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.

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 Suppresses the α-Melanocyte-Stimulating Hormone-Induced Melanogenic Signaling Pathway.

Pinostrobin is a dietary flavonoid found in several plants that possesses pharmacological properties, such as anti-cancer, anti-virus, antioxidant, anti-ulcer, and anti-aromatase effects. However, it is unclear if pinostrobin exerts anti-melanogenic properties and, if so, what the underlying molecular mechanisms comprise. Therefore, we, in this study, investigated whether pinostrobin inhibits melanin biosynthesis in vitro and in vivo, as well as the potential associated mechanism. Pinostrobin reduced mushroom tyrosinase activity in vitro in a concentration-dependent manner, with an IC50 of 700 µM. Molecular docking simulations further revealed that pinostrobin forms a hydrogen bond, as well as other non-covalent interactions, between the C-type lectin-like fold and polyphenol oxidase chain, rather than the previously known copper-containing catalytic center. Additionally, pinostrobin significantly decreased α-melanocyte-stimulating hormone (α-MSH)-induced extracellular and intracellular melanin production, as well as tyrosinase activity, in B16F10 melanoma cells. More specifically, pinostrobin inhibited the α-MSH-induced melanin biosynthesis signaling pathway by suppressing the cAMP-CREB-MITF axis. In fact, pinostrobin also attenuated pigmentation in α-MSH-stimulated zebrafish larvae without causing cardiotoxicity. The findings suggest that pinostrobin effectively inhibits melanogenesis in vitro and in vivo via regulation of the cAMP-CREB-MITF axis.

Anthocyanin-enriched polyphenols from Hibiscus syriacus L. (Malvaceae) exert anti-osteoporosis effects by inhibiting GSK-3ß and subsequently activating ß-catenin.

BACKGROUND: The bark and petal of Hibiscus syriacus L. (Malvaceae) have been used to relieve pain in traditional Korean medicine. Recently, we identified anthocyanin-enriched polyphenols from the petal of H. syriacus L. (AHs) and determined its anti-melanogenic, anti-inflammatory, and anti-oxidative properties. Nevertheless, the osteogenic potential of AHs remains unknown. PURPOSE: This study was aimed to investigating the effect of AHs on osteoblast differentiation and osteogenesis in osteoblastic cell lines and zebrafish larvae. Furthermore, we investigated whether AHs ameliorates prednisolone (PDS)-induced osteoporosis. STUDY DESIGN AND METHODS: Cell viability was assessed by cellular morphology, MTT assay, and flow cytometry analysis, and osteoblast differentiation was measured alizarin red staining, alkaline phosphatase (ALP) activity, and osteoblast-specific marker expression. Osteogenic and anti-osteoporotic effects of AHs were determined in zebrafish larvae. RESULTS: AHs enhanced calcification and ALP activity concomitant with the increased expression of osterix (OSX), runt-related transcription factor 2 (RUNX2), and ALP in MC3T3-E1 preosteoblast and MG-63 osteosarcoma cells. Additionally, AHs accelerated vertebral formation and mineralization in zebrafish larvae, concurrent with the increased expression of OSX, RUNX2a, and ALP. Furthermore, PDS-induced loss of osteogenic activity and vertebral formation were restored by treatment with AHs, accompanied by a significant recovery of calcification, ALP activity, and osteogenic marker expression. Molecular docking studies showed that 16 components in AHs fit to glucagon synthase kinase-3ß (GSK-3ß); particularly, isovitexin-4'-O-glucoside most strongly binds to the peptide backbone of GSK-3ß at GLY47(O), GLY47(N), and ASN361(O), with a binding score of -7.3. Subsequently, AHs phosphorylated GSK-3ß at SER9 (an inactive form) and released ß-catenin into the nucleus. Pretreatment with FH535, a Wnt/ß-catenin inhibitor, significantly inhibited AH-induced vertebral formation in zebrafish larvae. CONCLUSION: AHs stimulate osteogenic activities through the inhibition of GSK-3ß and subsequent activation of ß-catenin, leading to anti-osteoporosis effects.

Flavonoid Glycosides from Ziziphus jujuba var. inermis (Bunge) Rehder Seeds Inhibit α-Melanocyte-Stimulating Hormone-Mediated Melanogenesis.

