Effects of coconut oil long-term supplementation in Wistar rats during metabolic syndrome - regulation of metabolic conditions involving glucose homeostasis, inflammatory signals, and oxidative stress.

This study was designed to evaluate the long-term effects of Fructose (20%) feeding in rats, simulating metabolic syndrome (MetS), and the effects of coconut oil (C.O.) supplementation when administered in a MetS context. MetS is a cluster of systemic conditions that represent an increased chance of developing cardiovascular diseases and type 2 diabetes in the future. C.O. has been the target of media speculation, and recent studies report inconsistent results. C.O. improved glucose homeostasis and reduced fat accumulation in Fructose-fed rats while decreasing the levels of triglycerides (TGs) in the liver. C.O. supplementation also increased TGs levels and fructosamine in serum during MetS, possibly due to white adipose tissue breakdown and high fructose feeding. Pro-inflammatory cytokines IL-1ß and TNF-α were also increased in rats treated with Fructose and C.O. Oxidative stress marker nitrotyrosine is increased in fructose-fed animals, and C.O. treatment did not prevent this damage. No significant changes were observed in lipoperoxidation marker 4-Hydroxynonenal; however, fructose feeding increased total conjugated dienes and caused conjugated dienes to switch their conformation from cis-trans to trans-trans, which was not prevented by C.O. treatment. Potential benefits of C.O. have been reported with inconsistent results, and indeed we observed some benefits of C.O. supplementation in aiding weight loss, fat accumulation, and improving glucose homeostasis. Nonetheless, we also demonstrated that long-term C.O. supplementation could present some problematic effects with higher risk for individuals suffering MetS, including increased TGs and fructosamine levels and conformational changes in dienes.

Hyperoxia by short-term promotes oxidative damage and mitochondrial dysfunction in rat brain.

Oxygen (O2) therapy is used as a therapeutic protocol to prevent or treat hypoxia. However, a high inspired fraction of O2 (FIO2) promotes hyperoxia, a harmful condition for the central nervous system (CNS). The present study evaluated parameters of oxidative stress and mitochondrial dysfunction in the brain of rats exposed to different FIO2. Male Wistar rats were exposed to hyperoxia (FIO2 40 % and 60 %) compared to the control group (FIO2 21 %) for 2 h. Oxidative stress, neutrophilic infiltration, and mitochondrial respiratory chain enzymes were determined in the hippocampus, striatum, cerebellum, cortex, and prefrontal cortex after O2 exposure. The animals exposed to hyperoxia showed increased lipid peroxidation, formation of carbonyl proteins, N/N concentration, and neutrophilic infiltration in some brain regions, like hippocampus, striatum, and cerebellum being the most affected. Furthermore, CAT activity and activity of mitochondrial enzyme complexes were also altered after exposure to hyperoxia. Rats exposed to hyperoxia showed increase in oxidative stress parameters and mitochondrial dysfunction in brain structures.

Treatment with isolated gold nanoparticles reverses brain damage caused by obesity.

In this study, we performed two experiments. In the first experiment, the objective was to link gold nanoparticles (GNPs) with sodium diclofenac and/or soy lecithin and to determine their concentration in tissues and their toxicity using hepatic and renal analyzes in mice to evaluate their safety as therapeutic agents in the subsequent treatment of obesity. In the second experiment, we evaluated the effect of GNPs on inflammatory and biochemical parameters in obese mice. In the first experiment, we synthesized and characterized 18 nm GNPs that were administered intraperitoneally in isolation or in association with sodium diclofenac and/or soy lecithin in mice once daily for 1 or 14 days. Twenty-four hours after the single or final administration, the animals were euthanized, following which the tissues were removed for evaluating the concentration of GNPs, and serum samples were collected for hepatic and renal analysis. Hepatic damage was evaluated based on the levels of alanine aminotransferase (ALT), whereas renal damage was evaluated based on creatinine levels. A higher concentration of GNPs was detected in the tissues upon administration for 14 days, and there were no signs of hepatic or renal damage. In the second experiment, the mice were used as animal models of obesity and were fed a high-fat diet (obese group) and control diet (control group). After eight weeks of high-fat diet administration, the mice were treated with saline or with GNPs (average size of 18 nm) at a concentration of 70 mg/L (70 mg/kg) once a day, for 14 days, for 10 weeks. Body weight and food intake were measured frequently. After the experiment ended, the animals were euthanized, serum samples were collected for glucose and lipid profile analysis, the mesenteric fat content was weighed, and the brains were removed for inflammatory and biochemical analysis. In obese mice, although GNP administration did not reduce body and mesenteric fat weight, it reduced food intake. The glucose levels were reversed upon administration of GNPs, whereas the lipid profile was not altered in any of the groups. GNPs exerted a beneficial effect on inflammation and oxidative stress parameters, without reverting mitochondrial dysfunction. Our results indicate that the intraperitoneal administration of GNPs for 14 days results in a significant GNP concentration in adipose tissues, which could be an interesting finding for the treatment of inflammation associated with obesity. Based on the efficacy of GNPs in reducing dietary intake, inflammation, and oxidative stress, they can be considered potential alternative agents for the treatment of obesity.

