Cannabidiol protects keratinocyte cell membranes following exposure to UVB and hydrogen peroxide

Abstract

Keratinocytes, the major cell type of the epidermis, are particularly sensitive to environmental factors including exposure to sunlight and chemical agents. Since oxidative stress may arise as a result of these factors, compounds are actively sought that can act as protective agents. Recently, cannabidiol (CBD), a phytocannabinoid found in Cannabis Sativa L., has gained increased interest due to its anti-inflammatory and antioxidant properties, and absence of psychoactive effects. This prompted us to analyze the protective effects of CBD on keratinocytes exposed to UVB irradiation and hydrogen peroxide. Here we show, using liquid chromatography mass spectrometry, that CBD was able to penetrate keratinocytes, and accumulated within the cellular membrane. CBD reduced redox balance shift, towards oxidative stress, caused by exposure UVB/hydrogen peroxide, estimated by superoxide anion radical generation and total antioxidant status and consequently lipid peroxidation level. CBD was found to protect keratinocytes by preventing changes in the composition of the cellular membrane, associated with UVB/hydrogen peroxide damages which included reduced polyunsaturated fatty acid levels, increased sialic acid and lipid peroxidation products (malondialdehyde and 8-isoprostanes) levels. This maintains cell membranes integrity and prevents the release of lactate dehydrogenase. In addition, CBD prevented UVB/hydrogen peroxide-induced reduction of keratinocyte size and zeta potential, and also decreased activity of ATP-binding cassette membrane transporters. Together, these findings suggest that CBD could be a potential protective agent for keratinocytes against the harmful effects of irradiation and chemical environmental factors that cause oxidative stress.

Keywords: Antioxidative defense; Cannabidiol; Hydrogen peroxide; Keratinocyte cell membrane; Oxidative stress; UVB.

Graphical abstract

Fig. 1

The redox status estimated through pro-oxidant enzymes activity (XO - A; NOX - B), superoxide anion generation (C) and Total Antioxidant Status (D) in groups of keratinocytes: I. control; II. cultured with CBD (4uM) for 24h; III. cultured with CBD (4uM) for 48h; VI. irradiated with UVB [60 mJ/cm²]; V. irradiated with UVB [60 mJ/cm²] and cultured with CBD (4uM) for 24h after irradiation; VI. irradiated with UVB [60 mJ/cm²] and cultured with CBD for 24h befor irradiation and 24h after irradiation; VII. exposed to H₂O₂ [200μM]; VIII. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h after exposer to H₂O₂; IX. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h before and 24h after exposer to H₂O₂. Mean values ± SD of five independent experiments and statistically significant differences for p < 0.05 are presented: a - differences vs. group I/group IV/group VII; b - differences vs. group II/group V/group VIII; x - differences between group IV/group VII and group I.

Fig. 2

Sialic acid level in groups of keratinocytes: I. control; II. cultured with CBD (4uM) for 24h; III. cultured with CBD (4uM) for 48h; VI. irradiated with UVB [60 mJ/cm²]; V. irradiated with UVB [60 mJ/cm²] and cultured with CBD (4uM) for 24h after irradiation; VI. irradiated with UVB [60 mJ/cm²] and cultured with CBD for 24h befor irradiation and 24h after irradiation; VII. exposed to H₂O₂ [200μM]; VIII. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h after exposer to H₂O₂; IX. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h before and 24h after exposer to H₂O₂. Mean values ± SD of five independent experiments and statistically significant differences for p < 0.05 are presented:a - differences vs. group I/group IV/group VII; b - differences vs. group II/group V/group VIII; x - differences between group IV/group VII and group I.

