Oral intake of lingonberry and amla fruit extract improves skin conditions in healthy female subjects: A randomized, double-blind, placebo-controlled clinical trial.

In this study, we examined the effect of ingestion of lingonberry and amla fruit extract (LAE) on several human skin conditions. To conduct a randomized, double-blinded, placebo-controlled study, we randomly divided 99 healthy female subjects into three groups; the first group received a drink containing 25 mg of lingonberry extract and 30 mg of amla fruit extract; the second group received a drink containing double the volume of extracts received by the first group; and the third group received a placebo drink. Each participant drank 50 mL of their assigned drink once daily for 12 weeks. The primary endpoint was skin elasticity, and the secondary endpoints included skin thickness, stratum corneum water content, and degree of wrinkles around the eyes. After 12 weeks of LAE drink intake, skin elasticity showed significant, dose-dependent improvements (P < 0.01). Skin thickness, stratum corneum water content, and the degree of wrinkles also significantly improved (P < 0.001) in a dose-dependent manner. The improvements in skin elasticity and thickness, as well as in the stratum corneum water content and the degree of wrinkles, observed upon oral intake of LAE indicate that LAE may be considered a candidate anti-aging agent for preventing skin weakening.

Lycii fructus extract ameliorates hydrogen peroxide-induced cytotoxicity through indirect antioxidant action.

Lycii Fructus is the dried ripe fruits of Lycium chinense and L. barbarum, which has long been used as a traditional food material in East Asia. The purpose of this study was to investigate the role of the indirect antioxidative action in the Lycii fructus extract (LFE)-induced cytoprotective effect in vitro. LFE significantly enhanced the expression of the drug-metabolizing enzyme genes and intracellular glutathione level in mouse hepatoma Hepa1c1c7 cells. LFE stimulated the nuclear translocation of aryl hydrocarbon receptor as well as nuclear factor (erythroid-derived 2)-like 2. The pretreatment of LFE for 24 h, but not for 30 min, completely inhibited the cytotoxic effect of hydrogen peroxide. Furthermore, chlorogenic acid, one of the main constituents of LFE, showed cytoprotection against hydrogen peroxide with the enhanced phase 2 enzyme gene expression. These results suggested that LFE exhibits a cytoprotective effect, possibly through the enhancement of the antioxidant gene expression. ABBREVIATIONS: LFE: Lycii Fructus extract; GSH: glutathione; NQO1: NAD(P)H:quinone oxidoreductase 1; HO-1: heme oxygenase-1; GCLC: glutamate-cysteine ligase, catalytic subunit; xCT: a component of cysteine/glutamate antiporter (cystine/glutamate exchanger); CYP1A1: cytochrome P450 1A1; GSH: glutathione; AhR: aryl hydrocarbon receptor; Nrf2: nuclear factor (erythroid-derived 2)-like 2; CGA: chlorogenic acid; RT-PCR: reverse transcription-polymerase chain reaction; DTT: dithiothreitol; PMSF: phenylmethylsulfonyl fluoride; ARE: antioxidant response element; XRE: xenobiotic responsive element.

Changes in fibrillin-1 expression, elastin expression and skin surface texture at sites of cultured epithelial autograft transplantation onto wounds from burn scar excision.

This study investigated the recovery process during which grafted cultured epithelium generated skin elasticity and skin surface microarchitecture. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6 weeks (stage 1), 5 samples after 6 weeks and within 6 months (stage 2), 6 samples after 6 months and within 18 months (stage 3) and 7 samples beyond 18 months (stage 4) of transplantation. These samples were evaluated by taking replicas of skin surface, and histological changes of fibrillin-1 and elastin. The expression patterns were classified using a grading scale. The grade of skin surface texture was significantly higher at stage 3 and marginally significantly higher at stage 4 compared with stage 1. The grade of fibrillin-1 was marginally significantly higher at stage 3 and significantly higher at stage 4 compared with stage 1. The grade of elastin was marginally significantly higher at stage 4 compared with stage 1. These results showed that it is important for patients to have skin care and avoid external forces for at least 18 months after transplantation.

Changes in the expression of epidermal differentiation markers at sites where cultured epithelial autografts were transplanted onto wounds from burn scar excision.

