Isolation, Characterization, Genome Annotation, and Evaluation of Hyaluronidase Inhibitory Activity in Secondary Metabolites of Brevibacillus sp. JNUCC 41: A Comprehensive Analysis through Molecular Docking and Molecular Dynamics Simulation.

Brevibacillus sp. JNUCC 41, characterized as a plant-growth-promoting rhizobacterium (PGPR), actively participates in lipid metabolism and biocontrol based on gene analysis. This study aimed to investigate the crucial secondary metabolites in biological metabolism; fermentation, extraction, and isolation were performed, revealing that methyl indole-3-acetate showed the best hyaluronidase (HAase) inhibitory activity (IC50: 343.9 µM). Molecular docking results further revealed that the compound forms hydrogen bonds with the residues Tyr-75 and Tyr-247 of HAase (binding energy: -6.4 kcal/mol). Molecular dynamics (MD) simulations demonstrated that the compound predominantly binds to HAase via hydrogen bonding (MM-PBSA binding energy: -24.9 kcal/mol) and exhibits good stability. The residues Tyr-247 and Tyr-202, pivotal for binding in docking, were also confirmed via MD simulations. This study suggests that methyl indole-3-acetate holds potential applications in anti-inflammatory and anti-aging treatments.

Isolation, Characterization, Genome Annotation, and Evaluation of Tyrosinase Inhibitory Activity in Secondary Metabolites of Paenibacillus sp. JNUCC32: A Comprehensive Analysis through Molecular Docking and Molecular Dynamics Simulation.

A potential strain, Paenibacillus sp. JNUCC32, was isolated and subjected to whole-genome sequencing. Genome functional annotation revealed its active metabolic capabilities. This study aimed to investigate the pivotal secondary metabolites in the biological system. Fermentation and extraction were performed, resulting in the isolation of seven known compounds: tryptophol (1), 3-(4-hydroxyphenyl)propionic acid (2), ferulic acid (3), maculosin (4), brevianamide F (5), indole-3-acetic acid (6), and butyric acid (7). Tryptophol exhibited favorable pharmacokinetic properties and demonstrated certain tyrosinase inhibitory activity (IC50 = 999 µM). For further analysis of its inhibition mechanism through molecular docking and molecular dynamics (MD) simulation, tryptophol formed three hydrogen bonds and a pro-Michaelis complex with tyrosinase (binding energy = -5.3 kcal/mol). The MD simulation indicated favorable stability for the tryptophol-mushroom tyrosinase complex, primarily governed by hydrogen bond interactions. The crucial residues VAL-283 and HIS-263 in the docking were also validated. This study suggests tryptophol as a potential candidate for antibrowning agents and dermatological research.

Anti-Photoaging Effects of Upcycled Citrus junos Seed Anionic Peptides on Ultraviolet-Radiation-Induced Skin Aging in a Reconstructed Skin Model.

Side streams and byproducts of food are established sources of natural ingredients in cosmetics. In the present study, we obtained upcycled low-molecular-weight anionic peptides (LMAPs) using byproducts of the post-yuzu-juicing process by employing an enzyme derived from Bacillus sp. For the first time, we isolated anionic peptides less than 500 Da in molecular weight from Citrus junos TANAKA seeds via hydrolysis using this enzyme. The protective effect of LMAPs against UVR-induced photoaging was evaluated using a reconstructed skin tissue (RST) model and keratinocytes. The LMAPs protected the keratinocytes by scavenging intracellular reactive oxygen species and by reducing the levels of paracrine cytokines (IL-6 and TNF-α) in UVR (UVA 2 J/cm2 and UVB 15 mJ/cm2)-irradiated keratinocytes. Additionally, the increase in melanin synthesis and TRP-2 expression in RST caused by UVR was significantly inhibited by LMAP treatment. This treatment strongly induced the expression of filaggrin and laminin-5 in UVR-irradiated RST. It also increased type I collagen expression in the dermal region and in fibroblasts in vitro. These results suggest that a hydrolytic system using the enzyme derived from Bacillus sp. can be used for the commercial production of LMAPs from food byproducts and that these LMAPs can be effective ingredients for improving photoaging-induced skin diseases.

