Topical Application of M89PF Containing Vichy Mineralising Water and Probiotic Fractions Prevents Cutaneous Damage Induced by Exposure to UV and O3.

Purpose: Exposure of the skin to ultraviolet radiation (UV) or ozone (O3) results in stressed skin, leading to the alteration of the skin physical barrier and defence functions. In this work, the preventive benefit of a dermocosmetic, M89PF, containing Vichy mineralising water, probiotic fractions, antioxidant vitamins and hyaluronic acid, in the alteration of skin physical barrier and skin defence functions after exposure to O3 and UV, alone or combined, was assessed. Methods: Untreated and treated (M89PF) skin explants were exposed to O3, to UV rays or to O3+UV. Immunofluorescence was performed for skin barrier, oxidative stress, and inflammatory markers after one and four days of exposure to the pollutants. Results: M89PF significantly (p≤0.05) prevented the decrease of the expression level of different skin barrier markers, and significantly (p≤0.05) prevented the induction of OxInflammatory markers and inflammasome components by UV, O3, or both combined. Conclusion: M89PF prevents skin barrier damage, as well as oxidative stress and inflammatory markers induced by exposome factors, such as UV, O3, or both combined.

The role of potassium current in the pulmonary response to environmental oxidative stress.

Exposure of human lung epithelial cells (A549 cell line) to the oxidant pollutant ozone (O3) alters cell membrane currents inducing its decrease, when the cell undergoes to a voltage-clamp protocol ranging from -90 to +70mV. The membrane potential of these cells is mainly maintained by the interplay of potassium and chloride currents. Our previous studies indicated the ability of O3 to activate ORCC (Outward Rectifier Chloride Channel) and consequently increases the chloride current. In this paper our aim was to understand the response of potassium current to oxidative stress challenge and to identify the kind potassium channel involved in O3 induced current changes. After measuring the total membrane current using an intracellular solution with or without potassium ions, we obtained the contribution of potassium to the overall membrane current in control condition by a mathematical approach. Repeating these experiments after O3 treatment we observed a significant decrease of Ipotassium. Treatment of the cells with Iberiotoxin (IbTx), a specific inhibitor of BK channel, we were able to verify the presence and the functionality of BK channels. In addition, the administration of 4-Aminopyridine (an inhibitor of voltage dependent K channels but not BK channels) and Tetraethylammonium (TEA) before and after O3 treatment we observed the formation of BK oxidative post-translation modifications. Our data suggest that O3 is able to inhibit potassium current by targeting BK channel. Further studies are needed to better clarify the role of this BK channel and its interplay with the other membrane channels under oxidative stress conditions. These findings can contribute to identify the biomolecular pathway induced by O3 allowing a possible pharmacological intervention against oxidative stress damage in lung tissue.

The Lesson Learned from the COVID-19 Pandemic: Can an Active Chemical Be Effective, Safe, Harmless-for-Humans and Low-Cost at a Time? Evidence on Aerosolized Hypochlorous Acid.

The COVID-19 pandemic has underlined the importance of disinfectants as tools to prevent and fight against coronavirus spreading. An ideal disinfectant and sanitizer must be nontoxic to surface contact, noncorrosive, effective, and relatively inexpensive as it is hypochlorous acid (HOCl). The present work intended to evaluate, on different surfaces, the bactericidal and virucidal effectiveness of nebulized HOCl and test its safety usage in 2D and 3D skin and lung models. Our data showed that HOCl at the dose of 300 ppm did not affect cellular and tissue viability, not their morphology. The HOCl bactericidal properties varies with the surface analyzed: 69% for semi-porous, 96-99.9% for flat and porous. This discrepancy was not noticed for the virucidal properties. Overall, this study showed that nebulized HOCl can prevent virus and bacteria growth without affecting lung and skin tissues, making this compound a perfect candidate to sanitize indoor environments.

Ubiquitination as a key regulatory mechanism for O3-induced cutaneous redox inflammasome activation.

