each poster 3' presentation + 2' discussion
SFRR-E PP 3.1 [YIA]:
Methods to measure the effect of air pollution in skin
Phuong Thao Tran | Charite-Universitätsmedizin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin | Germany
The amount of air pollutants is increasing globally and causing serious health problems; according to the World Health Organization, more than 7 million people die each year from air pollution. Pollution not only damages the lining of the lungs; it also affects skin health and has been linked to the development of skin diseases. Promotion of the development of oxidative stress can lead to premature skin aging, impaired skin barrier, pigment disturbances, and cellular damage caused by free radicals. Existing symptoms may also worsen. To date, there is no established method to clearly assess the degree of risk of skin contact or the protective effect of the substances used.
With the help of electron paramagnetic resonance (EPR) spectroscopy, a new method was established to assess the potential risk of air pollution in the skin and to verify the effectiveness of products designed to protect the skin. Spin-labeled PCA (3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy) was used to study free radical formation, with cigarette smoke as a model pollutant. To make the stress effect measurable, UVA light was used as an additional external stressor.
In addition, the method of confocal Raman microscopy was applied which allows an assessment without the use of an additional marker and external stressor.
For subjecting excised pig ear skin samples reproducibly to cigarette smoke, an exposure chamber for ex vivo and in vivo studies was developed. For quantification of the smoke exposure, the deposit of the nicotine concentration next to the investigated area was used as a marker substance. Initial studies have shown that cigarette smoke promotes the production of free radicals in the skin, and there is a positive correlation between nicotine concentration and free radical production. These results will help to establish a new way to measure pollutant effectiveness and preventive measures.
SFRR-E PP 3.2 [YIA]:
Melatonin modulates Nrf2/NFkB activity to prevent cadmium-induced H2O2 production and reductive stress in porcine pre-pubertal Sertoli cells.
Dr. Desirée Bartolini | Università degli Studi di Perugia | Italy
Melatonin (MLT) is a cytoprotection agent holding potential to prevent cadmium (Cd) toxicity, a pro-oxidant heavy metal reported to interfere with testicular function and fertility. However, its efficacy in Sertoli cells remains unexplored. Porcine Sertoli cells (SCs) were used in the present study to explore this cytoprotection function of MLT during Cd toxicity; investigated parameters included cellular levels of H2O2, redox-sensitive transcription factors such as Nrf2, c-Jun and NFkB, and downstream detoxification and antioxidant effectors, such as the H2O2-scavenging enzyme catalase (CAT) and some inducible components of the glutathione stress response system.
Cd toxicity in SCs resulted in impaired viability and function, that was associated with increased H2O2 generation and induction of reductive stress by the upregulation of Nrf2 expression and activity, cystine uptake, cellular glutathione biosynthesis and efflux, and GSTP expression, whereas cellular protein glutathionylation decreased. MLT produced a potent cytoprotection effect in Cd-exposed SCs, with significant restoration of cell viability and function, and inhibition of H2O2 generation and efflux. Mechanistically, these effects of MLT were associated with increased CAT activity and NFkB phosphorylation, and with marked reduction of Nrf2 activation and GSTP expression to confirm a restoration effect on the cellular redox; at the higher dose of Cd investigated in this study, MLT significantly induced c-Jun, xCT protein expression, and GSH biosynthesis and efflux.
In conclusion, MLT is a cytoprotective in SCs exposed to Cd toxicity with efficient activity in modulating Nrf2 and other genes important to control the cellular flux of H2O2 and the reductive stress response.
