Digested Cinnamon (Cinnamomum verum J. Presl) Bark Extract Modulates Claudin-2 Gene Expression and Protein Levels under TNFα/IL-1ß Inflammatory Stimulus.

Epigenetic changes, host-gut microbiota interactions, and environmental factors contribute to inflammatory bowel disease (IBD) onset and progression. A healthy lifestyle may help to slow down the chronic or remitting/relapsing intestinal tract inflammation characteristic of IBD. In this scenario, the employment of a nutritional strategy to prevent the onset or supplement disease therapies included functional food consumption. Its formulation consists of the addition of a phytoextract enriched in bioactive molecules. A good candidate as an ingredient is the Cinnamon verum aqueous extract. Indeed, this extract, subjected to a process of gastrointestinal digestion simulation (INFOGEST), exhibits beneficial antioxidant and anti-inflammatory properties in an in vitro model of the inflamed intestinal barrier. Here, we deepen the study of the mechanisms related to the effect of digested cinnamon extract pre-treatment, showing a correlation between transepithelial electrical resistance (TEER) decrement and alterations in claudin-2 expression under Tumor necrosis factor-α/Interleukin-1ß (TNF-α/IL-1) ß cytokine administration. Our results show that pre-treatment with cinnamon extract prevents TEER loss by claudin-2 protein level regulation, influencing both gene transcription and autophagy-mediated degradation. Hence, cinnamon polyphenols and their metabolites probably work as mediators in gene regulation and receptor/pathway activation, leading to an adaptive response against renewed insults.

Coffee-Derived Phenolic Compounds Activate Nrf2 Antioxidant Pathway in I/R Injury In Vitro Model: A Nutritional Approach Preventing Age Related-Damages.

Age-related injuries are often connected to alterations in redox homeostasis. The imbalance between free radical oxygen species and endogenous antioxidants defenses could be associated with a growing risk of transient ischemic attack and stroke. In this context, a daily supply of dietary antioxidants could counteract oxidative stress occurring during ischemia/reperfusion injury (I/R), preventing brain damage. Here we investigated the potential antioxidant properties of coffee-derived circulating metabolites and a coffee pulp phytoextract, testing their efficacy as ROS scavengers in an in vitro model of ischemia. Indeed, the coffee fruit is an important source of phenolic compounds, such as chlorogenic acids, present both in the brewed seed and in the discarded pulp. Therefore, rat brain endothelial cells, subjected to oxygen and glucose deprivation (OGD) and recovery (ogR) to mimic reperfusion, were pretreated or not with coffee by-products. The results indicate that, under OGD/ogR, the ROS accumulation was reduced by coffee by-product. Additionally, the coffee extract activated the Nrf2 antioxidant pathway via Erk and Akt kinases phosphorylation, as shown by increased Nrf2 and HO-1 protein levels. The data indicate that the daily intake of coffee by-products as a dietary food supplement represents a potential nutritional strategy to counteract aging.

Systemic Exposure to Air Pollution Induces Oxidative Stress and Inflammation in Mouse Brain, Contributing to Neurodegeneration Onset.

In northern Italy, biomass burning-derived (BB) particles and diesel exhaust particles (DEP) are considered the most significant contributors to ultrafine particle (UFP) emission. However, a comparison between their impact on different brain regions was not investigated until now. Therefore, male BALB/c mice were treated with a single or three consecutive intratracheal instillations using 50 µg of UFPs in 100 µL of isotonic saline solution or 100 µL of isotonic saline solution alone, and brains were collected and analyzed. Proteins related to oxidative stress and inflammation, as well as Alzheimer's disease markers, were examined in the hippocampus, cerebellum, and the rest of the brain (RoB). Histopathological examination of the brain was also performed. Moreover, correlations among different brain, pulmonary, and cardiovascular markers were performed, allowing us to identify the potentially most stressful UFP source. Although both acute exposures induced inflammatory pathways in mouse brain, only DEP showed strong oxidative stress. The sub-acute exposure also induced the modulation of APP and BACE1 protein levels for both UFPs. We observed that DEP exposure is more harmful than BB, and this different response could be explained by this UFP's different chemical composition and reactivity.

In Vivo Comparative Study on Acute and Sub-acute Biological Effects Induced by Ultrafine Particles of Different Anthropogenic Sources in BALB/c Mice.

