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The purpose of this paper is to propose a novel transfer learning regularization method based on knowledge distillation. Recently, transfer learning methods have been used in various fields. However, problems such as knowledge loss still occur during the process of transfer learning to a new target dataset. To solve these problems, there are various regularization methods based on knowledge distillation techniques. In this paper, we propose a transfer learning regularization method based on feature map alignment used in the field of knowledge distillation. The proposed method is composed of two attention-based submodules: self-pixel attention (SPA) and global channel attention (GCA). The self-pixel attention submodule utilizes both the feature maps of the source and target models, so that it provides an opportunity to jointly consider the features of the target and the knowledge of the source. The global channel attention submodule determines the importance of channels through all layers, unlike the existing methods that calculate these only within a single layer. Accordingly, transfer learning regularization is performed by considering both the interior of each single layer and the depth of the entire layer. Consequently, the proposed method using both of these submodules showed overall improved classification accuracy than the existing methods in classification experiments on commonly used datasets.
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The biochemical properties of 2,4'-dihydroxybenzophenone (DHP) have not been extensively studied. Therefore, this study aimed to investigate whether DHP could alleviate inflammatory responses induced by lipopolysaccharide (LPS) and endotoxemia. The results indicated that DHP effectively reduced mortality and morphological abnormalities, restored heart rate, and mitigated macrophage and neutrophil recruitment to inflammatory sites in LPS-microinjected zebrafish larvae. Additionally, the expression of pro-inflammatory mediators, including inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12), was significantly reduced in the presence of DHP. In RAW 264.7 macrophages, DHP inhibited the LPS-induced inflammatory response by downregulating pro-inflammatory mediators and decreasing the expression of myeloid differentiation primary response 88 (MyD88), phosphorylation of IL-1 receptor-associated protein kinase-4 (p-IRAK4), and nuclear factor-κB (NF-κB). Molecular docking analysis demonstrated that DHP occupies the hydrophobic pocket of myeloid differentiation factor 2 (MD2) and blocks the dimerization of Toll-like receptor 4 (TLR4). A molecular dynamics simulation confirmed that DHP stably bound to the hydrophobic pocket of MD2. Furthermore, the DHP treatment inhibited mitochondrial reactive oxygen species (mtROS) production during the LPS-induced inflammatory response in both RAW 264.7 macrophages and zebrafish larvae, which was accompanied by stabilizing mitochondrial membrane potential. In conclusion, our study highlights the therapeutic potential of DHP in alleviating LPS-induced inflammation and endotoxemia. The findings suggest that DHP exerts its anti-inflammatory effects by inhibiting the TLR4/MD2 signaling pathway and reducing the level of mtROS production. These results contribute to a better understanding of the biochemical properties of DHP and support its further exploration as a potential therapeutic agent for inflammatory conditions and endotoxemia.
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2,4'-Dihydroxybenzophenone (DHP) is an organic compound derived from Garcinia xanthochymus, but there have been no reports on its biochemical functions and bioavailability. In this study, we evaluated whether DHP affects osteoblast differentiation and activation in MC3T3-E1 preosteoblast cells, as well as antiosteoporotic activity in zebrafish larvae. Nontoxic concentrations of DHP-treated MC3T3-E1 preosteoblast cells increased alkaline phosphatase (ALP) activation and mineralization in a concentration-dependent manner, accompanied by higher expression of osteoblast-specific markers, including Runt-related transcription factor 2 (RUNX2), osterix, and ALP. Consistent with the data in MC3T3-E1 preosteoblast cells, DHP upregulated osteoblast-specific marker genes in zebrafish larvae and simultaneously enhanced vertebral formation. We also revealed that DHP increased the phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) at Ser9 and the total expression of ß-catenin in the cytosol and markedly increased the localization of ß-catenin into the nucleus. Furthermore, DHP restored the prednisolone (PDS)-induced marked decrease in ALP activity and mineralization, as well as osteoblast-specific marker expression. In PDS-treated zebrafish, DHP also alleviated PDS-induced osteoporosis by restoring vertebral formation and osteoblast-related gene expression. Taken together, these results suggest that DHP is a potential osteoanabolic candidate for treating osteoporosis by stimulating osteoblast differentiation.
