Purinergic Enzymes Research Articles

Liposome preparation of alpha-arbutin: stability and toxicity assessment using mouse B16F10 melanoma cells

ABSTRACT Melanoma is the most aggressive type of skin cancer, with few therapeutic alternatives following metastasis development. In recent years, drug delivery-associated nanotechnology has shown promising targeted results with diminished adverse effects compared to conventional treatments. This study aimed to (1) examine the effects of plant-derived α-arbutin, a natural compound and (2) compare these findings with bioactively developed liposomes containing α-arbutin utilizing the B16-F10 murine melanoma cell line as a model. Liposomes were obtained through reversed-phase evaporation by applying a spray dryer to assess their stability. The following biologic assays were measured cytotoxicity/antiproliferative (MTT, Neutral Red, and dsDNA PicoGreen). In addition, the levels of melanin and purinergic enzymes were also measured. The production of reactive oxygen species (ROS) and nitric oxide (NO) was determined as a measure of oxidative state. Treatment with nano-liposome containing alpha-arbutin induced a significant 68.4% cytotoxicity, similar to the positive control, in the B16-F10 murine melanoma cell line at 72 hr. Further, arbutin and liposomes containing alpha-arbutin increased levels of ROS and nitrite formation at 72 hr at the highest concentration (100 and 300 µg/ml) of treatments. Arbutin and liposomes containing alpha-arbutin reduced melanin levels at all tested concentrations. In addition, arbutin and alpha-arbutin containing liposomes lowered nucleotides (AMP, ADP, and ATP) and nucleoside (adenosine) levels in melanoma cells. Evidence suggests that α-arbutin containing liposome can be considered as an alternative immunosuppressive agent stimulated in melanoma treatment.

Caffeic acid attenuates memory dysfunction and restores the altered activity of cholinergic, monoaminergic and purinergic in brain of cadmium chloride exposure rats.

This study aims to evaluate the neuroprotective effect of caffeic acid (CAF) against cadmium chloride (CdCl2) in rats via its effect on memory index as well as on altered enzymatic activity in the brain of CdCl2-induced neurotoxicity. The experimental rats were divided into seven groups (n=6 rats per group) of healthy rats (group 1), CdCl2 -induced (CD) (3 mg/kg BW) rats (group 2), CD rats + Vitamin C (group 3), CD rats + CAF (10 and 20 mg/kg BW respectively) (group 4 & 5), and healthy rat + CAF (10 and 20 mg/kg BW respectively) (group 6 & 7). Thereafter, CdCl2 and CAF were administered orally to the experimental rats in group 2 to group 5 on daily basis for 14days. Then, the Y-maze test was performed on theexperimental rats to ascertain their memory index. CdCl2 administration significantly altered cognitive function, the activity of cholinesterase, monoamine oxidase, arginase, purinergic enzymes, nitric oxide (NOx), and antioxidant status of Cd rats (untreated) when compared with healthy rats. Thereafter, CD rats treated with vitamin C and CAF (10 and 20 mg/kg BW) respectively exhibited an improved cognitive function, and the observed altered activity of cholinesterase, monoamine oxidase, arginase, purinergic were restored when compared with untreated CD rats. Also, the level of brain NOx and antioxidant status were significantly (p<0.05) enhanced when compared with untreated CD rats. In the same vein, CAF administration offers neuro-protective effect in healthy ratsvis-à-vis improved cognitive function, reduction in the activity of some enzymes linked to the progression of cognitive dysfunction, and improved antioxidant status when compared to healthy rats devoid of CAF. This study demonstrated the neuroprotective effect of CAF against CdCl2 exposure and in healthy rats.