SFRR-E PP 3.4:
Mechanisms of NLRP3 inflammasome activation in macrophages by air pollution fine particulate matter (PM2.5)
Dr. Timoteo Marchini | Cardiology and Angiology, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany | Germany
Particulate matter (PM2.5) exposure aggravates cardiorespiratory diseases by inflammatory cytokine secretion from alveolar macrophages (AMs). Inhalation of silica particles and asbestos triggers NLRP3 inflammasome activation and IL-1β release. However, the mechanisms involved in NLRP3 engagement by PM2.5 remain unclear. To study this process, THP1-ASC-GFP cells were incubated for 6 or 24 h with 0, 1, 10, or 100 µg/mL of PM2.5 surrogates of variable chemical composition, including ROFA, CAPs, SRM1648a, and SRM2975. NLRP3 priming and specks formation was detected by flow cytometry after incubation with ROFA and SRM1648a, which was confirmed by imaging ROFA-exposed bone marrow derived macrophages (BMDMs) from C57BL/6 ASC-Citrine transgenic mice. No LDH release following PM2.5 stimulation indicated conserved cell viability in our experimental conditions. Increased IL-1β was detected by ELISA in cell culture supernatants of ROFA-exposed BMDMs and AMs from wild type mice, but not from Nlrp3-/- or Casp1-/- mice, or after pre-incubation with the NLRP3-specific inhibitor MCC950. Upregulation of Tnf gene expression and increased TNF-α levels in cell culture supernatants were also observed. Pre-incubation with anti-TNF-α antibody resulted in decreased IL-1β release from ROFA-exposed AMs. Moreover, increased mitochondrial O2●- production by MitoSOX fluorescence was found in ROFA-exposed BMDMs, together with decreased maximal respiration by the Seahorse MitoStress Test. Accordingly, inhibition of mitochondrial complex I site responsible for O2●- production by S1QEL resulted in decreased IL-1β levels in ROFA-exposed BMDMs. K+ efflux contribution on NLRP3 activation was evident in ROFA-exposed BMDMs incubated with increasing concentrations of KCl. Lysosomal leakage in BMDMs was also observed after ROFA exposure. In conclusion, PM2.5 induces NLRP3 inflammasome priming and activation in macrophages, leading to inflammatory mediator release. TNF-α, mitochondrial O2●- production, K+ efflux, and lysosomal disruption were identified as potential drivers of inflammasome engagement after PM2.5 exposure. These findings unravel the mechanisms by which PM2.5 promotes cardiorespiratory inflammation and disease.
SFRR-E PP 3.3:
Keratinocyte-derived paracrine factors modulates UVB-induced stress response of melanocytes
Prof. Dr. Uraiwan Panich | Mahidol University Faculty of Medicine Siriraj Hospital | Thailand
Ultraviolet radiation (UVR) plays a role in skin photodamage through triggering various biological responses of skin cells including apoptosis, oxidative stress and melanogenesis. The microenvironment created by epidermal keratinocytes (KC) potentially influences the survival and function of melanocytes (MC) in response to various exogenous insults including UVB. In this study, we identified the candidate paracrine factors derived from UVB-irradiated KC that exerted the regulatory roles in cellular responses of MC to UVB irradiation using in vitro and in vivo models. We observed that conditioned media (CM) from UVB-irradiated KC potentially mitigated apoptosis and oxidative stress as well as stimulated melanogenesis in UVB-irradiated MC. RNA-sequening and functional experiments revealed that, among the upregulated transcripts in UVB-irradiated KC, the GCSF (granulocyte colony stimulating factor) and CCL20 (chemokine (C-C Motif) Ligand 20) mRNA were highly expressed in correlation with the KC’s pararince effects on the stress responses of MC to UVB. Then, treatment of MC with the recombinant GCSF and CCL20 revealed the strongest modulatory effects on UVB-induced MC responses including apoptosis, ROS formation and melanogenesis. Exposure of KC to UVB led to a substantial increase in secretion of both GCSF and CCL20. To demonstrate the in vivo relevance of the in vitro findings, immunofluorescence analysis revealed a correlation between protein expressions of the GCSF and CCL20, in KC and tyrosinase in MC in mouse skin exposed to UVB irradiation. In conclusion, GCSF and CCL20 might be the candidate paracrine factors secreted from KC that play a regulatory role in the responses of MC to UVB. Our findings might give novel insight into development of the UVB-responsive genes as epidermal biomarkers for predicting susceptibility of the skin to photodamage.
SFRR-E PP 3.5:
Effects of early life protein restriction on mitochondrial biogenesis and antioxidant defence capacity in skeletal muscle in mice
Ufuk Ersoy | University of Liverpool | United Kingdom
There is a strong relation between early life environment and age-related diseases in later life, including sarcopenia. Skeletal muscle development is especially prone to nutritional deficiency. With age, skeletal muscle has higher generation of reactive oxygen species that can cause oxidative stress and can potentially cause muscle atrophy. Also, mitochondrial dysfunction is among the most frequently reported mechanisms of aging muscle atrophy. Hence, we aimed to investigate the association between suboptimal early life nutrition and oxidative stress and mitochondrial turnover in skeletal muscle.
Female mice were fed either a normal (20%) or a low-protein (8%) diet prior to mating and during pregnancy. New-born pups were cross-fostered to different lactating dams (maintained on a 20% or 8% diet) within 24h after birth. At 21 days, they moved on to either a 20% or 8% protein diet, creating 3 groups: control (NNN), Normal-Low-Normal (NLN), and Normal-Low-Low (NLL). Mice were maintained for up to 3 months. Antioxidant defence capacity and mitochondrial biogenesis-related targets were measured by Western blotting and qPCR. Citrate synthase activity also measured.
