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. 2012 May;21(5):337-40.
doi: 10.1111/j.1600-0625.2012.01455.x.

Topical Hesperidin Improves Epidermal Permeability Barrier Function and Epidermal Differentiation in Normal Murine Skin

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Free PMC article

Topical Hesperidin Improves Epidermal Permeability Barrier Function and Epidermal Differentiation in Normal Murine Skin

Maihua Hou et al. Exp Dermatol. .
Free PMC article

Abstract

Orange peel extract appears to exhibit beneficial effects on skin whitening, inflammation, UVB protection, as well as keratinocyte proliferation. In the present study, we determine whether topical hesperidin influences epidermal permeability barrier function and its underlying mechanisms. Hairless mice were treated topically with 2% hesperidin or 70% ethanol alone twice daily for 6 days. At the end of treatment, basal transepidermal water loss (TEWL) was measured 2 and 4 h post barrier disruption. Epidermal proliferation and differentiation were evaluated by immunohistochemical staining and Western blot analysis. Additionally, lamellar body density and secretion were assessed by electron microscopy. Although there were no significant differences in basal barrier function, in comparison with control animals, topical hesperidin significantly accelerated barrier recovery at both 2 and 4 h after acute barrier abrogation. Enhanced barrier function in hesperidin-treated skin correlated with stimulation of both epidermal proliferation and differentiation, as well as enhanced lamellar body secretion. These results indicate that topical hesperidin enhances epidermal permeability barrier homeostasis at least in part due to stimulation of epidermal proliferation, differentiation, as well as lamellar body secretion.

Figures

Figure 1
Figure 1. Topical Hesperidin Accelerates Epidermal Permeability Barrier Recovery in Normal Mouse Skin
Hairless mice were topically treated with 60 μl of 2% hesperidin in 70% ethanol or 70% ethanol alone twice daily for 6 days. Basal epidermal permeability barrier function, skin surface pH and stratum corneum (SC) hydration were assessed with a MPA5 (CK electronic GmbH, Cologne, Germany) connected to TM 300, pH905 and Corneometer 825. Two readings were taken from each mouse for basal TEWL, hydration, as well as pH. For barrier recovery, TEWL was measured at 0, 2 and 4 hours after tape stripping, which results in a 10-fold increase in TEWL, and percent barrier recovery rates were calculated as described earlier (30). Figure 1a is basal TEWL (n=10); 1b, SC hydration (n=10); 1c, skin surface pH (n=10).
Figure 2
Figure 2. Topical Hesperidin Stimulates Epidermal Proliferation in Normal Murine Skin
Skin samples were from mice treated with either vehicle alone (2a, c) or 2% hesperidin (2b, d) twice daily for 6 days. 5 μm sections were incubated with the primary antibodies (1:500 dilutions) overnight at 4°C. After washing, sections were treated with DAB for 20 sec. Sections were examined with a Zeiss microscope (Jena, Germany), and digital images were captured with AxioVision software (Carl Zeiss Vision, Munich, Germany). Figure 2a and b are H&E staining. Figure 2c and d are PCNA staining. Magnification bars represent 20 μm (a-d). Figure 2e represents epidermal thickness and f, PCNA positive cells. Epidermal thickness of the nucleated cell layer was measured on 1260 × H & E sections. The measurement was taken at every 2 cm points along the epidermis. The data are presented as the mean of all measured points (n=33 for both groups). The number of PCNA positive cells was counted on every 2 cm segment along the epidermis (n=32 for vehicle-treated and n=24 for hesperidin-treated group). The data are presented as the mean of all segments counted ± SEM. Unpaired two-tailed student t test with Welch’s correction was used to determine statistical differences. P<0.05 was considered as significant difference.
Figure 3
Figure 3. Changes in Epidermal Differentiation Protein Expression Induced by Topical Hesperidin
5μm paraffin sections were incubated with primary rabbit anti-mouse antibodies (Covance/BabCo. Berkely, CA) at the dilutions of 1:2000 for filaggrin, 1:1000 for involucrin, and 1:500 for loricrin, overnight at 4°C. After washing with 10mM citrate buffer, sections were incubated with goat anti-rabbit antibody (1:400) for 30 min at room temperature, followed by ABC-peroxidase (Vector, Burlingame, CA) reaction. For PCNA staining, sections were incubated with biotinylated monoclonal anti-PCNA antibody (CalTag Laboratories, Burlingame, CA) for 2 hours at room temperature. The sections were visualized with a Zeiss (Axioplan 2) microscope (Jena, Germany). Digital images were captured with AxioVision software 2.05 (Carl Zeiss Vision, Munich, Germany). Fig. 3a and b are filaggrin staining; c and d are involucrin staining; e and f are loricrin staining. Fig. 3a, c, e are vehicle-treated samples and b, d, f are hesperidin-treated samples. Magnifications are the same for all figures. Magnification bars represent 20 μm. For Western blot analysis, mouse epidermis was isolated following 6 day treatment with 2% hesperidin or vehicle alone. Epidermal proteins were fractionated by electrophoresis followed by transferring onto polyvinylidene fluoride membranes. The proteins on the membrane were probed with respective antibody. The corresponding bands were detected by enhanced chemiluminescence (Thermo Fisher Sci., Rockford, IL), and quantitated by scanning densitometry. Results were presented as percentage of vehicle-treated control, setting vehicle-treated as 100% (dotted line in Fig 3g). An unpaired t-test with Welch’s correction was used to determine the significance. (n=5 for all groups).

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