2,4'-Dihydroxybenzophenone: A Promising Anti-Inflammatory Agent Targeting Toll-like Receptor 4/Myeloid Differentiation Factor 2-Mediated Mitochondrial Reactive Oxygen Species Production during Lipopolysaccharide-Induced Systemic Inflammation.

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.

Inhibition of α-melanocyte-stimulating hormone-induced melanogenesis and molecular mechanisms by polyphenol-enriched fraction of Tagetes erecta L. flower.

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.

2,4'-Dihydroxybenzophenone Exerts Bone Formation and Antiosteoporotic Activity by Stimulating the ß-Catenin Signaling Pathway.

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.

Gamma-aminobutyric acid (GABA)-mediated bone formation and its implications for anti-osteoporosis strategies: Exploring the relation between GABA and GABA receptors.

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.

Investigating rutin as a potential transforming growth factor-ß type I receptor antagonist for the inhibition of bleomycin-induced lung fibrosis.

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.

Derrone Targeting the TGF Type 1 Receptor Kinase Improves Bleomycin-Mediated Pulmonary Fibrosis through Inhibition of Smad Signaling Pathway.

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.

Pinostrobin ameliorates lipopolysaccharide (LPS)-induced inflammation and endotoxemia by inhibiting LPS binding to the TLR4/MD2 complex.

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.

Acertannin attenuates LPS-induced inflammation by interrupting the binding of LPS to the TLR4/MD2 complex and activating Nrf2-mediated HO-1 activation.

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.

Fisetin Protects HaCaT Human Keratinocytes from Fine Particulate Matter (PM2.5)-Induced Oxidative Stress and Apoptosis by Inhibiting the Endoplasmic Reticulum Stress Response.

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.

Fisetin Inhibits NLRP3 Inflammasome by Suppressing TLR4/MD2-Mediated Mitochondrial ROS Production.

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.

Fisetin promotes osteoblast differentiation and osteogenesis through GSK-3ß phosphorylation at Ser9 and consequent ß-catenin activation, inhibiting osteoporosis.

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 Ⅰ 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.

Protective Effect of Anthocyanin-Enriched Polyphenols from Hibiscus syriacus L. (Malvaceae) against Ultraviolet B-Induced Damage.

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.

Fisetin inhibits lipopolysaccharide-induced inflammatory response by activating ß-catenin, leading to a decrease in endotoxic shock.

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.

Fermented Oyster Extract Promotes Osteoblast Differentiation by Activating the Wnt/ß-Catenin Signaling Pathway, Leading to Bone Formation.

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.

β-Hydroxyisovalerylshikonin promotes reactive oxygen species production in HCT116 colon cancer cells, leading to caspase-mediated apoptosis

ABSTRACT Although β-hydroxyisovalerylshikonin is suggested as a potential therapeutic agent for preventing various cancers, the underlying molecular mechanisms are not completely understood. In the present study, we investigated whether β-hydroxyisovalerylshikonin enhances apoptosis by triggering reactive oxygen species production in colon cancer HCT116 cells. β-Hydroxyisovalerylshikonin significantly inhibited the viability of HCT116 cells with maximum inhibition at 4 µM. Furthermore, treatment with β-hydroxyisovalerylshikonin subsequently increased sub-G1 cells and annexin-V+ cell population. Additionally, pretreatment with the caspase-8 inhibitor, z-IETD-fmk, and the caspase-9 inhibitor, z-LETD-fmk, significantly decreased β-hydroxyisovalerylshikonin-induced apoptosis, suggesting that β-hydroxyisovalerylshikonin promotes apoptosis through both the intrinsic and the extrinsic apoptotic pathways by activating caspase-8 and caspase-9. We also found that mitochondria played an important role in β-hydroxyisovalerylshikonin-mediated apoptosis via the intrinsic pathway. Accordingly, β-hydroxyisovalerylshikonin-induced reactive oxygen species production was evident after treatment with β-hydroxyisovalerylshikonin, and pretreatment with reactive oxygen species inhibitors, N-acetyl-L-cysteine and glutathione, significantly decreased β-hydroxyisovalerylshikonin-induced reactive oxygen species production, resulting in inhibition of apoptosis, which suggests that ROS generation is required for β-hydroxyisovalerylshikonin-mediated apoptosis. Taken together, these results demonstrated that the apoptotic effect of β-hydroxyisovalerylshikonin is enhanced in colon cancer HCT116 cells via reactive oxygen species generation and triggering of the caspase pathways, indicating that β-hydroxyisovalerylshikonin has potential as a therapeutic in the treatment of colon cancers.