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BACKGROUND: The pursuit for safe and efficacious skin-whitening agents has prompted a dedicated exploration of plant-derived compounds. Notably, Tagetes erecta L. flowers have been used as a medicinal extract and possessed in vitro mushroom tyrosinase activity. However, whether polyphenol-enriched fraction extracted from T. erecta L. flowers (TE) regulates melanogenesis within cellular and animal models has not yet been investigated. PURPOSE: This study aimed to investigate the effect of TE as a prospective inhibitor of melanogenesis. METHODS: Through advanced UPLC-QTof/MS analysis, the components of TE were analyzed. Anti-melanogenic effects of TE were evaluated in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells by measuring cell viability assay, extracellular and intracellular melanin biosynthesis, cyclic adenosine monophosphate (cAMP) production, and melanogenesis-related gene and protein expression. Zebrafish larvae were employed for in vivo studies, assessing both heart rate and melanogenesis. Furthermore, molecular docking analyses were employed to predict the interaction between TE components and the melanocortin 1 receptor (MC1R). Direct binding activity of TE components to MC1R was compared with [Nle4, d-Phe7]-MSH (NDP-MSH). RESULTS: TE was found to contain significant phenolic compounds such as patulitrin, quercetagetin, kaempferol, patuletin, and isorhamnetin. This study revealed that TE effectively inhibits melanin biosynthesis in both in vitro and in vivo models. This inhibition was attributed to interference of TE with the cAMP-cAMP response element-binding protein (CREB)-microphthalmia-associated transcription factor (MITF)-tyrosinase pathway, which plays a pivotal role in regulating melanogenesis. Importantly, TE exhibited the remarkable ability to curtail α-MSH-induced melanogenesis in zebrafish larvae without impacting heart rates. Molecular docking analyses predicted that the components of TE possibly interact with the melanocortin 1 receptor, suggesting their role as potential inhibitors of melanin biosynthesis. However, through the direct binding activity compared with NDP-MSH, any TE components did not directly bind to MC1R, suggesting that TE inhibits α-MSH-induced melanogenesis by inhibiting the cAMP-mediated intracellular signaling pathway. The assessment of anti-melanogenic activity, conducted both in vitro and in vivo, revealed that patulitrin and patuletin exhibited significant inhibitory effects on melanin formation, highlighting their potency as major contributors. DISCUSSION: This investigation demonstrated the considerable potential of TE as a natural remedy endowed with remarkable anti-melanogenic properties. The demonstrated capacity of TE to attenuate melanin production by modulating the cAMP-CREB-MITF-tyrosinase pathway underscores its central role in management of disorders associated with excessive pigmentation. Importantly, the implications of these findings extend to the cosmetics industry, where TE emerges as a prospective and valuable ingredient for the formulation of skin-whitening products. The elucidated interactions between TE components and MC1R not only provide insight into a potential mechanism of action but also elevate the significance of this study. In summary, this study not only contributes to our comprehension of pigmentation-related conditions but also firmly establishes TE as a secure and natural strategy for the regulation of melanin production. The innovative aspects of TE propel it into the forefront of potential interventions, marking a noteworthy advancement in the pursuit of effective and safe solutions for pigmentation disorders.