Stanniocalcin 1 Inhibits the Inflammatory Response in Microglia and Protects Against Sepsis-Associated Encephalopathy.

Sepsis-associated encephalopathy is a serious consequence of sepsis, triggered by the host response against an infectious agent, that can lead to brain damage and cognitive impairment. Several mechanisms have been proposed in this bidirectional communication between the immune system and the brain after sepsis as neuroinflammation, oxidative stress, and mitochondrial dysfunction. Stanniocalcin-1 (STC-1), an endogen neuroprotective protein, acts as an anti-inflammatory and suppresses superoxide generation through induction of uncoupling proteins (UCPs) in the mitochondria. Here, we demonstrated a protective role of STC-1 on inflammatory responses in vitro, in activated microglia stimulated with LPS, and on neuroinflammation, oxidative stress, and mitochondrial function in the hippocampus of rats subjected to an animal model of sepsis by cecal ligation and puncture (CLP), as well the consequences on long-term memory. Recombinant human STC-1 (rhSTC1) suppressed the pro-inflammatory cytokine production in LPS-stimulated microglia without changing the UCP-2 expression. Besides, rhSTC1 injected into the cisterna magna decreased acute hippocampal inflammation and oxidative stress and increased the activity of complex I and II activity of mitochondrial respiratory chain and creatine kinase at 24 h after sepsis. rhSTC1 was effective in preventing long-term cognitive impairment after CLP. In conclusion, rhSTC1 confers significant neuroprotection by inhibiting the inflammatory response in microglia and protecting against sepsis-associated encephalopathy in rats.

Quality control in veterinary blood banks: evaluation of canine platelet concentrates stored for five days.

BACKGROUND: Platelets undergo structural, biochemical and functional alterations when stored, and platelet storage lesions reduce platelet function and half-life after transfusion. The objective of this study was to evaluate stored canine platelet concentrates with platelet aggregation, flow cytometry and biochemistry assays. Twenty-two bags of canine platelet concentrates were obtained by the platelet-rich plasma method and were assessed on days 1, 3 and 5 after collection. Parameters such as platelet counts, residual leukocytes, platelet swirling, glucose, lactate, pH, CD62P expression (platelet activation), JC-1 (mitochondrial function) and annexin V (apoptosis and cell death) were assessed. RESULTS: Over the five days of storage there was a significant decrease in glucose, HCO3, pCO2, ATP, pH, swirling and mitochondrial function, associated with a significant increase in lactate levels and pO2. At the end of storage pH was 5.9 ± 0.6 and lactate levels were 2.8 ± 1.2 mmol/L. Results of the quality parameters evaluated were similar to those reported in human platelets studies. The deleterious effects of storage were more pronounced in bags with higher platelet counts (> 7.49 × 1010/unit), suggesting that canine platelet concentrates should not contain an excessive number of platelets. CONCLUSIONS: Quality parameters of canine platelets under standard storage conditions were similar to those observed in human platelets. Our results have potential to be used for the routine evaluation and quality control in veterinary blood banks.

Stanniocalcin-1 ameliorates cerebral ischemia by decrease oxidative stress and blood brain barrier permeability.

