COX Activity Research Articles

Rosiglitazone rescues human neural stem cells from amyloid-beta induced ER stress via PPARγ dependent signaling

Peroxisome proliferator-activated receptor gamma (PPARγ) belongs to a family of ligand-activated nuclear receptors known to regulate many crucial physiological and pathological conditions. Indeed, altered PPARγ transcriptional activity contributes to metabolic syndromes (obesity and hyperglycemia associated with type 2 diabetes mellitus), stroke and neurodegenerative diseases. Various studies suggest that PPARγ agonists influence neuronal deficits in Alzheimer's Disease (AD) patients and rodent models of AD. Expression of amyloid-beta (Aβ), a neuropathological marker associated with the pathogenesis of AD neuronal impairment, is inversely correlated with the activation of PPARγ-dependent neuroprotective responses. Nevertheless, molecular mechanisms by which the effects of PPARγ agonists in AD remain to be clarified. Here, we explore the PPARγ signaling pathways and networks that protect against Aβ-induced endoplasmic reticulum (ER) stress (e.g., caspase 4, Bip, CHOP, ASK1 and ER calcium), cell death (e.g., viability and cytochrome c) and mitochondrial deficiency (e.g., maximal respiratory function, COX activity, and mitochondrial membrane potential) events in the human neural stem cells (hNSCs) treated with Aβ. Co-treatment with GW9662 (an antagonist of PPARγ) effectively blocked these protective effects by rosiglitazone, providing strong evidence that PPARγ-dependent signaling rescues hNSCs from Aβ-mediated toxicity. Together, our data suggest activation of PPARγ pathway might be critical to protecting against AD-related ER stress, ER disequilibrium and mitochondrial deficiency. These findings also improve our understanding of the role of PPARγ in hNSCs, and may aid in the development and implementation of new therapeutic strategies for the treatment of AD.

Does exposure to reduced pH and diclofenac induce oxidative stress in marine bivalves? A comparative study with the mussel Mytilus galloprovincialis and the clam Ruditapes philippinarum

CO2-driven acidification and emerging contaminants, such as pharmaceuticals, pose new threats for the maintenance of natural populations of marine organisms by interfering with their normal biochemical pathways and defences. The combined effects of seawater acidification, as predicted in climate change scenarios, and an emerging contaminant (the non-steroidal anti-inflammatory drug, NSAID, diclofenac) on oxidative stress-related parameters were investigated in the Mediterranean mussel Mytilus galloprovincialis and the Manila clam Ruditapes philippinarum. A flow-through system was used to carry out a three-week exposure experiment with the bivalves. First, the animals were exposed to only three pH values for 7 days. The pH was manipulated by dissolving CO2 in the seawater to obtain two reduced pH treatments (pH −0.4 units and pH −0.7 units), which were compared with seawater at the natural pH level (8.1). Thereafter, the bivalves were concomitantly exposed to the three experimental pH values and environmentally relevant concentrations of diclofenac (0.00, 0.05 and 0.50 μg/L) for an additional 14 days. The activities of superoxide dismutase, catalase and cyclooxygenase, and lipid peroxidation and DNA strand-break formation were measured in both the gills and digestive gland after 7, 14 and 21 days of exposure to each experimental condition. The results show that the biochemical parameters measured in both the mussels and clams were more influenced by the reduced pH than by the contaminant or the pH*contaminant interaction, although the biomarker variation patterns differed depending on the species and tissues analysed. Generally, due to increases in its antioxidant defence, M. galloprovincialis was more resistant than R. philippinarum to both diclofenac exposure and reduced pH. Conversely, reduced pH induced a significant decrease in COX activity in both the gills and digestive gland of clams, possibly resulting in the increased DNA damage observed in the digestive gland tissue.

METABOLIC ENZYME ACTIVIVTIES IN YOUNG SALMON OF DIFFERENT AGE GROUPS FROM THE ZOLOTITSA RIVER (ARKHANGELSK REGION)