6-Methylcoumarin Promotes Melanogenesis through the PKA/CREB, MAPK, AKT/PI3K, and GSK3ß/ß-Catenin Signaling Pathways.

We investigated the effects of four coumarin derivatives, namely, 6-methylcoumarin, 7-methylcoumarin, 4-hydroxy-6-methylcoumarin, and 4-hydroxy-7-methylcoumarin, which have similar structures on melanogenesis in a murine melanoma cell line from a C57BL/6J mouse called B16F10. Our results showed that only 6-methylcoumarin significantly increased the melanin synthesis in a concentration-dependent manner. In addition, the tyrosinase, TRP-1, TRP-2, and MITF protein levels were found to significantly increase in response to 6-methylcoumarin in a concentration-dependent manner. To elucidate the molecular mechanism whereby 6-methylcoumarin-induced melanogenesis influences the melanogenesis-related protein expression and melanogenesis-regulating protein activation, we further assessed the B16F10 cells. The inhibition of the ERK, Akt, and CREB phosphorylation, and conversely, the increased p38, JNK, and PKA phosphorylation activated the melanin synthesis via MITF upregulation, which ultimately led to increased melanin synthesis. Accordingly, 6-methylcoumarin increased the p38, JNK, and PKA phosphorylation in the B16F10 cells, whereas it decreased the phosphorylated ERK, Akt, and CREB expressions. In addition, the 6-methylcoumarin activated GSK3ß and ß-catenin phosphorylation and reduced the ß-catenin protein level. These results suggest that 6-methylcoumarin stimulates melanogenesis through the GSK3ß/ß-catenin signal pathway, thereby affecting the pigmentation process. Finally, we tested the safety of 6-methylcoumarin for topical applications using a primary human skin irritation test on the normal skin of 31 healthy volunteers. We found that 6-methylcoumarin did not cause any adverse effects at concentrations of 125 and 250 µM. Our findings indicate that 6-methylcoumarin may be an effective pigmentation stimulator for use in cosmetics and the medical treatment of photoprotection and hypopigmentation disorders.

Syringetin Promotes Melanogenesis in B16F10 Cells.

Syringetin, an active compound present in red grapes, jambolan fruits, Lysimachia congestiflora, and Vaccinium ashei, is a dimethyl myricetin derivative which contains free hydroxyl groups at the C-2' and C-4' positions in ring B. Recent studies have revealed that syringetin possesses multiple pharmacological properties, such as antitumor, hepatoprotective, antidiabetic, antioxidative, and cytoprotective activities. To date, there has been no attempt to test the action of syringetin on melanogenesis. In addition, the molecular mechanism for the melanogenic effects of syringetin remains largely unknown. In this study, we investigated the effect of syringetin on melanogenesis in a murine melanoma cell line from a C57BL/6J mouse, B16F10. Our results showed that syringetin markedly stimulated melanin production and tyrosinase activity in a concentration-dependent manner in B16F10 cells. We also found that syringetin increased MITF, tyrosinase, TRP-1, and TRP-2 protein expression. Moreover, syringetin inhibited ERK and PI3K/Akt phosphorylation by stimulating p38, JNK, PKA phosphorylation levels, subsequently stimulating MITF and TRP upregulation, resulting in the activation of melanin synthesis. Furthermore, we observed that syringetin activated phosphorylation of GSK3ß and ß-catenin and reduced the protein level of ß-catenin, suggesting that syringetin stimulates melanogenesis through the GSK3ß/ß-catenin signal pathway. Finally, a primary skin irritation test was conducted on the upper backs of 31 healthy volunteers to determine the irritation or sensitization potential of syringetin for topical application. The results of the test indicated that syringetin did not cause any adverse effects on the skin. Taken together, our findings indicated that syringetin may be an effective pigmentation stimulator for use in cosmetics and in the medical treatment of hypopigmentation disorders.

