ABSTRACT
University of Modena and Reggio Emilia, 1Dept of Dermatology, Via del Pozzo 71;
2Dept of Biomedical Sci- Section of Pharmacology, Via G. Campi 287, Modena, Italy
UVB radiation is the major etiological factor in the pathogenesis of skin aging and cancer development. New approaches to prevent and reverse UVB damage are needed to avoid sun light induced skin cancer rates. The hydrophobic fraction of licorice root extract is rich in glabridin (GLB), a flavone that exerts an inhibitory effect on tyrosinase activity thus producing beneficial effects on the skin moreover, the main constituents of the hydrophilic fraction are glycyrrhizin (GL) and glycyrrhetinic acid (GA). Flavonoids like GLB, are recognized as potent antioxidants; GL and its aglycone: 18β-glycyrrhetinic acid (18β-GA) have a wide range of pharmacological activity. This study aims to investigate “in vitro” on a possible protective activity against UVB radiation damage on human keratinocytes by GL, 18β-GA and GLB. Materials and Methods: Keratinocyte cultures were prepared as follows: Skin samples were minced and trypsinized at 37°C for 30 minutes and keratinocytes were grown in 75cm2 culture flasks with 10 mg/ml mitomycin-treated 3T3 fibroblasts for 2hours at 37°C. Keratinocytes were cultured in Dulbecco's Modified Eagle's Medium/Ham's F12 Medium (DMEM/F12, 3:1) containing insulin 5μg/ml, transferrin 5μg/ml, triiodothyronine 2nM, hydrocortisone 0.4μg/ml, adenine 180mM, mouse epidermal growth factors (EGF) 10ng/ml, 10% foetal calf serum, 100U/ml Penicillin-100μg/ml streptomycin-amphotericin B (PSA). Sub-confluent primary culture were transferred in secondary culturein keratinocyte growth medium (KGM) containing bovine pituitary extract (BPE) until near confluence.Once cell growth was well established, the cells were plated at 104/cm2 into disposable 96-multiwell plate. MTT test was performed as viability test: the MTT was performed in triplicate on 12 and 24 hour cell cultures, added with equimolecular increasing concentrations of GL, 18β-GA and GLB (upper concentration: 120µM). In the following tests only non toxic concentrations were used, therefore GL 30µM, 18β-GA 30µM and GLB 10µM were added into keratinocyte medium. UVB treatment: GL, 18β-GA and GLB were added into keratinocyte basal medium (KBM). After 12 hour incubation, keratinocytes were exposed to UVB radiation (50mJ/cm2, 75mJ/cm2 and 100mJ/cm2). Subsequently, the cells were provided with fresh medium, added with GL, 18β-GA and GLB and left for an additional time (12 h). Two controls were prepared: the first was UVB irradiated but not GL, 18β-GA and GLB added; the second was added with GL, 18β-GA and GLB but without UVB irradiation. GL, 18β-GA and GLB effects after UVB treatment were evaluated by the Comet assay, by the measurement of intracellular ROS levels and by Western blot analysis. DNA damage was evaluated by the Comet assay, whereas intracellular ROS generation was measured by fluorescent 2’7’- dichlorodihydrofluorescein diacetate assay. In addition we analyzed the activation of p53, down-regulation of bcl-2 and PARP cleavage by Western blot analysis. Results: The treatment of human keratinocytes with 18β-glycyrrhetinic acid and glabridin prevents direct and indirect DNA damage avoiding apoptosis activation. In the present study we observed that after 18β-GA and GLB treatment (in the presence of UVB) p53 activation is inhibited, bcl-2 protein is up-regulated and PARP cleavage is inhibited. In conclusion, the study demonstrates that 18β-GA and GLB are potent antioxidants also able to prevent in UVB-irradiated NHK: a) oxidative DNA fragmentation; b) injury caused by ROS production and c) activation of apoptotic pathway. All these effects are observed at non toxic concentrations therefore, we suggest that 18β-GA and GLBmust be considered as important natural active principles to include among skin photoprotective agents with promising applications in dermatological clinical research.