Beta-caryophyllene is an odoriferous bicyclic sesquiterpene found in various herbs and spices. Recently, it was found that beta-caryophyllene is a ligand of the cannabinoid receptor 2 (CB2). Activation of CB2 will decrease pain, a major signal for inflammatory responses. We hypothesized that beta-caryophyllene can affect wound healing by decreasing inflammation. Here we show that cutaneous wounds of mice treated with beta-caryophyllene had enhanced re-epithelialization. The treated tissue showed increased cell proliferation and cells treated with beta-caryophyllene showed enhanced cell migration, suggesting that the higher re-epithelialization is due to enhanced cell proliferation and cell migration. The treated tissues also had up-regulated gene expression for hair follicle bulge stem cells. Olfactory receptors were not involved in the enhanced wound healing. Transient Receptor Potential channel genes were up-regulated in the injured skin exposed to beta-caryophyllene. Interestingly, there were sex differences in the impact of beta- caryophyllene as only the injured skin of female mice had enhanced re-epithelialization after exposure to beta-caryophyllene. Our study suggests that chemical compounds included in essential oils have the capability to improve wound healing, an effect generated by synergetic impacts of multiple pathways.

Copyright: © 2019 Koyama et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The CB2 receptor is expressed in neuronal cells, immune tissue, hair follicles, sebaceous glands, the dermomuscular layer, and vascular smooth muscle in intact skin [ 21 ]. Activation of CB2 by CB2 selective agonist GP1a improved re-epithelialization in wound healing [ 22 , 23 ]. Essential oil of Copaifera paupera, which contains BCP also improved wound healing [ 24 ], although it is not clear whether BCP had a direct or indirect role and whether the wound healing is promoted by the CB2 receptor. In this project, we examined whether BCP can improve re-epithelialization and, if so, whether the olfactory system is involved in the impact.

In addition, the route of administration can affect the efficacy of the odorants. Odorants of herbal plants are often called “aromas”, which suggests that the route is through the olfactory system. However, there are various routes that odorant chemicals can exert effects. First, olfactory receptors are expressed in both olfactory neurons and in non-olfactory tissues, such as skin and circulatory organs [ 18 , 19 ]. Furthermore, some odorants activate both olfactory receptors and non-olfactory receptors. For example, beta-caryophyllene (BCP), which is an odoriferous bicyclic sesquiterpene present in many herbs and spices, is a ligand of cannabinoid receptor 2 (CB2) [ 20 ] (Cannabinoid receptors G-protein receptors first found as the receptors for Δ 9 -tetrahydrocannabinol (THC), which is the main psychoactive constituent of Cannabis sativa (marijuana). Two receptors have been identified, cannabinoid receptors 1 and 2) as well as olfactory receptors. How odorants such as BCP act on distinct receptor classes is important for the most effective use of BCP.

When essential oils are used instead of single chemical compound, there have been challenges to examine their mechanism of action because of the large variances in the percentage each chemical compound present [ 7 ] depending on the extraction methods [ 8 ], the area of origin including differences in the geographic altitude of the areas [ 9 ], seasons the plants were harvested [ 10 ], and parts of the plants extracted [ 11 , 12 ]. It is thus important to utilize chemically-pure compounds or chemically-defined extracts as described in some recent studies [ 13 – 17 ]. These studies enable testing the roles of individual chemicals and obtaining clear mechanistic insights [ 13 , 14 ].

Odors from other conspecific individuals [ 1 ] (Technical terms in the study field of animal behavior and ethology, meaning the same species) and the environment [ 2 – 6 ] affect the behaviors and physiological status in many animals. The significance of olfactory stimuli tends to be underestimated in case of humans. However, we have a long history of actively utilizing odorants to heal or alter our physiological conditions. Various herbal plants’ and spices’ extracts have been used to reduce stress, pain, and to promote recovery from injury or illness [ 2 – 6 ]. In spite of such a long history of using herbal plant extracts and although an increasing number of scientists are starting to test natural products owing to their diverse molecular scaffolds and biologically active substructures, there is still a strong need to examine the influences of extracts. It is especially important to better understand their mechanisms of action.