3-(4-methoxyphenyl) acrylic acid halts redox imbalance and modulate purinergic enzyme activity in iron-induced testicular injury

Abstract Various derivatives of cinnamic acid have been reported to possess significant activities such as antioxidant and hepatoprotective, and neuroprotective activities. Interestingly, testicular toxicity has been linked to several causes, with oxidative damage being one of the pathophysiological mechanisms. 3-(4-methoxyphenyl) acrylic acid (1), a derivative of cinnamic acid, was synthesized and then investigated for its effects on iron-induced testicular injury and oxidative stress via ex vivo and in silico studies, respectively. Evaluations were done on KAD-1’s FRAP, DPPH free radical scavenging activity, and iron chelating potential. Through the ex vivo incubation of tissue supernatant and 0.1 mM FeSO4 for 30 min at 37 °C with different concentration of 1, oxidative testicular damage treatments were induced. The scavenging property of 1 increases significantly (p &lt; 0.05) as the concentration increases when compared with the standard quercetin. The MDA, CAT, ATPase, and ENTPDase activities were reduced when testicular damage was induced (p &lt; 0.05). The group treated with 30 mg/mL had the highest level of MDA. A significant rise in GSH level and activity of SOD were observed. The result obtained indicated that 1 has the potential to prevent oxidative testicular toxicity, as evidenced by its capacity to control nucleotide hydrolysis and reduce oxidative stress. Overall, the results of this experimental study point to some possible uses of 3-(4-methoxyphenyl) acrylic acid (1) in the prevention of oxidative testicular dysfunction. Therefore, 3-(4-methoxyphenyl) acrylic acid (1) would be a good product in developing a medication to alleviate male infertility.

Date (Phoenix dactylifera L. Mill) fruit enhances sexual performance via modulation of oxido-inflammatory mediators and purinergic signaling in hypertensive male rats

Introduction The present study aimed at investigating the effect of dietary supplementation of Phoenix dactylifera, an important component of aphrodisiac supplements, on sexual performance, oxido-inflammatory mediators and purinergic signaling system in hypertensive rats. Material and methods Hypertension was induced via oral administration of 40 mg/kg L-NAME. Thereafter, the sexual performance of the experimental animals was determined and the hypertensive rats with impaired sexual activities were placed on P. dactylifera-supplemented diet for 21 days, and the effects of the treatment on the overall sexual behavior, antioxidant status, oxido-inflammatory biomarkers, and enzyme activity of the purinergic system were assessed. Results Hypertensive rats showed a significant (p < 0.05) decrease in sexual performance, elevated level of oxido-inflammatory mediators, and altered purinergic enzymes activity when compared with the control. However, sub-chronic feeding with P. dactylifera-supplemented diet improved sexual performance, significantly lowered oxido-inflammatory biomarkers, and enhanced the activity of purinergic enzymes in hypertensive rats. Conclusion Findings presented in this study suggest that dietary inclusion of P. dactylifera could be useful in managing erectile dysfunction (ED) commonly observed in subjects with hypertension. Findings highlighted in this study thus provide the scientific basis supporting the folkloric use of P. dactylifera as a key ingredient in aphrodisiac supplements.

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-1β, 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 enzymes on extracellular vesicles: immune modulation on the go.

An increase in the extracellular concentration of ATP as a consequence of cellular stress or cell death results in the activation of immune cells. To prevent inflammation, extracellular ATP is rapidly metabolized to adenosine, which deploys an anti-inflammatory signaling cascade upon binding to P1 receptors on immune cells. The ectonucleotidases necessary for the degradation of ATP and generation of adenosine are present on the cell membrane of many immune cells, and their expression is tightly regulated under conditions of inflammation. The discovery that extracellular vesicles (EVs) carry purinergic enzyme activity has brought forward the concept of EVs as a new player in immune regulation. Adenosine-generating EVs derived from cancer cells suppress the anti-tumor response, while EVs derived from immune or mesenchymal stem cells contribute to the restoration of homeostasis after infection. Here we will review the existing knowledge on EVs containing purinergic enzymes and molecules, and discuss the relevance of these EVs in immune modulation and their potential for therapy.