To assess the level of the energy metabolism in the development of salmon Salmo sa lar Linnaeus, 1758 (Salmonidae) from the Zolotitsa River (arkhangelsk region), the activity of enzymes оf aerobic and anaerobic metabolism (cytochrome c oxidase, COX, 1.9.З.1, lactate dehydrogenase, LDh, EC 1.1.1.27 ) and carbohydrate metabolism (glu cose-6-phosphate dehydrogenase, G6PDh, EC 1.1.1.49 , glycerol-3-phosphate dehydrogenase 1 GPDh1, EC 1.1.1.8 , aldolase, ес 4.1.2.13 ) was studied in white muscles and liver of juveniles aged 0+, 1+, 2+ and 3+. age-related multidirectional changes were detected in the activity of the enzymes of aerobic and anaerobic energy metabolism in muscles. Muscular COX activity was high in parr at 0+, 1+, 2+ age, and decreased on the fourth year (3+ parr). The activity of LDh and aldolase in muscles was high in individuals of older groups. age-specific differences in the activity of enzymes in the liver, namely high activity of LDh and GPDh1 in third-year parr (2+), probably reflect changes in the involvement of carbohydrates in biosynthetic processes. These results were analyzed in comparison with those obtained previously for salmon parr from the Indera River (Kola Peninsula). There was a higher activity of LDh in muscles in the first and second years, and lower levels of muscular and hepatic COX and aldolase activities in all age groups from the Zolotitsa River compared to fish from the Indera River. The revealed differences in the activities of all the studied enzymes of energy and carbohydrate metabolism in salmon from the Indera and the Zolotitsa, as well as in their weight, are directly related to the different habitat conditions (foraging, hydrological) in these rivers.

Cardiomyopathy-associated mutation in the ADP/ATP carrier reveals translation-dependent regulation of cytochrome c oxidase activity.

How the absence of the major mitochondrial ADP/ATP carrier in yeast, Aac2p, results in a specific defect in cytochrome c oxidase (COX; complex IV) activity is a long-standing mystery. Aac2p physically associates with respiratory supercomplexes, which include complex IV, raising the possibility that its activity is dependent on its association with Aac2p. Here, we have leveraged a transport-dead pathogenic AAC2 point mutant to determine the basis for the reduced COX activity in the absence of Aac2p. The steady-state levels of complex IV subunits encoded by the mitochondrial genome are significantly reduced in the absence of Aac2p function, whether its association with respiratory supercomplexes is preserved or not. This diminution in COX amounts is not caused by a reduction in the mitochondrial genome copy number or the steady-state level of its transcripts, and does not reflect a defect in complex IV assembly. Instead, the absence of Aac2p activity, genetically or pharmacologically, results in an aberrant pattern of mitochondrial translation. Interestingly, compared with the complete absence of Aac2p, the complex IV–related defects are greater in mitochondria expressing the transport-inactive Aac2p mutant. Our results highlight a critical role for Aac2p transport in mitochondrial translation whose disturbance uniquely impacts cytochrome c oxidase.

Indomethacin and Indomethacin‐PC inhibit the growth of colon cancer under in vitro and in vivo conditions.

BackgroundNonsteroidal anti‐inflammatory drugs (NSAIDs) possess chemopreventive actions against cancer in both animal models and humans. We previously reported that aspirin and a novel aspirin derivative which is associated with phosphatidylcholine (PC) to provide protection of the GI tract against NSAID‐induced injury, are both effective cancer‐preventing agents in a rodent model of colon cancer (Cancer Prev Res 10: 142–152, 2017). Indomethacin (Indo) is highly potent NSAID that has previously been reported to have anti‐neoplastic activity in both rodents and humans (Eur J Cancer 26:811–814, 1990; Cancer Res 54: 5602–5606, 1994).AimIn this study, we performed in vitro and in vivo studies to compare the growth‐inhibitory effect of PC‐associated indomethacin (Indo‐PC) vs the unmodified NSAID on MC‐26 colon cancer cells and in a cancer cell xenograft animal model.MethodsA murine colon cancer cell line MC‐26 was used to compare the efficacy of Indo‐PC and Indo in both in vitro and in vivo systems. The MC‐26 cells were cultured in the presence and absence of the test drug formulations for 8 days with one medium change on the 4th day, at which time, the culture medium was collected and saved for prostaglandin (PGE2) assay as a measure of COX‐2 activity and on day‐8 cells were counted using the MTT assay (Cancer Chemother Pharmacol 57: 93–99, 2006). In the in vivo study, the MC‐26 cells were inoculated into splenic capsule of the syngeneic BALB/c mice. The mice were randomly distributed into three groups and intragastrically treated once daily with vehicle, Indo (2 mg/kg), or Indo‐PC (2 mg Indo + 4 mg PC/kg) for 28 days. We also had a naïve group with no cancer cell injection and no drug treatment. At the end of the study, the mice were euthanized and tissues were collected for analysis of tumor growth (spleen weight and tumor nodule), metastatic cancer cell spread (liver weight and nodule number), and possible GI injury/bleeding, as assessed by measuring hematocrit and fecal hemoglobin. Serum levels of Thromboxane B2 (TXB2) and splenic tissue PGE2 were measured as indices of COX activity.ResultsIndo and Indo‐PC significantly (p< 0.05) inhibited the colon cancer cell growth in culture with an ID50 of 20 μmol/L, and 10 μmol/L, respectively. Both Indo and Indo‐PC were potent inhibitors of PGE2 in culture medium, with Indo‐PC being somewhat more potent at the lowest dose tested (1.28 μmol/L). In vivo, treatment with Indo and Indo‐PC resulted in significant reductions (p<0.05) in splenic tumor nodules and spleen weight. Analysis of liver tissue revealed the presence of a number of metastatic tumor nodules, which tended to be reduced by treatment with Indo and Indo‐PC. Serum TXB2 showed 80–90% inhibition by both NSAID test drugs, supporting the NSAIDs' ability to inhibit prostaglandin formation. Splenic conc of PGE2 in vehicle‐treated controls were elevated over naïve controls which were significantly reduced by both test NSAIDs (~84%, P<0.05). Assessments of GI bleeding showed no differences between treatment and control groups, with hematocrit levels falling within a normal range of 0.43 to 0.47 and fecal hemoglobin also showing minimal alterations.ConclusionsIndo and Indo‐PC are highly potent and effective inhibitors of colon cancer cell growth, under both in vitro and in vivo conditions. These results support the possible use of Indo‐PC, a highly potent NSAID to block COX activity, for chemoprevention of colorectal cancer and possibly treatment.Support or Funding InformationSupported by NIH grants R03 CA171613This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The overexpression of cytochrome c oxidase subunit 6C activated by Kras mutation is related to energy metabolism in pancreatic cancer