Ziziphus jujuba extracts possess a broad spectrum of biological activities, such as antioxidant and anticancer activities in melanoma cancers. Nevertheless, the compounds contain high antioxidant capacities and anticancer activities in melanoma cells, shown to be effective in hyperpigmentation disorders, but whether flavonoid glycosides from Z. jujuba regulate anti-melanogenesis remains unclear. In this study, we evaluated the anti-melanogenic activity of five flavonoid glycosides from Z. jujuba var. inermis (Bunge) Rehder seeds, including jujuboside A (JUA), jujuboside B (JUB), epiceanothic acid (EPA), betulin (BTL), and 6'''-feruloylspinosin (FRS), in B16F10 melanoma cells and zebrafish larvae. According to our results, JUB, EPA, and FRS potently inhibited α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis and prevented hyperpigmentation in zebrafish larvae. In particular, under α-MSH-stimulated conditions, FRS most significantly inhibited α-MSH-induced intracellular and extracellular melanin content in B16F10 melanoma cells. Additionally, JUB, EPS, and FRS remarkably downregulated melanogenesis in α-MSH-treated zebrafish larvae, with no significant change in heart rate. Neither JUA nor BTA were effective in downregulating melanogenesis in B16F10 melanoma cells and zebrafish larvae. Furthermore, JUB, EPA, and FRS directly inhibited in vitro mushroom tyrosinase enzyme activity. JUB, EPA, and FRS also downregulated cyclic adenosine monophosphate (cAMP) levels and the phosphorylation of cAMP-response element-binding protein (CREB), and subsequent microphthalmia transcription factor (MITF) and tyrosinase expression. In conclusion, this study demonstrated that JUB, EPA, and FRS isolated from Z. jujuba var. inermis (Bunge) Rehder seeds exhibit potent anti-melanogenic properties by inhibition of the cAMP-CERB-MITF axis and consequent tyrosinase activity.

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.

Anthocyanins from Hibiscus syriacus L. Inhibit NLRP3 Inflammasome in BV2 Microglia Cells by Alleviating NF-κB- and ER Stress-Induced Ca2+ Accumulation and Mitochondrial ROS Production.

Anthocyanins from the petals of Hibiscus syriacus L. (PS) possess anti-inflammatory, antioxidant, and antimelanogenic activities. However, it remains unclear whether PS inhibit the NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation and assembly. This study is aimed at investigating whether PS downregulate NLRP3-mediated inflammasome by inhibiting nuclear factor-κB (NF-κB) and endoplasmic reticulum (ER) stress. BV2 microglia cells were treated with PS in the presence of lipopolysaccharide and adenosine triphosphate (LPS/ATP), and the NLRP3-related signaling pathway was investigated. In this study, we found that LPS/ATP treatment activated the NLRP3 inflammasome, which resulted in the release of interleukin-1ß (IL-1ß) and IL-18. Meanwhile, PS reduced LPS/ATP-mediated NLRP3 inflammasome at 12 h by inhibiting ER stress-mediated Ca2+ accumulation and subsequent mitochondrial reactive oxygen species (mtROS) production, which, in turn, decreased IL-1ß and IL-18 release. Furthermore, PS inhibited the NLRP3 inflammasome 1 h after LPS/ATP treatment by suppressing the NF-κB pathway, which downregulated Ca2+ accumulation and mtROS production. These data showed that PS negatively regulated activation of the NLRP3 inflammasome in a time-different manner by inhibiting the NF-κB signaling pathway in the early stage and the ER stress response in the late stage. The pathways shared Ca2+ accumulation-mediated mtROS production, which was significantly inhibited in the presence of PS. In conclusion, our results suggested that PS has potential as a supplement against NLRP3 inflammasome-related inflammatory disorders; nevertheless, further studies are needed to determine the effect of PS in the noncanonical NLRP3 inflammasome pathways and pathological conditions in vivo.

GSK-3ß-Targeting Fisetin Promotes Melanogenesis in B16F10 Melanoma Cells and Zebrafish Larvae through ß-Catenin Activation.