NLL mice were smaller than control mice. PGC-1α content (P < 0.0001) and mRNA expression (P=0.0074) and NRF1 mRNA expression (P=0.0030) were significantly decreased in NLL and NLN mice compared to controls, suggesting decreased mitochondrial biogenesis. Muscles from these mice also had reduced SOD2 content (P=0.0002) and mRNA expression (P=0.0079) and Prx3 content (P=0.0080) and mRNA expression (P=0.0031). Cat mRNA expression was only reduced in NLL mice (P=0.0007). These mice also had a significantly decreased (P=0.0065) citrate synthase activity suggesting a reduction in mitochondrial number. These observations suggest that early life protein restriction can affect mitochondrial biogenesis and antioxidant defence capacity at a young age which may explain the impact of maternal diet on skeletal muscle aging.
SFRR-E PP 3.6 [YIA]:
CYTOPROTECTION EFFECT OF NATURAL VITAMIN D IN HUMAN LIVER CELL LIPOTOXICITY: A TRANSCRIPTOMICS APPROACH
Dr. Desirée Bartolini | UNIVERSITA' DEGLI STUDI DI PERUGIA | Italy
The aim of this study is to assess the cytoprotection function and the corresponding transcriptomics fingerprint of vitamin D (VD; 10 nM) in human hepatocytes (HepaRG) exposed to steatosis and lipotoxicity by means of oleic and palmitic acids (OA-PA) supplementation. A natural source of vitamin D (a Shiitake Mushroom extract) was compared with a synthetic form (DIBASE) and the active metabolite (1,25(OH)2D) of the vitamin in protecting and the presence of lipotoxicity was assessed by the cellular production of the reactive oxygen species (ROS) and induction of specific transcriptomic changes.
Cell viability data demonstrated that the in vitro model of steatosis produced conditions of sub-maximal lipotoxicity and cellular damage. Transcriptional modifications indicated the modulation of genes associated with long-chain fatty acid -oxidation, ROS production, cholesterol synthesis, AMPK activity, and hepatocyte apoptosis, liver fibrosis and damage. the different VD formulations showed similar efficacy in improving both the levels of steatosis and ROS. However, formulation-specific modifications of the cellular transcriptome were observed. Transcriptional fingerprints and biological functions identified by “Ingenuity Pathway Analysis” (IPA) showed higher similarities when the mushroom extract was compared with 1,25(OH)2-D metabolite than DIBASE. In conclusion, VD showed efficient protection against lipotoxicity in HepaRG cells. Comparable cytoprotection activity was observed for the natural and synthetic formulations, but transcriptomics data highlighted some specificities in the mechanism of action.
SFRR-E PP 3.7:
Ex vivo blood culture assessment of anti-oxidative effects of virgin olive oils differing in their bioactive contents.
Dr. Juan de Dios Alché | Estación Experimental del Zaidín (CSIC) | Spain
Dr. Elena Lima-Cabello | Estación Experimental del Zaidín (CSIC) | Spain
In the present study, we have performed an ex vivo blood culture and challenging assay with different inducers or inflammation and oxidative stress conditions, optimized to assess the anti-inflammatory and anti-oxidative response of individuals participants of a trial. The trial encompasses the raw intake of olive oils differing in their composition, in comparison to a standard virgin olive oil. Up to date, additional health benefits based on this trial have been determined and described on inflammatory, metabolic syndrome and endothelial function biomarkers, mainly in vivo.
Now, we have determined several oxidative markers including iNOS, presence of Tyr-nitrated proteins and production of NO. This study strongly confirms the first level of evidence already obtained in vivo regarding the health benefits of olive oil components in healthy humans, now in a inflammatory and oxidative scenario induced ex vivo. As discussed, the experimental system also provides an excellent tool to dissect the effects of individual componentes present in the oil in a individual manner.
This research was funded by research projects BFU-2016-77243-P, PID2020-113324GB-100 and STED202100X129616SV0 of the Spanish Ministry of Science, Innovation and Universities (MICIIN)/ State Research Agency (AGE)/ European Regional Development Fund (ERDF)/ European Union (EU).