Blood brain barrier (BBB) permeability and oxidative stress have been reported to be important mechanisms for brain damage following ischemic stroke and stanniocalcin-1 (STC-1), a neuroprotective protein, has anti-inflammatory and anti-oxidative stress properties. Herein, we report the effect of STC-1 on BBB permeability and brain oxidative stress after stroke in an animal model. Male Wistar received an intracerebroventricularly injection of human recombinant STC-1 (100 ng/kg) or saline and were subjected to sham procedure or global cerebral ischemia/reperfusion (I/R) model. Six and 24 h after I/R, neurological evaluation was performed; at 24 h brain water content was evaluated in the total brain, and BBB permeability, nitrite/nitrate (N/N) concentration, lipid peroxidation, protein carbonyls formation, superoxide dismutase (SOD) and catalase (CAT) activity were determined in the hippocampus, cortex, prefrontal cortex, striatum and cerebellum. Rats exhibited neurological deficit at 6 and 24 h after I/R and STC-1 reduction at 24 h. After I/R there were an increase of brain water content, BBB permeability in the hippocampus, cortex and pre-frontal cortex and N/N in the hippocampus, and STC-1 decreased this level only in the hippocampus. STC-1 decreased lipid peroxidation in the hippocampus, cortex and prefrontal cortex and protein oxidative damage in the hippocampus and cortex. SOD activity decreased in the hippocampus, cortex and prefrontal cortex after I/R and STC-1 reestablished these levels in the hippocampus and cortex. CAT activity decreased only in the hippocampus and cortex and STC-1 increased the CAT activity in the hippocampus. Our data provide the first experimental demonstration that STC-1 reduced brain dysfunction associated with cerebral I/R in rats, by decreasing BBB permeability and oxidative stress parameters.

Comparison of JC-1 and MitoTracker probes for mitochondrial viability assessment in stored canine platelet concentrates: A flow cytometry study.

Mitochondria perform crucial roles in many biochemical processes, and mitochondrial depolarization is an early sign of platelet apoptosis. The mitochondrial membrane potential is usually evaluated through JC-1 probe, but it can also be assessed with MitoTracker probes. Our aim was to evaluate mitochondrial viability in stored canine platelet concentrates (PCs) with the fluorescent probes JC-1 and MitoTracker. Platelets from 22 canine PCs were stained with JC-1 and MitoTracker probes on days 1, 3, and 5 of storage. Data on metabolic parameters were also collected for correlation studies. Results of JC-1 and MitoTracker revealed a decrease in mitochondrial membrane potential in day 5 of storage compared to days 1 and 3, providing evidence of mitochondrial depolarization, a finding that was confirmed by the data on metabolic parameters. MitoTracker probes also added information regarding platelet swelling. In conclusion, MitoTracker probes offered a more complete mitochondrial analysis in the evaluation of stored canine PCs. © 2018 International Society for Advancement of Cytometry.

In Vitro Adult Astrocytes are Derived From Mature Cells and Reproduce in Vivo Redox Profile.

Astrocytes are versatile cells involved in synaptic information processing, energy metabolism, redox homeostasis, inflammatory response, and structural support of the brain. Recently, we established a routine protocol of cultured astrocytes derived from adult and aged Wistar rats, which present several different responses compared to newborn astrocytes, commonly used to characterize the role of the astrocytes in the central nervous system. Previous studies hypothesized that astrocyte cultures prepared from adult animals derive from immature precursors present in the adult tissue throughout life. Since our group has already demonstrated that the glial functionality of adult astrocytes differs from newborn cultures, the aim of this study was to confirm that our in vitro astrocytes were derived from mature cells. Therefore, we evaluated cytoskeleton proteins, such as glial fibrillary acidic protein and vimentin, as well as Sox10, an essential marker of immature glial cells, in ex vivo tissue and in in vitro astrocytes from the same animals (1, 90, and 180 days old). In addition, we examined the mitochondrial functionality and the cellular redox homeostasis. Our results suggest that adult and aged astrocytes are derived from mature cells and that changes in mitochondrial parameters in ex vivo tissue were reproduced in in vitro astrocytes. J. Cell. Biochem. 118: 3111-3118, 2017. © 2017 Wiley Periodicals, Inc.

