Melanogenesis inhibitory effect of fatty acid alkyl esters isolated from Oxalis triangularis.

Ten fatty acid alkyl esters isolated from Oxalis triangularis, were evaluated for the effects on melanogenesis using mouse B16 melanoma cells. Treatment of methyl linoleate, methyl linolenate, ethyl linoleate and ethyl linolenate significantly blocked forskolin-induced melanogenesis and inhibited tyrosinase activity. In addition, we found that they inhibited cAMP production, suggesting that their anti-melanogenic effect is mediated by the inhibition of cAMP production. We concluded that methyl/ethyl linoleate and linolenate isolated from Oxalis triangularis have pigment inhibition activity. These compounds may be useful as the cosmetic agent to stimulate skin whitening.

Mechanisms of melanogenesis inhibition by propafenone.

Melanogenesis is a physiological process that results in the synthesis of melanin pigments, which play a crucial protective role against skin photocarcinogenesis. The present study was conducted to determine the inhibitory effects of propafenone on melanogenesis and to elucidate the molecular events involved in the inhibition of melanogenesis by propafenone. To accomplish this, several experiments were conducted using human epidermal melanocyte cells. The melanin content and cAMP production were evaluated, and western blots for proteins involved in melanogenesis were conducted. The melanin content was significantly inhibited by propafenone in a concentration-dependent manner. To clarify the mechanism of the depigmenting property of propafenone, we examined the involvement of propafenone in cAMP signaling. In the cAMP production assay, the intracellular cAMP level was reduced by propafenone. The level of microphthalmia-associated transcription factor (MITF) protein, the upstream transcription factor of tyrosinase, was also reduced by propafenone. In addition, propafenone inhibited the expression of tyrosinase, TRP-1, and TRP-2. Taken together, the results of our study show that propafenone inhibits melanogenesis by suppressing cAMP production, which is involved in the expression of melanogenesis-related proteins and suggests that propafenone may be an effective inhibitor of hyperpigmentation.

Anti-adipogenesis by 6-thioinosine is mediated by downregulation of PPAR gamma through JNK-dependent upregulation of iNOS.

Adipocyte dysfunction is associated with the development of obesity. This study shows that 6-thioinosine inhibits adipocyte differentiation. The mRNA levels of PPAR gamma and C/EBPalpha, but not C/EBPbeta and delta, were reduced by 6-thioinosine. Moreover, the mRNA levels of PPAR gamma target genes (LPL, CD36, aP2, and LXRalpha) were down-regulated by 6-thioinosine. We also demonstrated that 6-thioinosine inhibits the transactivation activity and the mRNA level of PPAR gamma. Additionally, attempts to elucidate a possible mechanism underlying the 6-thioinosine-mediated effects revealed that 6-thioinosine induced iNOS gene expression without impacting eNOS expression, and that this was mediated through activation of AP-1, especially, JNK. In addition, 6-thioinosine was found to operate upstream of MEKK-1 in JNK activation signaling. Taken together, these findings suggest that the inhibition of adipocyte differentiation by 6-thioinosine occurs primarily through the reduced expression of PPAR gamma, which is mediated by upregulation of iNOS via the activation of JNK.

Phloridzin-induced melanogenesis is mediated by the cAMP signaling pathway.

Melanogenesis is a physiological process that results in the synthesis of melanin pigments, which play a crucial protective role in protection against skin photocarcinogenesis. Phloridzin is a phloretin 2'-glucoside that is found in many parts of the apple tree that reportedly increases tyrosinase activity and melanin contents through inhibition of protein kinase C (PKC) activity in B16 melanoma cells. In this study, we attempted to accurately determine the effects and mechanisms of action of phloridzin on melanogenesis. Specifically, we observed that phloridzin-induced a dose-dependent increase in tyrosinase activity and melanin contents, and that these changes were accompanied by an increase in the levels of tyrosinase and the tyrosinase-related proteins, TRP-1 and TRP-2. Furthermore, the cAMP-dependent protein kinase A (PKA) inhibitor H89 impaired the response of the tyrosinase activity and melanin synthesis to phloridzin. Additionally, phloridzin stimulated cAMP production and phosphorylation of the cAMP-response element binding protein (CREB). Taken together, the results of this study indicate that phloridzin increases tyrosinase gene expression through the cAMP signaling pathway, thereby leading to the stimulation of melanogenesis.

A cell-based system for screening hair growth-promoting agents.