Background: Kras mutation is frequently detected in pancreatic cancers and leads to altered energy metabolite. Here we investigated molecule markers related with Kras mutation, which could be used as developing new target for Kras mutant driven cancer. Methods: A knockin BxPC-3/ Kras G12D cell line was constructed by CRISPR/Cas9 system. Proliferation and metabolite characterization in BxPC-3/ Kras G12D was compared with wild type BxPC-3 by using colony formation assay and mitochondrial dyes. The differential genes were screened using mitochondrial metabolite-related genes PCR array. The expression of COX6C was confirmed by real time polymerase chain reaction (RT-PCR) and western blot. COX6C expression in 30 pairs of tissue microarray of pancreatic carcinoma and matched adjacent tissues was analyzed by immunohistochemistry. ATP production stimulated by metabolites substrate assay was carried out to investigate whether the decreased COX6C by siRNA transfection affected the metabolite characterization of pancreatic cancer cells. Results: The BxPC-3/ Kras G12D displayed faster proliferation, increased mitochondrial mass and ATP, elevated mitochondrial membrane potential than BxPC-3. Using mitochondrial metabolite-related genes array, we identified COX6C was an up-regulated gene driven by Kras G12D . There was a notable difference of COX enzyme activity between BxPC-3 and BxPC-3/ Kras G12D . The overexpression of COX6C was found in pancreatic cancer tissues. Using COX6C siRNA-mediated knockdown, the cell viability, COX enzyme activity and ATP production of BxPC-3/ Kras G12D cells significantly decreased. Conclusions: These results suggested that activation of Kras G12D in pancreatic cancer cells increased the COX activity and ATP production of mitochondrial via up-regulation of COX6C. This regulatory subunit of COX may have utility as a Ras effector target for development of anti-pancreatic cancer therapeutics.

Decreased expression of megalin and cubilin and altered mitochondrial activity in tenofovir nephrotoxicity

Tenofovir disoproxil fumarate (TDF) is a commonly used antiretroviral drug for HIV, rarely causing Fanconi syndrome and acute kidney injury. We retrospectively analyzed the clinico pathological presentation of 20 cases of tenofovir-induced tubulopathy, and investigated the renal expression of the megalin and cubilin proteins, as well as the mitochondrial respiratory chain activity. Estimated glomerular filtration rate (eGFR) before TDF exposure was 92 ml/min/1.73m2, decreasing to 27.5 ml/min/1.73m2 at the time of biopsy, with 30% of patients requiring renal replacement therapy. Proximal tubular expression of megalin and cubilin was altered in 19 and 18 cases, respectively, whereas it was preserved in patients exposed to TDF without proximal tubular dysfunction and in HIV-negative patients with acute tubular necrosis. Loss of megalin/cubilin was correlated with low eGFR and high urine retinol binding protein at the time of biopsy, low eGFR at last follow-up, and was more severe in patients with multifactorial toxicity. Patients with additional nephrotoxic conditions promoting tenofovir accumulation showed a lower eGFR at presentation and at last follow-up, and more severe lesions of acute tubular necrosis, than those with isolated tenofovir toxicity. Altered mitochondrial COX activity in proximal tubules was observed and may be an early cellular alteration in tenofovir nephrotoxicity. In conclusion, altered megalin/cubilin expression represents a distinctive feature in tenofovir-induced tubulopathy, and its severity is correlated with urine retinol binding protein loss and is associated with a poor renal prognosis. Concomitant exposure to other nephrotoxic conditions severely impacts the renal presentation and outcome.