The Effects of 2'-Hydroxy-3,6'-Dimethoxychalcone on Melanogenesis and Inflammation.

In this study, we demonstrated that 2'-hydroxy-3,6'-dimethoxychalcone (3,6'-DMC) alleviated α-MSH-induced melanogenesis and lipopolysaccharides (LPS)-induced inflammation in mouse B16F10 and RAW 264.7 cells. In vitro analysis results showed that the melanin content and intracellular tyrosinase activity were significantly decreased by 3,6'-DMC, without cytotoxicity, via decreases in tyrosinase and the tyrosinase-related protein 1 (TRP-1) and TRP-2 melanogenic proteins, as well as the downregulation of microphthalmia-associated transcription factor (MITF) expression through the upregulation of the phosphorylation of extracellular-signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K)/Akt, and glycogen synthase kinase-3ß (GSK-3ß)/catenin, and downregulation of the phosphorylation of p38, c-Jun N-terminal kinase (JNK), and protein kinase A (PKA). Furthermore, we investigated the effect of 3,6'-DMC on macrophage RAW264.7 cells with LPS stimulation. 3,6'-DMC significantly inhibited LPS-stimulated nitric oxide production. 3,6'-DMC also suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 on the protein level. In addition, 3,6'-DMC decreased the production of the tumor necrosis factor-α and interleukin-6. Successively, our mechanistic studies revealed that 3,6'-DMC also suppressed the LPS-induced phosphorylation of the inhibitor of IκBα, p38MAPK, ERK, and JNK. The Western blot assay results showed that 3,6'-DMC suppresses LPS-induced p65 translocation from cytosol to the nucleus. Finally, the topical applicability of 3,6'-DMC was tested through primary skin irritation, and it was found that 3,6'-DMC, at 5 and 10 µM concentrations, did not cause any adverse effects. Therefore, 3,6'-DMC may provide a potential candidate for preventing and treating melanogenic and inflammatory skin diseases.

A 7-Hydroxy 4-Methylcoumarin Enhances Melanogenesis in B16-F10 Melanoma Cells.

The objectives of this study were to investigate the melanogenetic potentials of the naturally occurring 7-hydroxy coumarin derivatives 7-hydroxy 5,6-dimethoxycoumarin (7H-5,6DM), 7-hydroxy 6,8-dimethoxycoumarin (7H-6,8DM), 7-hydroxy 6-methoxycoumarin (7H-6M), and 7-hydroxy 4-methylcoumarin (7H-4M) in the melanogenic cells model for murine B16F10 melanoma cells. The initial results indicated that melanin production and intracellular tyrosinase activity were significantly stimulated by 7H-4M but not by 7H-5,6DM, 7H-6,8DM, or 7H-6M. Therefore, our present study further investigated the melanogenic effects of 7H-4M in B16-F10 cells, as well as its mechanisms of action. In a concentration-dependent manner, 7H-4M increased intracellular tyrosinase activity, leading to the accumulation of melanin without affecting the viability of B16-F10 cells. Our study further investigated the effects of 7H-4M on melanogenesis, including its ability to promote tyrosinase activity, increase melanin content, and activate molecular signaling pathways. The results indicate that 7H-4M effectively stimulated tyrosinase activity and significantly increased the expression of melanin synthesis-associated proteins, such as microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP1), and TRP2. Based on our findings, we can conclude that 7H-4M has the ability to activate the melanogenesis process through the upregulation of cAMP-dependent protein kinase (PKA) and the cAMP response element-binding protein (CREB). Additionally, our study showed that 7H-4M induced melanogenic effects by downregulating the extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthesis kinase-3ß (GSK-3ß) cascades, while upregulating the JNK and p38 signaling pathways. Finally, the potential of using 7H-4M in topical applications was tested through primary human skin irritation tests. During these tests, no adverse reactions were induced by 7H-4M. In summary, our results indicate that 7H-4M regulates melanogenesis through various signaling pathways such as GSK3ß/ß-catenin, AKT, PKA/CREB, and MAPK. These findings suggest that 7H-4M has the potential to prevent the development of pigmentation diseases.