This study investigated the recovery process during which grafted cultured epithelium formed normal epidermis. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6 weeks (stage 1), 5 samples after 6 weeks and within 6 months (stage 2), 6 samples after 6 months and within 18 months (stage 3) and 7 samples beyond 18 months (stage 4) after transplantation. These samples were stained for monoclonal antibodies against filaggrin, transglutaminase (TG), cytokeratin 6 and involucrin. Their expressions were examined in the epidermis. The expression patterns were classified using a six-grade scale. The grades of filaggrin and TG were significantly higher at stage 3 and 4 compared with stage 1. There was a marginally significant increase in the grade of cytokeratin 6 at stage 3 and it was significantly higher at stage 4 compared with stage 1. These results showed that wound healing continued at a molecular level until the end of stage 3. The recovery of involucrin was delayed compared with that of other markers. TG and involucrin are thought to be regulated independently at the grafted sites.

Modulating effects of oral administration of Lycii Fructus extracts on UVB-induced skin erythema: A Randomized, placebo-controlled study.

Severe UV exposure induces skin inflammation, causing erythema. Lycii Fructus (Lycium barbarum and Lycium chinense) is a potential antioxidant agent with a high content of polyphenols, including rutin and chlorogenic acid. This study examined the effects of Lycii Fructus extract (LFE) on UVB-induced skin erythema in humans. Healthy volunteers were randomly assigned to one of two groups and received UVB irradiation at 1.5 minimal erythemal dose (MED) on day 0 at three designated sites on their backs, and the skin color was measured until day 7. After an 8-week treatment with LFE (900 mg/day) or placebo, UVB irradiation (l.5 MED) was applied again at different sites on day 63. Skin color was continuously measured in each group until day 69. LFE tablet administration for 8 weeks significantly inhibited UVB-induced erythema formation and increased the MED by 13%. Erythema formation peaked on the first day after UVB irradiation, but gradually dissipated over the next several days. LFE tended to accelerate erythema disappearance. To determine the polyphenol responsible for the protection against UVB-induced skin damage, the effects of LFE-derived polyphenols and their metabolites on UVB-induced cytotoxicity were examined in vitro. The major intestinal metabolite of rutin and LFE significantly attenuated phototoxicity and in human keratinocyte HaCaT cells. Quercetin enhanced intracellular glutathione levels in HaCaT cells, even though LFE did not increase it. Together, the results showed that LFE inhibited erythema formation and accelerated erythema dissipation, possibly through its direct antioxidative action.

Visualized procollagen Iα1 demonstrates the intracellular processing of propeptides.

The processing of type I procollagen is essential for fibril formation; however, the steps involved remain controversial. We constructed a live cell imaging system by inserting fluorescent proteins into type I pre-procollagen α1. Based on live imaging and immunostaining, the C-propeptide is intracellularly cleaved at the perinuclear region, including the endoplasmic reticulum, and subsequently accumulates at the upside of the cell. The N-propeptide is also intracellularly cleaved, but is transported with the repeating structure domain of collagen into the extracellular region. This system makes it possible to detect relative increases and decreases in collagen secretion in a high-throughput manner by assaying fluorescence in the culture medium, and revealed that the rate-limiting step for collagen secretion occurs after the synthesis of procollagen. In the present study, we identified a defect in procollagen processing in activated hepatic stellate cells, which secrete aberrant collagen fibrils. The results obtained demonstrated the intracellular processing of type I procollagen, and revealed a link between dysfunctional processing and diseases such as hepatic fibrosis.

Influence of heparan sulfate chains in proteoglycan at the dermal-epidermal junction on epidermal homeostasis.

Basement membrane (BM) plays important roles in skin morphogenesis and homeostasis by controlling dermal-epidermal interactions. However, it remains unclear whether heparan sulfate (HS) chains of proteoglycan in epidermal BM contribute to epidermal homeostasis. To explore the function of HS chains at the dermal-epidermal junction (DEJ), we used a skin equivalent (SE) model. This model lacked HS at the DEJ and showed abnormal expression of the differentiation markers filaggrin and loricrin; similar changes were seen in ultraviolet B-irradiated human skin. Perlecan (core-protein of HS proteoglycan) remained localized at the DEJ in both SE and UV-irradiated human skin. Heparanase, which degrades HS, was increased in epidermis of UV-irradiated skin, compared with unirradiated skin. We found that deposition of HS at the DEJ in the SE model was markedly augmented by a synthetic heparanase inhibitor, and release of HS into conditioned medium was suppressed. The inhibitor also increased filaggrin and loricrin expression. Moreover, the recovery of HS was associated with an increase of Ki67-positive basal cells, compared with control SE cultured without inhibitor. Comparative gene expression analysis in epidermis of SE cultured in the presence and absence of heparanase inhibitor, using DNA microarrays, showed that recovery of HS was associated with increased expression of differentiation-related genes and down-regulation of degradation-enzyme-related genes. These results indicate that degradation of HS at the DEJ by heparanase impairs epidermal homeostasis in SE, leading to abnormal differentiation and proliferation behaviour. Thus, HS chains in epidermal BM appear to play an important role in epidermal homeostasis.

