Pedro H. Doleski

Free and nanoencapsulated curcumin prevents cigarette smoke-induced cognitive impairment and redox imbalance.

Cigarette smoke-exposure promotes neurobiological changes associated with neurocognitive abnormalities. Curcumin, a natural polyphenol, have shown to be able to prevent cigarette smoke-induced cognitive impairment. Here, we investigated possible mechanisms involved in curcumin protection against cigarette smoke-induced cognitive impairment and, due to its poor bioavailability, we investigated the potential of using curcumin-loaded lipid-core nanocapsules (C-LNC) suspension. Rats were treated with curcumin and cigarette smoke, once a day, 5 days each week, for 30 days. Animals were divided into ten groups: I, control (vehicle/corn oil); II, curcumin 12.5mg/kg; III, curcumin 25mg/kg; IV, curcumin 50mg/kg; V, C-LNC 4 mg/kg; VI, tobacco exposed; VII, curcumin 12.5mg/kg along with tobacco exposure; VIII, curcumin 25mg/kg along with tobacco exposure; IX, curcumin 50mg/kg along with tobacco exposure; X, C-LNC 4 mg/kg along with tobacco exposure. Cigarette smoke-exposure impaired object recognition memory (P<0.001), indicated by the low recognition index, increased biomarkers of oxidative/nitrosative stress such as TBARS (P<0.05) and NOx (P<0.01), decreased antioxidant defenses such as NPSH content (P<0.01) and SOD activity (P<0.01) and inhibited the activities of enzymes involved in ion homeostasis such as Na(+),K(+)-ATPase and Ca(2+)-ATPase. Both curcumin formulations (free and nanoencapsulated) prevented the memory impairment, the redox imbalance and the alterations observed in the ATPases activities. Maintenance of ion homeostasis and redox balance is involved in the protective mechanism of curcumin against tobacco-induced cognitive impairment. Our results suggest that curcumin is a potential therapeutic agent for neurocognition and that C-LNC may be an alternative to its poor bioavailability.

Memory deficit, toxic effects and activity of Na+, K+-ATPase and NTPDase in brain of Wistar rats submitted to orally treatment with alpha-terpinene

The neurotoxic effects and activity of Na(+), K(+)-ATPase and NTPDase in Wistar rats after treatment with α-terpinene (daily oral administration of 0.5, 0.75 and 1.0mLkg(-1) for 10days) were examined. Results of the inhibitory avoidance task showed a memory deficit (p<0.05) in rats treated with all doses of α-terpinene. The evaluation of DNA damage in brain tissue revealed an increase (p<0.05) on frequency of damage and damage index in all concentrations. According to the cytotoxicity assay, doses of 0.5, 0.75 and 1.0mLkg(-1) increase the lactate dehydrogenase levels, and doses of 1.0mLkg(-1) also decrease (p<0.05) cell viability in brain cells. A decrease (p<0.05) on Na(+), K(+)-ATPase activity in brain tissue and on NTPDase activity in serum were observed in all concentrations of α-terpinene. These results suggest that the α-terpinene was cytotoxic and genotoxic to the brain cells by inducing loss of cell viability and DNA damage, as well as causing alterations in Na(+), K(+)-ATPase and NTPDase activity, what may contribute to the memory deficit of treated animals. Thus, α-terpinene cannot be consumed by the population at the doses studied.

Characterization of E‐NTPDase (EC 3.6.1.5) activity in hepatic lymphocytes: A different activity profile from peripheral lymphocytes

The activity of ectonucleoside triphosphate diphosphohydrolase (E‐NTPDase; EC 3.6.1.5) was characterized in hepatic lymphocytes (HL) of rats. For this purpose, a specific method for the isolation of lymphocytes from hepatic tissue was developed. Subsequently, E‐NTPDase activity of rat HL was compared with that of rat peripheral lymphocytes. The HL showed high cell count and viability. Also, the characterization test revealed that the optimal E‐NTPDase activities were attained at 37°C and pH 8.0 in the presence of Ca2+. In addition, in the presence of specific E‐NTPDase inhibitors (20mM sodium azide and 0.3mM suramin), there were significant inhibitions in nucleotide hydrolysis. However, there was no significant change in adenosine triphosphate (ATP) or adenosine diphosphate (ADP) hydrolysis in the presence of inhibitors of other E‐ATPase (0.1mM Ouabain, 0.5mM orthovanadate, and 1mM, 5mM, and 10mM sodium azide). Furthermore, the kinetic behavior of the enzyme in HL showed apparent Km of 134.90 ± 0.03μM and 214.40 ± 0.06μM as well as Vmax of 345.0 ± 28.32 and 242.0 ± 27.55 ƞmol Pi/min/mg of protein for ATP and ADP, respectively. The Chevillard plot revealed that ATP and ADP were hydrolyzed at the same active site of the enzyme. Our results suggest that the degradation of extracellular nucleotides in HL may have been primarily accomplished by E‐NTPDase. The higher E‐NTPDase activity observed in HL may be attributed to the important physiological functions of ATP and ADP in HL.