HEPATO-PROTECTIVE ROLE OF DIPHENYL DISELENIDE IN STREPTOZOTOCININDUCED DIABETIC RATS

Diabetic hepatopathy prevalence as a result of prolonged hyperglycemia is increasing with the expanding size of the diabetes population around the world. In this present study, the hepato-protective activity of diphenyl diselenide (DPDSe) in a persistent diabetic state was studied. Diabetes mellitus were induced with streptozotocin (STZ) (60 mg/kg) intraperitoneally and after 60 days without treatment, the rats were administered diphenyl dislenide (DPDSe) (10 mg/kg) orally for 35 days. Animals were euthanized liver and serum were used for experimental determinations such as glycemic level, non enzymic and enzymic redox statuses, hepatic biomarkers, hematological and lipid prole. The results showed that the fasting glucose levels and thiobarbituric reactive substances were reduced at a signicant level of p &lt; 0.05. Furthermore, a concomitant rise in the percentage of DPPH free radicals scavenged, ferric reducing antioxidants property, protein and non-protein thiols were observed in hepatic tissue. The activities of delta–aminolevulinate dehydratase and antioxidant enzymes were improved at a signicant level of p &lt; 0.05. Additionally, there was signicant reduction (p &lt; 0.05) in the activity of the purinergic enzymes, hepatic biomarkers while there was an increase in the albumin level. Furthermore, the hematological and lipid prole of were refurbished. This study showed that DPDSe was able to reverse the biochemical alterations related with hepatic tissue in STZ-induced diabetic rats.

Effect of alkaloid extract from Andrographis paniculata (Burm. f.) Nees and Phyllanthus amarus Schumach. & Thonn. on cognitive related biochemicals in the brain of streptozotocin-induced diabetic rats

IntroductionDiabetes mellitus (DM) is a chronic metabolic disorder linked to cognitive impairment and memory deficit, known as diabetes encephalopathy. This study examined the anti-diabetic and neuroprotective effects of alkaloid extracts from Andrographis paniculata (A. paniculata) and Phyllanthus amarus (P. amarus) leaves, a Traditional Chinese Medicine, on carbohydrate-metabolizing (α-amylase and α-glucosidase) enzyme, cholinesterase and purinergic enzyme activities, and antioxidants in the brains of streptozotocin (STZ)-induced diabetic rats. MethodsThe alkaloid extraction of A. paniculata and P. amarus was done. Male Wistar rats were divided into seven groups (n = 5) and were made diabetic by intraperitoneal injection of 50 mg/kg of STZ except Group I, which received saline (normal control). Group II-untreated diabetic rats (diabetic control), Group III-diabetic rats received 5 mg/kg Glibenclamide (standard drug); Group IV and V received 5 and 50 mg/kg P. amarus respectively; and Group VI and VII received 5 and 50 mg/kg A. paniculata, respectively. ResultsThe results showed that the elevated blood glucose and redox makers levels and enzyme activities were significantly increased in the untreated diabetic rats. Reduced antioxidant biomolecules (enzymatic and non-enzymatic antioxidant) levels were also observed. However, these were considerably ameliorated upon treatment with the alkaloid extracts in a dose-dependent manner. ConclusionThe study found that the alkaloid extract of P. amarus and A. paniculata leaves can modulate cognitive functioning and antioxidant profile in STZ-induced diabetic rats, suggesting it could be a promising alternative for managing diabetes and its complications, including cognitive impairment, in the treatment of diabetes and diabetes encephalopathy.

Resistance training reduces platelet activation in hypertensive women: the role of purinergic signaling.

Essential arterial hypertension is a risk factor for stroke, myocardial infarction, heart failure, and arterial aneurysm, which are related to the activation of platelets. Purinergic signaling has a central role in platelet aggregation. Although ATP and ADP can act as a proaggregant agent, adenosine inhibits platelet aggregation and reduces vascular injury. Physical exercise exhibits antiaggregant properties and can modulate purinergic system. The aim of this study was to evaluate the effect of 6 months of resistance training on purinergic system components in platelets and on platelet activation, hemodynamic and anthropometric parameters in hypertensive woman. A total of 31 hypertensive and 28 normotensive middle-aged sedentary women were submitted to 6 months of resistance training. Purinergic enzymes activities were assessed in platelets; ATP and Tromboxane B2 (TXB2) levels were measured in serum. Blood pressure (BP), BMI, and body fat were also measured. All variables were statistically analyzed, considering P value less than 0.05. Six months of resistance training was able to significantly reduce BP, ATP, and TXB2 levels as well as NTPDase, ecto-5'nucleotidase, and ADA activities in hypertensive group. After 6 months of resistance training, purinergic system components and TXB2 of hypertensive group were similar to normotensive group in platelets, demonstrating that resistance training was able to modulate platelet activation. A positive correlation was found between BP, enzyme activities, and levels of ATP and TXB2. Our findings demonstrated the relationship between purinergic signaling and platelet activation in hypertension and suggests that resistance training serve as tool to reduce platelet aggregation in hypertensive woman by modulating purinergic system.

