The effects of topical alpha-hydroxyacids on the normal skin barrier of hairless mice

BACKGROUND: alpha-hydroxyacids (AHA), such as glycolic acid and lactic acid, have recently been used in cosmetic and dermatological formulations. However, the mechanisms of action of these substances have not been well documented. OBJECTIVES: This study was done to investigate the effects of AHA on the skin barrier of hairless mice and to clarify the contribution of AHA to the formation and secretion of the lamellar bodies (LB), which are known to be the critical structure for barrier function in the epidermis. METHODS: 5% Lactic acid and 5% glycolic acid were applied to normal skin of the mice daily for 14 days. RESULTS: Changes in transepidermal water loss (TEWL), capacitance and electron microscopic findings of the epidermis of hairless mice were compared with those in which only the vehicle was applied. CONCLUSIONS: There were no significant differences in TEWL, capacitance or epidermal thickness between the epidermis of the mice to which AHA or vehicle only had been applied. On electron micrographs, the normal epidermis to which AHA had been applied showed an increase in the number and secretion of LB and a decrease in the number of stratum corneum (SC) layers in comparison with the epidermis to which the vehicle only had been applied. The lipid layers of the SC intercellular spaces and calcium gradient in both the epidermis with application of AHA and that with vehicle only were normal. These results suggest that AHA, in low concentration (5%), may improve the skin barrier in hairless mice by inducing enhanced desquamation, and by increasing the number and secretion of LB without increasing TEWL.     

Role of PKC-delta as a signal mediator in epidermal barrier homeostasis

The skin shows an important "epidermal permeability barrier homeostasis" in response to barrier disruption. Calcium ion (Ca(2+)), a major regulator in keratinocyte differentiation and proliferation, plays a crucial role in skin barrier homeostasis. Acute barrier disruption induces an immediate depletion of both extra- and intracellular calcium ions in the epidermis, especially in the upper granular layers, and results in the loss of normal epidermal calcium gradient. Currently, we hypothesize that the change in the intracellular calcium ion concentration triggers the barrier repair responses, such as lamellar body (LB) secretion and increased lipid synthesis in the epidermis. In this article, we suggest that PKC-delta is a signaling mediator for the changes in extracellular and intracellular calcium ion concentration.     

External negative electric potential accelerates exocytosis of lamellar bodies in human skin ex vivo

Exocytosis of lamellar bodies at the uppermost nucleated layer of the epidermis is a crucial process for epidermal permeability barrier homoeostasis. We have previously suggested that skin surface electric potential might be associated with barrier homoeostasis. Thus, we hypothesized that the potential might drive exocytosis of lamellar bodies. In this study, we tested this idea by applying negative electric potential (?0.5 V) to human skin samples ex vivo for 2 h and observing the ultrastructure of the uppermost layer. The secretion of lamellar bodies was accelerated in the potential‐applied skin, compared to that in untreated control skin. Multiphoton observation indicated that extracellular lipid domains were more extensive in treated skin than in control skin. Moreover, the calcium ion gradient was greater at the uppermost layer of the epidermis of treated skin, compared to that in control skin. These results indicate that electric potential may regulate lamellar body secretion in healthy human skin.     

Rottlerin, a specific inhibitor of protein kinase C-delta, impedes barrier repair response by increasing intracellular free calcium

Several signals have been suggested in maintaining skin barrier homeostasis, but epidermal calcium ions are currently thought to be a main signaling factor. It is not clear, however, exactly how an intracellular calcium level decreases in response to the loss of an extracellular calcium gradient. In this study, we investigated the effects of several broad-type and isozyme-specific protein kinase C (PKC) inhibitors on epidermal permeability barrier recovery. Topical application of chelerythrine chloride, a broad-type PKC inhibitor, and rottlerin, a PKCdelta-specific inhibitor, significantly impeded the barrier recovery rate at 3 and 6 hours after barrier disruption. A significant decrease in the number and secretion of lamellar bodies was also observed at the inhibitor-treated site. Calcium ion-capture cytochemistry showed that the epidermal calcium gradient was rapidly reformed in inhibitor-treated skin, though recovery of the corresponding barrier function was not observed. In cultured keratinocytes treated with either inhibitor, increased intracellular calcium did not return to the baseline concentration after extracellular calcium decreased. These results suggest that PKC inhibitors, especially a PKCdelta-specific inhibitor, delay barrier recovery by affecting the intracellular calcium concentration after a loss of the extracellular calcium gradient. Furthermore, PKCdelta is important in controlling a decrease in intracellular calcium concentration.     