NLRP1 is one of the major inflammasomes modulating the cutaneous inflammatory responses and therefore linked to a variety of cutaneous conditions. Although NLRP1 has been the first inflammasome to be discovered, only in the past years a significant progress was achieved in understanding the molecular mechanism and the stimuli behind its activation. In the past decades a crescent number of studies have highlighted the role of air pollutants as Particulate Matter (PM), Cigarette Smoke (CS) and Ozone (O3) as trigger stimuli for inflammasomes activation, especially via Reactive Oxygen Species (ROS) mediators. However, whether NLRP1 can be modulated by air pollutants via oxidative stress and the mechanism behind its activation is still poorly understood. Here we report for the first time that O3, one of the most toxic pollutants, activates the NLRP1 inflammasome in human keratinocytes via oxidative stress mediators as hydrogen peroxide (H2O2) and 4-hydroxy-nonenal (4HNE). Our data suggest that NLRP1 represents a target protein for 4HNE adduction that possibly leads to its proteasomal degradation and activation via the possible involvement of E3 ubiquitin ligase UBR2. Of note, Catalase (Cat) treatment prevented inflammasome assemble and inflammatory cytokines release as well as NLRP1 ubiquitination in human keratinocytes upon O3 exposure. The present work is a mechanistic study that follows our previous work where we have showed the ability of O3 to induce cutaneous inflammasome activation in humans exposed to this pollutant. In conclusion, our results suggest that O3 triggers the cutaneous NLRP1 inflammasome activation by ubiquitination and redox mechanism.

Dimethyl Fumarate-Loaded Transethosomes: A Formulative Study and Preliminary Ex Vivo and In Vivo Evaluation.

In this study, transethosomes were investigated as potential delivery systems for dimethyl fumarate. A formulative study was performed investigating the effect of the composition of transethosomes on the morphology and size of vesicles, as well as drug entrapment capacity, using cryogenic transmission electron microscopy, photon correlation spectroscopy, and HPLC. The stability of vesicles was evaluated, both for size increase and capability to control the drug degradation. Drug release kinetics and permeability profiles were evaluated in vitro using Franz cells, associated with different synthetic membranes. The in vitro viability, as well as the capacity to improve wound healing, were evaluated in human keratinocytes. Transmission electron microscopy enabled the evaluation of transethosome uptake and intracellular fate. Based on the obtained results, a transethosome gel was further formulated for the cutaneous application of dimethyl fumarate, the safety of which was evaluated in vivo with a patch test. It was found that the phosphatidylcholine concentration affected vesicle size and lamellarity, influencing the capacity to control dimethyl fumarate's chemical stability and release kinetics. Indeed, phosphatidylcholine 2.7% w/w led to multivesicular vesicles with 344 nm mean size, controlling the drug's chemical stability for at least 90 days. Conversely, phosphatidylcholine 0.9% w/w resulted in 130 nm sized unilamellar vesicles, which maintained 55% of the drug over 3 months. These latest kinds of transethosomes were able to improve wound healing in vitro and were easily internalised by keratinocytes. The selected transethosome gel, loading 25 mg/mL dimethyl fumarate, was not irritant after cutaneous application under occlusion, suggesting its possible suitability in the treatment of wounds caused by diabetes mellitus or peripheral vascular diseases.

Inflammasome involvement in CS-induced damage in HaCaT keratinocytes.

Cigarette smoke (CS) alters cutaneous biological processes such as redox homeostasis and inflammation response that might be involved in promoting skin inflammatory conditions. Exposure to CS has also been linked to a destabilization of the NLRP3 inflammasome in pollution target tissues such as the lung epithelium, resulting in a more vulnerable immunological response to several exogenous and endogenous stimuli related to oxidative stress. Thus, CS has an adverse effect on host defense, increasing the susceptibility to develop lung infections and pathologies. In the skin, another direct target of pollution, inflammasome disorders have been linked to an increasing number of diseases such as melanoma, psoriasis, vitiligo, atopic dermatitis, and acne, all conditions that have been connected directly or indirectly to pollution exposure. The inflammasome machinery is an important innate immune sensor in human keratinocytes. However, the role of CS in the NLRP1 and NLRP3 inflammasome in the cutaneous barrier has still not been investigated. In the present study, we were able to determine in keratinocytes exposed to CS an increased oxidative damage evaluated by 4-HNE protein adduct and carbonyl formation. Of note is that, while CS inhibited NLRP3 activation, it was able to activate NLRP1, leading to an increased secretion of the proinflammatory cytokines IL-1ß and IL-18. This study highlights the importance of the inflammasome machinery in CS that more in general, in pollution, affects cutaneous tissues and the important cross-talk between different members of the NLRP inflammasome family.

Evaluation of oxidative damage and Nrf2 activation by combined pollution exposure in lung epithelial cells.