Key role of heparan sulfate chains in assembly of anchoring complex at the dermal-epidermal junction.

Epidermal basement membrane forms anchoring complex composed of hemidesmosomes, anchoring filaments, lamina densa and anchoring fibrils to link epidermis to dermis. However, the anchoring complex is rarely formed in skin equivalent models, probably because of degradation of extracellular matrix (ECM) proteins and heparan sulfate chains by matrix metalloproteinases (MMPs) and heparanase, respectively. To explore the roles of ECM proteins and heparan sulfate in anchoring complex assembly, we used specific inhibitors of MMPs and heparanase, and the formation of anchoring complex was analysed in terms of polarized deposition of collagen VII, BP180 and ß4 integrin at the dermal-epidermal junction (DEJ) by means of immunohistochemistry and transmission electron microscopy (TEM). The deposition of collagen VII was polarized to the basal side by the addition of MMP inhibitor, and the staining intensity was increased by combined treatment with MMP inhibitor and heparanase inhibitor, which enhanced anchoring fibril formation as observed by TEM. BP180 was polarized to the basal side by heparanase inhibitor, which protects HS chains, but not by MMP inhibitor. MMP inhibitor improved the polarization of ß4 integrin. Hemidesmosomes were formed in the presence of each inhibitor, as observed by TEM, and formation was greatly enhanced by the combined treatment. These findings suggest that heparan sulfate chains, in addition to ECM proteins at the DEJ, play an important role in the assembly of anchoring complex, especially hemidesmosomes and anchoring fibrils.

Heparanase Inhibitors Facilitate the Assembly of the Basement Membrane in Artificial Skin.

Recent research suggests that the basement membrane at the dermal-epidermal junction of the skin plays an important role in maintaining a healthy epidermis and dermis, and repeated damage to the skin can destabilize the skin and accelerate the aging process. Skin-equivalent models are suitable for studying the reconstruction of the basement membrane and its contribution to epidermal homeostasis because they lack the basement membrane and show abnormal expression of epidermal differentiation markers. By using these models, it has been shown that reconstruction of the basement membrane is enhanced not only by supplying basement membrane components, but also by inhibiting proteinases such as urokinase and matrix metalloproteinase. Although matrix metalloproteinase inhibitors assist in the reconstruction of the basement membrane structure, their action is not sufficient to promote its functional recovery. However, heparanase inhibitors stabilize the heparan sulfate chains of perlecan (a heparan sulfate proteoglycan) and promote the regulation of heparan sulfate binding growth factors in the basement membrane. Heparan sulfate promotes effective protein-protein interactions, thereby facilitating the assembly of type VII collagen anchoring fibrils and elastin-associated microfibrils. Using both matrix metalloproteinase inhibitors and heparanase inhibitors, the basement membrane in a skin-equivalent model comes close to recapitulating the structure and function of an in vivo basement membrane. Therefore, by using an appropriate dermis model and suitable protease inhibitors, it may be possible to produce skin-equivalent models that are more similar to natural skin.

Possible involvement of gelatinases in basement membrane damage and wrinkle formation in chronically ultraviolet B-exposed hairless mouse.