Mode of action of glycolic acid on human stratum corneum: ultrastructural and functional evaluation of the epidermal barrier

Alpha-hydroxy acids (AHA) such as glycolic acid have recently been used extensively in cosmetic and dermatological formulas. In low concentration (2– 5%) glycolic acid is believed to facilitate progressive weakening of cohesion of the intercellular material of the stratum corneum (SC), resulting in uniform exfoliation of its outermost layers (the stratum disjunctum). Since thinning of the SC as well as changes of intercellular lipids could theoretically compromise the barrier functions of the skin, we investigated the mode of AHA action on the SC to determine whether enhanced desquamation compromises the barrier structures of the SC and changes transepidermal water loss (TEWL) values. Electron microscopy of the epidermis biopsied from the volar forearm of human volunteers after 3 weeks of treatment with a 4% glycolic acid formulation twice daily was employed to evaluate 1) epidermal morphology and thickness of the SC, (2) the lamellar body and SC lipid bilayer organization, and (3) desquamative events based on degradation of desmosomes. TEWL values and SC hydration were recorded prior to and at the end of the study. Electron microscopy revealed no ultrastructural changes in the nucleated layers of the epidermis. The lamellar body (LB) secretory system in the stratum granulosum (SG), and intercellular lipid lamellae in the SC in both vehicle- and glycolic acid-treated samples were comparable to normal human SC. Within the SC, enhanced desmosomal breakdown, promoting loss of cohesion and desquamation, was restricted to the stratum disjunctum while desmosomes of the stratum compactum were unaffected. Treated areas displayed histologically, a more compact appearing SC. TEWL values remained unchanged in glycolic acid- and vehicle-treated skin. Our findings indicate that the barrier structures of the SC are not disrupted by glycolic acid formulations at the concentration used. One of the mechanism of action of AHA on the SC seemed to be a „targeted“ desmosomal (corneosomal) action without compromising the barrier structures of the skin. Received: 20 November 1996     

Mite and cockroach allergens activate protease-activated receptor 2 and delay epidermal permeability barrier recovery

Protease-activated receptor-2 (PAR-2) is known to be involved in epidermal permeability barrier function homeostasis. PAR-2 activation occurs after barrier disruption and further activation of PAR-2 by activating peptide significantly delays barrier recovery rate. Cockroach and house dust mite allergens, both known to be associated with the development of asthma, allergic rhinitis, and atopic dermatitis, have protease activity, which can activate PAR-2. In this study, we investigated the effects of both allergens on the epidermal barrier function as well as on the epidermal calcium gradient. Both allergens, when topically applied on the barrier-disrupted site, increased protease activities in the epidermis and delayed barrier recovery and lamellar body secretion in murine skin. The topical application of PAR-2-specific antagonist or protease inhibitors normalized the barrier recovery. Cockroach allergens induced intracellular calcium oscillations in cultured human keratinocytes through PAR-2-involved pathway, which was confirmed by desensitization protocol. Abnormal calcium ion distribution after barrier disruption was also observed in allergens-applied skin. These results suggest that allergens with protease activity can influence the epidermal permeability barrier homeostasis through PAR-2 activation and consequent modulation of the calcium ions in skin.     

Unsaturated fatty acids induce calcium influx into keratinocytes and cause abnormal differentiation of epidermis