Hepatic and seric levels of purines in rats experimentally infected by Fasciola hepatica

The aim of this study was to evaluate hepatic and seric levels of purines, as well as their breakdown products in rats infected by Fasciola hepatica on days 15 and 87 post-infection (PI). Rats were divided into two groups: uninfected (n = 10) and infected (n = 20). On day 15 (n = 5 for uninfected group and n = 10 for infected group) and 87 PI (n = 5 for uninfected group and n = 10 for infected group), animals were euthanized for sampling to evaluate levels of purines by high-performance liquid chromatography. In serum, ATP increased (P < 0.05) and ADP decreased (P < 0.05) on days 15 and 87 PI, while AMP increased (P < 0.05) only on day 15 PI. Hypoxanthine levels increased (P < 0.05) on days 15 and 87 PI, while adenosine and xanthine levels decreased and increased (P < 0.05), respectively, on day 87 PI. No difference was observed regarding seric inosine and uric acid (P > 0.05). Hepatic ATP, adenosine, and uric acid levels decreased (P < 0.05) on days 15 and 87 PI. AMP levels decreased (P < 0.05) on day 87 PI, while xanthine levels increased (P < 0.05) on day 15 PI in the liver. Also in the liver, hypoxanthine levels increased (P < 0.05) on day 15 PI and decreased (P < 0.05) on day 87 PI. On the other hand, there was no difference on hepatic ADP and inosine levels (P > 0.05). Therefore, it is possible to conclude that F. hepatica infection can change purine levels, which may be associated with an inflammatory process, and these alterations may influence fasciolosis pathogenesis.

Effect of Uncaria tomentosa extract on purinergic enzyme activities in lymphocytes of rats submitted to experimental adjuvant arthritis model.

BackgroundConsidering that adjuvant arthritis is an experimental model of arthritis widely used for preclinical testing of numerous anti-arthritic agents, which were taken by a large number of patients worldwide, it is of great interest to investigate the therapeutic action of compounds with anti-inflammatory properties, such as Uncaria tomentosa extract. Moreover, there are no studies demonstrating the effect of U. tomentosa on the metabolism of adenine nucleotides published so far. Thus, the purpose of the present study is to investigate the effects of U. tomentosa extract on E-NTPDase and E-ADA activities in lymphocytes of Complete Freund’s Adjuvant (CFA) arthritis induced rats.MethodsTo evaluate the effect of U. tomentosa extract on the activity of E-NTPDase and ADA in lymphocytes, the rats were submitted to an experimental adjuvant arthritis model. Peripheral lymphocytes were isolated and E-NTPDase and E-ADA activities were determined. Data were analyzed by a one- or two-way ANOVA. Post hoc analyses were carried out by the Student-Newman-Keuls (SNK) Multiple Comparison Test.ResultsE-NTPDase activity was increased in arthritic untreated. Arthritic rats which received U. tomentosa extract, presented similar results to the control group. However, results obtained for adenosine hydrolysis by E-ADA were not altered in arthritic rats. U. tomentosa extract did not alter E-NTPDase and E-ADA activity in healthy animals.ConclusionsThe present investigation supports the hypothesis that the increased E-NTPDase activity verified in arthritic rats might be an attempt to maintain basal levels of ATP and ADP in the extracellular medium, since the arthritis induction causes tissue damage and, consequently, large amounts of ATP are released into this milieu. Also, it highlights the possibility to use U. tomentosa extract as an adjuvant to treat arthritis.

Purinergic ecto-enzymes participate in the thromboregulation in acute in mice infection by Trypanosoma cruzi

Coagulation disorders have been described in Chagas disease with thrombocytopenia as an important event. Several mechanisms may be related to this pathogenesis, such as enzymes of the purinergic system, purine, and receptors involved in the regulation and modulation of physiological events related to hemostasis. Therefore, the aim of this study was to evaluate the activities of E-NTPDase, E-5′nucleotidase, and ecto-adenosine deaminase (E-ADA) in platelets of mice experimentally infected by Trypanosoma cruzi. Twelve female mice were used, divided into two groups (n = 6): uninfected and infected. Mice of infected group were intraperitoneally inoculated with 104 trypomastigotes of T. cruzi (strain Y). On day 12 post-infection (PI), blood samples were collected for quantitation and separation of platelets. A significant reduction in the number of platelets of infected mice (P < 0.05) was observed. The activities of E-NTPDase (ATP and ADP substrates), E-5′nucleotidase, and E-ADA in platelets increased significantly (P < 0.05) in mice infected by T. cruzi compared with uninfected animals. A negative correlation (P < 0.01)was observed between the number of platelets and ATP hydrolysis (r = −0.64), and ADP hydrolysis (r = −0.69) by E-NTPDase. Therefore, there is a response from the purinergic system activating ecto-enzymes in platelets of mice T. cruzi infected, as a compensatory effect of thrombocytopenia.

