Fig 1.
Re-epithelialization measured by keratin 14.
(a) Immunofluorescence staining of K14 (red) and PCNA (green) with nuclear staining using Draq5 (blue) of skin with wound treated with BCP or with oil. (b) Higher magnification images of areas of boxes in (a). PCNA was localized in the nucleus and, when density of cells increased, it became localized in the cytoplasm (b). This was observed at the center of the wound. In: intact area, W: wounded area. (c, d) Length of K14+ staining area from the boundary of intact and wounded area to the center of the wound in skin harvested on post-surgery day 3 (c) and 4 (d). Dots indicate each datum and the height of bars indicates the median. Horizontal lines indicate quartiles. 3rd day, Kolmogorov-Smirnov test, P = 0.008, BCP, n = 20, Oil, n = 15; 4th day, Kolmogorov-Smirnov test, P = 0.002, *: P<0.05, BCP, n = 16, Oil, n = 16.
Fig 2.
Expression of filaggrin (red) and estrogen alpha (green) (a), and PDGFR (green) and vimentin (red) (b) in the wounded area of mice. (a) View at the wound edge. In: intact area, WE: wound edge, WB: wound bed. Scale bar is in μm. Dotted lines show the boundaries between the intact area and wound edge, and the wound edge and wound bed. Filaggrin staining of the BCP group was broader in width and longer in the distance from the wound edge. Estrogen alpha staining did not show obvious differences between groups. (b) PDGFR expression in the epidermis was stronger in the BCP group. Both the BCP and Oil group showed a large area of staining of PDGFR in the wound edge (WE) and wound bed (WB). Vementin staining did not show obvious differences between the BCP group and Oil group. In: intact area, WE: wound edge, WB: wound bed. Scale bars are in μm.
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.
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.
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, F1,8 = 7.16, P = 0.028, cell type, F1,8 = 0.365, P = 0.562, BCP, n = 3, DMSO, n = 3.
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, F3,53 = 5.51, P = 0.002) (ANOVA, F3,53 = 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.
Fig 7.
Results of RNA sequencing of post-surgery 17 hours skin and intact skin.
Heatmap showing the top 50 significant gene expressions in the skin exposed to BCP (n = 2) or oil (n = 3), 17 to 18 hours post-surgery (inflammation stage), and in the skin of mice without skin excision (NT group) (n = 3). Comparison between BCP vs. oil (a) showed a clear difference between BCP and Oil. (b) Volcano plot from the comparison between BCP vs. Oil. Y-axis shows log10 (p value) and X-axis shows log2 fold change value.
Table 1.
Top 50 genes altered in mice treated with oil and BCP.
Top 50 genes were all up-regulated in the BCP group compared to the Oil group.
Fig 8.
Comparison of the expression of hair follicle marker genes.
Hair follicle genes related to the bulge were significantly up-regulated in the mice exposed to BCP compared to the Oil group. P-Values show the results of comparison between the BCP and the Oil groups. *: P<0.05, **: P<0.01, ***: P<0.001.
Fig 9.
IL-1β, TNFα in the skin at the wound bed and close to the wound, and TUNEL staining of the skin at the wound bed and close to the wound.
(a) IL-1β (red and purple) expression in the BCP and the Oil group. Blue color as well as purple color is Draq 5+ cells. Green is autofluorescence. (b) TNFα expression (red and purple) expression in the BCP and Oil group. Blue color as well as purple color is Draq 5+ cells. Green is autofluorescence. (c) TUNEL staining with double staining with Draq 5. Green and light blue is TUNEL+ cells. Blue and light blue is Draq5+ cells. Scale bars are μm.
Table 2.
Expression of pro-inflammatory cytokines in the BCP group compared to the Oil group.
Table 3.
Expression of cannabinoid receptors and TRP channel genes.
Genes significantly up-regulated or down-regulated in the BCP group compared to the Oil group are written in in bold. NA in P-Value is an outlier compared to other genes presumably because of its extremely high expression in the BCP group compared to the Oil group.
Table 4.
Significantly up/down-regulated genes in shh, pcp, fgf, and wnt/beta-catenin signaling pathways after exposure to BCP.
Genes with log fold change over 1 are shown. NA in P-Values is an outlier compared to other genes presumably because of its extremely high expression in the BCP group compared to the Oil group.
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, F1,66 = 14.793, P<0.001; gender, F1,66 = 30.761, Tukey’s post-hoc test, ***: P<0.001; females, BCP, n = 16, oil, n = 16, males, BCP, n = 23, oil, n = 15.