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Melanoma Experimental , Tagetes , Animales , Melaninas , Monofenol Monooxigenasa/metabolismo , alfa-MSH/farmacología , alfa-MSH/metabolismo , Pez Cebra/metabolismo , Tagetes/metabolismo , Melanogénesis , Polifenoles/farmacología , Receptor de Melanocortina Tipo 1/metabolismo , Simulación del Acoplamiento Molecular , Línea Celular Tumoral , Factor de Transcripción Asociado a Microftalmía/metabolismo , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/metabolismoRESUMEN
Osteoporosis is a significant global health concern, linked to reduced bone density and an increased fracture risk, with effective treatments still lacking. This study explored the potential of gamma-aminobutyric acid (GABA) and its receptors as a novel approach to promote osteogenesis and address osteoporosis. GABA concentrations up to 10 mM were well-tolerated by MC3T3-E1 preosteoblast, stimulating osteoblast differentiation and mineralization in a concentration- and time-dependent manner. In vivo experiments with zebrafish larvae demonstrated the ability of GABA to improve vertebral formation and enhanced bone density, indicating the potential therapeutic value for osteoporosis. Notably, GABA countered the adverse effects of prednisolone on vertebral formation, bone density, and osteogenic gene expression in zebrafish larvae, suggesting a promising therapeutic solution to counteract corticosteroid-induced osteoporosis. Moreover, our study highlighted the involvement of GABA receptors in mediating the observed osteogenic effects. By using GABAA, GABAB, and GABAC receptor antagonists, we demonstrated that blocking these receptors attenuated GABA-induced osteoblast differentiation and vertebral formation in both MC3T3-E1 cells and zebrafish larvae, underscoring the importance of GABA receptor interactions in promoting bone formation. In conclusion, these findings underscore the osteogenic potential of GABA and its ability to mitigate the detrimental effects of corticosteroids on bone health. Targeting GABA and its receptors could be a promising strategy for the development of novel therapeutic interventions to address osteoporosis. However, further investigations are warranted to fully elucidate the underlying molecular mechanism of GABA and its clinical applications in treating osteoporosis.
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Osteoporosis , Receptores de GABA , Animales , Receptores de GABA/metabolismo , Osteogénesis , Pez Cebra , Ácido gamma-Aminobutírico/farmacología , Ácido gamma-Aminobutírico/metabolismo , Osteoporosis/metabolismo , Diferenciación Celular , Osteoblastos/metabolismoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a chronic lung condition characterized by the abnormal regulation of extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT). In this study, we investigated the potential of rutin, a natural flavonoid, in attenuating transforming growth factor-ß (TGF-ß)-induced ECM regulation and EMT through the inhibition of the TGF-ß type I receptor (TßRI)-mediated suppressor of mothers against decapentaplegic (SMAD) signaling pathway. We found that non-toxic concentrations of rutin attenuated TGF-ß-induced ECM-related genes, including fibronectin, elastin, collagen 1 type 1, and TGF-ß, as well as myoblast differentiation from MRC-5 lung fibroblast cells accompanied by the downregulation of α-smooth muscle actin. Rutin also inhibited TGF-ß-induced EMT processes, such as wound healing, migration, and invasion by regulating EMT-related gene expression. Additionally, rutin attenuated bleomycin-induced lung fibrosis in mice, thus providing a potential therapeutic option for IPF. The molecular docking analyses in this study predict that rutin occludes the active site of TßRI and inhibits SMAD-mediated fibrotic signaling pathways in lung fibrosis. These findings highlight the potential of rutin as a promising anti-fibrotic prodrug for lung fibrosis and other TGF-ß-induced fibrotic and cancer-related diseases; however, further studies are required to validate its safety and effectiveness in other experimental models.
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Transforming growth factor-ß (TGF-ß) has a strong impact on the pathogenesis of pulmonary fibrosis. Therefore, in this study, we investigated whether derrone promotes anti-fibrotic effects on TGF-ß1-stimulated MRC-5 lung fibroblast cells and bleomycin-induced lung fibrosis. Long-term treatment with high concentrations of derrone increased the cytotoxicity of MRC-5 cells; however, substantial cell death was not observed at low concentrations of derrone (below 0.05 µg/mL) during a three-day treatment. In addition, derrone significantly decreased the expressions of TGF-ß1, fibronectin, elastin, and collagen1α1, and these decreases were accompanied by downregulation of α-SMA expression in TGF-ß1-stimulated MRC-5 cells. Severe fibrotic histopathological changes in infiltration, alveolar congestion, and alveolar wall thickness were observed in bleomycin-treated mice; however, derrone supplementation significantly reduced these histological deformations. In addition, intratracheal administration of bleomycin resulted in lung collagen accumulation and high expression of α-SMA and fibrotic genes-including TGF-ß1, fibronectin, elastin, and collagen1α1-in the lungs. However, fibrotic severity in intranasal derrone-administrated mice was significantly less than that of bleomycin-administered mice. Molecular docking predicted that derrone potently fits into the ATP-binding pocket of the TGF-ß receptor type 1 kinase domain with stronger binding scores than ATP. Additionally, derrone inhibited TGF-ß1-induced phosphorylation and nuclear translocations of Smad2/3. Overall, derrone significantly attenuated TGF-ß1-stimulated lung inflammation in vitro and bleomycin-induced lung fibrosis in a murine model, indicating that derrone may be a promising candidate for preventing pulmonary fibrosis.
