Modulation of Redox and Inflammatory Signaling in Human Skin Cells Using Phytocannabinoids Applied after UVA Irradiation: In Vitro Studies

Abstract

UVA exposure disturbs the metabolism of skin cells, often inducing oxidative stress and inflammation. Therefore, there is a need for bioactive compounds that limit such consequences without causing undesirable side effects. The aim of this study was to analyse in vitro the effects of the phytocannabinoids cannabigerol (CBG) and cannabidiol (CBD), which differ in terms of biological effects. Furthermore, the combined use of both compounds (CBG+CBD) has been analysed in order to increase their effectiveness in human skin fibroblasts and keratinocytes protection against UVA-induced alternation. The results obtained indicate that the effects of CBG and CBD on the redox balance might indeed be enhanced when both phytocannabinoids are applied concurrently. Those effects include a reduction in NOX activity, ROS levels, and a modification of thioredoxin-dependent antioxidant systems. The reduction in the UVA-induced lipid peroxidation and protein modification has been confirmed through lower levels of 4-HNE-protein adducts and protein carbonyl groups as well as through the recovery of collagen expression. Modification of antioxidant signalling (Nrf2/HO-1) through the administration of CBG+CBD has been proven to be associated with reduced proinflammatory signalling (NFκB/TNFα). Differential metabolic responses of keratinocytes and fibroblasts to the effects of the UVA and phytocannabinoids have indicated possible beneficial protective and regenerative effects of the phytocannabinoids, suggesting their possible application for the purpose of limiting the harmful impact of the UVA on skin cells.

Keywords: UVA radiation; fibroblasts; inflammation; keratinocytes; phytocannabinoids; redox balance.

Figure 1

The effects of cannabigerol (CBG) (1 µM) or/and cannabidiol (CBD) (5 µM) on NADPH oxidase (NOX) and reactive oxygen species (ROS) generation in keratinocytes and fibroblasts in control groups and cells exposed to UVA radiation (keratinocytes-30 J/cm² and fibroblasts-20 J/cm²). Mean values ± SD of five independent experiments are presented. The statistically significant differences in keratinocytes and fibroblasts are expressed as follows: a—indicates a comparison to the control group (control vs. all groups); p ≤ 0.05; b—indicates a comparison to the CBG group (only in no radiation part); p ≤ 0.05; c—indicates a comparison to the CBD group (only in no radiation part); p ≤ 0.05; x—indicates a comparison to the UVA radiation (only in UVA part); p ≤ 0.05; y—indicates a comparison to the UVA+CBG (only in UVA part); p ≤ 0.05; z—indicates a comparison to the UVA+CBD (only in UVA part); p ≤ 0.05.

Figure 2

Effects of cannabigerol (CBG) (1 µM) or/and cannabidiol (CBD) (5 µM) on phorylated Nrf2 (p-Nrf2) and heme oxygenase-1 (HO-1) generation in keratinocytes and fibroblasts exposed to UVA radiation (30 J/cm² and 20 J/cm², respectively). The mean values ± SD of five independent experiments are presented. The statistically significant differences in keratinocytes and fibroblasts are expressed as follows: a—indicates a comparison to the control group; p ≤ 0.05; b—indicates a comparison to the CBG group; p ≤ 0.05; c—indicates a comparison to the CBD group; p ≤ 0.05; x—indicates a comparison to the UVA radiation; p ≤ 0.05; y—indicates a comparison to the UVA+CBG (only in UVA part); p ≤ 0.05; z—indicates a comparison to the UVA+CBD; p ≤ 0.05.

Figure 3

Effects of cannabigerol (CBG) (1 µM) or/and cannabidiol (CBD) (5 µM) on superoxide dismutase (SOD-1 and SOD-2) and catalase (CAT) activity in keratinocytes and fibroblasts exposed to UVA radiation (30 J/cm² and 20 J/cm²), respectively. The mean values ± SD of five independent experiments are presented. The statistically significant differences in keratinocytes and fibroblasts are expressed as follows: a—indicates a comparison to the control group; p ≤ 0.05; b—indicates a comparison to the CBG group; p ≤ 0.05; c—indicates a comparison to the CBD group; p ≤ 0.05; x—indicates a comparison to the UVA radiation; p ≤ 0.05; y—indicates a comparison to the UVA+CBG (only in UVA part); p ≤ 0.05; z—indicates a comparison to the UVA+CBD; p ≤ 0.05.