Effects of clopidogrel bisulfate on B16-F10 cells and tumor development in a murine model of melanoma.

Metastatic melanoma is a very aggressive skin cancer. Platelets are constituents of the tumor microenvironment and, when activated, contribute to cancer progression, especially metastasis and inflammation. P2Y12 is an adenosine diphosphate receptor that triggers platelet activation. Inhibition of P2Y12 by clopidogrel bisulfate (CB) decreases platelet activation, which is also controlled by the extracellular concentration and the metabolism of purines by purinergic enzymes. We evaluated the effects of CB on the viability and proliferation of cultured B16-F10 cells. We also used a metastatic melanoma model with C57BL-6 mice to evaluate cancer development and purine metabolism modulation in platelets. B16-F10 cells were administered intraperitoneally to the mice. Two days later, the animals underwent a 12-day treatment with CB (30mg/kg by gavage). We have found that CB reduced cell viability and proliferation in B16-F10 culture in 72h at concentrations above 30µm. In vivo, CB decreased tumor nodule counts and lactate dehydrogenase levels and increased platelet purine metabolism. Our results showed that CB has significant effects on melanoma progression.

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 1β 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.

Hydrogel delivery of purinergic enzymes improves cardiac ischemia/reperfusion injury

RationaleThe innate immune response contributes to cardiac injury in myocardial ischemia/reperfusion (MI/R). Neutrophils are an important early part of the innate immune response to MI/R. Adenosine, an endogenous purine, is a known innate immune modulator and inhibitor of neutrophil activation. However, its delivery to the heart is limited by its short half-life (<30 s) and off-target side effects. CD39 and CD73 are anti-inflammatory homeostatic enzymes that can generate adenosine from phosphorylated adenosine substrate such as ATP released from injured tissue. ObjectiveWe hypothesize that hydrogel-delivered CD39 and CD73 target the local early innate immune response, reduce neutrophil activation, and preserve cardiac function in MI/R injury. Methods and resultsWe engineered a poly(ethylene) glycol (PEG) hydrogel loaded with the adenosine-generating enzymes CD39 and CD73. We incubated the hydrogels with neutrophils in vitro and showed a reduction in hydrogen peroxide production using Amplex Red. We demonstrated availability of substrate for the enzymes in the myocardium in MI/R by LC/MS, and tested release kinetics from the hydrogel. On echocardiography, global longitudinal strain (GLS) was preserved in MI/R hearts treated with the loaded hydrogel. Delivery of purinergic enzymes via this synthetic hydrogel resulted in lower innate immune infiltration into the myocardium post-MI/R, decreased markers of macrophage and neutrophil activation (NETosis), and decreased leukocyte-platelet complexes in circulation. ConclusionsIn a rat model of MI/R injury, CD39 and CD73 delivered via a hydrogel preserve cardiac function by modulating the innate immune response.

Epidermal melanocytes metabolize extracellular nucleotides by purinergic enzymes.

The human epidermal melanocyte (hEM) are melanin-producing cells that provide skin pigmentation and protection against ultraviolet radiation. Although purinergic signaling is involved in skin biology and pathology, the presence of NTPDase members, as well as the rate of nucleotides degradation by melanocytes were not described yet. Therefore, in this study, we analyzed the expression of ectonucleotidases in hEM derived from discarded foreskin of male patients. The expression of purinergic enzymes was confirmed by mRNA and flow cytometry. Among the ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1) and ecto-5´-nucleotidase were the ectoenzymes with higher expressions. The hydrolysis rate for ATP, ADP, and AMP was low in comparison to other primary cells already investigated. The amount of ATP in the culture medium was increased after a scratch wound and decreased to basal levels in 48 h, while the NTPDase1 and P2X7 expressions increased. Therefore, it is possible to suggest that after cell injury, the ATP released by hEM into the extracellular space will be hydrolyzed by ectonucleotidases as the NTPDase1 that will control the levels of nucleotides in the skin micro-environment.