Exposure to ultrafine particles (UFPs) leads to adverse effects on health caused by an unbalanced ratio between UFPs deposition and clearance efficacy. Since air pollution toxicity is first direct to cardiorespiratory system, we compared the acute and sub-acute effects of diesel exhaust particles (DEP) and biomass burning-derived particles (BB) on bronchoalveolar Lavage Fluid (BALf), lung and heart parenchyma. Markers of cytotoxicity, oxidative stress and inflammation were analysed in male BALB/c mice submitted to single and repeated intra-tracheal instillations of 50 µg UFPs. This in-vivo study showed the activation of inflammatory response (COX-2 and MPO) after exposure to UFPs, both in respiratory and cardiovascular systems. Exposure to DEP results also in pro- and anti-oxidant (HO-1, iNOS, Cyp1b1, Hsp70) protein levels increase, although, stress persist only in cardiac tissue under repeated instillations. Statistical correlations suggest that stress marker variation was probably due to soluble components and/or mediators translocation of from first deposition site. This mechanism, appears more important after repeated instillations, since inflammation and oxidative stress endure only in heart. In summary, chemical composition of UFPs influenced the activation of different responses mediated by their components or pro-inflammatory and pro-oxidative molecules, indicating DEP as the most damaging pollutant in the comparison.

Lipid Reshaping and Lipophagy Are Induced in a Modeled Ischemia-Reperfusion Injury of Blood Brain Barrier.

Ischemic-reperfusion (I/R) injury induced a remodeling of protein and lipid homeostasis, under oxidative stress and inflammatory status. Starvation occurring during I/R is a condition leading to autophagy activation, which allows abnormal material clearance or amino acid, or both, and fatty acid (FA) recycling essential for survival. This study investigated the lipid reshaping, peroxidation, and related-signaling pathways, in rat brain endothelial cells (RBE4) subjected to 3 h of oxygen and glucose deprivation (OGD) and restoration of standard condition (I/R in vitro model). Lipids and proteins were analyzed after 1 or 24 h of oxygen and nutrient restoration. Together with the oxidative stress and inflammatory status, I/R injury induced a reshaping of neutral lipids and biogenesis of lipid droplets (LD) with excessive lipid storage. The increase of LC3-II/LC3-I ratio, an autophagy marker, and LC3 co-localization with LD suggest the activation of lipophagy machinery to counteract the cell engulfment. Lipophagy leads to cholesterol ester (CE) hydrolysis, increasing free cholesterol (FC) secretion, which occurred by specific transporters or unconventional exocytosis pathways, or both. Here, we propose that an unconventional spreading of FC and other lipid metabolites may influence the neurovascular unit (NVU) cells, contributing to Blood brain barrier (BBB) alteration or adaptation, or both, to the cumulative effects of several transient ischemia.

Effects of PM2.5 Exposure on the ACE/ACE2 Pathway: Possible Implication in COVID-19 Pandemic.

Particulate matter (PM) is a harmful component of urban air pollution and PM2.5, in particular, can settle in the deep airways. The RAS system plays a crucial role in the pathogenesis of pollution-induced inflammatory diseases: the ACE/AngII/AT1 axis activates a pro-inflammatory pathway counteracted by the ACE2/Ang(1-7)/MAS axis, which in turn triggers an anti-inflammatory and protective pathway. However, ACE2 acts also as a receptor through which SARS-CoV-2 penetrates host cells to replicate. COX-2, HO-1, and iNOS are other crucial proteins involved in ultrafine particles (UFP)-induced inflammation and oxidative stress, but closely related to the course of the COVID-19 disease. BALB/c male mice were subjected to PM2.5 sub-acute exposure to study its effects on ACE2 and ACE, COX-2, HO-1 and iNOS proteins levels, in the main organs concerned with the pathogenesis of COVID-19. The results obtained show that sub-acute exposure to PM2.5 induces organ-specific modifications which might predispose to greater susceptibility to severe symptomatology in the case of SARS-CoV-2 infection. The novelty of this work consists in using a molecular study, carried out in the lung but also in the main organs involved in the disease, to analyze the close relationship between exposure to pollution and the pathogenesis of COVID-19.

LSEA Evaluation of Lipid Mediators of Inflammation in Lung and Cortex of Mice Exposed to Diesel Air Pollution.

