Induction of Browning in White Adipocytes: Fucoidan Characterization and Gold Nanoparticle Synthesis from Undaria pinnatifida Sporophyll Extract.

Seaweed extracts and their specific polysaccharides are widely known for their ability to act as reducing and capping agents during nanoparticle synthesis. Their application is highly favored in green synthesis methods, owing to their eco-friendliness, cost-effectiveness, and remarkable time and energy efficiency. In this study, fucoidan extracted from Undaria pinnatifida sporophyll (UPS) is introduced as a polysaccharide that effectively serves as a dual-function reducing and capping agent for the synthesis of gold nanoparticles (AuNPs). Results from various analyses indicate that AuNPs derived from UPS extract display a uniform spherical shape with an average size of 28.34 ± 1.15 nm and a zeta potential of -37.49 ± 2.13 mV, conclusively confirming the presence of Au. The FT-IR spectra distinctly revealed the characteristic fucoidan bands on the stabilized UPS-AuNPs surface. A 1H-NMR analysis provided additional confirmation by revealing the presence of specific fucoidan protons on the UPS-AuNPs surface. To comprehensively evaluate the impact of UPS extract, UPS-AuNPs, and fucoidan on the biological properties of adipocytes, a rigorous comparative analysis of lipid droplet formation and morphology was conducted. Our findings revealed that adipocytes treated with UPS extract, fucoidan, and UPS-AuNPs, in that order, exhibited a reduction in the total lipid droplet surface area, maximum Ferret diameter, and overall Nile red staining intensity when compared to mature white adipocytes. Furthermore, our analysis of the effects of UPS extracts, UPS-AuNPs, and fucoidan on the expression of key markers associated with white adipose tissue browning, such as UCP1, PGC1a, and PRDM16, demonstrated increased mRNA and protein expression levels in the following order: UPS-AuNPs > fucoidan > UPS extracts. Notably, the production of active mitochondria, which play a crucial role in enhancing energy expenditure in beige adipocytes, also increased in the following order: UPS-AuNPs > fucoidan > UPS extract. These findings underscore the pivotal role of UPS extract, fucoidan, and UPS-AuNPs in promoting adipocyte browning and subsequently enhancing energy expenditure.

OGD/R-induced ferroptosis and pyroptosis in retinal pigment epithelium cells: Role of PLD1 and PLD2 modulation.

This study investigated the role of phospholipase D (PLD) in retinal ischemia-reperfusion (I/R) injury using an oxygen-glucose deprivation/reperfusion (OGD/R) model commonly used in retinal I/R injury research. To create an in vitro cellular I/R model, pharmacological inhibitors and small interfering RNA (siRNA) were used to target PLD1 and PLD2 in retinal pigment epithelial (RPE) cells. Treatment with PLD inhibitors and siRNA reduced reactive oxygen species (ROS) and malondialdehyde (MDA) induced by OGD/R in RPE cells and increased the levels of superoxide dismutase (SOD) and glutathione (GSH), indicating a reduction in oxidative damage and improvement in the antioxidant system. Next, we showed that inhibiting PLD1 or PLD2 reduced intracellular iron levels and lipid peroxidation, which are critical factors in ferroptosis. Additionally, PLD1 and PLD2 modulated the expression of proteins involved in the regulation of ferroptosis, including GPX4, SLC7A11, FTH1, and ACSL4. We also investigated the roles of PLD1 and PLD2 in preventing pyroptosis, another form of programmed cell death associated with inflammation. Our study found that OGD/R significantly increased the production of pro-inflammatory cytokines and activated caspase-1, NLRP3, ASC, cleaved-caspase 1 (C-caspase-1), and GSDMD-N in RPE cells, indicating pyroptosis induction. However, PLD1 and PLD2 inhibition or knockdown significantly inhibited the production of pro-inflammatory cytokines and activation of the NLRP3 inflammasome, Taken together, our findings support the hypothesis that the PLD signaling pathway plays a key role in OGD/R-induced ferroptosis and pyroptosis induction and may be a potential therapeutic target for preventing or treating retinal dysfunction and degeneration.

Cucurbitacin B-, E-, and I-Induced Browning of White Adipocytes Is Promoted by the Inhibition of Phospholipase D2.