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-3ß (GSK-3ß). Therefore, we evaluated whether fisetin negatively regulated GSK-3ß, which subsequently activates ß-catenin, resulting in melanogenesis. As expected, fisetin increased the expression of ß-catenin, which was subsequently translocated into the nucleus. In the functional assay, FH535, a Wnt/ß-catenin inhibitor, significantly inhibited fisetin-mediated melanogenesis in zebrafish larvae. Our data suggested that fisetin inhibits GSK-3ß, which activates ß-catenin, resulting in melanogenesis through the revitalization of MITF and tyrosinase.

Anthocyanins from Hibiscus syriacus L. Inhibit Oxidative Stress-Mediated Apoptosis by Activating the Nrf2/HO-1 Signaling Pathway.

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.

Anthocyanins from Hibiscus syriacus L. Inhibit Melanogenesis by Activating the ERK Signaling Pathway.

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.

p53-Mediated Oxidative Stress Enhances Indirubin-3'-Monoxime-Induced Apoptosis in HCT116 Colon Cancer Cells by Upregulating Death Receptor 5 and TNF-Related Apoptosis-Inducing Ligand Expression.

Indirubin-3'-monoxime (I3M) exhibits anti-proliferative activity in various cancer cells; however, its anti-cancer mechanism remains incompletely elucidated. This study revealed that I3M promotes the expression of death receptor 5 (DR5) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in HCT116 p53+/+ cells, resulting in caspase-mediated apoptosis. However, this study demonstrated that HCT116 p53-/- cells were insensitive to I3M-mediated apoptosis, indicating that I3M-induced apoptosis depends on the p53 status of HCT116 cells. Additionally, in HCT116 p53-/- cells, I3M significantly increased Ras expression, while in HCT116 p53+/+ cells, it reduced Ras expression. Furthermore, I3M remarkably increased the production of reactive oxygen species (ROS), which were reduced in transient p53 knockdown, indicating that I3M-mediated apoptosis was promoted by p53-mediated ROS production. Our results also showed that I3M enhanced transcription factor C/EBP homologous protein (CHOP) expression, resulted in endoplasmic reticulum (ER) stress-mediated DR5 expression, which was upregulated by ROS production in HCT116 p53+/+ cells. Moreover, co-treatment with I3M and TRAIL enhanced DR5 expression, thereby triggering TRAIL-induced apoptosis of HCT116 p53+/+ cells, which was interfered by a DR5-specific blocking chimeric antibody. In summary, I3M potently enhances TRAIL-induced apoptosis by upregulating DR5 expression via p53-mediated ROS production in HCT116 p53+/+ cells. However, HCT116 p53-/- cells were less sensitive to I3M-mediated apoptosis, suggesting that I3M could be a promising anti-cancer candidate against TRAIL-resistant p53+/+ cancer cells. Additionally, this study also revealed that I3M sensitizes colorectal cancer cells such as HT29 and SW480 to TRAIL-mediated apoptosis.

Euodia daniellii Hemsl. (Bee-Bee Tree) Oil Attenuates Palmitate-Induced Lipid Accumulation and Apoptosis in Hepatocytes.

Hepatic lipid accumulation and apoptosis is elevated in patients with non-alcoholic steatohepatitis and is closely associated with severity. Saturated fatty acid palmitate stimulates lipid accumulation and apoptosis in hepatocytes. In the present study, we examined bee-bee tree oil (BO)-mediated protective effects on palmitate-induced lipid accumulation and apoptosis in mouse primary hepatocytes. Cells were cultured in a control media or the same media containing 150 or 300 µmol/L of albumin-bound palmitate for 24 h. BO concentrations used were 0, 0.1, 0.2, or 0.5%. Palmitate induced lipid accumulation and mRNA expression of lipogenic genes such as SREBP1c and SCD1. However, BO prevented these changes. Furthermore, palmitate stimulated caspase-3 activity and decreased cell viability in the absence of BO. BO reduced palmitate-induced activation of caspase-3 and cell death in a dose-dependent manner. AMP-activated protein kinase inhibitors abolished the effects of BO. Furthermore, BO suppressed palmitate-induced c-Jun N-terminal kinase (JNK) phosphorylation through the 5' adenosine monophosphate-activated protein kinase (AMPK)-dependent pathway. In conclusion, BO attenuated palmitate-induced hepatic steatosis and apoptosis through AMPK-mediated suppression of JNK signaling. These data suggest that BO is an important determinant of saturated fatty acid-induced lipid accumulation and apoptosis, and may be an effective therapeutic strategy for treatment of obesity-mediated liver diseases.