SFRR-E PP 3.8:
Role of oxidative stress in the cytotoxic action of extracts of vegetables and medicinal herbs on human ovary cancer cells
Prof. Dr. Izabela Sadowska-Bartosz | University of Rzeszow | Poland
Natural products often contain compounds, which may be cytotoxic for cancer cells. The aim of this study was to compare the cytotoxic action of 1:10 (w/v) phosphate-buffered saline extracts of several vegetables, teas and medicinal plants on PEO1 and SKOV3 cancer ovary cells and to examine whether oxidative stress contributes to their effects. We found that the extracts of green tea, garlic, and black tea were the most cytotoxic to PEO1 cells (IC50 of 1.66, 2.01 and 3.25 vol%, respectively), while extracts of green tea, horseradish and black tea were the most cytotoxic to SKOV3 cells (IC50 of 1.66, 4.34 and 10.56 vol% when estimated with Neutral Red). The presence of catalase (10 µg/ml) in the cell medium partly protected against the cytotoxicity of the extracts of teas, garlic, curly kale, Cistus incanus, Gingko biloba, and Betula pendula, evidencing the contribution of hydrogen peroxide formed by the extracts to their cytotoxic effects. Tea and horseradish but not garlic extracts increased the intracellular ROS levels detected with d5hydroethidine. These results indicate that oxidative stress, due mainly to the generation of hydrogen peroxide, participated in the cytotoxic effects of the extracts but its contribution depended on the kind of the extract.
SFRR-E PP 3.9:
Regulation of murine macrophage viability by the long-chain metabolite of vitamin E through targeting intracellular lipid composition via SREBP1/SCD1.
Sijia Liao | Friedrich Schiller University Jena | Germany
Recent studies suggest that lipid metabolism has strong associated with many cellular stress responses including cell survival as well as cell death. For this reason, compounds targeting intracellular lipid homeostasis are of high interest. α-13′-carboxychromanol (α-13‘-COOH), a long-chain metabolite (LCM) of vitamin E, has emerged as a new regulatory molecule exhibiting more potent or even different effects compared with its metabolic precursor α-tocopherol. Here, we present new insight into the interaction of α-13‘-COOH with the cellular lipid homeostasis in murine macrophages RAW264.7 cells.
Methods and results
Using cell extraction assays and Western Blot analyses the translocation of the transcriptional factor sterol regulatory element-binding protein-1 (SREBP1) we found that the treatment with α-13'-COOH suppressed significantly the formation of SREBP1 active fragment in the cytoplasmic fraction and also decreased its translocation into the nucleus. We also observed that the level of the enzyme stearoyl-CoA desaturase-1 (SCD1), a target gene of SREBP1, was significantly downregulated by the LCM. Considering the function of SCD1 as a desaturase enzyme, we hypothesized that the lipid desaturation ratio could be changed by the LCM. Using UPLC-MS/MS-based lipidomics, we could show that α-13'-COOH modulates the intracellular lipid composition of macrophages: In both, triglycerides and phospholipids, the amount of species with saturated fatty acid was increased, and the amount of species with mono-unsaturated fatty acid decreased. Next, we observed that α-13‘-COOH modulated cell proliferation, cell cycle arrest and cell apoptosis/necrosis using photometric and FACS analyses, with effects that are consistent with the SCD1 antagonist, CAY10566.
Our results provide evidence that the LCM α-13‘-COOH can modulate the viability of macrophages through the regulation of cellular lipid desaturation via SREBP1/SCD1.
SFRR-E PP 3.10:
Induced inflammation modulation by olive tree dietary byproducts in zebrafish.
Dr. Elena Lima-Cabello | Estación Experimental del Zaidín (CSIC) | Spain
Functional molecules from natural extracts may possess specific properties capable of improving health and reducing disease. Incorporation of such molecules into foods is a promising strategy for the food industry. The olive growing sector, widely spread in Mediterranean countries, generates large amounts of byproducts which include seeds with a high agri-food potential due to their high nutritional value and the presence of bioactive compounds. Zebrafish is a well-established model, widely used in various fields, including the study of human pathologies and immune responses. Also, oxidative and inflammatory responses can be easily induced in a reproducible manner, and visualized both in early developmental stages and in adult fishes. The main objective of this work is to evaluate the anti-inflammatory capacity of olive seeds, by optimizing commercial diets enriched with this material, in an experimental model of inflammation by LPS treatment in adult zebrafish. A control fish group was fed three times a day (once with Artemia and twice with standard fish chow, while the olive seed fish group was fed with standard fish chow enriched with olive seeds at 20%. After 8 weeks, inflammation was induced with lipopolysaccharide (LPS) for 8h. After the inductions, liver was extracted, and expression of inflammation and oxidative biomarkers were analyzed. Results exhibited a drastic drop in the levels of inflammation-related biomarkers in the group of animals fed with the olive seed-enriched diet, compared to the control group. Results point to a possible protective and anti-inflammatory effect of the olive seed-enriched diet. This research was funded by research projects BFU-2016-77243-P, PID2020-113324GB-100 and STED202100X129616SV0 of the Spanish Ministry of Science, Innovation and Universities (MICIIN)/ State Research Agency (AGE)/ European Regional Development Fund (ERDF)/ European Union (EU).