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Fibrosis Pulmonar , Ratones , Animales , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Bleomicina/toxicidad , Elastina/metabolismo , Fibronectinas/metabolismo , Simulación del Acoplamiento Molecular , Pulmón/patología , Transducción de Señal , Fibroblastos/metabolismo , Adenosina Trifosfato/metabolismo , Ratones Endogámicos C57BLRESUMEN
Acertannin (ACTN) is a polyphenol known for its powerful anticancer and antioxidant effects. However, its anti-inflammatory effects have not been investigated at the molecular levels. Therefore, to evaluate anti-inflammatory effects of ACTN and its signaling pathway, the expression of proinflammatory markers was measured in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Molecular docking predicted the binding site of ACTN to the TLR4/MD2 complex. Moreover, in LPS-microinjected zebrafish, we investigated whether ACTN reduces nitric oxide and reactive oxygen species (ROS) production. ACTN significantly attenuated LPS-induced proinflammatory cytokines and mediators by inhibiting nuclear factor-kappa B (NF-κB) activation. ACTN also reduced LPS-induced ROS production and activated nuclear factor E2-related factor 2 and heme oxygenase-1 (HO-1). In addition, zinc protoporphyrin, an HO-1 inhibitor, markedly abolished the anti-inflammatory and antioxidant effects of ACTN in LPS-stimulated zebrafish larvae. Moreover, molecular docking predictions verified that ACTN forms a conventional hydrogen bond with LYS91 in myeloid differentiation factor-2 (MD2) and interrupts LPS binding to the Toll-like receptor 4 (TLR4)/MD2 complex. In addition, ACTN forms many non-covalent bonds, such as π-π stacking, π-alkyl, unfavorable donor-donor, and van der Waals interactions, with the TLR4/MD2 complex. Furthermore, the binding of ACTN to the TLR4/MD2 complex inhibited the recruitment of intracellular adaptor proteins, including myeloid differentiation primary response 88 and interleukin-1 receptor-associated kinase 4, and consequently attenuated NF-κB-mediated inflammatory responses. The conclusion of this study is that ACTN is a potent anti-inflammatory agent in LPS-mediated inflammation, such as endotoxemia.
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Hemo-Oxigenasa 1 , Lipopolisacáridos , Animales , Lipopolisacáridos/farmacología , Hemo-Oxigenasa 1/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Receptor Toll-Like 4/metabolismo , FN-kappa B/metabolismo , Pez Cebra , Especies Reactivas de Oxígeno/metabolismo , Simulación del Acoplamiento Molecular , Antioxidantes/uso terapéutico , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéuticoRESUMEN
Pinostrobin is a natural flavonoid with valuable pharmacological properties, including anti-cancer, anti-viral, and anti-oxidant activities. However, the anti-inflammatory effects of pinostrobin have not been well studied. In this study, we investigated whether pinostrobin attenuates lipopolysaccharide (LPS)-induced inflammation and endotoxemia. Additionally, the target molecule of pinostrobin was identified through molecular docking simulation. Pinostrobin decreased LPS-induced nitric oxide (NO) and prostaglandin E2 production, and reduced the expression of inducible NO synthase and cyclooxygenase-2. Furthermore, pinostrobin inhibited the production of proinflammatory cytokines, including interleukin-12 and tumor necrosis factor-α in LPS-stimulated RAW 264.7 macrophages accompanied by inhibiting nuclear translocation of nuclear factor-κB. The anti-inflammatory effect of pinostrobin was further confirmed in LPS-microinjected zebrafish larvae by diminishing the recruitment of macrophages and neutrophils, and proinflammatory gene expression. Moreover, LPS-microinjected zebrafish larvae showed a decrease in heart rate and an increase in mortality and abnormalities. However, pinostrobin significantly attenuated these adverse effects. Molecular docking showed that pinostrobin fits into myeloid differentiation factor (MD2) and Toll-like receptor 4 (TLR4) with no traditional hydrogen bonds (pose 1). The 2D ligand interaction diagram showed that pinostrobin forms a carbon hydrogen bond with LYS89 in MD2 and many non-covalent interactions, including π-alkyl or alkyl and van der Waals interactions, indicating that pinostrobin hinders LPS binding between MD2 and TLR4 and consequently inhibits TLR4/MD2-mediated inflammatory responses. These data suggest that pinostrobin attenuates LPS-induced inflammation and endotoxemia by binding to the TLR4/MD2 complex.
