Hibiscus syriacus L. is distributed widely throughout Eastern and Southern Asia and considered as the national flower of South Korea. The extraction of several plant parts of H. syriacus L. is currently used as a natural remedy for several diseases, including breast and lung cancer, microbial infection, and chronic inflammation. However, the effect of the anthocyanin extract of H. syriacus L. petals (PS) in oxidative stress conditions has not been studied. In this study, we evaluated the cytoprotective effect of PS against H₂O₂-induced oxidative stress in HaCaT keratinocytes. In this study, we found that PS significantly inhibited H₂O₂-induced apoptosis of HaCaT keratinocytes. We also revealed that PS mediated-cytoprotective effect was associated with the increased expression of heme oxygenase-1 (HO-1) arising from the activation of nuclear factor erythroid 2-related factor-2 (Nrf2). PS also decreased H₂O₂-induced excessive intracellular ROS generation and restored H₂O₂-induced mitochondrial depolarization through the downregulation of mitochondrial ROS production. Furthermore, H₂O₂-induced Bax and caspase-3 expression was markedly abolished in the presence of PS. The inhibition of HO-1 by zinc protoporphyrin significantly attenuated the cytoprotective effect of PS in H₂O₂-treated HaCaT keratinocytes along with ROS generation, indicating that HO-1 crucially affects PS-mediated cytoprotective properties. Collectively, our results suggested that, under H₂O₂-mediated oxidative stress conditions, PS sustained a normal level of mitochondrial membrane potential and ROS generation in HaCaT keratinocytes by activating the Nrf2/HO-1 axis, exerting cytoprotective effects against oxidative stress.

Flumequine is a well-known second generation quinolone antibiotic that induces phototoxicity. However, the effect of flumequine on skin melanogenesis is unclear. Therefore, we, for the first time, investigated whether flumequine regulates melanogenesis. The present study showed that flumequine slightly inhibited in vitro mushroom tyrosinase activity but significantly increased extracellular and intracellular melanin content in B16F10 cells and promoted the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. Additionally, flumequine remarkably increased melanin pigmentation in zebrafish larvae without any toxicity. We also found that flumequine stimulated p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation; inhibition of p38 MAPK and JNK resulted in significant downregulation of extracellular and intracellular melanin content in B16F10 cells and pigmentation of zebrafish larvae accompanied with suppression of MITF and tyrosinase expression, indicating that flumequine-mediated p38 and JNK promote melanogenesis in vitro and in vivo. According to the molecular docking prediction, flumequine targeted dual-specificity MAPK phosphatase 16 (DUSP16), which is a major negative regulator of p38 MAPK and JNK. Our findings demonstrate that flumequine induces an increase in melanin content in B16F10 cells and zebrafish larvae by activating p38 MAPK and JNK. These data show the potential of flumequine for use as an anti-vitiligo agent.

Hibiscus syriacus L. exhibited promising potential as a new source of food and colorants containing various anthocyanins. However, the function of anthocyanins from H. syriacus L. has not been investigated. In the current study, we evaluated whether anthocyanins from the H. syriacus L. varieties Pulsae and Paektanshim (PS and PTS) inhibit melanin biogenesis. B16F10 cells and zebrafish larvae were exposed to PS and PTS in the presence or absence of α-melanocyte-stimulating hormone (α-MSH), and melanin contents accompanied by its regulating genes and proteins were analyzed. PS and PTS moderately downregulated mushroom tyrosinase activity in vitro, but significantly decreased extracellular and intracellular melanin production in B16F10 cells, and inhibited α-MSH-induced expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. PS and PTS also attenuated pigmentation in α-MSH-stimulated zebrafish larvae. Furthermore, PS and PTS activated the phosphorylation of extracellular signal-regulated kinase (ERK), whereas PD98059, a specific ERK inhibitor, completely reversed PS- and PTS-mediated anti-melanogenic activity in B16F10 cells and zebrafish larvae, which indicates that PS- and PTS-mediated anti-melanogenic activity is due to ERK activation. Moreover, chromatography data showed that PS and PTS possessed 17 identical anthocyanins as a negative regulator of ERK. These findings suggested that anthocyanins from PS and PTS inhibited melanogenesis in vitro and in vivo by activating the ERK signaling pathway.