Autophagy induced by purple pitanga (Eugenia uniflora L.) extract triggered a cooperative effect on inducing the hepatic stellate cell death.

Activated hepatic stellate cells (HSC) are the major source of collagen I in liver fibrosis. Eugenia uniflora L. is a tree species that is widely distributed in South America. E. uniflora L. fruit-popularly known as pitanga-has been shown to exert beneficial properties. Autophagy contributes to the maintenance of cellular homeostasis and survival under stress situation, but it has also been suggested to be an alternative cell death pathway. Mitochondria play a pivotal role on signaling cell death. Mitophagy of damaged mitochondria is an important cell defense mechanism against organelle-mediated cell death signaling. We previously found that purple pitanga extract induced mitochondrial dysfunction, cell cycle arrest, and death by apoptosis and necrosis in GRX cells, a well-established activated HSC line. We evaluated the effects of 72-h treatment with crescent concentrations of purple pitanga extract (5 to 100 µg/mL) on triggering autophagy in GRX cells, as this is an important mechanism to cells under cytotoxic conditions. We found that all treated cells presented an increase in the mRNA expression of autophagy-related protein 7 (ATG7). Concomitantly, flow cytometry and ultrastructural analysis of treated cells revealed an increase of autophagosomes/autolysosomes that consequentially led to an increased mitophagy. As purple pitanga extract was previously found to be broadly cytotoxic to GRX cells, we postulated that autophagy contributes to this scenario, where cell death seems to be an inevitable fate. Altogether, the effectiveness on inducing activated HSC death can make purple pitanga extract a good candidate on treating liver fibrosis.

Phosphatidylinositol 3-Kinase/AKT Pathway Inhibition by Doxazosin Promotes Glioblastoma Cells Death, Upregulation of p53 and Triggers Low Neurotoxicity.

Glioblastoma is the most frequent and malignant brain tumor. Treatment includes chemotherapy with temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide, as well as the human glioblastoma cell line U138-MG. We investigated doxazosin's (an antihypertensive drug) activity against glioblastoma cells (C6 and U138-MG) and its neurotoxicity on primary astrocytes and organoptypic hippocampal cultures. For this study, the following methods were used: citotoxicity assays, flow cytometry, western-blotting and confocal microscopy. We showed that doxazosin induces cell death on C6 and U138-MG cells. We observed that doxazosin's effects on the PI3K/Akt pathway were similar as LY294002 (PI3K specific inhibitor). In glioblastoma cells treated with doxasozin, Akt levels were greatly reduced. Upon examination of activities of proteins downstream of Akt we observed upregulation of GSK-3ß and p53. This led to cell proliferation inhibition, cell death induction via caspase-3 activation and cell cycle arrest at G0/G1 phase in glioblastoma cells. We used in this study Lapatinib, a tyrosine kinase inhibitor, as a comparison with doxazosin because they present similar chemical structure. We also tested the neurocitotoxicity of doxazosin in primary astrocytes and organotypic cultures and observed that doxazosin induced cell death on a small percentage of non-tumor cells. Aggressiveness of glioblastoma tumors and dismal prognosis require development of new treatment agents. This includes less toxic drugs, more selective towards tumor cells, causing less damage to the patient. Therefore, our results confirm the potential of doxazosin as an attractive therapeutic antiglioma agent.

STC1 interference on calcitonin family of receptors signaling during osteoblastogenesis via adenylate cyclase inhibition.

Stanniocalcin 1 (STC1) and calcitonin gene-related peptide (CGRP) are involved in bone formation/remodeling. Here we investigate the effects of STC1 on functional heterodimer complex CALCRL/RAMP1, expression and activity during osteoblastogenesis. STC1 did not modify CALCRL and ramp1 gene expression during osteoblastogenesis when compared to controls. However, plasma membrane spatial distribution of CALCRL/RAMP1 was modified in 7-day pre-osteoblasts exposed to either CGRP or STC1, and both peptides induced CALCRL and RAMP1 assembly. CGRP, but not STC1 stimulated cAMP accumulation in 7-day osteoblasts and in CALCRL/RAMP1 transfected HEK293 cells. Furthermore, STC1 inhibited forskolin stimulated cAMP accumulation of HEK293 cells, but not in CALCRL/RAMP1 transfected HEK293 cells. However, STC1 inhibited cAMP accumulation in calcitonin receptor (CTR) HEK293 transfected cells stimulated by calcitonin. In conclusion, STC1 signals through inhibitory G-protein modulates CGRP receptor spatial localization during osteoblastogenesis and may function as a regulatory factor interacting with calcitonin peptide members during bone formation.