Peripheral blood mononuclear cells from rat model of pleurisy: The effects of hesperidin on ectoenzymes activity, apoptosis, cell cycle and reactive oxygen species production

The present study investigates the effect of hesperidin; a flavonone commonly found in citrus fruits, on the ectoenzymes (ectonucleotidase and ecto-adenosine deaminase) activity, cell viability, apoptosis, cell cycle arrest and reactive oxygen species production in peripheral blood mononuclear cells (PBMCs) from rat model of pleurisy. Wistar rats were pretreated with either saline or hesperidin (80mg/kg) by oral gavage for 21days and injected intrapleurally with 2% carrageenan or saline on the 22nd day. PBMCs were subsequently prepared after 4h of carrageenan induction. The results revealed that hesperidin may exhibit its anti-inflammatory effects through possible modulation of ectonucleotidase (E-NTPDase) and ecto-adenosine deaminase (E-ADA) activities, reduction of intracellular reactive oxygen species, prevention of DNA damage and modulation of apoptosis as well as activation of cell cycle arrest. This study suggests some possible underlying anti-inflammatory mechanisms of hesperidin on PBMCs in acute inflammatory condition. Furthermore, hesperidin may minimize oxidative injury mediated pleurisy in rat.

Enzymes that hydrolyze adenine nucleotides in a model of hypercholesterolemia induced by Triton WR-1339: protective effects of β-caryophyllene

Purinergic system has been proven to play a critical role in the inflammatory process and to represent an important therapeutic target to improve the immune response during hypercholesterolemia. β-caryophyllene, a phytocannabinoid compound, has a powerful hypolipidemic and anti-inflammatory actions. However, the effects of β-caryophyllene on seric enzymes of purinergic system have not been evaluated. The purpose of this study was to investigate whether β-caryophyllene is able to ameliorate the seric activities of NTPDase and adenosine deaminase (ADA) in a model of hypercholesterolemia induced by Triton WR-1339. The activities of NTPDase and ADA were evaluated enzymatically, and the seric levels of β-caryophyllene were evaluated by high-performance liquid chromatography. We found that treatment with β-caryophyllene ameliorates the enzymatic activities of NTPDase and ADA in serum of hypercholesterolemic rats, in a concentration-dependent manner. These results indicated that β-caryophyllene treatment could improve the immune response during hypercholesterolemia through purinergic pathway.

Metaphylactic effect of minerals on immunological and antioxidant responses, weight gain and minimization of coccidiosis of newborn lambs

The aim of this study was to evaluate the metaphylactic effect of minerals on immunological and antioxidant responses, as well as performance and prevention of coccidiosis in newborn lambs. We divided 110 newborn lambs into two groups (55/group): control (untreated) and treated with two doses of 0.33 mL/kg of a mineral complex (zinc, copper, selenium, and manganese) on day of life (DOL) 1 and 30. Total blood was collected at DOL 1, 15, 30 and 45 to measure antioxidant enzymes, biochemical and immunology analyses, and haemogram. Treated animals were heavier (P < .05) than untreated lambs on DOL 15 and 45, but not on DOL 30 due to a coccidiosis outbreak. Catalase activity did not differ between groups, while superoxide dismutase and xanthine oxidase activities were higher (P < .05) in treated lambs compared with control animals. Serum levels of total protein and globulins were higher (P < .05) in treated animals (DOL 15, 30 and 45). A significant increased on the number of lymphocytes (DOL 45), as well as on seric levels of immunoglobulins (IgM and IgG) was observed in treated animals (DOL 15 and 30). Serum Ig levels remained constant throughout the experiment in the treated group, but fluctuated in the control group. Serum glucose levels were greater in treated animals (DOL 15 and 30). It is possible to conclude that subcutaneous administration of minerals has beneficial effects on lambs by increasing antioxidant and immunological defenses, reflected by greater weight gain, which could mitigate the impact of coccidiosis.

Xanthine oxidase activity exerts pro-oxidative and pro-inflammatory effects in serum of silver catfish fed with a diet contaminated with aflatoxin B1

Several studies have associated the involvement of xanthine oxidase (XO) activity, a source of uric acid and reactive oxygen species (ROS), to pro-oxidative and pro-inflammatory effects during pathological conditions. Considering this, the aim of this study was to evaluate whether upregulation on seric XO activity may be a pathway involved in the oxidative stress in fish exposed to a diet contaminated with aflatoxin B1 (AFB1 ), as well as whether it may be considered a pathway involved in ROS and NOx production. Xanthine oxidase activity, as well as the uric acid, ROS and NOx levels increased in serum of fish fed with a AFB1 -contaminated diet on days 14 and 21 post-feeding compared to fish fed with a basal diet. Based on these evidences, upregulation of seric XO activity induces pro-oxidant and pro-inflammatory profiles in serum of fish fed with a AFB1 -contaminated diet due to excessive formation on uric acid. Also, the excessive uric acid induces the release of pro-oxidant and pro-inflammatory mediators, as ROS and NOx, also contributing to oxidative and inflammatory profiles. In summary, the upregulation on seric XO activity may be considered a pathway involved in the oxidative stress of fish exposed to a diet contaminated with AFB1 .