The lungs are one the main organs exposed to environmental pollutants, such as tropospheric ozone (O3) and particulate matter (PM), which induce lung pathologies through similar mechanisms, resulting in altered redox homeostasis and inflammation. Although numerous studies have investigated the effects of these pollutants in the respiratory tract, there are only a few evidences that have evaluated the combined effects of outdoor stressors, despite the fact that humans are consistently exposed to more pollutants simultaneously. In this study, we wanted to investigate whether exposure to PM and O3 could have an additive, noxious effect in lung epithelial cells by measuring oxidative damage and the activity of redox-sensitive nuclear factor erythroid 2-related factor 2 (Nrf2) which is a master regulator of cellular antioxidant defenses. First, we measured the cytotoxic effects of O3 and PM individually and in combination. We observed that both pollutants alone increased LDH release 24 h post-exposure. Interestingly, we did observe via TEM that combined exposure to O3 and PM resulted in increased cellular penetration of PM particles. Furthermore, we found that levels of 4-hydroxy-nonenal (4HNE), a marker of oxidative damage, significantly increased 24 h post-exposure, in response to the combined pollutants. In addition, we observed increased levels of Nrf2, in response to the combined pollutants vs. either pollutant, although this effect was not followed by the increase in Nrf2-responsive genes expression HO1, SOD1, GPX, or GR nor enzymatic activity. Despite these observations, our study suggests that O3 exposure facilitate the cellular penetration of the particles leading to an increased oxidative damage, and additive defensive response.

Ethosomes for Coenzyme Q10 Cutaneous Administration: From Design to 3D Skin Tissue Evaluation.

Ethosome represents a smart transdermal vehicle suitable for solubilization and cutaneous application of drugs. Coenzyme Q10 is an endogenous antioxidant whose supplementation can counteract many cutaneous disorders and pathologies. In this respect, the present study describes the production, characterization, and cutaneous protection of phosphatidylcholine based ethosomes as percutaneous delivery systems for coenzyme Q10. CoQ10 entrapment capacity in ethosomes was almost 100%, vesicles showed the typical 'fingerprint' structure, while mean diameters were around 270 nm, undergoing an 8% increase after 3 months from production. An ex-vivo study, conducted by transmission electron microscopy, could detect the uptake of ethosomes in human skin fibroblasts and the passage of the vesicles through 3D reconstituted human epidermis. Immunofluorescence analyses were carried on both on fibroblasts and 3D reconstituted human epidermis treated with ethosomes in the presence of H2O2 as oxidative stress challenger, evaluating 4-hydroxynonenal protein adducts which is as a reliable biomarker for oxidative damage. Notably, the pretreatment with CoQ10 loaded in ethosomes exerted a consistent protective effect against oxidative stress, in both models, fibroblasts and in reconstituted human epidermis respectively.

Alterations of mitochondrial bioenergetics, dynamics, and morphology support the theory of oxidative damage involvement in autism spectrum disorder.

Autism spectrum disorder (ASD) has been hypothesized to be a result of the interplay between genetic predisposition and increased vulnerability to early environmental insults. Mitochondrial dysfunctions appear also involved in ASD pathophysiology, but the mechanisms by which such alterations develop are not completely understood. Here, we analyzed ASD primary fibroblasts by measuring mitochondrial bioenergetics, ultrastructural and dynamic parameters to investigate the hypothesis that defects in these pathways could be interconnected phenomena responsible or consequence for the redox imbalance observed in ASD. High levels of 4-hydroxynonenal protein adducts together with increased NADPH (nicotinamide adenine dinucleotide phosphateoxidase) activity and mitochondrial superoxide production coupled with a compromised antioxidant response guided by a defective Nuclear Factor Erythroid 2-Related Factor 2 pathway confirmed an unbalanced redox homeostasis in ASD. Moreover, ASD fibroblasts showed overactive mitochondrial bioenergetics associated with atypical morphology and altered expression of mitochondrial electron transport chain complexes and dynamics-regulating factors. We suggest that many of the changes observed in mitochondria could represent compensatory mechanisms by which ASD cells try to adapt to altered energy demand, possibly resulting from a chronic oxinflammatory status.

Proinflammatory properties and oxidative effects of atmospheric particle components in human keratinocytes.