Autophagy inhibition improves the efficacy of curcumin/temozolomide combination therapy in glioblastomas.

Glioblastoma is a devastating primary brain tumor resistant to conventional therapies. In this study, we tested the efficacy of combining temozolomide with curcumin, a phytochemical known to inhibit glioblastoma growth, and investigated the mechanisms involved. The data showed that synergy between curcumin and temozolomide was not achieved due to redundant mechanisms that lead to activating protective autophagy both in vitro and in vivo. Autophagy preceded apoptosis, and blocking this response with autophagy inhibitors (3-methyl-adenine, ATG7 siRNA and chloroquine) rendered cells susceptible to temozolomide and curcumin alone or combinations by increasing apoptosis. While curcumin inhibited STAT3, NFκB and PI3K/Akt to affect survival, temozolomide-induced autophagy relied on the DNA damage response and repair components ATM and MSH6, as well as p38 and JNK1/2. However, the most interesting observation was that both temozolomide and curcumin required ERK1/2 to induce autophagy. Blocking this ERK1/2-mediated temozolomide and curcumin induced autophagy with resveratrol, a blood-brain barrier permeable drug, improved temozolomide/curcumin efficacy in brain-implanted tumors. Overall, the data presented demonstrate that autophagy impairs the efficacy of temozolomide/curcumin, and inhibiting this phenomenon could provide novel opportunities to improve brain tumor treatment.

Effects of four additive solutions on canine leukoreduced red cell concentrate quality during storage.

BACKGROUND: Additive solutions (AS) and prestorage leukoreduction (LR) are important tools used to maintain erythrocyte viability during storage and avoid transfusion reactions in recipients, respectively. OBJECTIVES: The purpose of the study was to determine the efficacy of a WBC filter (Immugard IIIRC) and compare the effect of 4 AS (phosphate-adenine-glucose-guanosine-gluconate-mannitol [PAGGGM], saline-adenine-glucose-mannitol [SAGM], Adsol, Optisol) on the in vitro quality of canine leukoreduced packed RBC units (pRBC) stored for 41 days. METHODS: Five hundred milliliters of blood were collected from 8 healthy dogs each into 70 mL of citrate-phosphate-dextrose (CPD) solution, and were leukoreduced by a polyurethane filter. pRBC of each dog were divided equally into 4 bags containing a different AS. Bags were stored for 41 days at 4°C and evaluated every 10 days. Variables analyzed included pH, PCV, and% hemolysis, and lactate, glucose, potassium, sodium, ATP, and 2,3-diphosphoglycerate (2,3-DPG) concentrations. RESULTS: The LR resulted in residual WBC counts comparable to human standards. During storage, pH, and glucose, 2,3-DPG, and ATP concentrations decreased, and hemolysis, and lactate, sodium, and potassium concentrations increased (P < .05). Significant differences between AS were seen in the glucose and sodium concentrations, due to the composition of AS. Also, the pH maintained by PAGGGM at day 21 was significantly higher than that seen with SAGM or Adsol. CONCLUSIONS: All AS used gave satisfactory results during the first 21 days of storage based on the degree of hemolysis, and on ATP and 2,3-DPG concentrations. When compared with day 1 values, significant changes were seen in these variables by day 31 with all AS.

Antiproliferative and cytotoxic effects of purple pitanga (Eugenia uniflora L.) extract on activated hepatic stellate cells.