Antagonistic effects of acetylshikonin on LPS-induced NO and PGE2 production in BV2 microglial cells via inhibition of ROS/PI3K/Akt-mediated NF-κB signaling and activation of Nrf2-dependent HO-1.

Although acetylshikonin (ACS) is known to have antioxidant and antitumor activities, whether ACS regulates the expression of proinflammatory mediators in lipopolysaccharide (LPS)-stimulated microglial cells remains unclear. In this study, it was found that ACS isolated from Lithospermum erythrorhizon inhibits LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) release by suppressing the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in BV2 microglial cells. Furthermore, ACS reduced the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) and subsequently suppressed iNOS and COX-2 expression. Consistent with these data, ACS attenuated the phosphorylation of PI3K and Akt and suppressed the DNA-binding activity of NF-κB by inducing the generation of reactive oxygen species (ROS) in LPS-stimulated cells. In addition, ACS enhanced heme oxygenase-1 (HO-1) expression via nuclear factor-erythroid 2-related factor 2 (Nrf2) activation. Zinc protoporphyrin, a specific HO-1 inhibitor, partially attenuated the antagonistic effects of ACS on LPS-induced NO and PGE2 production. By contrast, the presence of cobalt protoporphyrin, a specific HO-1 inducer, potently suppressed LPS-induced NO and PGE2 production. These data indicate that ACS downregulates proinflammatory mediators such as NO and PGE2 by suppressing PI3K/Akt-dependent NF-κB activity induced by ROS as well as inducing Nrf2-dependent HO-1 activity. Taken together, ACS might be a good candidate to regulate LPS-mediated inflammatory diseases.

Neuroprotection against 6-OHDA-induced oxidative stress and apoptosis in SH-SY5Y cells by 5,7-Dihydroxychromone: Activation of the Nrf2/ARE pathway.

AIMS: The aim of this study was to prove the neuroprotective effect of 5,7-Dihydroxychromone (DHC) through the Nrf2/ARE signaling pathway. To elucidate the mechanism, we investigated whether 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in SH-SY5Y cells could be attenuated by DHC via activating the Nrf2/ARE signal and whether DHC could down-regulate 6-OHDA-induced excessive ROS generation MAIN METHODS: To evaluate the neuroprotective effect of DHC against 6-OHDA-induced apoptosis, FACS analysis was performed using PI staining. The inhibitory effect of DHC against 6-OHDA-induced ROS generation was evaluated by DCFH-DA staining assay. Additionally, translocation of Nrf2 to the nucleus and increased Nrf2/ARE binding activity, which subsequently resulted in the up-regulation of the Nrf2-dependent antioxidant gene expressions including HO-1, NQO1, and GCLc, were evaluated by Western blotting and EMSA. KEY FINDINGS: Pre-treatment of DHC, one of the constituents of Cudrania tricuspidata, significantly protects 6-OHDA-induced neuronal cell death and ROS generation. Also, DHC inhibited the expression of activated caspase-3 and caspase-9 and cleaved PARP in 6-OHDA-induced SH-SY5Y cells. DHC induced the translocation of Nrf2 to the nucleus and increased Nrf2/ARE binding activity which results in the up-regulation of the expression of Nrf2-dependent antioxidant genes, including HO-1, NQO1, and GCLc. The addition of Nrf2 siRNA abolished the neuroprotective effect of DHC against 6-OHDA-induced neurotoxicity and the expression of Nrf2-mediated antioxidant genes. SIGNIFICANCE: Activation of Nrf2/ARE signal by DHC exerted neuroprotective effects against 6-OHDA-induced oxidative stress and apoptosis. This finding will give an insight that activating Nrf2/ARE signal could be a new potential therapeutic strategy for neurodegenerative disease.

New compound, 5-O-isoferuloyl-2-deoxy-D-ribono-γ-lacton from Clematis mandshurica: Anti-inflammatory effects in lipopolysaccharide-stimulated BV2 microglial cells.