Acenocoumarol Exerts Anti-Inflammatory Activity via the Suppression of NF-κB and MAPK Pathways in RAW 264.7 Cells.

The repurposing of already-approved drugs has emerged as an alternative strategy to rapidly identify effective, safe, and conveniently available new therapeutic indications against human diseases. The current study aimed to assess the repurposing of the anticoagulant drug acenocoumarol for the treatment of chronic inflammatory diseases (e.g., atopic dermatitis and psoriasis) and investigate the potential underlying mechanisms. For this purpose, we used murine macrophage RAW 264.7 as a model in experiments aimed at investigating the anti-inflammatory effects of acenocoumarol in inhibiting the production of pro-inflammatory mediators and cytokines. We demonstrate that acenocoumarol significantly decreases nitric oxide (NO), prostaglandin (PG)E2, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß levels in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Acenocoumarol also inhibits the expression of NO synthase (iNOS) and cyclooxygenase (COX)-2, potentially explaining the acenocoumarol-induced decrease in NO and PGE2 production. In addition, acenocoumarol inhibits the phosphorylation of mitogen-activated protein kinases (MAPKs), c-Jun N terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK), in addition to decreasing the subsequent nuclear translocation of nuclear factor κB (NF-κB). This indicates that acenocoumarol attenuates the macrophage secretion of TNF-α, IL-6, IL-1ß, and NO, inducing iNOS and COX-2 expression via the inhibition of the NF-κB and MAPK signaling pathways. In conclusion, our results demonstrate that acenocoumarol can effectively attenuate the activation of macrophages, suggesting that acenocoumarol is a potential candidate for drug repurposing as an anti-inflammatory agent.

Natural Products for Cosmetic Applications.

Natural products provide an interesting and largely unexplored source for the development of potential new cosmetic ingredients [...].

Imperatorin Positively Regulates Melanogenesis through Signaling Pathways Involving PKA/CREB, ERK, AKT, and GSK3ß/ß-Catenin.

The present study investigated the melanogenic effects of imperatorin and isoimperatorin and the underlying mechanisms of imperatorin using a mouse melanoma B16F10 model. Interestingly, treatment with 25 µM of either imperatorin or isoimperatorin, despite their structural differences, did not produce differences in melanin content and intracellular tyrosinase activity. Imperatorin also activated the expression of melanogenic enzymes, such as tyrosinase (TYR) and tyrosinase-related proteins TYRP-1 and TYRP-2. Mechanistically, imperatorin increases melanin synthesis through the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA)/cAMP-responsive element-binding protein (CREB)-dependent upregulation of microphthalmia-associated transcription factor (MITF), which is a key transcription factor in melanogenesis. Furthermore, imperatorin exerted melanogenic effects by downregulating extracellular signal-regulated kinase (ERK) and upregulating phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/glycogen synthesis kinase-3ß (GSK-3ß). Moreover, imperatorin increased the content of ß-catenin in the cell cytoplasm and nucleus by reducing the content of phosphorylated ß-catenin (p-ß-catenin). Finally, we tested the potential of imperatorin in topical application through primary human skin irritation tests. These tests were performed on the normal skin (upper back) of 31 volunteers to determine whether 25 or 50 µM of imperatorin had irritation or sensitization potential. During these tests, imperatorin did not induce any adverse reactions. Taken together, these findings suggest that the regulation of melanogenesis by imperatorin can be mediated by signaling pathways involving PKA/CREB, ERK, AKT, and GSK3ß/ß-catenin and that imperatorin could prevent the pathogenesis of pigmentation diseases when used as a topical agent.