Androgen-inducible transforming growth factor beta (TGF-beta1) derived from dermal papilla cells (DPCs) is a catagen inducer that mediates hair growth suppression in androgenetic alopecia (AGA). In this study, a cell-based assay system was developed to monitor TGF-beta1 promoter activity and then used to evaluate the effects of activated TGF-beta1 promoter in human epidermal keratinocytes (HaCaT). To accomplish this, a pMetLuc-TGF-beta1 promoter plasmid that expresses the luciferase reporter gene in response to TGF-beta1 promoter activity was constructed. Treatment of HaCaT with dihydrotestosterone, which is known to be a primary factor of AGA, resulted in a concentration-dependent increase in TGF-beta1 promoter activity. However, treatment of HaCaT with the TGF-beta1 inhibitor, curcumin, resulted in a concentration-dependant decrease in TGF-beta1 expression. Subsequent use of this assay system to screen TGF-beta1 revealed that HaCaT that were treated with apigenin showed decreased levels of TGF-beta1 expression. In addition, treatment with apigenin also significantly increased the proliferation of both SV40T-DPCs (human DPCs) and HaCaT cells. Furthermore, apigenin stimulated the elongation of hair follicles in a rat vibrissa hair follicle organ culture. Taken together, these findings suggest that apigenin, which is known to have antioxidant, anti-inflammatory, and anti-tumor properties, stimulates hair growth through downregulation of the TGF-beta1 gene. In addition, these results suggest that this assay system could be used to quantitatively measure TGF-beta1 promoter activity in HaCaT, thereby facilitating the screening of agents promoting hair growth.

6-Benzylaminopurine stimulates melanogenesis via cAMP-independent activation of protein kinase A.

Melanogenesis is a physiological process that results in the synthesis of melanin pigments, which play a crucial protective role against skin photocarcinogenesis. The present study was designed to determine the effects of 6-benzylaminopurine (6-BAP) on melanogenesis and elucidate the molecular events of melanogenesis induced by 6-BAP. To elucidate the pigmenting effect of 6-BAP and its mechanism, several experiments were performed in B16 melanoma cells. Melanin content, tyrosinase activity, cAMP production, and Western blots for proteins which are involved in melanogenesis were introduced in this study. Melanin content and tyrosinase activity increased in response to treatment with 6-BAP in a concentration-dependent manner. The tyrosinase, TRP-1, TRP-2 and MITF protein levels were found to increase significantly in response to 6-BAP in a time-dependent manner. In addition, Western blot analysis revealed that 6-BAP increased the phosphorylated level of CRE-binding protein. The increased melanin synthesis that was induced by treatment with 6-BAP treatment was reduced significantly in response to co-treatment with H-89 [a protein kinase A (PKA) inhibitor], whereas co-treatment with SB203580 (a p38 MAPK inhibitor) and Ro-32-0432 (a PKC inhibitor) did not attenuate the increase in melanin content levels that was induced by 6-BAP. In a cAMP production assay, 6-BAP did not increase the intracellular cAMP level. These findings suggest that 6-BAP activates PKA via a cAMP-independent pathway and subsequently stimulates melanogenesis by up-regulating MITF and tyrosinase expression.

Isorhamnetin represses adipogenesis in 3T3-L1 cells.

Adipocyte dysfunction is strongly associated with the development of obesity, which is a major risk factor for many disorders including diabetes, hypertension, and heart disease. It is generally accepted that the regulation of adipogenesis or adipokines expression prevents obesity. In this study, we show that isorhamnetin inhibits adipocyte differentiation, as evidenced by reduced triglyceride (TG) accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity. At the molecular level, the mRNA expression levels of peroxidase proliferator-activated receptor-gamma (PPAR-gamma) and CCAAT/enhancer-binding protein-alpha (C/EBP-alpha), which are the major adipogenic transcription factors, were markedly reduced by isorhamnetin. However, the mRNA levels of C/EBP-beta and -delta, the upstream regulators of PPAR-gamma and C/EBP-alpha, were not reduced by isorhamnetin. Moreover, the mRNA levels of PPAR-gamma target genes such as lipoprotein lipase (LPL), CD36, aP2, and liver X receptor-alpha (LXR-alpha) were downregulated by isorhamnetin. We also showed that isorhamnetin inhibits the expression and secretion of adiponectin, and the results of adiponectin promoter assays suggest the inhibition of PPAR-gamma expression as a possible mechanism underlying the isorhamnetin-mediated effects. Taken together, these results indicate that isorhamnetin inhibits adipogenesis through downregulation of PPAR-gamma and C/EBP-alpha.

Matrine inhibits PMA-induced MMP-1 expression in human dermal fibroblasts.

Matrix metalloproteinase-1 (MMP-1) plays an important role in the maintenance and turnover of extracellular matrix (ECM) macromolecules. Remodelling of extracellular matrix by MMPs is a hallmark feature of physiological and pathological processes. In this study, in order to establish the therapeutic potential of matrine, we investigated its effect on MMP-1 expression in human dermal fibroblast cells. We found that matrine inhibited both MMP-1 mRNA and protein expression induced by PMA (phorbol myristate acetate). Therefore, we characterized the inhibitory mechanism of matrine on PMA-induced MMP-1 expression. Matrine inhibited PMA-induced activation of the AP-1 promoter, an important nuclear transcription factor in MMP-1 expression. Additionally, we detected that matrine suppressed the PMA-induced phosphorylation of two mitogen-activated protein kinases, extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but did not suppress the PMA-induced phosphorylation of p38 kinase. These results suggest that matrine suppresses PMA-induced MMP-1 expression through inhibition of the AP-1 signaling pathway and also may be beneficial for treatment of some inflammatory skin disorders.

Effect of Camellia japonica oil on human type I procollagen production and skin barrier function.