A Possible Link between Dysregulated Mitochondrial Calcium Homeostasis and Citrullination in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is characterized by citrullination of peptides and proteins mediated by calciumdependent peptidyl arginine deiminase enzymes (PADs). Various mechanisms of intracellular and extracellular protein citrullination have been elucidated so far. Here, we highlight one more important possible mechanism that could lead to intracellular citrullination due to dysregulated mitochondrial Ca2+ homeostasis i.e., inability of mitochondrial uptake of Ca2+ during physiological signaling and triggering the generation of autoimmune disease. Spontaneous secretion of intracellular PAD may be the result of high cytosolic Ca2+ due to a disturbance in mitochondrial calcium homeostasis secondary to oxidative damage without any compromise to the cell membrane. Various environmental triggers like air pollutants also induce oxidative stress (OS) which may compromise the mitochondrial integrity and disturb Ca2+ homeostasis. Adoption of simple lifestyle modification like yoga and meditation optimizes reactive oxygen species (ROS) levels and maintains mitochondrial integrity by increasing COX activity, thus may curb citrullination process and its sequelae.

A tiered approach to assess effects of diclofenac on the brown mussel Perna perna: A contribution to characterize the hazard

Pharmaceutical discharges into the aquatic ecosystem are of environmental concern and sewage treatment plants (STPs) have been pointed out as the major source of these compounds to coastal zones, where oceanic disposal of sewage occurs through submarine outfalls. Diclofenac (DCF) is one of the most frequently detected pharmaceuticals in water, but little is known about the effects on marine organisms. In this study, we employed a tiered approach involving the determination of environmental concentrations of DCF in marine water and the adverse biological effects for fertilization, embryo-larval development and biomarker responses of the mussel Perna perna. Results indicate that effects in fertilization rate and embryo-larval development were found in the order of mg·L−1. However, low concentrations of DCF (ng·L−1) significantly decreased the lysosomal membrane stability and COX activity, as well as triggered DNA damage, oxidative stress and changes in antioxidant defenses. Our results point to an environmental hazard at coastal ecosystems and suggest the need for improvements in the treatment of domestic wastewater aiming to reduce DCF concentrations, as well as regulation on current environmental legislation and monitoring of aquatic ecosystems.

Prospective Controlled Trial on Endurance Exercise Training in Adult Sickle Cell Disease Patients

▪B Gellen and L Messonnier, and L Feasson and P Bartolucci equally contributed to this workIntroduction Sickle cell disease (SCD) is the most common inherited disease worldwide. Because favoring the risk of vaso-occlusive crises (VOC), SCD patients are banned from strenuous exercise. We hypothesized that a well-controlled moderate-intensity endurance exercise training (EET) program could improve SCD patients without provoking VOC.Patients and Methods: Weconducted aprospective, multicenter, controlled study with individually tailored Blood lactate-guided (BL) endurance training on a cycloergometer for 8 weeks in adult SCD patients. The primary end point was the submaximal incremental cardio-pulmonary exercise testing (CPET) at a BL concentration of 4 mmol.L-1. Secondary endpoints were: CPET at a BL 2.5 mmol/L and at the first lactic threshold (LT1), BL levels at given workload level (Female: 40W and Male: 60W), safety (pain, VOC and acute chest syndrome), biological and echocardiography parameters, pulmonary function test (PFT), 6-minute walk test (6MWT), quality of life (QoL) and muscle properties and vascularization (biopsy of vastus lateralis muscle).Results: Forty homozygous SCD patients (33±10 years, 23 male) were included. Four patients (3 EET and 1 control) were lost to follow-up. Data of 33 patients (15 EET and 18 controls) were analyzed. EET and control patients were comparable at baseline with regard to CPET, clinical, biological, and resting cardiorespiratory parameters. EET patients presented a significant increase of CPET workload at BL4 (p=0.031), BL2.5 (p=0.003) and LT1 (p<0.0001) compared to the control group that remained unchanged. BL levels at a given level decreased significantly by 18% in the EET group compared to control group (p=0.01). Among the 40 randomized patients, 5 MAE occurred in the control group and 2 in the EET group. Quality of life improved for vitality (p=0.04) and physical activity (p=0.03) in the EET group compared to control group. Thigh biopsy showed in the EET group a significant increase of the proportion of oxidative type I fibers and of COX activity, paralleled by a significant increase in capillary density. No significant alterations were found in the control group. No change was found according to biological and echocardiography parameters, 6MWT and pulmonary function test.Conclusion In SCD patients, EET based on Blood lactate guidance is safe and yields a significant functional benefit. This benefit is linked essentially to improved oxidative function of the peripheral skeletal musculature. DisclosuresGalactéros:Addmedica: Membership on an entity's Board of Directors or advisory committees. Bartolucci:GBT: Membership on an entity's Board of Directors or advisory committees; Addmedica: Research Funding; Fondation Fabre: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.