The skin is one of the main organs exposed to airborne particulate matter (PM), which may contain various pollutants linked to a wide range of adverse health endpoints. In the present work, we analyzed the proinflammatory and oxidative effects of some PM components leading to inflammatory responses, cell proliferation or cell death. We investigated four redox-active chemicals, such as Cu (II) metal and quinones generated from polycyclic aromatic hydrocarbons (PAHs), i.e., 9,10 phenanthrenequinone and isomers 1,2 and 1,4 naphthoquinone. We performed in vitro biological tests on human keratinocyte (HaCaT) cells and also acellular assays based on the oxidation of dithiothreitol and ascorbic acid, antioxidants to assess the oxidative potential (OP). We found that treated keratinocytes showed increased activation of the redox-sensitive transcription factor NFκB and increased transcript levels of the NFκB-dependent gene IL8. Moreover, the treatment with Cu(II) and quinones increased the activities and the expression of genes involved in the redox response, SOD1 and GPX, suggesting that PM components induced cellular damage due to redox imbalances. Finally, we found alteration of the mitochondrial ultrastructure and increased apoptosis after 24 h of treatment. The results presented suggest that all of the analyzed pollutant components are able to modulate similar signal transduction pathways, resulting in activation of inflammatory processes in the skin, followed by oxidative damage. Altogether these observations indicate that exposure of skin to air pollutants modifies the redox equilibrium of keratinocytes, which could explain the increased skin damage observed in populations that live in high-pollution cities.

Involvement of the TREK-1 channel in human alveolar cell membrane potential and its regulation by inhibitors of the chloride current.

K+ channels of the alveolar epithelium control the driving force acting on the ionic and solvent flow through the cell membrane contributing to the maintenance of cell volume and the constitution of epithelial lining fluid. In the present work, we analyze the effect of the Cl- channel inhibitors: (4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-inden-5-yl)oxy] butanoic acid (DCPIB) and 9-anthracenecarboxylic acid (9-AC) on the total current in a type II pneumocytes (A549 cell line) model by patch clamp, immunocytochemical, and gene knockdown techniques. We noted that DCPIB and 9-AC promote the activation of K conductance. In fact, they significantly increase the intensity of the current and shift its reversal potential to values more negative than the control. By silencing outward rectifier channel in its anoctamin 6 portion, we excluded a direct involvement of Cl- ions in modulation of IK and, by means of functional tests with its specific inhibitor spadin, we identified the TREK-1 channel as the presumable target of both drugs. As the activity of TREK-1 has a key role for the correct functioning of the alveolar epithelium, the identification of DCPIB and 9-AC molecules as its activators suggests their possible use to build new pharmacological tools for the modulation of this channel.

SR-B1 involvement in keratinocytes in vitro wound closure.

Skin represents the most extended organ of human body, having as main function the protection of our body from outdoor stressors. Its protective ability is compromised when the skin is disrupted as a consequence of mechanical insults. For this purpose, cutaneous tissue is equipped with an efficient and fine mechanism involved in repairing the wounded area. Among the numerous players that take part in the wound healing process, SR-B1 has been recently shown to have a role in keratinocyte re-epithelialization. SR-B1 is a mediator of cholesterol uptake from HDLs, whereas it is implicated in other cellular processes such as vitamins absorption, vesicle trafficking or pathogen identification. The aim of this study was to investigate the mechanisms involved in SR-B1 role in skin wound closure. Our in vitro data demonstrated that SR-B1 influenced keratinocyte proliferation and migration through a downregulation of nuclear cyclin D1 levels and active MMP9 expression respectively possibly in an NF-kB-dependent mechanism. In addition, SR-B1 was also able to modulate keratinocyte morphology into a pro-migratory cytoskeleton rearrangement. The present in vitro study suggests a new role of SRB1 as a possible new key player in cutaneous wound healing mechanism.

Tropospheric ozone affects SRB1 levels via oxidative post-translational modifications in lung cells.

Exposure to air pollution is associated with increased respiratory morbidities and susceptibility to lung dysfunction. Ozone (O3) is commonly recognized as one of the most noxious air pollutant and has been associated with several lung pathologies. It has been demonstrated that decreased lung disorder severity and incidence are connected with the consumption of a diet rich in fruits and vegetables, suggesting that higher intake of dietary micronutrients and phytoactive compounds can be beneficial. However, dietary supplementation - i.e. vitamin E (α-tocopherol) or vitamin A - has not always been effective in improving pulmonary function. Recently, research on the role of nutritional antioxidants on human health has focused more on studying their uptake at the cellular level rather than their effective ability to scavenge reactvive oxygen species (ROS). The Scavenger Receptor B1 (SRB1) has been shown to play a prominent role in the uptake, delivery and regulation of vitamin E in the lung. Given the importance of SRB1 in maintaining lung tissue in a healthy condition, we hypothesize that its expression could be modulated by pollution exposure, which thus could indirectly affect the uptake and/or delivery of lipophilic substances, such as vitamin E. To characterize the molecular mechanism involved in the redox modulation of SRB1, its cellular levels were assessed in human alveolar epithelial cells after O3 exposure. The results demonstrated that O3 induced the loss of SRB1 protein levels. This decline seems to be driven by hydrogen peroxide (H2O2) as a consequence of an increased activation of cellular NADPH oxidase (NOX), as demonstrated by the use of NOX inhibitors or catalase that reversed this effect. Furthermore, O3 caused the formation of SRB1-aldheyde adducts (4-hydroxy-2-nonenal) and the consequent increase of its ubiquitination, a mechanism that could account for SRB1 protein loss.