Microglia are main immune cells to exacerbate neural disorders in persistent overactivating. Therefore, it is a good strategy to regulate microglia for the treatment of neural disorders. In the present study, we isolated and characterized a novel compound, 5-O-isoferuloyl-2-deoxy-D-ribono-γ-lacton (5-DRL) from Clematis mandshurica, and evaluated its anti-inflammatory effect in lipopolysaccharide (LPS)-treated BV2 microglial cells. 5-DRL inhibited the expression of LPS-stimulated proinflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2), as well as their regulatory genes inducible NO syntheses (iNOS) and cyclooxygenase-2 (COX-2). 5-DRL also downregulated the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) through suppression of the nuclear translocation of the NF-κB subunits, p65 and p50. Consistent with the inhibition of iNOS and COX-2 via NF-κB activity with 5-DRL, an inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), also led to the suppression of LPS-induced iNOS and COX-2 expression. Additionally, 5-DRL corresponding with antioxidants, N-acetylcysteine (NAC) and glutathione (GSH), remarkably inhibited reactive oxygen species (ROS) generation. Both NAC and GSH, thus attenuated the expression of iNOS and COX-2 by suppressing NF-κB activation, indicating that 5-DRL suppresses LPS-induced iNOS and COX-2 expression through downregulation of the ROS-dependent NF-κB signaling pathway. The present study also indicated that 5-DRL suppresses NO and PGE2 production by inducing heme oxygenase-1 (HO-1) via nuclear factor erythroid 2-related factor 2 (Nrf2). Taken together, the present data indicate that 5-DRL attenuates the production of proinflammatory mediators such as NO and PGE2 as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting ROS-dependent NF-κB activation and stimulating the Nrf2/HO-1 signal pathway. These data may be implicated in the application of 5-DRL in LPS-stimulated inflammatory disease.

Isobutyrylshikonin inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in BV2 microglial cells by suppressing the PI3K/Akt-mediated nuclear transcription factor-κB pathway.

Microglia are important macrophages to defend against pathogens in the central nervous system (CNS); however, persistent or acute inflammation of microglia lead to CNS disorders via neuronal cell death. Therefore, we theorized that a good strategy for the treatment of CNS disorders would be to target inflammatory mediators from microglia in disease. Consequently, we investigated whether isobutyrylshikonin (IBS) attenuates the production of proinflammatory mediators, such as nitric oxide (NO) and prostaglandin E2, in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Treatment with IBS inhibited the secretion of NO and prostaglandin E2 (as well as the expression of their key regulatory genes), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2). Isobutyrylshikonin also suppressed LPS-induced DNA-binding activity of nuclear transcription factor-κB (NF-κB), by inhibiting the nuclear translocation of p50 and p65 in addition to blocking the phosphorylation and degradation of IκBα. Pretreatment with pyrrolidine dithiocarbamate, a specific NF-κB inhibitor, showed the down-regulation of LPS-induced iNOS and COX-2 messenger RNA by suppressing NF-κB activity. This indirectly suggests that IBS-mediated NF-κB inhibition is the main signaling pathway involved in the inhibition of iNOS and COX-2 expression. In addition, IBS attenuated LPS-induced phosphorylation of PI3K and Akt, which are upstream molecules of NF-κB, in LPS-stimulated BV2 microglial cells. The functional aspects of the PI3K/Akt signaling pathway were analyzed with LY294002, which is a specific PI3K/Akt inhibitor that attenuated LPS-induced iNOS and COX-2 expression by suppressing NF-κB activity. These data suggest that an IBS-mediated anti-inflammatory effect may be involved in suppressing the PI3K/Akt-mediated NF-κB signaling pathway.

Anti-inflammatory mechanism of α-viniferin regulates lipopolysaccharide-induced release of proinflammatory mediators in BV2 microglial cells.