Istradefylline modulates purinergic enzymes and reduces malignancy-associated factors in B16F10 melanoma cells.

ATP and adenosine exert pivotal roles in the development, maintenance, and metastatic spreading of melanoma. The action of such key melanoma tumor microenvironment (TME) constituents might be complementary or opposed, and their effects are not exclusive to immune cells but also to other host cells and tumor cells. The effects of ATP are controlled by the axis CD39/73, resulting in adenosine, the main actor in the TME, and A2A is the crucial mediator of its effects. We evaluated ATP and adenosine signaling through A2A on B16F10 melanoma cells using istradefylline (IST) (antiparkinsonian A2A antagonist) and caffeine (CAF) treatments after exposure to ATP and adenosine. Adenosine increased melanoma cell viability and proliferation in a concentration-dependent manner. ATP increases viability only as a substrate by CD39 to produce adenosine. Both IST and CAF are toxic to B16F10 cells, but only IST potentialized paclitaxel-induced cytotoxic effects, even decreasing its IC50 value. IST positively modulated CD39 and CD73 expression. CD39 activity was increased, and E-ADA was reduced, indicating that the melanoma cells promoted compensatory feedback in the production and maintenance of adenosine levels. A2A antagonism by IST reduced the factors associated with malignancy, like migration, adhesion, colony formation, and the capacity to produce melanin. Moreover, IST significantly increases nitric oxide (NO) production, which correlates to a decline in melanoma cell viability by apoptotic events. Altogether, our results suggest that adenosine signaling through A2A is essential for B16F10 cells, and its inhibition by IST causes compensatory purinergic enzymatic modulations. Furthermore, IST is a promising therapy that provides new ways to improve current melanoma treatments.

CHARACTERIZATION OF DIFFERENTIAL EXPRESSION PATTERNS OF THE EXTRACELLULAR PURINERGIC ENZYMES IN COLORECTAL CANCER

The aim of this study is to characterize tumor cell specific expression of purinergic ecto-enzymes CD39 and CD73, and to associate prognostic significance of these expression patterns in colorectal cancer (CRC) patients. Protein and gene expression of the target genes in various CRC cell lines were assessed via Western Blot (WB) analysis and Real Time PCR (RT-PCR). Additionally, tumor vs stromal cell expression of the target genes was analyzed from publicly available patient expression datasets. Finally, the correlation between CD39 and CD73 expression with patient prognosis was analyzed via The Cancer Genome Atlas (TCGA) datasets. In CRC cell lines, CD39 was found to be not expressed at all while CD73 was expressed extensively in most cell lines via WB and RT-PCR analyses. Patient microarray expression data confirmed the results from CRC cell lines that CD39 expression was very low in epithelial/tumor cells relative to other stromal cell types yet CD73 was expressed abundantly in every cell type within patient tumor samples. Interestingly, CD39 expression in patient tumors was correlated with favorable prognosis while CD73 expression was associated with worse prognosis. Although CD39 and CD73 are related enzymes involved in extracellular purinergic signaling, their expression patterns in tumor cells and prognostic effects in patients show opposing outcomes. Therefore, better insights in understanding the functional involvement of purinergic ecto-enzymes in colorectal tumor development is needed via further mechanistic studies.

Insights of ethyl acetate fraction from Vassobia breviflora in multidrug-resistant bacteria and cancer cells: from biological to therapeutic