Results and discussion

BCP treatment enhanced cell proliferation To determine the rate and location of cell proliferation in the wounded tissue, 5-bromo-2’-deoxyuridine (BrdU) was injected twice every 2 hours and tissues were harvested 2 hours after the second injection (see Materials andMethods for details). Significantly more BrdU+ cells were found on the basal layer of the interfollicular epidermis and hair follicles, and in the papillary dermis [37–39] in the BCP-treated wounds. In both the BCP-treated and controls, there were many BrdU+ cells at the wound margin and wound bed (Fig 3). Previous studies have shown that proliferation takes place at a proliferative hub, which is from 2 mm outside of the wound edge until 3 mm from the wound edge [40]. We observed that cell proliferation in the controls took place highly at the wound margin and at the basal layer of the interfollicular epidermis (Fig 3F, Oil). In contrast, the BCP-treated wounds had higher cell proliferation in the interfollicular epidermis, in the dermis, at the hair follicles, and in the wound bed (Fig 3F, BCP). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. BrdU+ cells in the epidermis and dermis. (a) BrdU+ cells in the epidermis (epi in (f)) (Student T-test, T = 3.077, df = 8, P = 0.015, BCP, n = 5, Oil, n = 5), (b) BrdU+ cells in the wound margin (wm in (f)) (Student T-test, T = 1.343, df = 8, P = 0.216, BCP, n = 5, Oil, n = 5), (C) BrdU+ cells at the hair follicle (h in (f)) (Student T-test, T = 3.038, df = 8, P = 0.015, BCP, n = 5, Oil, n = 5). (d) BrdU+ cells at the dermis excluding wound margin (Student T-test, T = 4.645, df = 8, P = 0.002, BCP, n = 5, Oil, n = 5). (e) BrdU+ cells at the BrdU+ cells at the wound bed (Student T-test, T = 2.197, df = 8, P = 0.059, BCP, n = 5, Oil, n = 5). (f) Representative views of combined photos used in quantification for each group. h: hair follicle, epi: epidermis, der: dermis, wm: wound margin, wb: wound bed, scale bar: 200 μm. Numbers below and above the photos are operationally assigned numbers for explanation in the text on locations in the wound. Dotted lines show the boundaries between epidermis and dermis, dermis and wound margin zone, and wound margin zone and wound bed. https://doi.org/10.1371/journal.pone.0216104.g003 Stem cells in the hair follicle contribute to re-epithelialization by migrating from the hair follicle bulge to epidermis and then to the center of the wound [41]. The strong BrdU staining of hair follicles in the BCP-treated wounds compared to the controls suggest that a larger number of hair follicle stem cells may have contributed to the enhanced re-epithelialization in BCP group. If enhanced re-epithelialization was due to a reduction of inflammation and an early shift to the cell proliferation/migration stage, then BCP should not impact cultured cells. To test this hypothesis, we conducted time-lapse imaging for 7 hours and enumerated cell division. We conducted this with different concentrations of BCP. We found a peak of cell proliferation at 26 μM BCP, which decreased at higher concentrations (Fig 4). In higher concentrations we observed less cell proliferation. These results suggested that BCP treatment was on cell proliferation and enhanced re-epithelialization rather than the suppression of inflammation. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Proliferating primary fibroblast cells exposed to BCP at various concentrations. Number of dividing and apoptotic cells during 7 hours of exposure to BCP. https://doi.org/10.1371/journal.pone.0216104.g004