Anti-Inflammatory Effects of Spiramycin in LPS-Activated RAW 264.7 Macrophages.

Drug repurposing is a simple concept with a long history, and is a paradigm shift that can significantly reduce the costs and accelerate the process of bringing a new small-molecule drug into clinical practice. We attempted to uncover a new application of spiramycin, an old medication that was classically prescribed for toxoplasmosis and various other soft-tissue infections; specifically, we initiated a study on the anti-inflammatory capacity of spiramycin. For this purpose, we used murine macrophage RAW 264.7 as a model for this experiment and investigated the anti-inflammatory effects of spiramycin by inhibiting the production of pro-inflammatory mediators and cytokines. In the present study, we demonstrated that spiramycin significantly decreased nitric oxide (NO), interleukin (IL)-1ß, and IL-6 levels in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Spiramycin also inhibited the expression of NO synthase (iNOS), potentially explaining the spiramycin-induced decrease in NO production. In addition, spiramycin inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs); extracellular signal-regulated kinase (ERK) and c-Jun N terminal kinase (JNK) as well as the inactivation and subsequent nuclear translocation of nuclear factor κB (NF-κB). This indicated that spiramycin attenuates macrophages' secretion of IL-6, IL-1ß, and NO, inducing iNOS expression via the inhibition of the NF-κB and MAPK signaling pathways. Finally, we tested the potential application of spiramycin as a topical material by human skin primary irritation tests. It was performed on the normal skin (upper back) of 31 volunteers to determine whether 100 µM and µM of spiramycin had irritation or sensitization potential. In these assays, spiramycin did not induce any adverse reactions. In conclusion, our results demonstrate that spiramycin can effectively attenuate the activation of macrophages, suggesting that spiramycin could be a potential candidate for drug repositioning as a topical anti-inflammatory agent.

Anti-Inflammatory Effects of Psoralen Derivatives on RAW264.7 Cells via Regulation of the NF-κB and MAPK Signaling Pathways.

Using repositioning to find new indications for existing functional substances has become a global target of research. The objective of this study is to investigate the anti-inflammatory potential of psoralen derivatives (5-hydroxypsoralen, 5-methoxypsoralen, 8-hydroxypsoralen, and 8-methoxypsoralen) in macrophages cells. The results indicated that most psoralen derivatives exhibited significantly inhibited prostaglandin E2 (PGE2) production, particularly for 8-hydroxypsoralen (xanthotoxol) in lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells. In addition, xanthotoxol treatment decreased the PGE2, IL-6, and IL-1ß production caused by LPS stimulation in a concentration-dependent manner. Moreover, Western blot results showed that the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which activated with LPS treatment, were decreased by xanthotoxol treatment. Mechanistic studies revealed that xanthotoxol also suppressed LPS-stimulated phosphorylation of the inhibitor of κBα (IκBα), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) in RAW 264.7 cells. The Western blot assay results show that xanthotoxol suppresses LPS-induced p65 translocation from cytosol to the nucleus in RAW 264.7 cells. Moreover, we tested the potential application of xanthotoxol as a cosmetic material by performing human skin patch tests. In these tests, xanthotoxol did not induce any adverse reactions at a 100 µΜ concentration. These results demonstrate that xanthotoxol is a potential therapeutic agent for topical application that inhibits inflammation via the MAPK and NF-κB pathways.

Mechanistic Insights into the Ameliorating Effect of Melanogenesis of Psoralen Derivatives in B16F10 Melanoma Cells.