Type I collagen is the primary component of the skin dermis. Both the quantity and quality of extracellular collagen are primarily related to skin ageing. In this study, we investigated the possibility that Camellia japonica oil (CJ oil) may be introduced as an anit-wrinkle agent. As a first step to this end, human COL1A2 promoter luciferase assay was performed in human dermal fibroblast cells. CJ oil was determined to activate human COL1A2 promoter in a concentration-dependent manner. In consistency with this result, while matrix metalloproteinase (MMP)-1 activity was inhibited by CJ oil, human type I procollagen synthesis was also induced by CJ oil. These results suggest the possibility that CJ oil may be involved in the skin ageing. For the evaluation of CJ oil's safety and efficiency on human skin, human skin primary irritation test and trans-epidermal water loss (TEWL) were performed. Transepidermal water loss (TEWL) was measured before treatment then, 1h and 2h after treatment; the forearm site was selected to measure TEWL. Also, a human skin primary irritation test was performed on the normal skin (upper back) in 30 volunteers to see if a certain material included in CJ oil has irritation or sensitization potential. In these assays, CJ oil reduced trans-epidermal water loss (TEWL) and did not induce any adverse reactions. Therefore, based on these results, we suggest the possibility that CJ oil may be considered as possible wrinkle-reducing candidates for topical application.

Panax ginseng induces human Type I collagen synthesis through activation of Smad signaling.

Skin aging appears to be principally related to a decrease in levels of Type I collagen, the primary component of the dermal layer of skin. It is important to introduce an efficient agent for effective management of skin aging; this agent should have the fewest possible side effects and the greatest wrinkle-reducing effect. In the course of screening collagen production-promoting agents, we obtained Panax ginseng C.A. Meyer. This study was designed to investigate the possible collagen production-promoting activities of Panax ginseng C.A. Meyer root extract (PGRE) in human dermal fibroblast cells. As a first step to this end, human COL1A2 promoter luciferase assay was performed in human dermal fibroblast cells. In this assay, PGRE activated human COL1A2 promoter activity in a concentration-dependent manner. Human Type I procollagen synthesis was also induced by PGRE. These results suggest that PGRE promotes collagen production in human dermal fibroblast cells. Additionally, we have attempted to characterize the mechanism of action of PGRE in Type I procollagen synthesis. PGRE was found to induce the phosphorylation of Smad2, an important transcription factor in the production of Type I procollagen. When applied topically in a human skin primary irritation test, PGRE did not induce any adverse reactions. Therefore, based on these results, we suggest the possibility that PGRE may be considered as an attractive, wrinkle-reducing candidate for topical application.

A cell-based assay system for high-throughput screening of anti-wrinkle agents in human dermal fibroblast transfectant cells.

A cell-based assay system for monitoring COL1A2 [alpha2(I) collagen gene] promoter activity was developed to determine the influence of activated COL1A2 promoter in human dermal fibroblast cells. A pLuc-COL1A2 promoter plasmid that expresses the luciferase reporter gene in response to COL1A2 promoter activity was constructed. The pLuc-COL1A2 promoter plasmid and pCI-neo plasmid containing the NPT (neomycin phosphotransferase) gene for Geneticin resistance in host cells were co-transfected into human dermal fibroblast cells. COL1A2 promoter activities were measured by luciferase reporter gene assay using a luminescence detection method. Fibroblast cell transfectants treated with TNFalpha (tumour necrosis factor alpha), known to be an inhibitor of COL1A2 promoter expression, showed a reduction of COL1A2 promoter activity in a concentration-dependent manner, whereas TGF-beta (transforming growth factor-beta), known to be a stimulator of COL1A2 promoter expression, increased COL1A2 activity in a concentration-dependent manner. This assay system could be used to quantitatively measure COL1A2 promoter activity in human dermal fibroblast cells and allow the screening of anti-wrinkle agents from various synthetic chemicals and natural products.

Emodin inhibits TNF alpha-induced MMP-1 expression through suppression of activator protein-1 (AP-1).

Matrix metalloproteinases (MMPs) are the proteases involved in the degradation of the extracellular matrix. MMP-1 is thought to be one of the key enzymes acting in fibrolysis, a process closely related to tissue remodeling. In this study, we found that emodin, an anthraquinone which has been isolated from the rhizome of Rheum palmatum, significantly inhibited TNF alpha-induced MMP-1 gene expression in a concentration-dependent manner. Therefore, we have attempted to characterize the inhibitory mechanism of emodin in TNF alpha-induced MMP-1 expression. Emodin was determined to inhibit TNF alpha-induced activation of AP-1 promoter, an important nuclear transcription factor in MMP-1 expression. Additionally, we detected that emodin suppressed the TNF alpha-induced phosphorylation of two mitogen-activated protein kinases, extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but it did not suppress the TNF alpha-induced phosphorylation of p38 kinase. In a consistent result, the TNF alpha-induced MMP-1 expression was inhibited by PD98059 (MEK/ERK inhibitor) and SP600125 (JNK inhibitor), but was not inhibited by SB203580, a p38 MAPK inhibitor. Taken together, these results show that emodin suppresses TNF alpha-induced MMP-1 expression through the inhibition of the AP-1 signaling pathway.