Production and Characterization of Nanoparticle Based Hyaluronate Gel Containing Retinyl Palmitate for Wound Healing.

BACKGROUND: Wound healing is a biological process that can get in a state of pathologic inflammation, requiring the use of specific medications able to promote proper tissue repair. OBJECTIVE: The study describes the production and characterization of nanoparticle based gel for wound healing treatment designed to deliver hyaluronic acid and retinyl palmitate onto the skin. METHODS: Tristearin solid lipid nanoparticles and nanostructured lipid carriers based on a tristearin and caprylic/capric triglyceride mixture were produced and characterized. Gel spreadability and viscosity were investigated. Drug diffusion and "in vitro" wound healing were assessed by Franz cell and scratch wound assay in keratinocytes. RESULTS: Cryogenic transmission electron microscopy evidenced flat discoid nanoparticles. Photon correlation spectroscopy analysis indicated homogeneous dimensional distribution and mean diameter 132±46 nm. X-ray evidenced a lamellar inner structure of lipid nanoparticles. Nanostructured lipid carriers, being based on a heterogeneous solid/ liquid lipid mixture, could better solubilize retinyl palmitate and control its stability. The hyaluronic acid directly added into nanoparticles' dispersion enabled to obtain a shear-thinning gel suitable for cutaneous administration. Retynil palmitate diffusion was slower from the nanoparticulate gel with respect to the plain nanoparticle dispersion. The "wound healing" effect of nanoparticulate gel containing retinyl palmitate and hyaluronic acid, analyzed in HaCaT cells, showed significant differences in wounded areas between treated and control cells during the first 24 h postwounding suggesting a synergic effect of retinyl palmitate and hyaluronic acid in "in vitro" wound healing. CONCLUSIONS: This study suggests that a nanoparticle based hyaluronate gel containing retinyl palmitate can be efficiently used for wound healing.

Role of Nrf2 in preventing oxidative stress induced chloride current alteration in human lung cells.

The lung tissue is one of the main targets of oxidative stress due to external sources and respiratory activity. In our previous work, we have demonstrated in that O3 exposure alters the Cl- current-voltage relationship, with the appearance of a large outward rectifier component mainly sustained by outward rectifier chloride channels (ORCCs) in human lung epithelial cells (A549 line). In the present study, we have performed patch clamp experiments, in order to identify which one of the O3 byproducts (4hydroxynonenal (HNE) and/or H2 O2 ) was responsible for chloride current change. While 4HNE exposition (up to 25 µM for 30' before electrophysiological analysis) did not reproduce O3 effect, H2 O2 produced by glucose oxidase 10 mU for 24 hr before electrophysiological analysis mimicked O3 response. This result was confirmed treating the cell with catalase (CAT) before O3 exposure (1,000 U/ml for 2 hr): CAT was able to rescue Cl- current alteration. Since CAT is regulated by Nrf2 transcription factor, we pre-treated the cells with the Nrf2 activators, resveratrol and tBHQ. Immunochemical and immunocytochemical results showed Nrf2 activation with both substances that lead to prevent OS effect on Cl- current. These data bring new insights into the mechanisms involved in OS-induced lung tissue damage, pointing out the role of H2 O2 in chloride current alteration and the ability of Nfr2 activation in preventing this effect.

Keratinocytes oxidative damage mechanisms related to airbone particle matter exposure.

