Department: Department of Inorganic and Analytical Chemistry, Medical University of Bialystok, Poland
Public PhD Defence date: 19.09.2022
Doctoral Thesis title: Protective effects of cannabidiol on skin keratinocytes in an oxidative microcellular environment induced by UVA/B radiation or exposure to hydrogen peroxide
Prof. dr hab. Elżbieta Skrzydlewska (Department of Inorganic and Analytical Chemistry, Medical University of Bialystok, Poland) and Prof. dr hab. Pedro Domingues (Department of Chemistry, University of Aveiro, Portugal)
Prof. dr hab. Błażej Rubiś (Department of Clinical Chemistry and Molecular Diagnostics, Poznań University of Medical Sciences), Prof. dr hab. Krzysztof Jóźwiak (Department of Boipharmacy, Medical University of Lublin) and Prof. dr hab. Adam Buciński (Department of Biopharmacy, University of Nicolaus Copernicus in Torun)
Post-doctoral Research Assistant in Laboratory of Intracellular Immunity, The Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, Warsaw
Plans for the Future:
Development of new therapeutic approaches, by focusing high-throughput proteomic analyses, as well as by taking into account its reflection on the pharmaceutical application.
Abstract of the Doctoral Thesis:
Hydrogen peroxide (H2O2) and ultraviolet (UV) radiation, including UVA and UVB, are two types of external oxidative stressors, one chemical and the other physical, which are widely used in the clinical practice due to their antiseptic and cell-arresting/anti-proliferative properties, respectively. These two factors cause oxidative stress and associated chronic inflammation on skin cells, in particular keratinocytes, which are the cells most exposed to external factors. However, overproduction of reactive oxygen species (ROS), it is known to cause changes in cellular metabolism that can lead to changes in differentiation or cell death signalling.
The results of this study indicate that H2O2 and UV radiation shift the redox balance towards oxidative stress, intensifying the process of lipid peroxidation, causing changes in the structure and functions of membrane phospholipids. This process is also accompanied by changes in the proteome of cell membranes, which ultimately changes the integrity of these membranes. It was found that H2O2 mainly affects the level of proteins involved in the regulation of the proteostasis network, while UVA/B radiation primarily induces changes in the level of proteins mainly involved in the regulation of apoptotic and inflammatory signaling. In order to prevent the harmful effects of H2O2 and UV radiation on skin cells, there is a need for protective compounds, especially natural ones, to counteract adverse metabolic changes. Therefore, the effect of cannabidiol (CBD), the main non-psychoactive phytocannabinoid of Cannabis sativa L., on metabolic changes in skin keratinocytes was investigated. The main goal was to evaluate the effect of CBD on cellular metabolism linked to redox homeostasis, particularly with regard to changes in the phospholipid and protein profiles of the cell membrane, under the conditions of the oxidative microenvironment caused by the exposure to H2O2 or UV radiation. Two CBD treatments were also compared: short-term (using CBD after exposure to stressors) and long-term (using CBD before and after exposure to stressors).
The results obtained using electron spin resonance (ESR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) indicate the protective effect of CBD in counteracting the oxidative stress caused by the action of H2O2 and UVA/B radiation. CBD has been shown to prevent over-production of ROS and augmentation of lipid peroxidation products and their adducts with proteins such as MDA/4-HNE/4-ONE-protein adducts, evaluated by changes in intensity of adducted proteins obtained from nano-high performance LC-tandem mass spectrometry (nanoHPLC-QorbiTrap). By decreasing the oxidative metabolism of phospholipids, CBD reduces the structural and functional changes of phospholipids, as observed in the phospholipid profile obtained by GC-MS, which helps to maintain the integrity of damaged keratinocyte membranes following the pro-oxidative action of H2O2 and UV radiation. This situation has been confirmed by the significant decrease in lactate dehydrogenase (LDH) leakage from keratinocytes induced by CBD.
Moreover, the results of this study comparing two types of CBD use show that long-term use of CBD is more effective in preventing oxidative stress compared to short-term use of CBD, due to more intense reduction of lipid modifications and preservation of membrane integrity. Also, the data showing alterations in the membrane proteome indicate that this specific treatment is found to be more effective in maintaining protein homeostasis in biological membranes, by preventing changes in protein expression induced by the effect of H2O2 or UVB. In order to assess the impacts of the protective effect of CBD closer to real medical practice, the effectiveness of CBD used in vivo on the skin of nude rats subjected to a 4-week UVA/UVB treatment, often used in the treatment of skin diseases, was studied to uncover changes in the membrane and cytosolic proteome. Comparison of the protein intensities obtained with nanoHPLC-QorbiTrap shows that CBD significantly prevents the UVA/B induced overexpression of proteins involved in the regulation of redox balance, inflammation, and apoptosis.
Finally, the results presented in this study indicate two potential effects of CBD on keratinocytes under oxidative conditions induced by H2O2 or UV: pro-survival activity of CBD in keratinocytes exposed to H2O2 by reducing in oxidative stress and maintaining the proteostasis network; modulatoring effect of CBD on apoptotic signaling and regulation of redox balance and inflammatory signaling in keratinocytes exposed to UV radiation. Thus, it is possible to suggest that CBD may be used as a protective compound for skin keratinocytes against oxidative damage of cell metabolism caused by the effect of H2O2 or UV radiation.