AM404, paracetamol metabolite, prevents prostaglandin synthesis in activated microglia by inhibiting COX activity

BackgroundN-arachidonoylphenolamine (AM404), a paracetamol metabolite, is a potent agonist of the transient receptor potential vanilloid type 1 (TRPV1) and low-affinity ligand of the cannabinoid receptor type 1 (CB1). There is evidence that AM404 exerts its pharmacological effects in immune cells. However, the effect of AM404 on the production of inflammatory mediators of the arachidonic acid pathway in activated microglia is still not fully elucidated.MethodIn the present study, we investigated the effects of AM404 on the eicosanoid production induced by lipopolysaccharide (LPS) in organotypic hippocampal slices culture (OHSC) and primary microglia cultures using Western blot, immunohistochemistry, and ELISA.ResultsOur results show that AM404 inhibited LPS-mediated prostaglandin E2 (PGE2) production in OHSC, and LPS-stimulated PGE2 release was totally abolished in OHSC if microglial cells were removed. In primary microglia cultures, AM404 led to a significant dose-dependent decrease in the release of PGE2, independent of TRPV1 or CB1 receptors. Moreover, AM404 also inhibited the production of PGD2 and the formation of reactive oxygen species (8-iso-PGF2 alpha) with a reversible reduction of COX-1- and COX-2 activity. Also, it slightly decreased the levels of LPS-induced COX-2 protein, although no effect was observed on LPS-induced mPGES-1 protein synthesis.ConclusionsThis study provides new significant insights about the potential anti-inflammatory role of AM404 and new mechanisms of action of paracetamol on the modulation of prostaglandin production by activated microglia.

Alpha-tocopherol succinate increases cyclooxygenase-2 activity: Tissue-specific action in pregnant rat uterus in vitro

AimsLipid soluble vitamin E plays a role in several physiological mechanisms, however, the mechanism of this action is controversial. We investigated how tocopherol (α-tocopherol acid succinate) influences the effects of cyclooxygenase inhibitors (COXi) in the smooth muscles. Main methodsThe contractility of the samples from 22-day-pregnant myometrium and non-pregnant myometrium and trachea was determined in an isolated organ bath in vitro. The activity of cyclooxygenase enzymes (COX) was also measured in the tissues. Key findingsDiclofenac (10−9–10−5M) and rofecoxib (10−10–10−5M) decreased the contractions in non-pregnant and 22-day-pregnant uteri. Tocopherol (10−7M) increased the relaxant effect only in pregnant uteri. Both diclofenac (10−9–10−5M) and rofecoxib (10−10–10−5M) reduced the tracheal tones, while they were slightly intensified by pretreatment with tocopherol (10−7M). Tocopherol enhanced the contractions of pregnant uteri. Tocopherol (10−7M) itself can induce the cyclooxygenase activity and shift the COX-1 and COX-2 ratio to COX-2. The lowest COX activity was found in non-pregnant uteri, while the highest one was in the trachea. SignificanceThe COX enzymes, especially COX-2, play an important role in the contraction of pregnant uteri in rat. Tocopherol has a tissue specific COX-2 activity increasing effect in pregnant rat uterus but has no such action in non-pregnant uteri or tracheal tissue. Hereby, tocopherol may intensify selectively the uterine relaxing effect of COX-2 inhibitors in preterm contractions. However, tocopherol can enhance the contractile response of pregnant uterus that may increase the risk of premature contractions.