Abnormal follicular keratinization is involved in comedogenesis in acne vulgaris. We recently demonstrated that calcium influx into epidermal keratinocytes is associated with impaired skin barrier function and epidermal proliferation. Based on these results, we hypothesized that sebum components affect calcium dynamics in the keratinocyte and consequently induce abnormal keratinization. To test this idea, we first observed the effects of topical application of sebum components, triglycerides (triolein), saturated fatty acids (palmitic acid and stearic acid), and unsaturated fatty acids (oleic acid and palmitoleic acid) on hairless mouse skin. Neither triglyceride nor saturated fatty acids affected the skin surface morphology or epidermal proliferation. On the other hand, application of unsaturated fatty acids, oleic acid, and palmitoleic acid induced scaly skin, abnormal keratinization, and epidermal hyperplasia. Application of triglycerides and saturated fatty acids on cultured human keratinocytes did not affect the intracellular calcium concentration ([Ca(2+)](i)), whereas unsaturated fatty acids increased the [Ca(2+)](i) of the keratinocytes. Moreover, application of oleic acid on hairless mouse skin induced an abnormal calcium distribution in the epidermis. These results suggest that unsaturated fatty acids in sebum alter the calcium dynamics in epidermal keratinocytes and induce abnormal follicular keratinization.     

Modulations in epidermal calcium regulate the expression of differentiation-specific markers

Mammalian epidermis normally displays a distinctive calcium gradient, with low levels in the basal/spinous layers and high levels in the stratum granulosum. Although changes in stratum granulosum calcium regulate the lamellar body secretory response to permeability barrier alterations, whether modulations in calcium also regulate the expression of differentiation-specific proteins in vivo remains unknown. As acute barrier perturbations reduce calcium levels in stratum granulosum, we studied the regulation of murine epidermal differentiation after loss of calcium accompanying acute barrier disruption and by exposure of such acutely perturbed skin sites to either low (0.03 M) or high (1.8 M) calcium. Three hours after acute barrier disruption, coincident with reduced calcium and ultrastructural evidence of accelerated lamellar body secretion, both northern analyses and in situ hybridization revealed decreased mRNA levels for loricrin, profilaggrin, and involucrin in the outer epidermis, but protein levels did not change significantly. Moreover, exposure of acutely disrupted skin sites to low calcium solutions sustained the reduction in mRNA levels, whereas exposure to high calcium solutions restored normal mRNA levels (blocked by the L-type calcium channel inhibitor, nifedipine). Finally, with prolonged exposure to a low (<10% relative humidity) or high (>80% relative humidity) humidity, calcium levels increased and declined, respectively. Accordingly, mRNA and protein levels of the differentiation-specific markers increased and decreased at low and high relative humidity, respectively. These results provide direct evidence that acute and sustained fluctuations in epidermal calcium regulate expression of differentiation-specific proteins in vivo, and demonstrate that modulations in epidermal calcium coordinately regulate events late in epidermal differentiation that together form the barrier.     

Influx of calcium and chloride ions into epidermal keratinocytes regulates exocytosis of epidermal lamellar bodies and skin permeability barrier homeostasis

In the nervous system, influx of calcium and chloride ions into neurons regulates the signaling system by excitation and inhibition, respectively. In this study, we demonstrated the effects of the ion influx into epidermal keratinocytes in the permeability barrier repair process of the skin after damage. Topical application of the neurotransmitters glutamate and nicotine, which activate the calcium channel in neurons, delayed the barrier repair after tape stripping. In contrast, the neurotransmitters GABA and glycine, which activate the chloride channel in neurons, accelerated barrier repair. Topical application of the calcium ionophore ionomycin delayed barrier recovery and chloride ionophore 1 accelerated barrier repair after barrier disruption by tape stripping and acetone treatment. Ionomycin increased the intracellular calcium concentration in cultured keratinocytes whereas the chloride ionophore 1 increased the intracellular chloride ion concentration. In vivo light microscopy and electron microscopy observation showed acceleration of the exocytosis of lipid-containing lamellar bodies by the chloride ionophore and delay of the exocytosis by the calcium ionophore. These results suggest that, like the nervous system, influx of calcium and chloride ions into epidermal keratinocytes through ionotropic receptors plays a crucial role in cutaneous barrier homeostasis.     