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Endotoxemia , Receptor Toll-Like 4 , Animales , Receptor Toll-Like 4/metabolismo , Lipopolisacáridos/farmacología , Pez Cebra/metabolismo , Endotoxemia/inducido químicamente , Endotoxemia/tratamiento farmacológico , Simulación del Acoplamiento Molecular , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , FN-kappa B/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéuticoRESUMEN
The purpose of this paper is to propose a novel noise removal method based on deep neural networks that can remove various types of noise without paired noisy and clean data. Because this type of filter generally has relatively poor performance, the proposed noise-to-blur-estimated clean (N2BeC) model introduces a stage-dependent loss function and a recursive learning stage for improved denoised image quality. The proposed loss function regularizes the existing loss function so that the proposed model can better learn image details. Moreover, the recursive learning stage provides the proposed model with an additional opportunity to learn image details. The overall deep neural network consists of three learning stages and three corresponding loss functions. We determine the essential hyperparameters via several simulations. Consequently, the proposed model showed more than 1 dB superior performance compared with the existing noise-to-blur model.
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Procesamiento de Imagen Asistido por Computador , Redes Neurales de la Computación , Aprendizaje , Relación Señal-RuidoRESUMEN
Fine particulate matter (PM2.5) originates from the combustion of coal and is found in the exhaust of fumes of diesel vehicles. PM2.5 readily penetrates the skin via the aryl hydrocarbon receptor, causing skin senescence, inflammatory skin diseases, DNA damage, and carcinogenesis. In this study, we investigated whether fisetin, a bioactive flavonoid, prevents PM2.5-induced apoptosis in HaCaT human keratinocytes. The results demonstrated that fisetin significantly downregulated PM2.5-induced apoptosis at concentrations below 10 µM. Fisetin strongly inhibited the production of reactive oxygen species (ROS) and the expression of pro-apoptotic proteins. The PM2.5-induced apoptosis was associated with the induction of the endoplasmic reticulum (ER) stress response, mediated via the protein kinase R-like ER kinase (PERK)-eukaryotic initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4)-CCAAT-enhancer-binding protein (C/EBP) homologous protein (CHOP) axis. Additionally, the cytosolic Ca2+ levels were markedly increased following exposure to PM2.5. However, fisetin inhibited the expression of ER stress-related proteins, including 78 kDa glucose-regulated protein (GRP78), phospho-eIF2α, ATF4, and CHOP, and reduced the cytosolic Ca2+ levels. These data suggest that fisetin inhibits PM2.5-induced apoptosis by inhibiting the ER stress response and production of ROS.
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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-1ß. 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.