A number of studies indicate that matrix metalloproteinase might be involved in photoaging, but little is known about their direct contribution to ultraviolet-induced histologic and morphologic changes in the skin in vivo. This study reports the relationship between changes of matrix metalloproteinase activities and ultraviolet B-induced skin changes in hairless mouse. The role of matrix metalloproteinase in the skin changes was studied by topical application of a specific matrix metalloproteinase inhibitor. The backs of mice were exposed to ultraviolet B three times a week for 10 wk. Histologic studies showed that the basement membrane structure was damaged, with epidermal hyperplasia, in the first 2 wk of ultraviolet B irradiation, followed by the appearance of wrinkles, which gradually extended in the latter half of the ultraviolet B irradiation period. We observed enhancement of type IV collagen degradation activity, but not collagenase or matrix metalloproteinase-3 activity, in extracts of ultraviolet B-irradiated, wrinkle-bearing skin. Gelatin zymographic analysis revealed that gelatinases, matrix metalloproteinase-9 and matrix metalloproteinase-2, were significantly increased in the extract. In situ zymographic study clarified that the activity was specifically localized in whole epidermis of ultraviolet B-irradiated, wrinkled skin in comparison with normal skin. The activity was induced around the basal layer of the epidermis by a single ultraviolet exposure of at least one minimal erythema dose. Furthermore, topical application of a specific matrix metalloproteinase inhibitor, CGS27023A, inhibited ultraviolet B-induced gelatinase activity in the epidermis, and its repeated application prevented ultraviolet B-induced damage to the basement membrane, as well as epidermal hyperplasia and dermal collagen degradation. Ultraviolet B-induced wrinkles were also prevented by administration of the inhibitor. These results, taken together, suggest that ultraviolet B-induced enhancement of gelatinase activity in the skin contributes to wrinkle formation through the destruction of basement membrane structure and dermal collagen in chronically ultraviolet B-exposed hairless mouse, and thus topical application of matrix metalloproteinase inhibitors may be an effective way to prevent ultraviolet B-induced wrinkle formation.

Cultured human dermal papilla cells secrete a chemotactic factor for melanocytes.

Large numbers of pigmented melanocytes are located in human hair follicles, predominantly around the dermal papillae, and the number of melanocytes and the melanogenic activity of the hair follicles are closely related to the hair cycle. We found that cultured human dermal papilla cells secreted a melanocyte chemoattractant into the medium. Skin fibroblasts also showed weak chemoattraction of melanocytes, while skin keratinocytes and melanocytes did not. Since this chemotactic activity was heat-and protease-sensitive and was present in the relatively high molecular weight fraction (130-200 kDa), it may be due to extracellular matrix (ECM) that proteins secreted from the cultured dermal papilla cells. This chemotactic signal between dermal papillae and melanocytes may control the localization and migration of hair melanocytes in vivo.

Immunohistochemical survey of the distribution of epidermal melanoblasts and melanocytes during the development of UVB-induced pigmented spots.

BACKGROUND: Repeated exposures to ultraviolet B radiation (UVB) induce pigmented spots on dorsal skin of (HR-1 x HR/De) F(1) hairless mouse. We showed previously that this mouse is suitable for studies of melanocyte function. OBJECTIVE: To clarify the mechanism of development of pigmented spots induced by chronic UVB exposure. METHODS: We used light and fluorescence microscopy to quantify changes in the numbers of differentiated melanocytes containing melanin pigments (MM) and melanoblasts/melanocytes immunohistochemically positive for tyrosinase-related protein (TRP)-1, TRP-2 (dopachrome tautomerase), and c-kit in epidermis during the development of pigmented spots in hairless mice chronically exposed to UVB (99 mJ/cm(2), 3 times/week, 8 weeks). RESULTS: The change in the number of TRP-1-positive cells during chronic UVB exposure was similar to that of MM: both increased dramatically during the stage of acute pigmentation, then decreased sharply after cessation of UVB, concomitantly with depigmentation; subsequently they increased gradually with the development of pigmented spots. In contrast, after two UVB exposures, no c-kit-positive cells were detected, then the number gradually increased during UVB irradiation, and continued to increase after cessation of irradiation; TRP-2-positive cells showed a rather similar pattern, except that they did not disappear initially. CONCLUSION: Our results indicate that chronic UVB irradiation induces differentiation and proliferation of melanoblasts, followed by an increase of differentiated melanocytes, leading to the development of pigmented spots. The sequence of expression of markers appeared to be c-kit, TRP-2, TRP-1, and finally melanin, as it is during normal melanocyte differentiation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the proliferation and differentiation of mouse epidermal melanocytes from pigmented spots induced by ultraviolet radiation B.

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB-induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor-free medium supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF induced the proliferation and differentiation of melanocytes in those keratinocyte-depleted cultures. Moreover, an antibody to GM-CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV-irradiated mice, but not from control mice. Further, the GM-CSF antibody inhibited the proliferation and differentiation of melanocytes co-cultured with keratinocytes derived from UV-irradiated mice, but not from control mice. The quantity of GM-CSF secreted from keratinocytes derived from the pigmented spots of UV-irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM-CSF in keratinocytes derived from the pigmented spots of skin in UV-irradiated mice, but not from normal skin in control mice. These results suggest that GM-CSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB-induced pigmented spots.