The presence of phenolic compounds in fruit- and vegetable-rich diets has attracted researchers' attention due to their health-promoting effects. The objective of this study was to evaluate the effects of purple pitanga (Eugenia uniflora L.) extract on cell proliferation, viability, mitochondrial membrane potential, cell death and cell cycle in murine activated hepatic stellate cells (GRX). Cell viability by 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was significantly decreased on cells treated with 50 and 100 µg ml(-1) of purple pitanga extract for 48 and 72 h, and the percentage of dead cell stained with 7-amino-actinomycin D was significantly higher in treated cells. The reduction of cell proliferation was dose dependent, and we also observed alterations on cell cycle progression. At all times studied, GRX cells treated with 50 and 100 µg ml(-1) of purple pitanga showed a significant reduction in cellular mitochondrial content as well as a decrease in mitochondrial membrane potential. Furthermore, our results indicated that purple pitanga extract induces early and late apoptosis/necrosis and necrotic death in GRX cells. This is the first report describing the antiproliferative, cytotoxic and apoptotic activity for E. uniflora fruits in hepatic stellate cells. The present study provides a foundation for the prevention and treatment of liver fibrosis, and more studies will be carried to elucidate this effect.

Major components of energy drinks (caffeine, taurine, and guarana) exert cytotoxic effects on human neuronal SH-SY5Y cells by decreasing reactive oxygen species production.

SCOPE: To elucidate the morphological and biochemical in vitro effects exerted by caffeine, taurine, and guarana, alone or in combination, since they are major components in energy drinks (EDs). METHODS AND RESULTS: On human neuronal SH-SY5Y cells, caffeine (0.125-2 mg/mL), taurine (1-16 mg/mL), and guarana (3.125-50 mg/mL) showed concentration-dependent nonenzymatic antioxidant potential, decreased the basal levels of free radical generation, and reduced both superoxide dismutase (SOD) and catalase (CAT) activities, especially when combined together. However, guarana-treated cells developed signs of neurite degeneration in the form of swellings at various segments in a beaded or pearl chain-like appearance and fragmentation of such neurites at concentrations ranging from 12.5 to 50 mg/mL. Swellings, but not neuritic fragmentation, were detected when cells were treated with 0.5 mg/mL (or higher doses) of caffeine, concentrations that are present in EDs. Cells treated with guarana also showed qualitative signs of apoptosis, including membrane blebbing, cell shrinkage, and cleaved caspase-3 positivity. Flow cytometric analysis confirmed that cells treated with 12.5-50 mg/mL of guarana and its combinations with caffeine and/or taurine underwent apoptosis. CONCLUSION: Excessive removal of intracellular reactive oxygen species, to nonphysiological levels (or "antioxidative stress"), could be a cause of in vitro toxicity induced by these drugs.

Estudo ultrassonográfico morfométrico do fígado e trato biliar de suínos submetidos a obstrução biliar experimental / Sonographic morphometry of the liver and biliary tract in porcine models submitted to experimental biliary obstruction

OBJETIVO: Comparar as alterações anatômicas decorrentes de um quadro de icterícia obstrutiva experimental induzida em suínos nos períodos pré e pós-operatório por meio de exame ultrassonográfico.MATERIAIS E MÉTODOS: Seis suínos da raça Landrace, com 36 dias de idade, foram submetidos a obstrução biliar completa mediante ligadura do ducto colédoco por cirurgia videolaparoscópica.RESULTADOS: Não ocorreram dificuldades na execução dos procedimentos obstrutivos e a recuperação cirúrgica foi eficiente. Decorridos sete dias, os animais apresentaram icterícia, bilirrubinúria e acolia fecal. O exame ultrassonográfico comparativo permitiu visualizar hepatomegalia, colecistomegalia e aumento no calibre do ducto colédoco em todos os animais, assim como alterações decorrentes da colestase. A avaliação morfométrica revelou aumento significativo nos diâmetros da vesícula biliar e do lobo hepático lateral esquerdo.CONCLUSÃO: Os suínos representam um modelo experimental adequado de icterícia obstrutiva, e o exame ultrassonográfico demonstrou-se sensível e relevante no diagnóstico das alterações decorrentes de obstrução biliar extra-hepática nesses animais.