α-Viniferin is an oligostilbene of trimeric resveratrol and has anticancer activity; however, the molecular mechanism underlying the anti-inflammatory effects of α-viniferin has not been completely elucidated thus far. Therefore, we determined the mechanism by which α-viniferin regulates lipopolysaccharide (LPS)-induced expression of proinflammatory mediators in BV2 microglial cells. Treatment with α-viniferin isolated from Clematis mandshurica decreased LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2). α-Viniferin also downregulated the LPS-induced expression of proinflammatory genes such as iNOS and COX-2 by suppressing the activity of nuclear factor kappa B (NF-κB) via dephosphorylation of Akt/PI3K. Treatment with a specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), indirectly showed that NF-κB is a crucial transcription factor for expression of these genes in the early stage of inflammation. Additionally, our results indicated that α-viniferin suppresses NO and PGE2 production in the late stage of inflammation through induction of heme oxygenase-1 (HO-1) regulated by nuclear factor erythroid 2-related factor (Nrf2). Taken together, our data indicate that α-viniferin suppresses the expression of proinflammatory genes iNOS and COX-2 in the early stage of inflammation by inhibiting the Akt/PI3K-dependent NF-κB activation and inhibits the production of proinflammatory mediators NO and PGE2 in the late stage by stimulating Nrf2-mediated HO-1 signaling pathway in LPS-stimulated BV2 microglial cells. These results suggest that α-viniferin may be a potential candidate to regulate LPS-induced inflammation.

Potential chemoprevention of LPS-stimulated nitric oxide and prostaglandin E2 production by α-L-rhamnopyranosyl-(1→6)-ß-D-glucopyranosyl-3-indolecarbonate in BV2 microglial cells through suppression of the ROS/PI3K/Akt/NF-κB pathway.

α-l-Rhamnopyranosyl-(1→6)-ß-d-glucopyranosyl-3-indolecarbonate (RG3I) is a chemical constituent isolated from the commonly used Asian traditional medicinal plant, Clematis mandshurica; however, no studies have been reported on its anti-inflammatory properties. In the present study, we found that RG3I attenuates the lipopolysaccharide (LPS)-induced DNA-binding activity of nuclear factor-κB (NF-κB) via the dephosphorylation of PI3K/Akt in BV2 microglial cells, leading to a suppression of nitric oxide (NO) and prostaglandin E2 (PGE2) production, along with that of their regulatory genes, inducible NO synthase (iNOS) and cyclooxygenase-2 (Cox-2). Further, the PI3K/Akt inhibitor, LY294002 diminished the expression of LPS-stimulated iNOS and COX-2 genes by suppressing NF-κB activity. Moreover, RG3I significantly inhibited LPS-induced reactive oxygen species (ROS) generation similar to the ROS inhibitors, N-acetylcysteine (NAC) and glutathione (GSH). Notably, NAC and GSH abolished the LPS-induced expression of iNOS and Cox-2 in BV2 microglial cells by inhibiting NF-κB activity. Taken together, our data indicate that RG3I suppresses the production of proinflammatory mediators such as NO and PGE2 as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting the PI3K/Akt- and ROS-dependent NF-κB signaling pathway, suggesting that RG3I may be a good candidate to regulate LPS-induced inflammatory response.

Downregulation of NO and PGE2 in LPS-stimulated BV2 microglial cells by trans-isoferulic acid via suppression of PI3K/Akt-dependent NF-κB and activation of Nrf2-mediated HO-1.

Little is known about whether trans-isoferulic acid (TIA) regulates the production of lipopolysaccharide (LPS)-induced proinflammatory mediators. Therefore, we examined the effect of TIA isolated from Clematis mandshurica on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in BV2 microglial cells. We found that TIA inhibited the production of LPS-induced NO and PGE2 without accompanying cytotoxicity in BV2 microglial cells. TIA also downregulated the expression levels of specific regulatory genes such as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) by suppressing LPS-induced NF-κB activity via dephosphorylation of PI3K/Akt. In addition, we demonstrated that a specific NF-κB inhibitor PDTC and a selective PI3K/Akt inhibitor, LY294002 effectively attenuated the expression of LPS-stimulated iNOS and COX-2 mRNA, while LY294002 suppressed LPS-induced NF-κB activity, suggesting that TIA attenuates the expression of these proinflammatory genes by suppressing PI3K/Akt-mediated NF-κB activity. Our results showed that TIA suppressed NO and PGE2 production through the induction of nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent heme oxygenase-1 (HO-1). Taken together, our data indicate that TIA suppresses the production of proinflammatory mediators such as NO and PGE2, as well as their regulatory genes, in LPS-stimulated BV2 microglial cells, by inhibiting PI3K/Akt-dependent NF-κB activity and enhancing Nrf2-mediated HO-1 expression.