CBD has been reported to have improving effects on various skin diseases [17,18,19,20]. In this study, we verified the skin moisturizing effect of CBD and analyzed its mechanism.

Laura Casares et al. have shown that the application of a 1% CBD solution to the skin of 6-month-old mice induces keratinocyte proliferation, which may be involved in wound healing [26]. Therefore, as in a previous report, a 1% CBD solution was applied to mouse skin every day, and various examinations were conducted. The results showed that dermal water content in the CBD-treated group increased approximately 1.3 times compared with that in the control group, and a significant difference was observed (Figure 2A). Moreover, no skin inflammation was observed (Figure 1), and the skin barrier function was normal (Figure 2B). These findings show that CBD exhibits a skin moisturizing effect and may be considered useful as a cosmetic material. When a 3% CBD solution was applied to mouse skin, no inflammatory reaction was observed, but TEWL increased and dermal water content decreased to the same level as in the control group (data not shown). Therefore, an optimal concentration was considered when applying CBD to the skin.

Dermal water content is regulated by moisturizing factors such as loricrin, filaggrin, collagen, hyaluronic acid, and ceramide. Therefore, we investigated the effect of CBD on the expression of genes involved in these processes. CBD did not affect the mRNA expression levels of loricrin, filaggrin, collagen, hyaluronan degrading enzyme, hyaluronan synthase, ceramide degrading enzyme, or ceramide synthase in the skin (Figure 3). Therefore, it is unlikely that CBD exerted a skin moisturizing effect by changing the amount of these moisturizing factors.

In recent years, it has been reported that the water channel known as AQP plays an important role in skin water retention [9,10], and active research is being conducted. AQP is a water-permeable protein expressed on the cell membrane, and 15 isoforms of AQPs are known to exist in mammals. Aqp1, Aqp2, Aqp3, Aqp4, Aqp7, Aqp9, and Aqp11 mRNA were detected in the skin of the mice in this study (Figure 4). Of these, it was revealed that only the mRNA expression level of Aqp3 was significantly increased by the application of CBD compared with that in the control group (Figure 4). The protein expression level of AQP3 was also significantly higher in the CBD-treated group than in the control group (Figure 5). AQP3 is predominantly expressed in the cell membrane of skin keratinocytes and is classified as an aquaglyceroporin, which is responsible for transporting glycerol as well as water [27]. It has been reported that AQP3-knockout mice have dry skin and reduced skin elasticity compared to wild-type mice [10]. In addition, the expression level of AQP3 in the skin of aged mice was lower than that in young mice, suggesting that AQP3 may be involved in the decrease in dermal water content in old age [28]. Furthermore, AQP3 is decreased at the onset of various pathological conditions (diabetes mellitus [29,30], vitiligo [31,32], and psoriasis [33]) in which dry skin is observed. In addition, when mice are treated with the anticancer drug, which causes marked dry skin as a side effect, dry skin and a decrease in AQP3 were also observed [34]. Thus, it can be hypothesized that AQP3 is a key molecule that plays an important role in skin water retention. Since CBD increased the expression of AQP3 in the skin, it was thought that this effect may be involved in the skin moisturizing effect of CBD.

AQP3-induced glycerol transport contributes not only skin water retention but also to skin protection. AQP3 facilitates glycerol movement in several cells of skin layers, including the epidermal keratinocytes [10,35] or hypodermal adipocytes [36]. It was reported that topic or systemic glycerol administration or AQP3 re-expression in keratinocytes reverse the phenotype of skin abnormalities in Aqp3-deficient mice [35,37]. In this study, although the skin glycerol concentration in CBD-treated mice was not measured, it is possible that the increase in glycerol associated with the increase in AQP3 is also involved in the increase in dermal water content.

AQP3 is known to be present not only in keratinocytes but also in hypodermal adipocytes. However, its expression level is very low, and it is a fact that the epidermal layer is strongly stained when the skin is performed immunohistochemistry of AQP3 [11,28]. As AQP3 in the epidermal layer is mainly involved in the increase in dermal water content, we believe that the increase in dermal water content due to CBD treatment is mainly due to the increase in AQP3 in the epidermal layer.

Why did the application of CBD increase AQP3 in the skin? So far, various cytokines, such as tumor necrosis factor-α (TNF-α) or transforming growth factor-β (TGF-β), can change the expression levels of aquaglyceroporins AQP3, AQP7, and AQP11 in subcutaneous adipocytes [38,39]. In this study, it was clarified that the application of CBD to mouse skin did not change the cutaneous Tnfa mRNA expression level, and at this time, only Aqp3 increased; no correlation was found between Tnfa and Aqps transcript levels. Therefore, it is unlikely that these cytokines are involved in the increased Aqp3 expression.

In general, CBD acts on cannabinoid receptors to exert various pharmacological actions [40]. On the other hand, it has also been reported that CBD increases the activity of peroxisome proliferator-activated receptor-γ (PPARγ) and exerts a pharmacological effect on various cells [41,42]. PPARγ is expressed in skin keratinocytes, and it has been revealed that PPARγ agonists increase the expression level of AQP3 in keratinocytes [43]. In this study, there was no change in the mRNA expression level of Pparg in mouse skin treated with CBD (data not shown). Although the mechanism by which CBD increases AQP3 in mouse skin is unclear, it is possible that CBD activates PPARγ in keratinocytes and increases AQP3, which requires further study.

The results of this study show that CBD has a skin moisturizing effect. In addition, it is possible that the increased expression of AQP3, which plays an important role in skin water retention, is involved as one of the mechanisms of action. Currently, there is an active search for substances and natural products that increase cutaneous AQP3 expression. CBD is expected to be applied in the future as a cosmetic material that has the unique effect of increasing AQP3.