The overlap between the geographic distribution of COVID-19 outbreaks and pollution levels suggested a strong correlation between exposure to atmospheric particulate matter and an increased risk of developing severe forms of disease. This correlation has been highlighted by several epidemiological studies, indicating the existence of shared molecular mechanisms. Emerging evidence has highlighted the important role of lipid rafts in facilitating viral entry into cells. Specifically, the receptor binding domain of the SARS-CoV-2 spike protein interacts with sialylated glycans of the monosialic ganglioside GM1 and GM2 that are particularly enriched in lipid rafts. This interaction has been proposed to facilitate ACE2 recognition by the spike protein and may contribute to early events involved in viral attachment and entry. Here, we reveal that A549 alveolar lung cells, after DEP exposure, exhibit a significant shift in ACE2 into lipid rafts, accompanied by an increase in the immature form of ADAM17, the sheddase responsible for ACE2 cleavage. Additionally, DEP exposure results in a significant increase in IL-6 release, while no changes were observed in IL-8 and sACE2 release. This treatment does not cause significant alterations in protein levels or membrane redistribution of COX-2 and HO-1, proteins involved in the inflammatory response and oxidative stress following exposure to air pollution, and linked to COVID-19 pathogenesis. Finally, lipidomic analysis by UHPLC-MS revealed that DEP exposure induces a significant increase in GM2 levels, and a concomitant decrease in GM1 and GM3 levels. Together, these results indicate that DEP exposure remodels lipid raft-associated molecular features in A549 cells, including ACE2 membrane redistribution, altered ganglioside composition, and increased IL-6 release. Although these changes may be relevant to cellular mechanisms associated with SARS-CoV-2 susceptibility, the present study does not directly assess viral binding, viral entry, or infection, and further functional studies are required.

Inflammatory bowel disease (IBD) incidence has increased in the last decades due to changes in dietary habits. IBDs are characterized by intestinal epithelial barrier disruption, increased inflammatory mediator production and excessive tissue injury. Since the current treatments are not sufficient to achieve and maintain remission, complementary and alternative medicine (CAM) becomes a primary practice as a co-adjuvant for the therapy. Thus, the intake of functional food enriched in vegetal extracts represents a promising nutritional strategy. This study evaluates the anti-inflammatory effects of artichoke, caihua and fenugreek vegetal extract original blend (ACFB) in an in vitro model of gut barrier mimicking the early acute phases of the disease. Caco2 cells cultured on transwell supports were treated with digested ACFB before exposure to pro-inflammatory cytokines. The pre-treatment counteracts the increase in barrier permeability induced by the inflammatory stimulus, as demonstrated by the evaluation of TEER and CLDN-2 parameters. In parallel, ACFB reduces p65NF-κB pro-inflammatory pathway activation that results in the decrement of COX-2 expression as PGE2 and IL-8 secretion. ACFB properties might be due to the synergistic effects of different flavonoids, indicating it as a valid candidate for new formulation in the prevention/mitigation of non-communicable diseases.

The overlap between the geographic distribution of COVID-19 outbreaks and pollution levels confirmed a correlation between exposure to atmospheric particulate matter (PM) and the SARS-CoV-2 pandemic. The RAS system is essential in the pathogenesis of inflammatory diseases caused by pollution: the ACE/AngII/AT1 axis activates a pro-inflammatory pathway, which is counteracted by the ACE2/Ang(1-7)/MAS axis, which activates an anti-inflammatory and protective pathway. However, ACE2 is also known to act as a receptor through which SARS-CoV-2 enters host cells to replicate. Furthermore, in vivo systems have demonstrated that exposure to PM increases ACE2 expression. In this study, the effects of acute and sub-acute exposure to ultrafine particles (UFP), originating from different anthropogenic sources (DEP and BB), on the levels of ACE2, ACE, COX-2, HO-1, and iNOS in the lungs and other organs implicated in the pathogenesis of COVID-19 were analyzed in the in vivo BALB/c male mice model. Exposure to UFP alters the levels of ACE2 and/or ACE in all examined organs, and exposure to sub-acute DEP also results in the release of s-ACE2. Furthermore, as evidenced in this and our previous works, COX-2, HO-1, and iNOS levels also demonstrated organ-specific alterations. These proteins play a pivotal role in the UFP-induced inflammatory and oxidative stress responses, and their dysregulation is linked to the development of severe symptoms in individuals infected with SARS-CoV-2, suggesting a heightened vulnerability or a more severe clinical course of the disease. UFP and SARS-CoV-2 share common pathways; therefore, in a “risk stratification” concept, daily exposure to air pollution may significantly increase the likelihood of developing a severe form of COVID-19, explaining, at least in part, the greater lethality of the virus observed in highly polluted areas.

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.

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-1β (TNF-α/IL-1) β 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.

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.

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.

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.

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-β-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.

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.

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.

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.

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.

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.

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.

Background: Short term increases in air pollution aggravate pre-existing respiratory diseases, increasing hospital admissions and leading to reduction of life expectancy in some subjects. Recently, attention has been directed to smaller particles (UFPs < 100 nm). Aim: The aim of this study is to disclose the pulmonary short-term effects induced by UFPs derived from two different anthropogenic sources (DEP, diesel exhaust particles; BC, biomass combustion derived particles). Methods: Male BALB/c mice were submitted to intratracheal instillation of 50 µg of BC or DEP in 100 µl of isotonic saline solution or 100 µl of isotonic saline solution (sham) by means of MicroSprayer® Aerosolizer system (Penn Century). After 3h, mice have been euthanized; Broncho Alveolar Lavage fluid (BALf) pellets and supernatants have been collected, and lungs were homogenated. Different pro-inflammatory, cytotoxic and oxidative stress markers have been analysed using biochemical methods such as total and differential cell counts, EF-SDS-PAGE and immunodecoration with specific antibodies, Luminex and colorimetric assays. Results: In the BALf, DEP treatment induced a significant increase in total cells count and PMNs %, while AMs % decreased. Moreover, total protein and ET-1 concentrations were higher in BALf after DEP treatment, comparing to sham. Finally, LDH activity resulted induced by BC and DEP treatment, and also MPO levels were increased by both BC and DEP. In lung parenchyma, DEP induced increased levels of HO-1, Cyp1b1 and COX2, while BC was not influent. Conclusions: DEP is more harmful than BC in inducing inflammation and oxidative stress after a single instillation in BALB/c mice, while both UFPs were equally cytotoxic.

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-1β, 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.