Energy response and modulation of AMPK pathway of the olive flounder Paralichthys olivaceus in low-temperature challenged

Cold resistance is important for fish in natural and aquaculture environments. Modulation of energy metabolism has been proved to be vital for fish in cold. However, the interaction of energy response and the known AMPK pathway remains vague in fish coping with cold stress. In the present study, the olive flounders Paralichthys olivaceus were subject to low-temperature condition (0.2±0.2°C). The energy metabolism and modulation of AMPK pathway were analyzed. Results showed that flounders increased ATP-coupled ion transport, as indicated by Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities. Nevertheless, higher levels of aerobic and anaerobic metabolism, respectively indicated by the elevation of COX and LDH activities, contributed to the stable energy homeostasis during cold exposure. The three subunits of AMPK responded differently to cold, and AMPKα and AMPKβ might be more sensitive in the early stage while AMPKγ might be more sensitive in the later stage. The transcriptional response of LKB1/CaMKKβ-AMPK-NRF1 axis might play an important role in helping flounders against the early cold stress. However, flounders differentiated in cold resistance with the stress time elapsed. Concomitantly a greater reliance on LKB1-AMPK-NRF1 axis for cold-tolerant flounders compared to those sensitive ones to cold was observed, and this axis was speculated to be related with flounder cold resistance. To our knowledge, this is the first time to explore the possible response of AMPK pathway to cold in fish. Our data showed that the flounder relied on metabolic regulation and modulation of AMPK pathway including not only higher expression levels of genes in this pathway but also changes in its related function axes to maintain energy homeostasis.

Determination of dietary iron requirements by full expression of iron-containing cytochrome c oxidase in the heart of broilers from 22 to 42 d of age

The present study was carried out to determine dietary Fe requirements for the full expression of Fe-containing enzyme in broilers chicks from 22 to 42 d of age. At 22 d of age, 288 Arbor Acres male chicks were randomly assigned to one of six treatments with six replicates and fed a basal maize-soyabean-meal diet (control, containing 47·0 mg Fe/kg) or the basal diet supplemented with 20, 40, 60, 80 or 100 mg Fe/kg from FeSO4.7H2O for 21 d. Regression analysis was performed to estimate the optimal dietary Fe level using quadratic models. Liver cytochrome c oxidase (Cox), heart Cox and kidney succinate dehydrogenase mRNA levels as well as heart COX activity were affected (P<0·08) by dietary Fe level, and COX mRNA level and activity in heart of broilers increased quadratically (P<0·03) as dietary Fe level increased. The estimates of dietary Fe requirements were 110 and 104 mg/kg for the full expression of Cox mRNA and for its activity in the heart of broilers, respectively. The results from this study indicate that COX mRNA level and activity in the heart are new and sensitive criteria to evaluate the dietary Fe requirements of broilers, and the dietary Fe requirements would be 104-110 mg/kg to support the full expression of COX in the heart of broiler chicks from 22 to 42 d of age, which are higher than the current National Research Council Fe requirement (80 mg/kg) of broiler chicks from 1 to 21 d or 22 to 42 d of age.

Lack of Additive Effects of Resveratrol and Energy Restriction in the Treatment of Hepatic Steatosis in Rats.

The aims of the present study were to analyze the effect of resveratrol on liver steatosis in obese rats, to compare the effects induced by resveratrol and energy restriction and to research potential additive effects. Rats were initially fed a high-fat high-sucrose diet for six weeks and then allocated in four experimental groups fed a standard diet: a control group, a resveratrol-treated group, an energy restricted group and a group submitted to energy restriction and treated with resveratrol. We measured liver triacylglycerols, transaminases, FAS, MTP, CPT1a, CS, COX, SDH and ATP synthase activities, FATP2/FATP5, DGAT2, PPARα, SIRT1, UCP2 protein expressions, ACC and AMPK phosphorylation and PGC1α deacetylation. Resveratrol reduced triacylglycerols compared with the controls, although this reduction was lower than that induced by energy restriction. The mechanisms of action were different. Both decreased protein expression of fatty acid transporters, thus suggesting reduced fatty acid uptake from blood stream and liver triacylglycerol delivery, but only energy restriction reduced the assembly. These results show that resveratrol is useful for liver steatosis treatment within a balanced diet, although its effectiveness is lower than that of energy restriction. However, resveratrol is unable to increase the reduction in triacylglycerol content induced by energy restriction.

Regulation of the autophagy system during chronic contractile activity-induced muscle adaptations.