Ultraviolet B-induced alterations of the skin barrier and epidermal calcium gradient

Ultraviolet irradiation induces a variety of cutaneous changes, including epidermal permeability barrier disruption. In the present study, we assessed the effects of ultraviolet B (UVB) irradiation in epidermal barrier function and calcium distribution in murine epidermis. Adult hairless mice were exposed to a single dose of UVB (0.15 J/cm(2)). Barrier function was evaluated by transepidermal water loss (TEWL), lanthanum perfusion. The morphological alterations were examined by histology, immunohistochemistry and electron microscopy using ruthenium tetroxide (RuO(4)) postfixation. For evaluation of the effect on epidermal calcium distribution, the ion-capture cytochemistry was employed. UVB irradiation caused a significant increase in TEWL, which peaked at day 4. In parallel, the increased number of sunburn cells and the changes in epidermal hyperplasia and proliferation were observed. Electron microscopic observation demonstrated that the water-soluble lanthanum tracer was present in the extracellular stratum corneum domains, and the increased intercellular permeability was correlated with defective organization of the extracellular lipid lamellar bilayers of the stratum corneum. Moreover, UVB irradiation also caused an appearance of calcium precipitates in the stratum corneum and transitional cell layers as well as the increased cytosolic calcium in the lower epidermis, reflecting the alterations of the epidermal calcium gradient. These results suggest that the changes of the epidermal calcium distribution pattern may correlate with the perturbation of the epidermal barrier induced by UVB irradiation.     

Effects of alpha-hydroxy acids on the human skin of Japanese subjects: the rationale for chemical peeling

Alpha-hydroxy acid (AHA) agents, such as glycolic acid and lactic acid, have been used as therapeutic agents for more than a quarter of a century. Recently, they have been used as agents to rejuvenate photo-aged skin. It is believed that these AHA agents induce the epidermis to remodel and accelerate desquamation, thus exerting their therapeutic effects. In this study, we investigated the histological differences in skin treated with glycolic, lactic, citric and acetic acids once daily for 6 weeks. The melanin pigments in the basal layer were less prominent in the glycolic and lactic acid-treated skin than in the citric and acetic acid-treated skin. The melanin deposits in the horny layers were equal for all AHA. However, the melanin deposits in the squamous layers were less prominent in the glycolic and lactic acid-treated skins than in the citric and acetic acid-treated skins; this was analogous to observations of the basal layers. Collagen I and procollagen I were increased after treatment with glycolic, lactic and citric acid in the upper dermis, but were not increased with acetic acid treatment. However, the staining of the epidermis and dermis for matrix metalloproteinase-1 (MMP-1) after treatment was not significantly different among the agents. Our data suggest that longer treatment intervals with glycolic and lactic acid can cause improvements in both the epidermal and dermal components and support the usefulness of AHA for rejuvenating photo-damaged skin.     

The effect of wet-wrap dressing on epidermal barrier in patients with atopic dermatitis

BACKGROUND: Wet-wrap dressing has been shown to be effective for atopic dermatitis; however, the therapeutic mechanism of wet-wrap dressing is only the hypothesis based on the recovery of decreased epidermal barrier function. OBJECTIVES: To examine the therapeutic efficacy as well as the mechanism of wet-wrap dressing in atopic dermatitis patients. METHODS: To examine the difference of non-lesional and lesional atopic skin and to evaluate the change between epidermal barrier function before and after the treatment, SCORAD, epidermal water content, transepidermal water loss, the lipid amount of skin surface, immunohistochemical staining of filaggrin and loricrin, transmission electron microscopic examination, and calcium ion capture cytochemistry method were done in 10 severe form atopic dermatitis patients. RESULTS: In atopic dermatitis patients, SCORAD was clearly decreased, epidermal water content was increased, and transepidermal water loss was decreased after wet-wrap dressing. After wet-wrap dressing, increased release of lamellar body and the recovery of the damaged lamellar structure of intercellular lipid were observed; nevertheless, neither the change in keratinocyte differentiation nor the change of calcium ion gradient was detected. A week after the termination of wet-wrap dressing, increased water content and decreased transepidermal water loss were still maintained. CONCLUSION: We confirmed the abnormality of the epidermal barrier in atopic dermatitis, and the effects of wet-wrap were associated with the recovery of epidermal barrier. In atopic lesions, wet-wrap dressing induced clinical improvement by the release of lamellar body and the restoration of intercellular lipid lamellar structure.     