The objectives of this study were to investigate the melanogenetic potential of the psoralen derivatives 5-hydroxypsoralen, 5-methoxypsoralen, 8-hydroxypsoralen, 8-methoxypsoralen, and 5,8-dimethoxypsoralen in B16F10 melanoma cells. The results indicated that melanin production is significantly stimulated in B16F10 melanoma cells with 5-methoxypsoralen, 8-methoxypsoralen, and 5,8-dimethoxypsoralen, especially for 5-methoxypsoralen (bergapten), as reported previously. In addition, Western blot results showed that the protein levels of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2) increase after bergapten treatment for the first time. The results also showed that bergapten promotes the phosphorylation of Akt at Ser 473 and glycogen synthase kinase-3ß at Ser 9. Moreover, bergapten increased the content of ß-catenin in the cell cytoplasm and nucleus by reducing the phosphorylated ß-catenin (p-ß-catenin) content. The results also indicated that bergapten regulates melanogenesis by upregulating the phosphorylation of p38 and JNK-mitogen-activated protein kinase. Taken together, these findings suggest that the regulation of melanogenesis by bergapten may be mediated by the ß-catenin and MAPK signaling pathways and that bergapten might provide new insights into the pathogenesis of pigmented diseases.

Miglitol, an Oral Antidiabetic Drug, Downregulates Melanogenesis in B16F10 Melanoma Cells through the PKA, MAPK, and GSK3ß/ß-Catenin Signaling Pathways.

Hyperpigmentation is a common condition that causes darker spots or patches on the skin, which often look brown, black, gray, red, or pink. This results in unresolved psychological impact due to high anxiety, depression, and somatoform disorder. We aimed to repurpose an antidiabetic drug, miglitol, as an effective compound against hyperpigmentation when applied as a cosmeceutical agent. The present study investigated the antimelanogenic effects of miglitol and the trehalase inhibitor validamycin A. Miglitol in isolation exhibited no cytotoxicity and significantly reduced the melanin production and intracellular tyrosinase activity in B16F10 melanoma cells. The Western blotting results showed that miglitol reduces the expression of melanogenic regulatory factors, including tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, and microphthalmia-associated transcription factor (MITF). Mechanistically, miglitol appears to suppress melanin synthesis through cAMP-dependent protein kinase (PKA)-dependent downregulation of MITF, a master transcription factor in melanogenesis. The antimelanogenic effects of miglitol was mediated by downregulation of the p38 signaling pathway and upregulation of extracellular signal-regulated kinase (ERK). Moreover, miglitol decreases P-GSK3ß and ß-catenin levels compared to those in the untreated group. However, miglitol activated P-ß-catenin expression compared to that in the untreated group. Finally, we tested the potential of miglitol in topical application through primary human skin irritation tests on the normal skin (upper back) of 33 volunteers. In these assays, miglitol (125 and 250 µM) did not induce any adverse reactions. Taken together, these findings suggest that the regulation of melanogenesis by miglitol may be mediated by the PKA, MAPK, and GSK3ß/ß-Catenin signaling pathways and that miglitol might provide new insights into drug repurposing for the treatment of hyperpigmentation symptoms.

Inhibitory Effects of Pinostilbene on Adipogenesis in 3T3-L1 Adipocytes: A Study of Possible Mechanisms.

Resveratrol is a phytoalexin with multiple bioactive properties, including antioxidative, neuroprotective, cardioprotective, and anticancer effects. However, resveratrol exhibits structural instability in response to UV irradiation, alkaline pH, and oxygen exposure. Thus, resveratrol derivatives have attracted considerable research interest. In this study, we aimed to evaluate the anti-adipogenic effects of pinostilbene hydrate (PH), a methylated resveratrol derivative, in 3T3-L1 cells. We also evaluated the mechanisms underlying the effects of PH on adipogenesis in 3T3-L1 adipocytes. Oil Red O staining, lipid accumulation assay, and triglyceride (TG) content assay revealed that PH significantly inhibited lipid and TG accumulation without cytotoxicity. In addition, we determined that PH decreased the expression of adipogenesis-related transcription factors, such as PPARγ, C/EBPα, SREBP-1c, and FABP4, and the phosphorylation of MAPK and protein kinase B (AKT). Moreover, PH attenuated the expression of CREB and C/EBPß, while increasing the phosphorylation of AMPK and ACC, and decreasing the expression of fatty acid synthase and FABP4. Based on these results, we suggest that PH suppresses adipogenesis in 3T3-L1 cells via the activation of the AMPK signaling pathway and the inhibition of the MAPK and AKT insulin-dependent signaling pathways.