ABSTRACT Cancer and infectious diseases are among the leading causes of death in the world. Despite the diverse array of treatments available, challenges posed by resistance, side effects, high costs, and inaccessibility persist. In the Solanaceae plant family, few studies with Vassobia breviflora species relating to biological activity are known, but promising results have emerged. The phytochemicals present in the ethyl acetate fraction were obtained using ESI-MS-QTOF, and the antioxidants assays 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical capture (ABTS), plasma ferric reduction capacity (FRAP), and total antioxidant capacity (TAC). Cytotoxic activity was evaluated by MTT, Neutral Red, and lactate dehydrogenase (LDH) released. The production of reactive oxygen species, nitric oxide, and purinergic enzymes was also investigated. Antibacterial activity was measured through minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and antibiofilm activity, in addition to genotoxicity in plasmid DNA. Five major masses were identified D-glucopyranose II, allyl disulfide, γ-lactones, pharbilignoside, and one mass was not identified. V. breviflora exhibited relevant antioxidant and cytotoxic activity against the HeLa cell line and enhanced expression effect in modulation of purinergic signaling. Antibacterial activities in the assays in 7 ATCC strains and 8 multidrug-resistant clinical isolates were found. V. breviflora blocked biofilm formation in producing bacteria at the highest concentrations tested. However, there was no plasmid DNA cleavage at the concentrations tested. Data demonstrated that V. breviflora exhibited an antioxidant effect through several methods and proved to be a promising therapeutic alternative for use against tumor cells via purinergic signaling and multidrug-resistant microorganisms, presenting an anti-biofilm effect.

Impact of gallic acid on tumor suppression: Modulation of redox homeostasis and purinergic response in in vitro and a preclinical glioblastoma model

Glioblastoma (GBM) is the deadliest primary brain tumor in adults due to the high rate of relapse with current treatment. Therefore, the search for therapeutic alternatives is urgent. Gallic acid (GA), a potent natural antioxidant, has antitumor and modulatory actions on purinergic signaling. In this study, we investigated the cytotoxic effects of GA on the rat GBM (C6) cell line and on astrocyte culture and analyzed its role in regulating oxidative stress and purinergic enzymes involved in GBM proliferation. Cells were exposed to GA from 50 to 400 µM for 24 and/or 48 h. Next, the effect of GA was evaluated in the preclinical model of GBM. Wistar rats were treated with 50 or 100 mg/kg of GA for 15 days, and cerebral and systemic redox status and degradation of adenine nucleotides and nucleosides in circulating platelets, lymphocytes, and serum were evaluated. Our results demonstrated that GA has selective anti-glioma activity in vitro, without inducing cytotoxicity in astrocyte. Furthermore, GA prevented oxidative stress and changes in the hydrolysis of nucleotides in GBM cells. The anti-glioma effect was also observed in vivo, as GA reduced tumor volume by 90%. Interestingly, GA decreased the oxidative damage induced by a tumor in the brain, serum, and platelets, and, also prevented changes in the degradation of nucleotides and nucleosides in lymphocytes, platelets, and serum. These results indicate, for the first time, the therapeutic potential of GA in a preclinical model of GBM, whose effects may be related to its role in redox and purinergic modulation.

Composite biscuits from sandpaper and acha flour restore the altered activity of arginase, cholinergic, and purinergic enzymes in hypertensive-diabetic rats.

Hypertension is one of the common co-morbidities in diabetes. Thus, the present study sought to study the effects of composite biscuits from the mixture of acha (Digitaria exilis) and sandpaper (Fiscus exasperata) leaf flours (ASLF) on mean arterial blood pressure (MABP), arginase, cholinergic, purinergic enzymatic cascade, and nitric oxide (NO) levels as well as oxidative status in streptozotocin (STZ)/L-NG -nitro arginine methyl ester (L-NAME)-induced hypertensive/diabetic rats. Experimental rats were distributed randomly into 7 groups (n=5). Group I-III rats were placed on the basal diet; IV-VII rats were placed on composite biscuits designated as A, B, C, and D respectively for 14 days. On the 13th day, the MABP of the experimental rats was monitored and recorded. Thereafter, the rats were sacrificed, tissues of interest were harvested, and homogenized. Subsequently, the activity of arginase cholinesterase and purinergic enzymes, as well as NO levels were evaluated in the experimental rats. However, hypertensive/diabetic rats placed on the formulated diet exhibited reduced MABP when compared with the untreated hypertensive/diabetic rats. Also, altered activity of arginase, cholinergic and purinergic were restored in diet-treated hypertensive/diabetic rats when compared with hypertensive/diabetic rats. Similarly, the NO level and antioxidant status of the treated hypertensive/diabetic rats were notably enhanced when compared with hypertensive/diabetic rats. It could be inferred that composite biscuits exhibited an ameliorative effect in hypertensive/diabetic states via their reductive effect on the MABP, arginase, cholinesterase, and purinergic enzymes and enhanced NO levels in hypertensive/diabetic rats. Meanwhile, the biscuit designated as D had seems better when their effects were compared holistically. PRACTICAL APPLICATIONS: Acha grains and sandpaper leaf have been used in the folklore for disease treatment. However, the production of composite biscuits from these naturally available recipes for the management of hypertensive diabetics proved therapeutic since their effect on hypertensive diabetic rats is positive. Therefore, the composite biscuit will offer nutraceutical benefits to both healthy and disease individuals.