Fisetin is found in many fruits and plants such as grapes and onions, and exerts anti-inflammatory, anti-proliferative, and anticancer activity. However, whether fisetin regulates melanogenesis has been rarely studied. Therefore, we evaluated the effects of fisetin on melanogenesis in B16F10 melanoma cell and zebrafish larvae. The current study revealed that fisetin slightly suppressed in vitro mushroom tyrosinase activity; however, molecular docking data showed that fisetin did not directly bind to mushroom tyrosinase. Unexpectedly, fisetin significantly increased intracellular and extracellular melanin production in B16F10 melanoma cells regardless of the presence or absence of α-melanocyte stimulating hormone (α-MSH). We also found that the expression of melanogenesis-related genes such as tyrosinase and microphthalmia-associated transcription factor (MITF), were highly increased 48 h after fisetin treatment. Pigmentation of zebrafish larvae by fisetin treatment also increased at the concentrations up to 200 µM and then slightly decreased at 400 µM, with no alteration in the heart rates. Molecular docking data also revealed that fisetin binds to glycogen synthase kinase-3β (GSK-3β). Therefore, we evaluated whether fisetin negatively regulated GSK-3β, which subsequently activates β-catenin, resulting in melanogenesis. As expected, fisetin increased the expression of β-catenin, which was subsequently translocated into the nucleus. In the functional assay, FH535, a Wnt/β-catenin inhibitor, significantly inhibited fisetin-mediated melanogenesis in zebrafish larvae. Our data suggested that fisetin inhibits GSK-3β, which activates β-catenin, resulting in melanogenesis through the revitalization of MITF and tyrosinase.

The Pacific oyster, Crassostrea gigas, is well-known as a nutritious food. Recently, we revealed that fermented extract of C. gigas (FO) inhibited ovariectomy-induced osteoporosis, resulting from suppression of osteoclastogenesis. However, since the beneficial effect of FO on osteogenesis is poorly understood, it was examined in mouse preosteoblast MC3T3-E1 cells, human osteosarcoma MG-63 osteoblast-like cells, and zebrafish larvae in this study. We found that FO increased mitochondrial activity from days 1 to 7; however, total cell number of MC3T3-E1 cells gradually decreased without any change in cell viability, which suggests that FO stimulates the differentiation of MC3T3-E1 cells. FO also promoted the expression of osteoblast marker genes, including runt-related transcription factor 2 (mRUNX2), alkaline phosphatase (mALP), collagen type I α1 (mCol1α1), osteocalcin (mOCN), osterix (mOSX), bone morphogenetic protein 2 (mBMP2), and mBMP4 in MC3T3-E1 cells accompanied by a significant increase in ALP activity. FO also increased nuclear translocation of RUNX2 and OSX transcription factors, ALP activity, and calcification in vitro along with the upregulated expression of osteoblast-specific marker proteins such as RUNX2, ALP, Col1α1, OCN, OSX, and BMP4. Additionally, FO enhanced bone mineralization (calcein intensity) in zebrafish larvae at 9 days post-fertilization comparable to that in the β-glycerophosphate (GP)-treated group. All the tested osteoblast marker genes, including zRUNX2a, zRUNX2b, zALP, zCol1a1, zOCN, zBMP2, and zBMP4, were also remarkably upregulated in the zebrafish larvae in response to FO. It also promoted tail fin regeneration in adult zebrafish as same as the GP-treated groups. Furthermore, not only FO positively regulate β-catenin expression and Wnt/β-catenin luciferase activity, but pretreatment with a Wnt/β-catenin inhibitor (FH535) also significantly decreased FO-mediated bone mineralization in zebrafish larvae, which indicates that FO-induced osteogenesis depends on the Wnt/β-catenin pathway. Altogether, the current study suggests that the supplemental intake of FO has a beneficial effect on osteogenesis.

Fisetin has numerous therapeutic properties, such as anti-inflammatory, antioxidative, and anticancer effects. However, the mechanism by which fisetin inhibits NLRP3 inflammasome remains unclear. In this study, we observed that fisetin bound to TLR4 and occluded the hydrophobic pocket of MD2, which in turn inhibited the binding of LPS to the TLR4/MD2 complex. This prevented the initiation of scaffold formation by the inhibition of MyD88/IRAK4 and subsequently downregulated the NF-κB signaling pathway. The result also demonstrated that fisetin downregulated the activation of the NLRP3 inflammasome induced by LPS and ATP (LPS/ATP) and the subsequent maturation of IL-1β. Fisetin also activated mitophagy and prevented the accumulation of damaged mitochondria and the excessive production of mitochondrial reactive oxygen species. The transient knockdown of p62 reversed the inhibitory activity of fisetin on the LPS/ATP-induced formation of the NLRP3 inflammasome. This indicated that fisetin induces p62-mediated mitophagy for eliminating damaged mitochondria. Recently, the existence of inflammasomes in non-mammalian species including zebrafish have been identified. Treatment of an LPS/ATP-stimulated zebrafish model with fisetin aided the recovery of the impaired heart rate, decreased the recruitment of macrophage to the brain, and gradually downregulated the expression of inflammasome-related genes. These results indicated that fisetin inhibited the TLR4/MD2-mediated activation of NLRP3 inflammasome by eliminating damaged mitochondria in a p62-dependent manner.