OBJECTIVE: To compare, by means of ultrasonography, pre- and postoperative anatomical changes arising from experimentally induced obstructive jaundice in porcine models.MATERIALS AND METHODS: Six 36-day-old Landrace pigs underwent laparoscopically induced complete biliary obstruction by common bile duct ligation.RESULTS: No difficulty was faced during the procedures and the surgical recovery was uneventful. After seven days, the animals showed jaundice, bilirubinuria and acholic stools. Comparative ultrasonography allowed visualization of hepatomegaly, cholecystomegaly and increased caliber of the common bile duct in all the animals, as well as changes resulting from cholestasis. The morphometric analysis revealed a significant increase in diameter of the gallbladders and left lateral liver lobes.CONCLUSION: Pigs represent appropriate experimental models for investigation of obstructive jaundice, and ultrasonography has shown to be sensitive, playing a relevant role in the diagnosis of extrahepatic biliary obstruction in such animals.

Vitamin A (retinol) downregulates the receptor for advanced glycation endproducts (RAGE) by oxidant-dependent activation of p38 MAPK and NF-kB in human lung cancer A549 cells.

As an essential component of the diet, retinol supplementation is often considered harmless and its application is poorly controlled. However, recent works demonstrated that retinol may induce a wide array of deleterious effects, especially when doses used are elevated. Controlled clinical trials have demonstrated that retinol supplementation increased the incidence of lung cancer and mortality in smokers. Experimental works in cell cultures and animal models showed that retinol may induce free radical production, oxidative stress and extensive biomolecular damage. Here, we evaluated the effect of retinol on the regulation of the receptor for advanced glycation end-products (RAGE) in the human lung cancer cell line A549. RAGE is constitutively expressed in lungs and was observed to be down-regulated in lung cancer patients. A549 cells were treated with retinol doses reported as physiologic (2 µM) or therapeutic (5, 10 or 20 µM). Retinol at 10 and 20 µM increased free radical production, oxidative damage and antioxidant enzyme activity in A549 cells. These doses also downregulated RAGE expression. Antioxidant co-treatment with Trolox®, a hydrophilic analog of α-tocopherol, reversed the effects of retinol on oxidative parameters and RAGE downregulation. The effect of retinol on RAGE was mediated by p38 MAPK activation, as blockade of p38 with PD169316 (10 µM), SB203580 (10 µM) or siRNA to either p38α (MAPK14) or p38ß (MAPK11) reversed the effect of retinol on RAGE. Trolox also inhibited p38 phosphorylation, indicating that retinol induced a redox-dependent activation of this MAPK. Besides, we observed that NF-kB acted as a downstream effector of p38 in RAGE downregulation by retinol, as NF-kB inhibition by SN50 (100 µg/mL) and siRNA to p65 blocked the effect of retinol on RAGE, and p38 inhibitors reversed NF-kB activation. Taken together, our results indicate a pro-oxidant effect of retinol on A549 cells, and suggest that modulation of RAGE expression by retinol is mediated by the redox-dependent activation of p38/NF-kB signaling pathway.

Comparative study between ligature with thread or metallic clamping by means of laparoscopy with the purpose of experimental biliary obstruction in swines.

PURPOSE: To induce a total extra-hepatic obstructive jaundice in swines, by ligation of the common bile duct by laparoscopic surgery. METHODS: Eight swines of the Landrace race, 36-day-old, originated from the same matrix, distributed in two groups. Group A: was used titanium metal clip to the common bile duct ligation in three animals; group B: were ligated with 2-0 cotton thread in five animals. RESULTS: The ligation of the biliary ducts was performed successfully in all animals, with easy identification of the common bile duct by laparoscopy. There weren't difficulties in the procedures, mainly due to the increased surgical field provided by the excellent quality of light and image of the appliance. The clinical signs of jaundice were evident in the animals in seven days. In group A, two animals showed bile duct perforation near the clip, probably due to ischemic necrosis, progressing to peritonitis and death. In group B, five animals showed obstructive jaundice without any amendment. CONCLUSION: Under the conditions of this study, we therefore recommend the use of unabsorbed wires to experimental biliary obstruction, in order to avoid complications, such as ischemia and necrosis, followed by perforation of the wall of the bile ducts.