Syzygium aromaticum (L.) Merr. & L.M.Perry mitigates iron-mediated oxidative brain injury via in vitro, ex vivo, and in silico approaches

Clove (Syzygium aromaticum) has been identified as a spice high in phytochemicals that could be exploited in drug development. The purpose of this study is to investigate the neuroprotective effect of S. aromaticum on Fe2+-mediated oxidative brain damage via in vitro, ex vivo, and in silico studies. We assessed the in vitro antioxidant activities of aqueous S. aromaticum (ASAE) using standard procedures. Oxidative damage was induced in rat brain tissues by incubating them with FeSO4 and treating them with different concentrations of ASAE. Biochemical parameters like CAT, SOD, GSH, MDA, and NO levels, cholinergic and purinergic enzyme activity, glucose-6-phosphatase (G6Pase), and fructose-1,6-bisphosphatase (F-1,6-BPase) were all measured in the brain tissues. Inducing brain tissue damage reduced GSH levels, SOD, CAT, and ENTPDase activity while increasing MDA and NO levels, ATPase, G6Pase, and F-1,6-BPase activity (p < .05). These levels and activities returned to near-normal levels after ASAE treatment (p < .05). In a molecular docking investigation, the two leading phytochemicals for AChE were ellagic acid and stigmasterol, with ellagic acid having a greater binding affinity for the BChE enzyme. Stigmasterol also had more binding energy than the BACE-1 protein. In addition, cholesterol had the highest binding energy to MAO-B and BACE-1. Rhamnetin exhibited strong binding to both BChE and the Na+/K+ ATPase. The analysis of the trajectories of led phytochemicals complexed to respective proteins in comparison with the unbound proteins revealed high degree of stability in 100 ns molecular dynamics. These data imply that ASAE can protect against oxidative brain damage by preventing oxidative stress, modifying cholinergic and purinergic activity, and controlling gluconeogenesis.

Neurotoxicity of anthracene and benz[a]anthracene involves oxidative stress-induced neuronal damage, cholinergic dysfunction and disruption of monoaminergic and purinergic enzymes.

In this study, the modulatory effects of anthracene (ANT) and benz[a]anthracene (BEN) on biochemical markers associated with neurodegeneration were assessed in mouse hippocampal neuronal cells (HT-22). Neuronal cells were cultured and exposed to ANT and BEN (25-125µM) for 5days, and the cell viability was determined via MTT assay. Morphological characteristics of the cells were assessed using a compound microscope. Biochemical parameters such as acetylcholinesterase (AChE), monoamine oxidase (MAO) and adenosine deaminase (ADA) activities as well as oxidative stress biomarkers (catalase [CAT], glutathione -S- transferase [GST] activities and Glutathione [GSH] levels) and nitric oxide [NO] levels were assessed after cells were treated with ANT and BEN for two days. The results showed that cell viability reduced with an increase in exposure time. After the fifth day of treatment, BEN and ANT (125µM) reduced percentage viability to 41 and 38.1%, respectively. Light micrographs showed shrinkage of cells, neuronal injury and cell death in cells treated with higher concentrations of BEN and ANT (50 and 125µM). Furthermore, AChE and MAO activities reduced significantly after treatment for 48h with ANT and BEN. A significant decrease in CAT and GST activities and low GSH levels were observed after treatment with BEN and ANT. However, both polycyclic aromatic hydrocarbons caused a significant increase in ADA activity and NO levels. These results suggest that ANT and BEN may induce neurodegeneration in neuronal cells via oxidative stress-induced-neuronal injury, disruption of cholinergic, monoaminergic and purinergic transmission, and increased nitric oxide levels.