Skeletal muscle is adaptable to exercise stimuli via the upregulation of mitochondrial biogenesis, and recent studies have suggested that autophagy also plays a role in exercise‐induced muscle adaptations. However, it is still obscure how muscle regulates autophagy over the time course of training adaptations. This study examined the expression of autophagic proteins in skeletal muscle of rats exposed to chronic contractile activity (CCA; 6 h/day, 9V, 10 Hz continuous, 0.1 msec pulse duration) for 1, 3, and 7 days (n = 8/group). CCA‐induced mitochondrial adaptations were observed by day 7, as shown by the increase in mitochondrial proteins (PGC‐1α, COX I, and COX IV), as well as COX activity. Notably, the ratio of LC3 II/LC3 I, an indicator of autophagy, decreased by day 7 largely due to a significant increase in LC3 I. The autophagic induction marker p62 was elevated on day 3 and returned to basal levels by day 7, suggesting a time‐dependent increase in autophagic flux. The lysosomal system was upregulated early, prior to changes in mitochondrial proteins, as represented by increases in lysosomal system markers LAMP1, LAMP2A, and MCOLN1 as early as by day 1, as well as TFEB, a primary regulator of lysosomal biogenesis and autophagy flux. Our findings suggest that, in response to chronic exercise, autophagy is upregulated concomitant with mitochondrial adaptations. Notably, our data reveal the surprising adaptive plasticity of the lysosome in response to chronic contractile activity which enhances muscle health by providing cells with a greater capacity for macromolecular and organelle turnover.

U46619‐induced contraction is impaired by COX activity in coronary arteries from renal hypertensive rats

IntroductionThe coronary vascular bed is responsible for the proper myocardial blood supply and cardiac work. The vascular endothelium controls the vascular tone by the production and/or release of relaxing factors and contractile factors. In cardiovascular diseases like hypertension this balance can be impaired with increased prostanoids production like thromboxane derived from COX and TP receptors activation. Therefore, this work aimed to evaluate the contraction induced by the TP receptors agonist tromboxane analogue (U46619) in renal hypertensive (2K‐1C) and normotensive (2K) coronary arteries. The procedures were approved by the Ethics Committee: USP‐2015.1.441.60.7.MethodsHypertension was induced by surgery in rats. After six weeks, the rats were killed, the heart was removed and placed in ice cold Krebs solution to isolate the septal coronary arteries. The vessels were mounted in microvessels myograph and stimulated with 120 mM KCl and concentration‐effect curves were performed to 5‐hydroxytryptamine (5‐HT, 10 nM–100 μM) following the relaxation to acetylcholine (10 μM). In addition, concentration‐effect curves were constructed to U46619 (10 nM–10 μM) in the absence (Control) or in the presence of COX inhibitor (Indomethacin, 10 μM), selective COX inhibitors of COX‐1 (SC560, 10 μM) and COX‐2 (SC236, 10 μM). The coronary arteries were collected and frozen at −80° C to measure COX‐1 and COX‐2 expression. Data were analyzed by student's t test and one way ANOVA with Dunnet's post‐test (P&lt;0.05).ResultsThe coronary diameter (2K: 337.4 ± 29.9 vs 2K‐1C: 260.4 ± 27.6 μm), the contraction induced by KCl (2K: 4.28 ± 0.65 vs 2K‐1C: 2.98 ± 0.58 mN) and the acetylcholine‐induced relaxation (2K: 77.5 ± 6.5% vs 2K‐1C: 68.2 ± 14.0%) were not different. COX‐1 expression was not different between 2K and 2K‐1C rats (P=0.054). However, COX‐2 expression was higher in 2K‐1C than in 2K (P=0.02). Contraction‐induced by U46619 presented similar pD2 values in 2K (6.83 ± 0.17, n=5) and 2K‐1C (6.52 ± 0.10, n=6) but the maximum effect (ME) was lower in 2K‐1C (87.5 ± 17.0%) than in 2K (149.8 ± 13.1%). All the COX inhibitors used did not change the pD2 and ME in 2K coronary arteries. However, indomethacin increased the ME in 2K‐1C to 162.9 ± 16.9%, n=4 and SC560 increased the pD2 (7.33 ± 0.20, n=5).ConclusionOur results indicate that the contractile response induced by U46619 is impaired in 2K‐1C coronary arteries due to the production of relaxant factor derived from COX activity.Support or Funding InformationFinancial Support: CAPES, CNPq, FAPESP

Pro-inflammatory immune-to-brain signaling is involved in neuroendocrine responses to acute emotional stress