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Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a dual role in carcinogenesis. We previously reported that Nrf2 deficiency enhances the anti-tumorigenic effect of 17ß-estradiol (E2) in an azoxymethane (AOM)/dextran sodium sulfate (DSS) model of colitis-associated cancer (CAC). Herein, we aimed to determine a possible explanation for our recent work and investigated the immune microenvironment represented by programmed death-ligand 1 (PD-L1) expression. One week after the AOM injection, mice were administered with DSS in drinking water for seven days; daily E2 injections were intraperitoneally administered during this period. The mice were sacrificed 16 weeks after AOM injection and analyzed for PD-L1 expression in the distal colon tissues using Western blotting and immunohistochemistry (IHC). Based on Western blotting results, PD-L1 expression was reduced in Nrf2 knockout (KO) female and E2-treated male mice when compared with their wild-type counterparts, following AOM/DSS treatment; this supports the association of PD-L1 expression with tumor progression. Additionally, this finding was in good agreement with the IHC results for PD-L1. Furthermore, we observed that PD-L1 is predominantly expressed in stromal cells rather than on epithelial cells in the colon. Western blotting revealed that PD-L1 expression in the colon positively correlates with expressions of inducible nitric oxide synthase (iNOS) (male, P = 0.002; female, P <0.001) and cyclooxygenase-2 (COX-2) (male, P <0.001; female, P <0.001). Collectively, our findings indicate that estrogen ameliorates the immune microenvironment represented by PD-L1 expression and enhances its effect in the absence of Nrf2.
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Fisetin is a bioactive flavonol that inhibits osteoclastogenesis and promotes osteoblastogenesis. However, the osteogenic activity of fisetin needs to be comprehensively elucidated. In the present study, we observed that fisetin significantly increased alkaline phosphatase (ALP) activity and bone mineralization in MC3T3-E1 preosteoblasts, accompanied by a significant increase in runt-related transcription factor 2 (RUNX2), ALP, collagen type â alpha 1 (Col1α1), osterix (OSX), osteocalcin (OCN), and bone morphogenetic protein 4 (BMP4) expression. Furthermore, fisetin promoted vertebral formation in zebrafish larvae, with the highest fisetin concentration comparable with that observed in ß-glycerophosphate treatment. Fisetin also inhibited prednisolone (PDS)-induced anti-osteoblastic genes, including nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), tartrate-resistant acid phosphatase-6 (ACP6), dendritic cell-specific transmembrane protein (DC-STAMP), and cathepsin K (CTSK). Fisetin potently mitigated the PDS-induced inhibition of ALP activity and bone mineralization, as well as vertebral resorption in zebrafish larvae. Moreover, we confirmed that fisetin-induced osteogenic effect was activated through phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) at Ser9, consequently releasing ß-catenin from the destructive complex to promote its nuclear translocation. ß-Catenin inhibition by FH535 and the stabilization of GSK-3ß by DOI hydrochloride remarkably inhibited fisetin-induced osteogenic activities, indicating that the GSK-3ß/ß-catenin signaling pathway plays a vital role in fisetin-induced osteogenesis. Collectively, our findings suggest that fisetin stimulates osteogenic activity and could be used as an effective strategy to prevent bone resorption.
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Diferenciación Celular/efectos de los fármacos , Flavonoles/farmacología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Osteogénesis/efectos de los fármacos , Osteoporosis/metabolismo , beta Catenina/metabolismo , Animales , Animales Modificados Genéticamente , Diferenciación Celular/fisiología , Línea Celular , Relación Dosis-Respuesta a Droga , Flavonoles/uso terapéutico , Ratones , Osteogénesis/fisiología , Osteoporosis/prevención & control , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Serina/metabolismo , Pez CebraRESUMEN
Anthocyanin-enriched polyphenols from the flower petals of H. syriacus L. (Malvaceae, AHs) possess anti-septic shock, anti-oxidant, and anti-melanogenic properties. However, whether AHs positively or negatively regulate ultraviolet B (UVB)-mediated photoaging and photodamage remains unclear. This study aims to investigate the protective effect of AHs against UVB-induced damage. We examined the photoprotective effects of AHs on UVB-induced apoptosis, endoplasmic reticulum (ER) stress, and mitochondrial reactive oxygen species (mtROS). AHs prevented UVB irradiation-induced apoptosis of HaCaT keratinocytes by inhibiting caspase activation and ROS production. Moreover, AHs restored the survival rate and the hatchability of UVB-irradiated zebrafish larvae without any abnormalities. Furthermore, AHs inhibited UVB-induced ER stress, resulting in a decrease in mtROS production via the stabilization of the mitochondrial membrane potential. Our results indicate that AHs inhibit UVB-induced apoptosis by downregulating total cytosolic ROof cytosolic CaS and ER-mediated mitoROS production in both HaCaT keratinocytes and zebrafish larvae. These findings provide evidence for the applications of AHs to protect skin from UVB-induced photodamage.