Epidemiological evidences have correlated airbone particulate matter (PM) to adverse health effects, mainly linking to pulmonary and cardiovascular disease. Nevertheless, only recently, some studies reported detrimental effects of PM on other organs such as skin. In a recent work, we have reported increased oxidative and inflammatory responses in Reconstituted Human Epidermis (RHE) exposed to ambient particles (CAPs) and we also demonstrated the ability of CAPs to penetrate the skin tissue. The present study was aimed to better understand the cellular mechanisms beyond the oxidative changes induced by CAPs (5-10-25µg/mL) in human immortalized keratinocytes (HaCaT). After 24h of treatment, CAPs were able to enter the cells leading to a decrease in viability, increased levels of 4-hydroxinonenal products (4-HNE) and IL-1α release. Overall these data, suggest lipid and protein oxidative damage, as well as an increase of inflammatory response after being challenged with CAPs. In addition, 3h after CAPs exposure we found a significant increase in NF-kB and Nrf2 translocation into the nucleus. In contrast, no differences in gene expression and enzymatic activity of Nrf2 target genes were detected. This last finding could be explained by the ability of CAPs to possibly alter the binding of Nrf2 to the ARE DNA sequence.

Potassium Ascorbate with Ribose: Promising Therapeutic Approach for Melanoma Treatment.

While surgery is the definitive treatment for early-stage melanoma, the current therapies against advanced melanoma do not yet provide an effective, long-lasting control of the lesions and a satisfactory impact on patient survival. Thus, research is also focused on novel treatments that could potentiate the current therapies. In the present study, we evaluated the effect of potassium ascorbate with ribose (PAR) treatment on the human melanoma cell line, A375, in 2D and 3D models. In the 2D model, in line with the current literature, the pharmacological treatment with PAR decreased cell proliferation and viability. In addition, an increase in Connexin 43 mRNA and protein was observed. This novel finding was confirmed in PAR-treated melanoma cells cultured in 3D, where an increase in functional gap junctions and a higher spheroid compactness were observed. Moreover, in the 3D model, a remarkable decrease in the size and volume of spheroids was observed, further supporting the treatment efficacy observed in the 2D model. In conclusion, our results suggest that PAR could be used as a safe adjuvant approach in support to conventional therapies for the treatment of melanoma.

Tooth wear pattern analysis in a sample of Italian Early Bronze Age population. Proposal of a 3-D sampling sequence.

OBJECTIVE: The purpose of this study was to assess the prevalence, distribution and intensity of tooth wear in a sample of an ancient Italian population in order to explain the pattern in terms of dietary habits and/or non-dietary tooth-use behaviors during the Early Bronze Age, with a focus on possible age-group and sex differences. DESIGN: Well-preserved permanent teeth of individuals from the Bronze Age site of Ballabio (Lecco) in northern Italy were examined for tooth wear by different methods. Eight 3D models of teeth at increasing severity of wear were created. RESULTS: In total, 357 permanent teeth belonging to male and female individuals were included in the study. Dental wear was present in 96.6% of the total sample. Males showed significantly greater levels of wear than females in the mandibular teeth. Both sexes exhibited a significantly different wear direction between the anterior (oblique and flat) and posterior (oblique and concave) teeth. Significant age differences were observed in the direction and level of wear in the incisors, canines and premolars, with higher wear in the older group. Complete and rotatable virtual 3D images of different wear patterns are proposed. CONCLUSIONS: The findings of the present study confirm the data from archaeological studies on this site and on northern Italian habits during the Early Bronze Age suggesting a diet rich in vegetables. The observed wear patterns can be related both to the diet of this Bronze age population, based on hard and abrasive food requiring vigorous mastication, and to sex differences in cultural practices.

SRB1 as a new redox target of cigarette smoke in human sebocytes.

For its critical location, the skin represents the major interface between the body and the environment, therefore is one of the major biological barriers against the outdoor environmental stressors. Among the several oxidative environmental stressors, cigarette smoke (CS) has been associated with the development and worsening of many skin pathologies such as acne, dermatitis, delayed wound healing, aging and skin cancer. In our previous work we have demonstrated that CS is able to affect genes involved in skin cholesterol trafficking, among which SRB1, a receptor involved in the uptake of cholesterol from HDL, seems to be very susceptible to the oxidative stress induced by CS. In the present work we wanted to investigate the presence of SRB1 in human sebocytes and whether CS can affect cholesterol cellular uptake via the redox modulation of SRB1. By using a co-culture system of keratinocytes/sebocytes, we found that CS exposure induced a SRB1 protein loss without affecting sebocytes viability. The decrease of SRB1 levels was a consequence of SRB1/HNE adducts formation that leads to SRB1 ubiquitination and degradation. Moreover, the CS-induced loss of SRB1 induced an alteration of sebocytes lipid content, also demonstrated by cholesterol quantification in SRB1 siRNA experiments. In conclusion, exposure to CS, induced SRB1 post-translational modifications in sebocytes and this might affect sebocytes/skin functionality.