Anti-Inflammatory Effects of 6-Methylcoumarin in LPS-Stimulated RAW 264.7 Macrophages via Regulation of MAPK and NF-κB Signaling Pathways.

Persistent inflammatory reactions promote mucosal damage and cause dysfunction, such as pain, swelling, seizures, and fever. Therefore, in this study, in order to explore the anti-inflammatory effect of 6-methylcoumarin (6-MC) and suggest its availability, macrophages were stimulated with lipopolysaccharide (LPS) to conduct an in vitro experiment. The effects of 6-MC on the production and levels of pro-inflammatory cytokines (interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α) and inflammatory mediators (nitric oxide (NO), prostaglandin E2 (PGE2)) in LPS-stimulated RAW 264.7 cells were examined. The results showed that 6-MC reduced the levels of NO and PGE2 without being cytotoxic. In addition, it was demonstrated that the increase in the expression of pro-inflammatory cytokines caused by LPS stimulation, was decreased in a concentration-dependent manner with 6-MC treatment. Moreover, Western blot results showed that the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which increased with LPS treatment, were decreased by 6-MC treatment. Mechanistic studies revealed that 6-MC reduced the phosphorylation of the mitogen-activated protein kinase (MAPK) family and IκBα in the MAPK and nuclear factor-kappa B (NF-κB) pathways, respectively. These results suggest that 6-MC is a potential therapeutic agent for inflammatory diseases that inhibits inflammation via the MAPK and NF-κB pathways.

Genome Analysis of Streptomyces nojiriensis JCM 3382 and Distribution of Gene Clusters for Three Antibiotics and an Azasugar across the Genus Streptomyces.

Streptomyces spp. have been major contributors of novel natural products that are used in many application areas. We found that the nojirimycin (NJ) producer JCM 3382 has antimicrobial activity against Staphylococcus aureus via cellular degradation. Genome analysis revealed 30 biosynthetic gene clusters, including those responsible for producing antibiotics, including an azasugar NJ. In-depth MS/MS analysis confirmed the production of 1-deoxynojirimycin (DNJ) along with NJ. In addition, the production of tambromycins, setomimycin, and linearmycins was verified by spectroscopic analyses, including LC-MS and NMR. The distribution of the clusters of genes coding for antibiotics in 2061 Streptomyces genomes suggested potential producers of tambromycin, setomimycin, and linearmycin. For a DNJ gene cluster, homologs of gabT1 and gutB1 were commonly found; however, yktC1 was identified in only 112 genomes. The presence of several types of clusters suggests that different strains may produce different types of azasugars. Chemical-profile-inspired comparative genome analysis may facilitate a more accurate assessment of the biosynthetic potential to produce secondary metabolites.

Myristoleic Acid Promotes Anagen Signaling by Autophagy through Activating Wnt/ß-Catenin and ERK Pathways in Dermal Papilla Cells.