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Fisetin is a naturally occurring flavonoid that possesses several pharmacological benefits including anti-inflammatory activity. However, its precise anti-inflammatory mechanism is not clear. In the present study, we found that fisetin significantly inhibited the expression of proinflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Additionally, fisetin attenuated LPS-induced mortality and abnormalities in zebrafish larvae and normalized the heart rate. Fisetin decreased the recruitment of macrophages and neutrophils to the LPS-microinjected inflammatory site in zebrafish larvae, concomitant with a significant downregulation of proinflammatory genes, such as inducible NO synthase (iNOS), cyclooxygenase-2a (COX-2a), IL-6, and TNF-α. Fisetin inhibited the nuclear localization of nuclear factor-kappa B (NF-κB), which reduced the expression of pro-inflammatory genes. Further, fisetin inactivated glycogen synthase kinase 3ß (GSK-3ß) via phosphorylation at Ser9, and inhibited the degradation of ß-catenin, which consequently promoted the localization of ß-catenin into the nucleus. The pharmacological inhibition of ß-catenin with FH535 reversed the fisetin-induced anti-inflammatory activity and restored NF-κB activity, which indicated that fisetin-mediated activation of ß-catenin results in the inhibition of LPS-induced NF-κB activity. In LPS-microinjected zebrafish larvae, FH535 promoted the migration of macrophages to the yolk sac and decreased resident neutrophil counts in the posterior blood island and induced high expression of iNOS and COX-2a, which was accompanied by the inhibition of fisetin-induced anti-inflammatory activity. Altogether, the current study confirmed that the dietary flavonoid, fisetin, inhibited LPS-induced inflammation and endotoxic shock through crosstalk between GSK-3ß/ß-catenin and the NF-κB signaling pathways.
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Antiinflamatorios/farmacología , Endotoxemia/prevención & control , Flavonoles/farmacología , Inflamación/prevención & control , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , beta Catenina/metabolismo , Animales , Endotoxemia/inducido químicamente , Endotoxemia/metabolismo , Endotoxemia/patología , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Pez Cebra , beta Catenina/genéticaRESUMEN
Colon tumors develop more frequently in male than in female. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays differential roles in the stage of tumorigenesis. The purpose of this study was to investigate the role of Nrf2 on colitis-associated tumorigenesis using Nrf2 knockout (KO) female mice. Azoxymethane (AOM) and dextran sulfate sodium (DSS)-treated wild-type (WT) and Nrf2 KO female mice were sacrificed at week 2 and 16 after AOM injection. Severity of colitis, tumor incidence, and levels of inflammatory mediators were evaluated in AOM/DSS-treated WT and Nrf2 KO mice. Furthermore, qRT-PCR, Western blot abnalysis, and ELISA were performed in colon tissues. At week 2, AOM/DSS-induced colon tissue damages were significantly greater in Nrf2 KO than in WT mice. At week 16, tumor numbers (> 2 mm size) were significantly lower in both the proximal and distal colon in Nrf2 KO compared to WT. The overall incidences of adenoma/cancer of the proximal colon and submucosal invasive cancer of the distal colon were reduced by Nrf2 KO. The mRNA and protein expression levels of NF-κB-related mediators (i.e., iNOS and COX-2) and Nrf2-related antioxidants (i.e., heme oxygenase-1 and glutamate-cysteine ligase catalytic subunit) were significantly lower in the Nrf2 KO than in WT mice. Interestingly, the protein level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was higher in AOM/DSS-treated Nrf2 KO than in WT mice. Our results support the oncogenic effect of Nrf2 in the later stage of carcinogenesis and upregulation of tumor suppressor 15-PGDH might contribute to the repression of colitis-associated tumorigenesis in Nrf2 KO female mice.