Activation of the hypothalamo-pituitary-adrenal (HPA) axis by inflammatory stressors (e.g., bacterial lipopolysaccharide) is thought to involve vascular transduction of circulating cytokines, with perivascular macrophages (PVMs) along with endothelia, effecting activation of HPA control circuitry via inducible (cyclooxygenase-2- or COX-2-dependent) prostaglandin synthesis. To test the stressor-specificity of this mechanism, we examined whether ablation of PVMs or pharmacologic blockade of COX activity affected HPA responses to a representative emotional stressor, restraint. Exposing rats to a single 30min acute restraint episode provoked increased plasma levels of at least one proinflammatory cytokine, IL-6, microglial activation and multiple indices of cerebrovascular activation, including COX-2 expression and increased brain prostaglandin E2 levels at 0–2h after stress. Pretreatment with the nonselective COX inhibitor, indomethacin, either icv (10μg in 5μl) or iv (1mg/kg) significantly reduced restraint-induced Fos expression in the paraventricular hypothalamic nucleus (PVH) by 45%, relative to vehicle-injected controls. A 75% reduction of the PVH activational response was seen in rats exposed to acute restraint 5–7days after ablation of brain PVMs by icv injection of liposomes encapsulating the bisphosphonate drug, clodronate. Basal plasma levels of ACTH and corticosterone were not altered in clodronate liposome-injected rats, but the peak magnitude of restraint-induced HPA secretory responses was substantially reduced, relative to animals pretreated with saline-filled liposomes. These findings support an unexpectedly prominent role for inducible prostaglandin synthesis by PVMs in HPA responses to acute restraint, a prototypic emotional stressor.

Differential expression of E-type prostanoid receptors 2 and 4 in microglia stimulated with lipopolysaccharide

BackgroundCyclooxygenase-2 (COX-2) is induced under inflammatory conditions, and prostaglandin E2 (PGE2) is one of the products of COX activity. PGE2 has pleiotropic actions depending on the activation of specific E-type prostanoid EP1-4 receptors. We investigated the involvement of PGE2 and EP receptors in glial activation in response to an inflammatory challenge induced by LPS.MethodsCultures of mouse microglia or astroglia cells were treated with LPS in the presence or absence of COX-2 inhibitors, and the production of PGE2 was measured by ELISA. Cells were treated with PGE2, and the effect on LPS-induced expression of TNF-α messenger RNA (mRNA) and protein was studied in the presence or absence of drug antagonists of the four EP receptors. EP receptor expression and the effects of EP2 and EP4 agonists and antagonists were studied at different time points after LPS.ResultsPGE2 production after LPS was COX-2-dependent. PGE2 reduced the glial production of TNF-α after LPS. Microglia expressed higher levels of EP4 and EP2 mRNA than astroglia. Activation of EP4 or EP2 receptors with selective drug agonists attenuated LPS-induced TNF-α in microglia. However, only antagonizing EP4 prevented the PGE2 effect demonstrating that EP4 was the main target of PGE2 in naïve microglia. Moreover, the relative expression of EP receptors changed during the course of classical microglial activation since EP4 expression was strongly depressed while EP2 increased 24 h after LPS and was detected in nuclear/peri-nuclear locations. EP2 regulated the expression of iNOS, NADPH oxidase-2, and vascular endothelial growth factor. NADPH oxidase-2 and iNOS activities require the oxidation of NADPH, and the pentose phosphate pathway is a main source of NADPH. LPS increased the mRNA expression of the rate-limiting enzyme of the pentose pathway glucose-6-phosphate dehydrogenase, and EP2 activity was involved in this effect.ConclusionsThese results show that while selective activation of EP4 or EP2 exerts anti-inflammatory actions, EP4 is the main target of PGE2 in naïve microglia. The level of EP receptor expression changes from naïve to primed microglia where the COX-2/PGE2/EP2 axis modulates important adaptive metabolic changes.

The Effects of Thai Herbal Ha-Rak Formula on COX Isoform Expression in Human Umbilical Vein Endothelial Cells Induced by IL-1β.

Objective To investigate the modulated effects of HRF on cyclooxygenase isoform expression and its activity, using the human umbilical vein endothelial cell (HUVEC) model induced by interleukin-1 beta (IL-1β). Methods Cells were treated with indomethacin (positive control), HRF, and its components at various concentrations prior to treatment with IL-1β at 24 h. Cell viability was determined by MTT assay. Moreover, the anti-inflammatory effects of HRF and its components through mRNA and protein expression were established using real-time quantitative PCR and Western blot, respectively. COX activity was identified via exogenous and endogenous PGE2 productions using the EIA. Result There was no cytotoxicity in HUVECs treated with HRF. None of the experimental conditions used in the study affected the expression of COX-1, but COX-2 protein expression was inhibited at concentrations under 10 µg/mL. Despite the significantly increased levels of exogenous PGE2, HRF had no effect on COX-2 mRNA expression. However, the production of PGE2 was lower at a concentration of 100 µg/mL HRF than at a concentration below 10 µg/mL. Interestingly, each component of HRF revealed different effects of the Ha-Rak formula. Conclusion Our preliminary findings suggest that HRF and its components provide diverse modulation of COX-2 and PGE2 at the in vitro level.