependent (SOD2) gene, generated by paraquat and porphyrin exposure, on the keratinocytes cell line (HaCaT) oxidative metabolism. Paraquat acts increasing superoxide (O2·-) levels, while porphyrin increases hydrogen peroxide (H2O2) levels, acting as VV-SOD2-like and AA-SOD2-like molecules, respectively. First of all, HaCAT cells were treated with different concentrations of paraquat and porphyrin (1; 10; 30, and 70 µM) to determine the concentration of both that causes imbalance. After defining the concentration of paraquat and porphyrin (70 µM), a time curve was performed (1, 3, 6, and 24 h) to evaluate ROS production levels. Other oxidative parameters, such as nitric oxide (NO), lipoperoxidation (TBARS) and protein carbonyl, were evaluated after 24 h of incubation, as well as genotoxic analyses, apoptosis detection, and gene expression. Our findings revealed that paraquat exposure decreased cell viability, increasing lipoperoxidation, DNA damage, and apoptosis. On the other hand, porphyrin treatment increased cell viability and proliferation, ROS and NO production, triggering protein and DNA damage. In addition, porphyrin up-regulated Keap1 and Nrf2 gene expression, while paraquat decreased Nrf2 gene expression. In this sense, we suggested that the superoxide-hydrogen peroxide imbalance differentially modulates oxidative stress on keratinocytes cell line via Keap1-Nrf2 gene expression pathway.

Antiproliferative and apoptotic effects of caffeic acid on SK-Mel-28 human melanoma cancer cells.

Cutaneous melanoma (CM) is an extremely aggressive cancer presenting low survival and high mortality. The vast majority of patients affected by this disease does not respond or show resistance to the chemotherapeutic drugs, which makes the treatment ineffective. In this sense, the necessity for the development of new agents to assist in CM therapy is extremely important. One of the sources of great interest in this search are compounds of natural origin. Among these compounds, caffeic acid has demonstrated a broad spectrum of pharmacological activities as well as antitumor effects in some types of cancer. Therefore, the objective of this work was to investigate the possible antitumor effect of caffeic acid on the SK-Mel-28 cell line, human CM cells. Cells were cultured in flasks with culture medium containing fetal bovine serum, antibiotic, and antifungal, and maintained in ideal conditions. Cells were treated with 25 µM, 50 µM, 100 µM, 150 µM and 200 µM of caffeic acid and dacarbazine at 1 mg/mL. We verified the effect on cell viability and cell death, apoptosis, cell cycle, colony formation and gene expression of caspases. Results showed a decrease in cell viability, cell death induction by apoptosis, inhibition of colony formation, modulation of cell cycle and alterations in gene expression of caspases after caffeic acid treatment. These results suggest an antitumor effect of the compound on SK-Mel-28 cells. This study provides original information on mechanisms by which caffeic acid may play a key role in preventing tumor progression in human melanoma cells.

High levels of extracellular ATP lead to chronic inflammatory response in melanoma patients.

Skin cancer represents a serious public health problem and melanoma is considered the most significant due to its high metastasis capacity. Evasion mechanisms are the main characteristic of these tumor cells to escape of immune response. Extracellular nucleotides and nucleosides play an important role in inflammatory and immune responses. In this study, we analyzed the expression and activity of purinergic system enzymes in platelets and lymphocytes, ATP levels quantification, as well the level of pro and anti-inflammatory interleukins in the serum of 23 patients with surgical melanoma removal (CM group) and 23 control subjects (CT group). Results showed a decrease in ATP, ADP, and AMP hydrolysis and an increase in ATP levels quantification in CM group. The pro-inflammatory cytokines were elevated in CM group when compared to CT group. These results suggest an inflammatory process, even after surgical removal, due to elevated extracellular ATP levels. Besides, CM group displayed an increase in IL-10 levels and an increased in ADA activity in platelets and lymphocytes. Once adenosine and IL-10 are anti-inflammatory molecules, these results indicate a down-regulation of immune system front to malignant process. The alteration in nucleotide and nucleoside hydrolysis reinforces the purinergic systems role in this cancer. Therefore, even after surgical removal, the purinergic system can develop a chronic inflammatory micro-environment that can influence directly on relapse or metastasis.

Hypothyroidism Enhanced Ectonucleotidases and Acetylcholinesterase Activities in Rat Synaptosomes can be Prevented by the Naturally Occurring Polyphenol Quercetin.

Thyroid hormones have an influence on the functioning of the central nervous system. Furthermore, the cholinergic and purinergic systems also are extensively involved in brain function. In this context, quercetin is a polyphenol with antioxidant and neuroprotective properties. This study investigated the effects of (MMI)-induced hypothyroidism on the NTPDase, 5'-nucleotidase, adenosine deaminase (ADA), and acetylcholinesterase (AChE) activities in synaptosomes of rats and whether the quercetin can prevent it. MMI at a concentration of 20 mg/100 mL was administered for 90 days in the drinking water. The animals were divided into six groups: control/water (CT/W), control/quercetin 10 mg/kg, control/quercetin 25 mg/kg, methimazole/water (MMI/W), methimazole/quercetin 10 mg/kg (MMI/Q10), and methimazole/quercetin 25 mg/kg (MMI/Q25). On the 30th day, hormonal dosing was performed to confirm hypothyroidism, and the animals were subsequently treated with 10 or 25 mg/kg quercetin for 60 days. NTPDase activity was not altered in the MMI/W group. However, treatment with quercetin decreased ATP and ADP hydrolysis in the MMI/Q10 and MMI/Q25 groups. 5'-nucleotidase activity increased in the MMI/W group, but treatments with 10 or 25 mg/kg quercetin decreased 5'-nucleotidase activity. ADA activity decreased in the CT/25 and MMI/Q25 groups. Furthermore, AChE activity was reduced in all groups with hypothyroidism. In vitro tests also demonstrated that quercetin per se decreased NTPDase, 5'-nucleotidase, and AChE activities. This study demonstrated changes in the 5'-nucleotidase and AChE activities indicating that purinergic and cholinergic neurotransmission are altered in this condition. In addition, quercetin can alter these parameters and may be a promising natural compound with important neuroprotective actions in hypothyroidism.