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Radiotherapy (RT) has been used for decades as one of the main treatment modalities for cancer patients. The therapeutic effect of RT has been primarily ascribed to DNA damage leading to tumor cell death. Besides direct tumoricidal effect, RT affects antitumor responses through immune-mediated mechanism, which provides a rationale for combining RT and immunotherapy for cancer treatment. Thus far, for the combined treatment with RT, numerous studies have focused on the immune checkpoint inhibitors and have shown promising results. However, treatment resistance is still common, and one of the main resistance mechanisms is thought to be due to the immunosuppressive tumor microenvironment where myeloid-derived suppressor cells (MDSCs) play a crucial role. MDSCs are immature myeloid cells with a strong immunosuppressive activity. MDSC frequency is correlated with tumor progression, recurrence, negative clinical outcome, and reduced efficacy of immunotherapy. Therefore, increasing efforts to target MDSCs have been made to overcome the resistance in cancer treatments. In this review, we focus on the role of MDSCs in RT and highlight growing evidence for targeting MDSCs in combination with RT to improve cancer treatment.
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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 ß-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 ß-catenin expression and Wnt/ß-catenin luciferase activity, but pretreatment with a Wnt/ß-catenin inhibitor (FH535) also significantly decreased FO-mediated bone mineralization in zebrafish larvae, which indicates that FO-induced osteogenesis depends on the Wnt/ß-catenin pathway. Altogether, the current study suggests that the supplemental intake of FO has a beneficial effect on osteogenesis.
Asunto(s)
Osteogénesis/efectos de los fármacos , Ostreidae/química , Extractos de Tejidos/farmacología , Proteínas de Pez Cebra/genética , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Femenino , Fermentación , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Larva/efectos de los fármacos , Ratones , Osteoblastos/efectos de los fármacos , Osteocalcina/química , Osteocalcina/farmacología , Osteosarcoma/genética , Osteosarcoma/patología , Factor de Transcripción Sp7/química , Factor de Transcripción Sp7/farmacología , Extractos de Tejidos/química , Vía de Señalización Wnt/efectos de los fármacos , Pez Cebra/genética , Proteínas de Pez Cebra/efectos de los fármacosRESUMEN
Proliferative and migratory abilities of fibroblasts are essential for wound healing at the skin surface. Cytoplasmic linker-associated protein-2 (CLASP2) was originally found to interact with cytoplasmic linker protein (CLIP)-170. CLASP2 plays an important role in microtubule stabilization and the microtubule-stabilizing activity of CLASP2 depends on its interactions with end binding (EB)-1 and CLIP-170. Although the microtubule-stabilizing role of CLASP2 is well established, the effects of CLASP2 on the migration and proliferation of fibroblasts remain unclear in the context of wound healing. Therefore, we tested the utilization of CLASP2 as a directly applied protein drug to improve wound healing by promoting the migration of effector cells, including skin fibroblasts, to the site of repair or injury using an in vivo excisional wound mouse model and in vitro Hs27 skin fibroblast model. Epidermal growth factor, which is a recognized contributor to cell proliferation and migration, was used as positive control. In vitro and in vivo, CLASP2 treatment significantly enhanced cell migration and accelerated wound closure. Furthermore, in vivo, the CLASP2-treated animal group displayed enhanced epidermal repair and collagen deposition. Next, we studied the mechanism of CLASP2 for wound healing. Increasing the abundance of intracellular free CLASP2 in skin fibroblasts by supplying exogenous CLASP2 seemed to stabilize microtubules through an interaction between CLASP2 and CLIP-170, as well as EB1. Exogenous CLASP2 also showed direct binding with IQGAP1, increasing both cyclic adenosine monophosphate activity and phosphorylation of glycogen synthase kinase 3ß, which in turn reinstated the binding between free CLASP2 and IQGAP1. In summary, exogenous CLASP2 increased Hs27 skin fibroblast migration by interacting with IQGAP1 and other cytoskeletal linker proteins, such as CLIP-170 and EB1. Our results strongly suggest that CLASP2 can be developed in wound healing drugs for skin repair and/or regenerating cosmetic products.