BCP enhanced cell migration The increased cell migration caused by BCP was not reported previously and we examined this further. To determine whether exposure to BCP can stimulate cell migration, we conducted in vitro assays of scratch tests and chemotaxis assays. Primary cultured fibroblasts and keratinocytes from C57BL/6 mice exposed to BCP (diluted with DMSO) for 24 hours showed higher chemotactic responses relative to the controls (fibroblasts, 2.1 times higher number of cells, and keratinocytes, 2.5 times higher number of cells, both compared to control condition cells exposed to DMSO) (Fig 5A, 5B and 5C). However, exposure to BCP did not stimulate chemotactic responses in fibroblasts isolated from CB2 knockout mice (1.2 times higher number of cells compared to control condition cells exposed to DMSO) (Fig 5D). These results suggest that activation of CB2 could lead to an increase in chemotactic responses. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. Influence of BCP on cell migration. (a) Representative results of chemotaxis assay. Cells showed chemotactic responses to BCP. Fibroblasts and keratinocytes were exposed either to BCP or to DMSO (control) in chemotaxis assay. WT cells exposed to BCP showed significantly more chemotactic responses toward the bottom of the inserts (fibroblasts, Student T-test, T = 2.564, df = 8, P = 0.033, BCP, n = 4, DMSO, n = 4, (b); keratinocytes, Student T-test, T = 2.456, df = 8, P = 0.04, BCP, n = 4, DMSO, n = 4, (c)), but cells isolated from CB2 knockout mice did not show differences between those exposed to BCP and those exposed to DMSO control (fibroblasts, Student T-test, T = 0.944, df = 4, P = 0.398, BCP, n = 3, DMSO, n = 3, (d)). (e, f, g) Results of scratch tests. Representative images on the influence of BCP (27 μM) on cell migration of fibroblasts. Six hours after scratch (e) and 1 day after scratch (f). Dotted white lines show the edge of scratched area. Scale bar = μm, (g) The distance cells migrated during the 6 hours shown as a comparison to the original width (% of shrink in width). O% means no change from original width. ANOVA, Medium type, F 1,8 = 7.16, P = 0.028, cell type, F 1,8 = 0.365, P = 0.562, BCP, n = 3, DMSO, n = 3. https://doi.org/10.1371/journal.pone.0216104.g005 In the chemotaxis assay above we found that cells show chemotactic responses toward BCP. We then conducted scratch tests to determine if exposure to BCP stimulates chemotactic responses to repopulate in petri dishes. After culture dish reached confluency, the culture media was replaced to culture media containing BCP (23μM/10 μL DMSO/10 mL cell culture media) or DMSO (10 μL/10mL cell culture media). Exposure to BCP increased cell migration to the scratched area (39.2% decrease in the width in 6 hours), relative to the control (6.7% decrease in the width in 6 hours). The cells from CB2 knockout mice exposed to BCP also showed a comparable rate of repopulating the devoided area (34.0% decrease in the width in 6 hours) (Fig 5E, 5F and 5G). These results suggest that BCP can stimulate cell migration, but not through CB2 pathway. We started this study with the hypothesis that BCP will positively impact wound healing by activating signaling by CB2. While BCP did improve re-epithelialization, the results of the cell migration studies showed that BCP’s effect on re-epithelialization may be more complicated than through the activation of CB2. Therefore, we examined whether CB2 antagonists and agonists will affect re-epithelialization. AM630, an antagonist of CB2 [42] was daily injected 20 min before the daily application of BCP, CB2 agonist JWH133 [43] was topically applied instead of BCP to determine if it generates a similar impact as BCP. Then tissues of the BCP group, the Oil group, the AMP630+BCP group, and the JWH133 group were harvested on the 5th day post-surgery. Sections were stained with K14 antibody and the distance of migration from the edge of the wound was measured. Consistent with our previous observations, topical application of BCP enhanced re-epithelialization. CB2 agonist JWH133 significantly enhanced re-epithelialization compared to the Oil group (Fig 6). When CB2 antagonist AM630 was injected daily into mice treated with BCP, the results were not clear. Re-epithelialization was not statistically different from the Oil group but the variance was large and there was a trend towards differences (P = 0.071) (Fig 6). These results suggest that there could be some other pathways involved in the BCP enhanced re-epithelization of the wounds. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 6. Re-epithelialization (K14+ distance from wound edge) in mice exposed to BCP, Oil, CB2 agonist JWH133, and BCP+CB2 antagonist AM633. (a) Length of K14+ staining area from the boundary of the intact and wounded area to the center of the wound in skin harvested on post-surgery day 5 from mice exposed to BCP, oil, JWH133 or injected CB2 antagonist AM630 daily before daily treatment with BCP. Dots indicate each data set and the height of bars indicates the median. Horizontal lines indicate quartiles. ANOVA, F 3,53 = 5.51, P = 0.002) (ANOVA, F 3,53 = 5.51, P = 0.002. Post-hoc pairwise analyses between BCP vs. oil, Tukey’s post-hoc pairwise comparison, P = 0.002, CB2 agonist JMW133 group vs. oil group, Tukey’s post-hoc pairwise comparison, P = 0.071, and CB2 antagonist AM630+BCP group vs. BCP group, Tukey’s pairwise comparison, P = 0.097; BCP, n = 9, Oil, n = 10, JWH133, n = 8, AM630+BCP, n = 7). (b) Representative images of JWH133 group and BCP+AM630 group. In: Intact area, W: wounded area. https://doi.org/10.1371/journal.pone.0216104.g006

Sex differences in the impact of BCP These results summarized above are based on experiments using female mice. Previous studies have shown sex differences in the morphology and physiology of mouse skin, with female mice having a 40% thicker epidermis than males [69]. In addition, androgen receptors have an inhibitory influence on wound healing in males [70], which may hinder the impact of BCP. We tested whether the influences of BCP on wound healing in male mice is similar to its influences in females and found no statistically significant differences in re-epithelialization between the BCP and Oil groups in males (based on K14 staining) (Fig 10). Whether males need a higher concentration of BCP to induce its impact or whether BCP does not affect males at all needs to be addressed in future. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 10. Influence of BCP on re-epithelialization in male and female mice. Length of K14+ staining area from the boundary of the intact and wounded area to the center of the wound in skin harvested on post-surgery day 4 from mice exposed to BCP or oil. Bar indicates median and line indicates 25 and 75% quartile. ANOVA, BCP vs. oil, F 1,66 = 14.793, P<0.001; gender, F 1,66 = 30.761, Tukey’s post-hoc test, ***: P<0.001; females, BCP, n = 16, oil, n = 16, males, BCP, n = 23, oil, n = 15. https://doi.org/10.1371/journal.pone.0216104.g010 Sex steroids are known to affect wound healing [71], androgen receptors inhibit wound healing [60] whereas estrogen receptors accelerate it [32]. The re-epithelialization in the Oil control group for males was similar to that of the Oil group of female mice (Fig 10), which suggests that sex hormones did not suppress re-epithelialization in males.