Airborne ultrafine particle (UFP) exposure is a great concern as they have been correlated to increased cardiovascular mortality, neurodegenerative diseases and morbidity in occupational and environmental settings. The ultrafine components of diesel exhaust particles (DEPs) represent about 25% of the emission mass; these particles have a great surface area and consequently high capacity to adsorb toxic molecules, then transported throughout the body. Previous in-vivo studies indicated that DEP exposure increases pro- and antioxidant protein levels and activates inflammatory response both in respiratory and cardiovascular systems. In cells, DEPs can cause additional reactive oxygen species (ROS) production, which attacks surrounding molecules, such as lipids. The cell membrane provides lipid mediators (LMs) that modulate cell-cell communication, inflammation, and resolution processes, suggesting the importance of understanding lipid modifications induced by DEPs. In this study, with a lipidomic approach, we evaluated in the mouse lung and cortex how DEP acute and subacute treatments impact polyunsaturated fatty acid-derived LMs. To analyze the data, we designed an ad hoc bioinformatic pipeline to evaluate the functional enrichment of lipid sets belonging to the specific biological processes (Lipid Set Enrichment Analysis-LSEA). Moreover, the data obtained correlate tissue LMs and proteins associated with inflammatory process (COX-2, MPO), oxidative stress (HO-1, iNOS, and Hsp70), involved in the activation of many xenobiotics as well as PAH metabolism (Cyp1B1), suggesting a crucial role of lipids in the process of DEP-induced tissue damage.

Study of the Antioxidant Effects of Coffee Phenolic Metabolites on C6 Glioma Cells Exposed to Diesel Exhaust Particles.

The contributing role of environmental factors to the development of neurodegenerative diseases has become increasingly evident. Here, we report that exposure of C6 glioma cells to diesel exhaust particles (DEPs), a major constituent of urban air pollution, causes intracellular reactive oxygen species (ROS) production. In this scenario, we suggest employing the possible protective role that coffee phenolic metabolites may have. Coffee is a commonly consumed hot beverage and a major contributor to the dietary intake of (poly) phenols. Taking into account physiological concentrations, we analysed the effects of two different coffee phenolic metabolites mixes consisting of compounds derived from bacterial metabolization reactions or phase II conjugations, as well as caffeic acid. The results showed that these mixes were able to counteract DEP-induced oxidative stress. The cellular components mediating the downregulation of ROS included extracellular signal-regulated kinase 1/2 (ERK1/2), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and uncoupling protein 2 (UCP2). Contrary to coffee phenolic metabolites, the treatment with N-acetylcysteine (NAC), a known antioxidant, was found to be ineffective in preventing the DEP exposure oxidant effect. These results revealed that coffee phenolic metabolites could be promising candidates to protect against some adverse health effects of daily exposure to air pollution.

Bicarbonate induces membrane reorganization and CBR1 and TRPV1 endocannabinoid receptor migration in lipid microdomains in capacitating boar spermatozoa.

Mammalian spermatozoa acquire full fertilizing ability only after a morphofunctional maturation called "capacitation." During this process the high level of bicarbonate present within the upper female genital tract or in culture medium induces a marked reorganization of sperm membranes characterized by a biphasic behavior: In a few minutes, it promotes membrane phospholipid scrambling preliminary to the apical translocation of sterol that, 2-4 h later, enables spermatozoa to recognize zona pellucida after albumin-mediated cholesterol extraction. In the present research it was demonstrated that spermatozoa incubated with bicarbonate in protein-free media underwent a marked reorganization of lipid microdomains present in a detergent-resistant membrane fraction (DRM) isolated by ultracentrifugation on sucrose density gradient. In fact, bicarbonate exposed sperm (ES) cells, compared with ejaculated spermatozoa (nonexposed sperm [nES] cells), displayed an increase in protein DRM content and, in particular, in Cav-1 and CD55, markers of caveolae and lipid rafts, as well in acrosin-2, a marker of the outer acrosomal membrane (OAM). Moreover, the amount of certain proteins involved in capacitation, such as the endocannabinoid system receptors cannabinoid receptor type 1 (CBR1) and transient receptor potential cation channel 1 (TRPV1), increased in DRM obtained from ES. These data allow us to hypothesize that sperm membrane reorganization takes place even in the absence of extracellular proteins; that not only the plasma membrane but also the OAM participate in this process; and that important molecules playing a key role in inside-out signaling, such as the endocannbinoid receptors TRPV1 and CBR1, are involved in this event, with potentially important consequences on sperm function.