Calcium and potassium inhibit barrier recovery after disruption, independent of the type of insult in hairless mice

Abstract Disruption of the cutaneous permeability barrier induces metabolic responses in the epidermis which result in barrier recovery. Barrier disruption by either solvent treatment or tape stripping results in the loss of the epidermal calcium gradient. Previous studies in acetone treated hairless mice have shown that maintaining this calcium gradient inhibits barrier repair, suggesting that alterations in the epidermal calcium concentration may be an important signal for barrier homeostasis. In the present study, we show that in hairless mice disruption of the barrier by treatment with the detergent. SDS, also results in the loss of the calcium gradient, as demonstrated both semi-quantitatively with ultrastructural cytochemical localization and quantitatively using proton induced X-ray emission (PIXE). Additionally, immersion in calcium containing solutions delays barrier repair after either detergent (SDS treatment) or mechanical (tape stripping) disruption of the barrier, as reported previously for acetone treated skin. These results indicate that barrier disruption, regardless of the insult, induces changes in the epidermal calcium gradient which may play an important role in signaling the metabolic changes required for barrier homeostasis.     

Negative electric potential induces alteration of ion gradient and lamellar body secretion in the epidermis, and accelerates skin barrier recovery after barrier disruption

Previous reports suggested that ion gradients of ions such as calcium and magnesium in the epidermis play a crucial part in skin barrier homeostasis. We hypothesized that external electric potential affects the ionic gradient and skin barrier homeostasis. We demonstrated here that application of a negative electric potential (0.50 V) on hairless mice skin accelerated the barrier recovery approximately 60.7% of the original level within 1 h compared with control (37.5%) after barrier disruption by acetone treatment. Even after the application of a negative potential, the skin showed accelerated repair for 6 h. On the contrary, the skin that was applied a positive potential for 1 h showed a significant delay in barrier recovery (25.3%) than the control. Ultrastructural studies by electron microscopy suggested that the extent of lamellar body exocytosis into the stratum corneum/stratum granulosum interface increased under a negative potential. Magnesium and calcium ion concentrations in the upper epidermis were relatively higher in the negative portion than in the portion where the positive potential was applied. Topical application of these ions on mice skin also accelerated the barrier recovery. These results suggest that the external electric potential affects the ionic gradients in the epidermis and also influences the skin barrier homeostasis.     

Structural and functional consequences of loricrin mutations in human loricrin keratoderma (Vohwinkel syndrome with ichthyosis)

Although loricrin is the predominant protein of the cornified envelope (CE) in keratinocytes, loss or gain of loricrin function in mouse models produces only modest skin phenotypes. In contrast, insertional mutations resulting in a frameshift in the C-terminal domain of loricrin produce the characteristic ichthyosis of loricrin keratoderma in mouse and man. To ascertain the basis for the loricrin keratoderma phenotype, we assessed epidermal structure and stratum corneum (SC) function in a previously genotyped human loricrin keratoderma kindred. Our studies revealed abnormal corneocyte fragility and basal permeability barrier function, but accelerated repair kinetics. Despite fragility, increased water loss occurred predominantly via extracellular domains, which correlated with disorganized lamellar bilayers that were linked spatially to discontinuities of the CE. Accelerated barrier recovery was explicable by amplified lamellar body secretion, while partial normalization of the CE in the outer SC correlated with persistence of abundant calcium in the extracellular spaces (positioned to activate transglutaminase-1). These results show that the barrier abnormality in loricrin keratoderma is linked to a defective CE scaffold, resulting in increased extracellular permeability, as shown previously for another "scaffold disorder", lamellar ichthyosis. But in contrast to lamellar ichthyosis, the CE scaffold partially normalizes in the outer SC in loricrin keratoderma.     

Omega-hydroxyceramides are required for corneocyte lipid envelope (CLE) formation and normal epidermal permeability barrier function