Fig. 3

Malondialdehyde (MDA) and F₂-isoprostanes level in groups of keratinocytes: I. control; II. cultured with CBD (4uM) for 24h; III. cultured with CBD (4uM) for 48h; VI. irradiated with UVB [60 mJ/cm²]; V. irradiated with UVB [60 mJ/cm²] and cultured with CBD (4uM) for 24h after irradiation; VI. irradiated with UVB [60 mJ/cm²] and cultured with CBD for 24h befor irradiation and 24h after irradiation; VII. exposed to H₂O₂ [200μM]; VIII. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h after exposer to H₂O₂; IX. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h before and 24h after exposer to H₂O₂. Mean values ± SD of five independent experiments and statistically significant differences for p < 0.05 are presented:a - differences vs. group I/group IV/group VII; b - differences vs. group II/group V/group VIII; x - differences between group IV/group VII and group I.

Fig. 4

Size [% of control cells] and zeta potential [mV] in groups of keratinocytes: I. control; II. cultured with CBD (4uM) for 24h; III. cultured with CBD (4uM) for 48h; VI. irradiated with UVB [60 mJ/cm²]; V. irradiated with UVB [60 mJ/cm²] and cultured with CBD (4uM) for 24h after irradiation; VI. irradiated with UVB [60 mJ/cm²] and cultured with CBD for 24h befor irradiation and 24h after irradiation; VII. exposed to H₂O₂ [200μM]; VIII. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h after exposer to H₂O₂; IX. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h before and 24h after exposer to H₂O₂. Mean values ± SD of five independent experiments and statistically significant differences for p < 0.05 are presented: a - differences vs. group I/group IV/group VII; b - differences vs. group II/group V/group VIII; x - differences between group IV/group VII and group I.

Fig. 5

Lactate dehydrogenase (LDH) activity in mediums of keratinocytes: I. control; II. cultured with CBD (4uM) for 24h; III. cultured with CBD (4uM) for 48h; VI. irradiated with UVB [60 mJ/cm²]; V. irradiated with UVB [60 mJ/cm²] and cultured with CBD (4uM) for 24h after irradiation; VI. irradiated with UVB [60 mJ/cm²] and cultured with CBD for 24h befor irradiation and 24h after irradiation; VII. exposed to H₂O₂ [200μM]; VIII. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h after exposer to H₂O₂; IX. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h before and 24h after exposer to H₂O₂. Mean values ± SD of five independent experiments and statistically significant differences for p < 0.05 are presented: a - differences vs. group I/group IV/group VII; b - differences vs. group II/group V/group VIII; x - differences between group IV/group VII and group I.

Fig. 6

Activities of ABC-cassette transporters (MDR1, MRP, BCRP) in groups of keratinocytes: I. control; II. cultured with CBD (4uM) for 24h; III. cultured with CBD (4uM) for 48h; VI. irradiated with UVB [60 mJ/cm²]; V. irradiated with UVB [60 mJ/cm²] and cultured with CBD (4uM) for 24h after irradiation; VI. irradiated with UVB [60 mJ/cm²] and cultured with CBD for 24h befor irradiation and 24h after irradiation; VII. exposed to H₂O₂ [200μM]; VIII. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h after exposer to H₂O₂; IX. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h before and 24h after exposer to H₂O₂. Mean values ± SD of five independent experiments and statistically significant differences for p < 0.05 are presented: a - differences vs. group I/group IV/group VII; b - differences vs. group II/group V/group VIII; x - differences between group IV/group VII and group I.

Fig. 7

CBD level [μg/mg protein] in groups of keratinocytes: II. cultured with CBD (4uM) for 24h; III. cultured with CBD (4uM) for 48h; V. irradiated with UVB [60 mJ/cm²] and cultured with CBD (4uM) for 24h after irradiation; VI. irradiated with UVB [60 mJ/cm²] and cultured with CBD for 24h befor irradiation and 24h after irradiation; VIII. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h after exposer to H₂O₂; IX. exposed to H₂O₂ [200μM] and cultured with CBD (4uM) for 24h before and 24h after exposer to H₂O₂. Mean values ± SD of five independent experiments and statistically significant differences for p < 0.05 are presented: b - differences vs.group II/group V/group VIII; x - differences between group IV/group VII and group I.