Graphene Oxide Improves in vitro Fertilization in Mice With No Impact on Embryo Development and Preserves the Membrane Microdomains Architecture.

During the latest years, human infertility worsened all over the world and is nowadays reputed as a global public health issue. As a consequence, the adoption of Assisted Reproductive Technologies (ARTs) such as In Vitro Fertilization (IVF) is undergoing an impressive increase. In this context, one of the most promising strategies is the innovative adoption of extra-physiological materials for advanced sperm preparation methods. Here, by using a murine model, the addition of Graphene Oxide (GO) at a specific concentration has demonstrated to increase the spermatozoa fertilizing ability in an IVF assay, finding that 0.5 µg/ml GO addition to sperm suspensions before IVF is able to increase both the number of fertilized oocytes and embryos created with a healthy offspring given by Embryo Transplantation (ET). In addition, GO treatment has been found more effective than that carried out with methyl-ß-cyclodextrin, which represents the gold standard in promoting in vitro fertility of mice spermatozoa. Subsequent biochemical characterization of its interaction with male gametes has been additionally performed. As a result, it was found that GO exerts its positive effect by extracting cholesterol from membranes, without affecting the integrity of microdomains and thus preserving the sperm functions. In conclusion, GO improves IVF outcomes in vitro and in vivo, defining new perspectives for innovative strategies in the treatment of human infertility.

Characterization of prion protein-enriched domains, isolated from rat cerebellar granule cells in culture.

The biological functions of prion protein (PrP(C)) and its possible interaction with other specific molecular membrane partners remain largely unknown. The aim of this study is to gain information on the molecular environment of PrP(C) by analyzing the lipid and protein composition of a PrP(C)-enriched membrane subfraction, called prion domain, PrD. This domain was obtained by immunoprecipitation of detergent-resistant microdomains (DRM) of rat cerebellar granule cells under conditions designed to preserve lipid-mediated membrane organization. The electrophoretic pattern of PrD, after staining with Coomassie blue, showed the enrichment of some protein bands in comparison with DRM. microLiquid chromatography-electrospray ionization-mass spectrometry (microLC-ESI-MS)/MS analysis showed that Thy-1 and different types of myosin were strongly enriched in PrD and, in a lesser extent, also OBCAM, LSAMP and tubulin, present altogether in a single band. Experiments using the chemical cross-linker BS(3) suggested the existence of an interaction between PrP(C) and neural cell adhesion molecule (NCAM). Concerning lipids, the comparison between PrD and DRM showed a similar phospholipid/sphingolipid ratio, a phospholipid/cholesterol ratio doubled, and a strong decrease of plasmenilethanolamine (19.7 +/- 3.5% vs. 38.3 +/- 1.2%). In conclusion, the peculiar lipid composition and in particular the presence of proteins involved in synaptic plasticity, cell adhesion, cytoskeleton regulation and signalling, suggest an important physiological role in neurons of Prion Domain.

Graphene Oxide increases mammalian spermatozoa fertilizing ability by extracting cholesterol from their membranes and promoting capacitation.

Graphene Oxide (GO) is a widely used biomaterial with an amazing variety of applications in biology and medicine. Recently, we reported the ability of GO to improve the in vitro fertilization (IVF) outcomes in swine, a validated animal model with a high predictive value for human fertility. For that reason, here we characterized the mechanisms involved in this positive interaction by adopting an experimental approach combining biological methods (confocal microscopy analysis on single cell, flow cytometry on cell populations and co-incubation with epithelial oviductal cells), physical-chemical techniques (Differential Scanning Calorimetry and Thermogravimetric Analysis), and chemical methods (mass spectrometry and lipid measurement). As a result, we propose a model in which GO is able to extract cholesterol from the spermatozoa membrane without causing any detrimental effect. In this way, the cholesterol extraction promotes a change in membrane chemical-physical properties that could positively affect male gamete function, modulating sperm signalling function and increasing in this way the fertilizing potential, without losing the ability to physiologically interact with the female environment. In conclusion, these data seem to suggest new intriguing possibilities in engineering sperm membrane for improving assisted reproduction technologies outcomes, even in human medicine.