Are glass fiber particles released during the use of electronic cigarettes? Development of a semi-quantitative approach to detect glass particle emission due to vaping.

This study investigated the emission characteristics of glass particles resulting from smoking electronic cigarettes (ECs). First, the most suitable filter for the collection of glass particles was explored by examining the performance (reliability) of various types of filters. A polycarbonate filter was determined as the optimum choice to collect glass particles in EC aerosol. A cartomizer was filled with EC refill solution composed of an equal volume of propylene glycol (PG) and vegetable glycol (VG). To simulate the potential conditions for glass particle emission, EC vaped aerosols were collected at three distinctive puffing intervals: (1) 0-10 puffs, (2) 101-110 puffs, and (3) 201-210 puffs (flow rate of 1 L min-1, 2 s per puff, and 10 puffs per sample). Glass particles were observed as early as after 100 times puffing from certain products, while after 200 from others. Thus, glass particles were generated by increasing the number of puffs and usage of the EC cartomizer. The analysis of glass particles collected onto polycarbonate filters by scanning electron microscopy (SEM)/energy dispersive X-ray spectroscopy (EDS) confirmed the presence of glass particles in samples collected after puffing 100-200 times. The study demonstrated that the possibility of glass particle emissions from the EC device increased considerably with the increasing number of total puffs.

Apigenin promotes TRAIL-mediated apoptosis regardless of ROS generation.

Apigenin is a bioactive flavone in several herbs including parsley, thyme, and peppermint. Apigenin possesses anti-cancer and anti-inflammatory properties; however, whether apigenin enhances TRAIL-mediated apoptosis in cancer cells is unknown. In the current study, we found that apigenin enhanced TRAIL-induced apoptosis by promoting caspase activation and death receptor 5 (DR5) expression and a chimeric antibody against DR5 completely blocked the apoptosis. Apigenin also upregulated reactive oxygen species (ROS) generation; however, intriguingly, ROS inhibitors, glutathione (GSH) or N-acetyl-l-cysteine (NAC), moderately increased apigenin/TRAIL-induced apoptosis. Additional results showed that an autophagy inducer, rapamycin, enhanced apigenin/TRAIL-mediated apoptosis by a slight increase of ROS generation. Accordingly, NAC and GSH rather decreased apigenin-induced autophagy formation, suggesting that apigenin-induced ROS generation increased autophagy formation. However, autophagy inhibitors, bafilomycin (BAF) and 3-methyladenine (3-MA), showed different result in apigenin/TRAIL-mediated apoptosis without ROS generation. 3-MA upregulated the apoptosis but remained ROS levels; however, no changes on apoptosis and ROS generation were observed by BAF treatment. Taken together, these findings reveal that apigenin enhances TRAIL-induced apoptosis by activating apoptotic caspases by upregulating DR5 expression regardless of ROS generation, which may be a promising strategy for an adjuvant of TRAIL.

Methanol extract of Codium fragile inhibits tumor necrosis factor-α-induced matrix metalloproteinase-9 and invasiveness of MDA-MB-231 cells by suppressing nuclear factor-κB activation.

OBJECTIVE: To evaluate whether the methanol extract of Codium fragile (MECF) regulates tumor necrosis factor-α (TNF-α)-induced invasion of human breast cancer MDA-MB-231 cells by suppressing matrix metalloproteinase-9 (MMP-9). METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were performed to analyze the expression of MMP-9 and nuclear factor-κB (NF-κB) subunits, p65 and p50, and IκB in MDA-MB-231 cells. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used for cell viability. MMP-9 activity and invasion were measured by gelatin zymography and a matrigel invasion assay, respectively. NF-κB activity was measured by an electrophoretic mobility shift assay and luciferase activity. RESULTS: MECF had no effect on cell viability up to a concentration of 100 µg/mL in human breast cancer MDA-MB-231 cells regardless of the presence of TNF-α. MDA-MB-231 cells that were stimulated with TNF-α showed a marked increase of invasion compared to the untreated control, whereas pretreatment with MECF downregulated the TNF-α-induced invasion of MDA-MB-231 cells. Additionally, zymography, western blot analysis, and RT-PCR confirmed that MECF decreased TNF-α-induced MMP-9 expression and activity which is a key regulator for cancer invasion. According to an electrophoretic morbidity shift assay, pretreatment with MECF in MDA-MB-231 cells significantly decreased the TNF-α-induced DNA-binding activity of NF-κB, which is an important transcription factor for regulating cancer invasion-related genes such as MMP-9. Furthermore, treatment with MECF sustained the expression of p65 and p50 in response to TNF-α in the cytosolic compartment. The luciferase assay demonstrated that MECF attenuated TNF-α-induced NF-κB luciferase activity. CONCLUSION: MECF exhibited its anti-invasive capability by downregulating TNF-α-induced MMP-9 expression, resulting from the suppression of NF-κB activity in the human breast cancer cell line MDA-MB-231.