Olfactory receptors are not involved in wound healing BCP is an odorant. When mice are topically treated with BCP, they are exposed to its smell and there is a possibility that the olfactory system has some role in the BCP mediated enhanced re-epithelialization. In addition, olfactory receptors are expressed in non-olfactory tissues. A synthetic analogue of sandalwood odorant, sandalore, is the ligand of the olfactory receptor gene OR2AT4 and that OR2AT4 receptors are expressed in human skin [72]. Thus odorants can directly produce an impact on non-olfactory tissues like skin tissues [72] other than through the route of the olfactory system to the brain, affecting emotional status [73]. There is a possibility that the impact of BCP was mediated by olfactory receptors expressed in the skin. To examine if the olfactory system and/or olfactory receptors expressed in the skin has some role in enhanced re-epithelialization by exposure to BCP, we conducted multiple experiments. First we tested if mice can smell BCP by testing the expression of an immediate early gene protein in the olfactory bulb of mice exposed to BCP and found clear expression of c-FOS protein, suggesting that it is highly likely that mice can smell BCP (S8 Fig). Then we tested whether exposure of injured mice to BCP only through the air would produce a similar impact as topical application, and found no improved re-epithelialization (S9 Fig). It was clear that the olfactory system was not significantly involved in the impact of BCP on re-epithelialization. To examine if olfactory receptors expressed in the non-olfactory system are involved, we first examined the olfactory epithelium after exposure of non-injured mice to BCP for one hour to identify the olfactory receptor genes for BCP using pS6-IP RNAseq [74]. We could narrow down the candidates of olfactory receptor genes for BCP (S2 Table). It is known that odorants stimulate a limited number of types of olfactory receptors [75, 76]. As the BCP we used was not 100% in purity, multiple olfactory receptor genes were up-regulated following exposure to BCP with Olfr340 showing the largest fold change and significant P-Value (S2 Table). After this step, we analyzed the results of RNA sequencing of the skin to determine if the olfactory receptor genes up-regulated in olfactory epithelium by exposure to BCP are expressed in skin and are up-regulated after topical application of BCP. There were many olfactory receptor genes expressed in the skin (S3 Table), and one of them overlapped with the olfactory receptor genes up-regulated in olfactory epithelium (Olfr111), but none of them were up-regulated specifically in the BCP group. From these results, we conclude that olfactory receptors expressed in skin are not involved in the enhanced re-epithelialization by exposure to BCP.

Synergetic influences by exposure to BCP Wound healing starts from the inflammatory stage and shifts to the cell proliferation stage. Since BCP is an agonist of CB2, we had assumed that it can improve wound healing by decreasing pain and reducing pro-inflammatory signals and speed up the shift to the cell proliferation stage. However, our results suggests that BCP may directly impact cell proliferation and cell migration concomittent with reduced inflammation. BCP also stimulates mitogenesis of hair follicle stem cells. Hair follicle stem cells participate in re-epithelialization when there is a cutaneous wound [41, 51], our study suggests that enhanced re-epithelialization by exposure to BCP is mediated by BCP stimulated production of hair follicle stem cells in the bulge, and their migration to epidermis and to wound bed. BCP is known to suppress apoptosis [77] and necroptosis [78], raising the possibility that suppression of cell death enabled higher cells survival and proliferation. The suppression of Septin4 (Sept4) has been shown to protect cells from cell death and promote wound healing [79], however, Sept4 was not up-regulated in our study and seemed to be not involved in BCP enhanced re-epithelialization. It is possible that exposure to BCP generates a specific wound healing environment by up-regulating the genes related to embryonic growth as was suggested from our results on transcriptome analyses. Our study focused on the early stage following skin excision. Studies have shown that oxidants from inflammatory cells are involved in fibrotic healing (scarring) [80, 81], which suggests that suppression of inflammation through activation of CB2 by BCP, in addition to up-regulation of genes related to embryonic growth would have a high possibility of reducing scar formation. These possibilities need to be addressed in future by testing the influence of exposure to BCP on the late stage after injury.