Hypoxia-induced modifications in plasma membranes and lipid microdomains in A549 cells and primary human alveolar cells.

We evaluated the response to mild hypoxia exposure of A549 alveolar human cells and of a continuous alveolar cell line from human excised lungs (A30) exposed to 5% O(2) for 5 and 24 h. No signs of increased peroxidation and of early apoptosis were detected. After 24 h of hypoxia total cell proteins/DNA ratio decreased significantly by about 20%. Similarly, we found a decrease in membrane phospholipid and cholesterol content. The membrane fluidity assessed by fluorescence anisotropy measurements was unchanged. We also prepared the detergent resistant membrane fraction (DRM) to analyze the distribution of the two types of lipid microdomains, caveolae and lipid rafts. The DRM content of Cav-1, marker of caveolae, was decreased, while CD55, marker of lipid rafts, increased in both cell lines. Total content of these markers in the membranes was unchanged indicating remodelling of their distribution between detergent-resistant and detergent-soluble fraction of the cellular membrane. The changes in protein markers distribution did not imply changes in the corresponding mRNA, except in the case of Cav-1 for A30 line. In the latter case we found a parallel decrease in Cav-1 and in the corresponding mRNA. We conclude that an exposure to a mild degree of hypoxia triggers a significant remodelling of the lipid microdomains expression, confirming that they are highly dynamic structures providing a prompt signalling platform to changes of the pericellular microenvironment.

Ischemic Conditions Affect Rerouting of Tau Protein Levels: Evidences for Alteration in Tau Processing and Secretion in Hippocampal Neurons.

The spreading of misfolded protein species contributes to the propagation of harmful mediators in proteinopathies, including Alzheimer's disease (AD). Cellular stress circumstances, such as abnormal protein accumulation or nutrient deprivation, elicit the secretion of soluble misprocessed proteins and insoluble aggregates via multiple mechanisms of unconventional secretion. One of them consists in the rerouting of autophagic vacuoles towards exocytosis, an unconventional type of autophagy mediated by caspase-3 activation under starvation. Ischemic injury is a starvation condition characterized by oxygen/nutrient deprivation, whose contribution in AD onset has definitely been endorsed. Thus, we investigated the effect of oxygen-glucose deprivation (OGD), an experimental condition mimicking cerebral ischemia, in search of alteration in Tau processing and secretion in hippocampal neurons primary cultures. Our results showed that OGD caused alterations in Tau phosphorylation and processing, paralleled by an induction of its secretion. Interestingly, together with caspase-3 activation, full-length (FL) and fragmented Tau forms were secreted by their own or through a heterogeneous population of microvesicles (MVs), including autophagosome marker LC3-positive vesicles. Accordingly, confocal microscopy revealed a partial colocalization of intracellular Tau and LC3. Summarizing, our findings indicate that OGD alters Tau intracellular levels and protein processing. Consequently, Tau clearance was stimulated through multiple mechanisms related to unconventional Tau secretion, including exophagy. However, the activation of this response represent a double edge sword, because it could contribute to the spreading of misfolded Tau, a neurodegeneration pathway in AD and other tauopathies.

Early evidence of stress in immortalized neurons exposed to diesel particles: the role of lipid reshaping behind oxidative stress and inflammation.

Diesel combustion is the major source of fine particle road emission, whose solid fraction is represented by diesel exhaust particles (DEP). Many studies indicate the contribution of DEP to the onset of different neurological diseases, such as Alzheimer's disease (AD), identifying oxidative stress and neuroinflammation as two cardinal processes of brain damage. This study aimed to investigate the effects of different concentrations of DEP (10 µg/ml and 50 µg/ml) on the mouse HT22 cells treated for 3 h or 24 h. Our results demonstrated that DEP contributed to an increased oxidative stress, defined by overexpression of HO-1, Hsp70 and Cyp1b1 protein levels. Moreover, an inflammatory-related processes were also observed, as COX-2 and iNOS levels were higher in treated cells when compared to the control. Furthermore, our investigations highlighted the alteration of fatty acid composition, total cholesterol content in cells and media, and of membrane fluidity, suggesting a lipid reshaping after DEP treatment. Finally, we detected APP and BACE1 increase after 24 h of treatment with 50 µg/ml of DEP. Indeed, our results propose a role of acute exposure in the onset of a deleterious mechanism for AD neurodegeneration, even though no differences were observed in p-APP Thr668 levels, BACE1 activity and APP C-terminal fragment beta amount.