Fulvic acid promotes extracellular anti-cancer mediators from RAW 264.7 cells, causing to cancer cell death in vitro.

Fulvic acid (FA) is known to promote electrochemical balance as a donor or a receptor possessing many biomedical functions. Nevertheless, the effect of FA on the anti-cancer activity has not been elucidated. In the current study, we first isolated FA from humus and investigated whether FA regulates immune-stimulating functions, such as production of nitric oxide (NO), in RAW 264.7 cells. Our data showed that FA slightly enhances cell viability in a dose-dependent manner and secretion of NO from RAW 264.7 cells. It upregulated the protein and mRNA expression of inducible NO synthesis (iNOS). In addition, FA enhanced the DNA-binding activity of nuclear factor-κB (NF-κB) in RAW 264.7 cells; the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) effectively attenuated the expression of FA-stimulated iNOS, suggesting that FA stimulates NF-κB to promote iNOS and NO production. Finally, FA-stimulated culture media (FA-CM) from RAW 264.7 cells were collected and MCA-102 fibrosarcoma cells were cultured in this media. The FA-CM augmented MCA-102 fibrosarcoma cell apoptosis; however, an NO inhibitor N(G)-monomethyl-l-arginine (NMMA) slightly inhibited the FA-CM-mediated MCA-102 fibrosarcoma cell apoptosis, which was accompanied by low levels of NO. In the present study, we found that FA induces the generation of NO and iNOS in RAW 264.7 cells by inducing NF-κB activation; however, NO did not significantly stimulate MCA-102 fibrosarcoma cell apoptosis in the current study. In addition, FA-CM enhanced cell death in various human cancer cells such as Hep3B, LNCaP, and HL60. Taken together, FA most likely stimulates immune-modulating molecules such as NO and induces cancer cell apoptosis.

Hydrangenol inhibits lipopolysaccharide-induced nitric oxide production in BV2 microglial cells by suppressing the NF-κB pathway and activating the Nrf2-mediated HO-1 pathway.

We previously demonstrated the anti-inflammatory effect of water extract of Hydrangea macrophylla in lipopolysaccharide (LPS)-stimulated macrophage cells. Here, we investigated whether hydrangenol, a bioactive component of H. macrophylla, attenuates the expression of nitric oxide (NO) and its associated gene, inducible NO synthase (iNOS), in LPS-stimulated BV2 microglial cells. Our data showed that low dosages of hydrangenol inhibited LPS-stimulated NO release and iNOS expression without any accompanying cytotoxicity. Hydrangenol also suppressed LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) subunits, consequently inhibiting DNA-binding activity of NF-κB. Additionally, the NF-κB inhibitors, pyrrolidine dithiocarbamate (PDTC) and PS-1145, significantly attenuated LPS-induced iNOS expression, indicating that hydrangenol-induced NF-κB inhibition might be a key regulator of iNOS expression. Furthermore, our data showed that hydrangenol suppresses NO production by inducing heme oxygenase-1 (HO-1). The presence of cobalt protoporphyrin, a specific HO-1 inducer, potently suppressed LPS-induced NO production. Hydrangenol also promoted nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and subsequently increased its binding activity at the specific antioxidant response element sites. Additionally, transient knockdown of Nrf2 significantly downregulated hydrangenol-induced HO-1 expression, indicating that hydrangenol-induced Nrf2 is an upstream regulator of HO-1. Taken together, these data suggest that hydrangenol attenuates NO production and iNOS expression in LPS-stimulated BV2 microglial cells by inhibiting NF-κB activation and by stimulating the Nrf2-mediated HO-1 signaling pathway. Therefore, hydrangenol is a promising therapeutic agent for treatment of LPS-mediated inflammatory diseases.