Omega-hydroxyceramides (omega-OHCer) are the predominant lipid species of the corneocyte lipid envelope in the epidermis. Moreover, their omega-esterified-derivatives (acylCer) are major components of the stratum corneum extracellular lamellae, which regulate cutaneous permeability barrier function. Because epidermal omega-OHCer appear to be generated by a cytochrome P450-dependent process, we determined the effects of a mechanism-based inhibitor of omega-hydroxylation, aminobenzotriazole (ABT), on epidermal omega-OH Cer formation and barrier function. We first ascertained that ABT, but not hydroxybenzotriazole (OHBT), a chemical relative with no P450 inhibitory activity, inhibited the incorporation of [14C]-acetate into the omega-OH-containing Cer species in cultured human keratinocytes (68.1% +/- 6.9% inhibition versus vehicle-treated controls; p < 0.001), without altering the synthesis of other Cer and fatty acid species. In addition, ABT significantly inhibited the omega-hydroxylation of very long-chain fatty acids in cultured human keratinocytes. Topical application of ABT, but not OHBT, when applied to the skin of hairless mice following acute barrier disruption by tape-stripping, resulted in a significant delay in barrier recovery (e.g., 38.3% delay at 6 h versus vehicle-treated animals), assessed as increased transepidermal water loss. The ABT-induced barrier abnormality was associated with: (i) a significant decrease in the quantities of omega-OHCer in both the unbound and the covalently bound Cer pools; (ii) marked alterations of lamellar body structure and contents; and (iii) abnormal stratum corneum extracellular lamellar membrane structures, with no signs of cellular toxicity. Furthermore, pyridine-extraction of ABT- versus vehicle-treated skin, which removes all of the extracellular lamellae, leaving the covalently attached lipids, showed numerous foci with absent corneocyte lipid envelope in ABT- versus vehicle-treated stratum corneum. These results provide the first direct evidence for the importance of omega-OHCer for epidermal permeability function, and suggest further that acylCer and/or corneocyte lipid envelope are required elements in permeability barrier homeostasis.     

Association of cyclic adenosine monophosphate with permeability barrier homeostasis of murine skin

Activation of Gs protein increases the intracellular cyclic adenosine monophosphate (cAMP) level, and the Gs protein-linked receptor has been implicated in the skin barrier homeostasis. In this study, we investigated the role of cAMP in epidermal barrier function. The barrier was disrupted by tape stripping or treatment with acetone. Immediately after barrier disruption, reagents affecting the cAMP level were topically applied. Topical application of forskolin, which activates cAMP synthesis delayed barrier recovery, whereas application of the antagonist of cAMP, cAMP-Rp, accelerated barrier recovery. Moreover, application of 9-cyclopentyladenine, an inhibitor of cAMP synthesis also accelerated barrier recovery. Tape stripping was found to increase the cAMP in the epidermis. Light and electron microscopic observations showed the delay of lamellar body secretion by forskolin and acceleration of the lamellar body secretion by cAMP-Rp. Application of an inhibitor of protein kinase A did not affect the barrier recovery rate. The delay of barrier recovery induced by forskolin was blocked by the voltage-gated calcium channel blockers, nifedipine and verapamil. In cultured keratinocytes, forskolin increased the intracellular calcium concentration and both nifedipine and verapamil blocked the increase. These results suggest that intracellular cAMP in the epidermis is involved in skin barrier homeostasis.     

Basis of occlusive therapy in psoriasis: correcting defects in permeability barrier and calcium gradient

BACKGROUND: Although occlusive dressings have great potential in the management of psoriasis vulgaris, the therapeutic mechanism is not completely understood. Occlusion artificially restores and corrects the defective barrier in psoriasis plaques. Additionally, occlusion is know to normalize the epidermal calcium gradients in hyperproliferative murine skin models. METHODS: To investigate the basis of the therapeutic effect of occlusion on psoriatic plaques, we investigated the ultrastructural morphology of intercorneocyte lipid layers, lamellar bodies, and calcium gradient in chronic plaque-type psoriasis after occlusion with a water vapor-impermeable membrane. The specimens were processed for electron microscopy using: (i) ruthenium tetroxide postfixation; and (ii) ion-capture cytochemistry for calcium localization. RESULTS: Occlusion for 7 days resulted in a nearly mature pattern of intercellular multilamellar structures, re-establishment of the near-normal epidermal calcium gradient, and disappearance of calcium precipitates from the stratum corneum interstices. CONCLUSIONS: The normalization of the permeability barrier and epidermal calcium gradient may play important roles in the therapeutic effects of occlusive dressings in chronic plaque-type psoriasis.     

Topical hesperidin improves epidermal permeability barrier function and epidermal differentiation in normal murine skin

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.