Aminopurvalanol A, a Potent, Selective, and Cell Permeable Inhibitor of Cyclins/Cdk Complexes, Causes the Reduction of in Vitro Fertilizing Ability of Boar Spermatozoa, by Negatively Affecting the Capacitation-Dependent Actin Polymerization.

The adoption of high-througput technologies demonstrated that in mature spermatozoa are present proteins that are thought to be not present or active in sperm cells, such as those involved in control of cell cycle. Here, by using an in silico approach based on the application of networks theory, we found that Cyclins/Cdk complexes could play a central role in signal transduction active during capacitation. Then, we tested this hypothesis in the vitro model. With this approach, spermatozoa were incubated under capacitating conditions in control conditions (CTRL) or in the presence of Aminopurvalanol A a potent, selective and cell permeable inhibitor of Cyclins/Cdk complexes at different concentrations (2, 10, and 20 µM). We found that this treatment caused dose-dependent inhibition of sperm fertilizing ability. We attribute this event to the loss of acrosome integrity due to the inhibition of physiological capacitation-dependent actin polymerization, rather than to a detrimental effect on membrane lipid remodeling or on other signaling pathways such as tubulin reorganization or MAPKs activation. In our opinion, these data could revamp the knowledge on biochemistry of sperm capacitation and could suggest new perspectives in studying male infertility.

Diesel exhaust particles (DEP) pre-exposure contributes to the anti-oxidant response impairment in hCMEC/D3 during post-oxygen and glucose deprivation damage.

Recently, air pollution has been identified as a significant modifiable risk factor to the increasing stroke burden. Diesel exhaust particles, characterized by high polycyclic aromatic hydrocarbons content, constitute an important component of outdoor air pollution and is known to cause oxidative stress, and could therefore contribute to and exacerbate the effects of ROS in post-ischemic injury. hCMEC/D3 cells have been submitted to 48h treatment with diesel exhaust particles (25µg/ml and 50µg/ml, DEP50) or alternatively to 3h of oxygen and glucose deprivation, followed by 1h of oxygen and glucose restoration. The combined treatment consisted in 48h of diesel exhaust particles (25µg/ml and 50µg/ml, DEP50) followed by 3h of oxygen and glucose deprivation and 1h of restoration. A panel of markers related to oxidative stress and inflammatory responses, such as transcription factors (Nrf2 and HIF-1α), anti-oxidant proteins (HO-1, SOD-1, Hsp70) and proteins potentially inducing further oxidative-stress or inflammation (Cyp1b1, iNOS, COX-2, TNF-α, IL-1α, IL-1ß, IL-8, VEGF), have been examined. Data obtained showed that diesel exhaust particles and oxygen and glucose deprivation treatments alone elicited the antioxidants response, each by means of a different transcription factor, while the combined treatment led to a dysregulation of the antioxidant response during ischemic injury reperfusion.

Biochemical and morphological changes in endothelial cells in response to hypoxic interstitial edema.