Figure 4

Effects of cannabigerol (CBG) (1 µM) or/and cannabidiol (CBD) (5 µM) on a glutathione (GSH, GSSG-R, GSH-Px) and thioredoxin (Trx, TrxR)-dependent system in keratinocytes and fibroblasts exposed to UVA radiation (30 J/cm² and 20 J/cm², respectively). The mean values ± SD of five independent experiments are presented. The statistically significant differences in keratinocytes and fibroblasts are expressed as follows: a—indicates a comparison to the control group; p ≤ 0.05; b—indicates a comparison to the CBG group; p ≤ 0.05; c—indicates a comparison to the CBD group; p ≤ 0.05; x—indicates a comparison to the UVA radiation; p ≤ 0.05; y—indicates a comparison to the UVA+CBG (only in UVA part); p ≤ 0.05; z—indicates a comparison to the UVA+CBD; p ≤ 0.05.

Figure 5

Effects of cannabigerol (CBG) (1 µM) or/and cannabidiol (CBD) (5 µM) on 4-hydroxynonenal (4-HNE) and 4-hydroxynonenal-protein (4-HNE-protein) generation, and the level of carbonyl group (CBO) in keratinocytes and fibroblasts exposed to UVA radiation (30 J/cm² and 20 J/cm², respectively). The mean values ± SD of five independent experiments are presented. The statistically significant differences in keratinocytes and fibroblasts are expressed as follows: a—indicates a comparison to the control group; p ≤ 0.05; b—indicates a comparison to the CBG group; p ≤ 0.05; c—indicates a comparison to the CBD group; p ≤ 0.05; x—indicates a comparison to the UVA radiation; p ≤ 0.05; y—indicates a comparison to the UVA+CBG (only in UVA part); p ≤ 0.05; z—indicates a comparison to the UVA+CBD; p ≤ 0.05.

Figure 6

Effects of cannabigerol (CBG) (1 µM) or/and cannabidiol (CBD) (5 µM) on nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB-p65 and NFκB-p52) and tumour necrosis factor-alpha (TNFα) generation in keratinocytes and fibroblasts exposed to UVA radiation (30 J/cm² and 20 J/cm², respectively). Mean values ± SD of five independent experiments are presented. The statistically significant differences in keratinocytes and fibroblasts are expressed as follows: a—indicates a comparison to the control group; p ≤ 0.05; b—indicates a comparison to the CBG group; p ≤ 0.05; c—indicates a comparison to the CBD group; p ≤ 0.05; x—indicates a comparison to the UVA radiation; p ≤ 0.05; y—indicates a comparison to the UVA+CBG (only in UVA part); p ≤ 0.05; z—indicates a comparison to the UVA+CBD; p ≤ 0.05.

Figure 7

Effects of cannabigerol (CBG) (1 µM) or/and cannabidiol (CBD) (5 µM) on collagen expression in keratinocytes (n = 3) and fibroblasts (n = 3) exposed to UVA radiation (30 J/cm² and 20 nJ/cm², respectively).

Figure 8

The effect of phytocannabinoids on keratinocytes and fibroblasts under standard conditions. Abbreviation: 4-HNE, 4-hydroxynonenal, 4-HNE-protein, 4-HNE-protein adducts; CAT, catalase; CBD, cannabidiol; CBG, cannabigerol; CBO, carbonyl groups; GSH, glutathione; GSH-Px, glutathione peroxidase; GSSG-R, glutathione reductase; HO-1, heme oxygenase-1; NF-κB, nuclear factor kappa-light-chain-enhancer; NOX, NADPH oxidase; Trx, hioredoxin; pNrf2, nuclear factor erythroid 2-related factor 2 phosphorylated form; ROS, reacctive oxygene species; SOD-1, cytosolic superoxide dismutase; SOD-2, mitochondrial superoxide dismutase; TNFα, tumor necrosis factor α; TrxR, thioredoxin reductase; UV, ultraviolet radiation.

Figure 9

The effect of phytocannabinoids on keratinocytes and fibroblasts used after UVA cell irradiation. Abbreviation: 4-HNE, 4-hydroxynonenal, 4-HNE-protein, 4-HNE-protein adducts; CAT, catalase; CBD, cannabidiol; CBG, cannabigerol; CBO, carbonyl groups; GSH, glutathione; GSH-Px, glutathione peroxidase; GSSG-R, glutathione reductase; HO-1, heme oxygenase-1; NF-κB, nuclear factor kappa-light-chain-enhancer; NOX, NADPH oxidase; Trx, hioredoxin; pNrf2, nuclear factor erythroid 2-related factor 2 phosphorylated form; ROS, reacctive oxygene species; SOD-1, cytosolic superoxide dismutase; SOD-2, mitochondrial superoxide dismutase; TNFα, tumor necrosis factor α; TrxR, thioredoxin reductase; UV, ultraviolet radiation.