Alopecia is a distressing condition caused by the dysregulation of anagen, catagen, and telogen in the hair cycle. Dermal papilla cells (DPCs) regulate the hair cycle and play important roles in hair growth and regeneration. Myristoleic acid (MA) increases Wnt reporter activity in DPCs. However, the action mechanisms of MA on the stimulation of anagen signaling in DPCs is not known. In this study, we evaluated the effects of MA on anagen-activating signaling pathways in DPCs. MA significantly increased DPC proliferation and stimulated the G2/M phase, accompanied by increasing cyclin A, Cdc2, and cyclin B1. To elucidate the mechanism by which MA promotes DPC proliferation, we evaluated the effect of MA on autophagy and intracellular pathways. MA induced autophagosome formation by decreasing the levels of the phospho-mammalian target of rapamycin (phospho-mTOR) and increasing autophagy-related 7 (Atg7) and microtubule-associated protein 1A/1B-light chain 3II (LC3II). MA also increased the phosphorylation levels of Wnt/ß-catenin proteins, such as GSK3ß (Ser9) and ß-catenin (Ser552 and Ser675). Treatment with XAV939, an inhibitor of the Wnt/ß-catenin pathway, attenuated the MA-induced increase in ß-catenin nuclear translocation. Moreover, XAV939 reduced MA-induced effects on cell cycle progression, autophagy, and DPC proliferation. On the other hand, MA increased the levels of phospho (Thr202/Tyr204)-extracellular signal regulated kinases (ERK). MA-induced ERK phosphorylation led to changes in the expression levels of Cdc2, Atg7 and LC3II, as well as DPC proliferation. Our results suggest that MA promotes anagen signaling via autophagy and cell cycle progression by activating the Wnt/ß-catenin and ERK pathways in DPCs.

Nojirimycin suppresses inflammation via regulation of NF-κ B signaling pathways.

Nojirimycin (NJ) is a compound in which the oxygen of the ring is replaced with an NH group in the D-glucose structure. NJ, which has a structure similar to D-glucose, is a powerful glucosidase inhibitor and an interesting compound. However, no anti-inflammatory effects of NJ have been reported. Therefore, to investigate its anti-inflammatory effect, the production and expression of inflammatory cytokines, as well as inflammatory mediators, such as iNOS and COX-2, were measured in LPS-stimulated RAW264.7 macrophages. In addition, the effects on the representative inflammatory signaling pathways, the suppression of NF-κ B, and the activation of MAPK were studied. The production of iNOS, COX-2, and inflammatory cytokines (PGE2, IL-6, IL-1ß, and TNF-α) after NJ treatment was significantly inhibited. In addition, NJ showed anti-inflammatory effects through suppression of LPS-induced NF-κ B activation. D-Glucose is structurally similar to NJ. The effects of these substances on RAW264.7 macrophages were evaluated. NJ reduced nitric oxide (NO) levels, whereas D-glucose had no significant effect. Overall, the results suggested that NJ is a potential anti-inflammatory compound.

Anti-melanogenic effects of hot-water extracts from Torreya nucifera via MAPKs and cAMP signaling pathway on B16F10 cells.

Torreya nucifera is an evergreen tree in the family Taxaceae, the seeds, leaves, and stems of which have long been used as edible products and herbal medicines in Korea. Previous studies of biological activity have shown that T. nucifera has antioxidant and anti-inflammatory effects. However, the effect of T. nucifera leaves on melanogenesis are yet to be studied. In this investigation, we used B16F10 melanoma cells to test the efficacy of T. nucifera leaf hot water extract (TLWE). α-melanocyte stimulating hormone (α-MSH) stimulated B16F10 melanoma cells were treated with various concentrations of TLWE (50, 100, and 200 µg/mL). The results showed that TLWE reduced the melanin content and cellular tyrosinase activity in a concentration-dependent manner. It also inhibited the phosphorylation of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) in the mitogen-activated protein kinase (MAPK) signaling pathway. The compounds catechin and ρ-coumaric acid, which are known to have a whitening effect on skin, were detected by HPLC analysis. These results suggest that TLWE has an anti-melanogenic effect. In addition, the safety of TLWE was tested. The results of the skin irritation test showed that TLWE is harmless to the human skin, even at higher concentrations than those used in the experiment. Therefore, we suggest that the water extract of T. nucifera leaves has potential for use as a skin-whitening agent.