BACKGROUND: A correlation between interstial pulmonary matrix disorganization and lung cellular response was recently documented in cardiogenic interstitial edema as changes in the signal-cellular transduction platforms (lipid microdomains: caveoale and lipid rafts). These findings led to hypothesize a specific "sensing" function by lung cells resulting from a perturbation in cell-matrix interaction. We reason that the cell-matrix interaction may differ between the cardiogenic and the hypoxic type of lung edema due to the observed difference in the sequential degradation of matrix proteoglycans (PGs) family. In cardiogenic edema a major fragmentation of high molecular weight PGs of the interfibrillar matrix was found, while in hypoxia the fragmentation process mostly involved the PGs of the basement membrane controlling microvascular permeability. Based on these considerations, we aim to describe potential differences in the lung cellular response to the two types of edema. METHODS: We analysed the composition of plasma membrane and of lipid microdomains in lung tissue samples from anesthetized rabbits exposed to mild hypoxia (12 % O2 for 3-5 h) causing interstitial lung edema. Lipid analysis was performed by chromatographic techniques, while protein analysis by electrophoresis and Western blotting. Lipid peroxidation was assessed on total plasma membranes by a colorimetric assay (Bioxytech LPO-586, OxisResearch). Plasma membrane fluidity was also assessed by fluorescence. Lipid microdomains were isolated by discontinuous sucrose gradient. We also performed a morphometric analysis on lung cell shape on TEM images from lung tissue specimen. RESULTS: After hypoxia, phospholipids content in plasma membranes remained unchanged while the cholesterol/phospholipids ratio increased significantly by about 9% causing a decrease in membrane fluidity. No significant increase in lipid peroxidation was detected. Analysis of lipid microdomains showed a decrease of caveolin-1 and AQP1 (markers of caveolae), and an increase in CD55 (marker of lipid rafts). Morphometry showed a significant decrease in endothelial cell volume, a marked increase in the cell surface/volume ratio and a decrease in caveolar density; epithelial cells did not show morphological changes. CONCLUSION: The biochemical, signaling and morphological changes observed in lung endothelial cell exposed to hypoxia are opposite to those previously described in cardiogenic edema, suggesting a differential cellular response to either type of edema.

Involvement of MEK-ERK1-2 pathway in the anti-oxidant response in C6 glioma cells after diesel exhaust particles exposure.

Ultrafine particles translocate to the central nervous system and activate oxidative stress-related pathways. The transcription factor Nrf2 activation by ERK1-2 has been suggested as a key regulator of cellular response to oxidative stress. C6 glioma cells have been treated with different doses of diesel exhaust particles (25µg/ml, DEP25, and 50µg/ml, DEP50), for different times. Cells have been screened for oxidative stress and inflammatory markers, and for the activation of the MEK-ERK1-2 pathway. The same markers have been examined after inhibition of MEK, the kinase upstream to ERK1-2. 3h and 24h of DEP25 and DEP50 induced a significant increase in HO-1 levels. After 24h, DEP25 and DEP50 induced an increase in HO-1 and Cyp1b1 levels, while increase in OGG1 level was observed only with DEP25. After 5h of treatment with DEP25, ERK1-2 resulted phosphorylated, concomitantly with a significant increase in HO-1 levels, no changes in iNOS levels, and decreased levels of anti-oxidant enzymes. After treatment with MEK inhibitor U0126, ERK1-2 showed no activation, with a consequent decrease in Nrf2, no increase in HO-1 and a significant increase of iNOS. MEK inhibitor is able to deplete anti-oxidant enzymes. In conclusion, the MEK-ERK1-2 pathway is involved in regulating the anti-oxidant strategies to compensate the oxidative status induced by DEP treatment.

Diets with different lipid contents do not modify the neuronal membrane lipid raft profile in a scrapie murine model.

UNLABELLED: In Transmissible Spongiform Encephalopathies (TSEs), the localization of the prion protein in the neuronal membrane lipid rafts (LR) seems to play a role in sustaining the protein misfolding. Changes in membrane properties, due to altered lipid composition, affect their organization and interaction between lipids and protein therein, and consequently also membrane resident protein functionality; dietary polyunsaturated fatty acids (PUFAs), gangliosides and cholesterol seem to influence these processes. AIMS: In this work, the influence of administration of different feed, able to change the composition of lipid membrane, on the clinical progression of prion disease was studied. MAIN METHODS: The activity of three diets (hyperlipidic with 6% fats; hypolipidic with 0.1% fats; and purified with 4% fats) was tested in CD1 mouse model experimentally infected with RML scrapie strain. Presence and distribution of typical central nervous system (CNS) lesions and deposits of PrP(sc) were evaluated by histopathological analysis and immunohistochemistry. Analysis of lipids was performed in homogenate and insoluble brain fraction of the neuronal membrane rich in LR. KEY FINDINGS: Results show that a diet with a different lipid level has not a significant role in the development of the scrapie disease. All infected mice fed with different diets died in the same time span. Histology, immunohistochemistry, and neuropathological analyses of the infected brains did not show significant differences between animals subjected to different diets. SIGNIFICANCE: Independently of the diet, the infection induced a significant modification of the lipid composition in homogenates, and a less noticeable one in insoluble brain fraction.