The mechanism of white adipose tissue browning is not well understood; however, naturally occurring compounds are known to play a positive role. The effects of cucurbitacins B, E, and I on the browning of mature white adipocytes were investigated. First, the cell viability exhibited by cucurbitacins B, E, and I in pre- and mature adipocytes was verified. Cucurbitacins B, E, and I had no effect on cell viability in pre- and mature adipocytes at concentrations up to 300 nM. To investigate the characteristics of representative beige adipocytes, the formation and morphology of cucurbitacin B, E, and I lipid droplets were verified. The total lipid droplet surface area, maximum Feret diameter, and total Nile red staining intensity of cucurbitacin B-, E-, and I-treated adipocytes were lower than those of mature white adipocytes. Furthermore, treatment of white mature adipocytes with cucurbitacin B, E, and I led to the formation of several small lipid droplets that are readily available for energy expenditure. We evaluated the effect of cucurbitacins B, E, and I on the expression of representative browning markers UCP1, PGC1a, and PRDM16, which participate in the browning of white adipose tissue. Cucurbitacins B, E, and I increased the mRNA and protein expression levels of UCP1, PGC1a, and PRDM16 in a concentration-dependent manner. To promote energy consumption by beige adipocytes, active mitochondrial biogenesis is essential. Next, we investigated the effects of cucurbitacin B, E, and I on mitochondrial biogenesis in mature adipocytes. Mitochondrial mass increased when mature adipocytes were treated with cucurbitacin B, E, and I. The degree of cucurbitacin B-, E- and I-induced transformation of white adipocytes into beige adipocytes was in the order of Cu E > Cu B > Cu I. To verify the effect of phospholipase D2 on the browning of white adipocytes, CAY10594­a PLD2 pharmacological inhibitor, and a knockdown system were used. PLD2 inhibition and knockdown improved the expression levels of UCP1, PGC1a, and PRDM16. In addition, PLD2 inhibition and knockdown in mature white adipocytes promoted mitochondrial biosynthesis. The effect of PLD2 inhibition and knockdown on promoting browning of white adipocytes significantly increased when Cu B, Cu E, and Cu I were co-treated. These data indicate that mature white adipocytes' beige properties were induced by cucurbitacins B, E, and I. These effects became more potent by the inhibition of PLD2. These findings provide a model for determining anti-obesity agents that induce browning and increase energy expenditure in mature white adipocytes.

Characterization of Plocamium telfairiae Extract-Functionalized Au Nanostructures and Their Anti-Adipogenic Activity through PLD1.

Here, Au nanostructure (AuNS) biosynthesis was mediated through ethanolic extract of Plocamium telfairiae (PT) without the use of stabilizers or surfactants. PT-functionalized AuNSs (PT-AuNSs) were analyzed using ultraviolet-visible spectroscopy, dynamic light scattering, high-resolution transmission electron microscopy, energy-dispersive spectroscopy, and Fourier-transform infrared spectroscopy. Stable monodisperse PT-AuNSs were synthesized, with a mean size of 15.36 ± 0.10 nm and zeta potential of -35.85 ± 1.36 mV. Moreover, biosynthetic AuNPs with a face-centered structure of PT-AuNS exhibited crystalline characteristics. In addition, many functional groups playing important roles in the biological reduction of PT extracts were adsorbed on the surface of PT-AuNSs. Furthermore, the effects of PT-AuNSs on adipogenesis in immature adipocytes were investigated. PT-AuNSs reduced morphological changes, lowered triglyceride content, and increased lipid accumulation by approximately 78.6% in immature adipocytes compared with the values in mature adipocytes (MDI-induced). PT-AuNS suppressed lipid accumulation by downregulating the transcript and protein expression of C/EBPα, PPARγ, SREBP 1, FAS, and aP2. Finally, PT-AuNS induced the transcript and protein expression of UCP1, PRDM16, and PGC1a, thereby increasing mitochondrial biogenesis in mature adipocytes and effectively inducing brown adipogenesis. In this study, the biosynthesized PT-AuNS was used as a potential therapeutic candidate because it conferred a potent anti-lipogenic effect. As a result, it can be used in various scientific fields such as medicine and the environment.

Nano-Graphene Oxide-Promoted Epithelial-Mesenchymal Transition of Human Retinal Pigment Epithelial Cells through Regulation of Phospholipase D Signaling.

Nano-graphene oxide (Nano-GO) is an extensively studied multifunctional carbon nanomaterial with attractive applications in biomedicine and biotechnology. However, few studies have been conducted to assess the epithelial-to-mesenchymal transition (EMT) in the retinal pigment epithelium (RPE). We aimed to determine whether Nano-GO induces EMT by regulating phospholipase D (PLD) signaling in human RPE (ARPE-19) cells. The physicochemical characterization of Nano-GO was performed using a Zetasizer, X-ray diffraction, Fourier-transform infrared spectroscopy, and transmission electron microscopy. RPE cell viability assays were performed, and the migratory effects of RPE cells were evaluated. RPE cell collagen gel contraction was also determined. Intracellular reactive oxygen species (ROS) levels were determined by fluorescence microscopy and flow cytometry. Immunofluorescence staining and western blot analysis were used to detect EMT-related protein expression. Phospholipase D (PLD) enzymatic activities were also measured. Nano-GO significantly enhanced the scratch-healing ability of RPE cells, indicating that the RPE cell migration ability was increased. Following Nano-GO treatment, the RPE cell penetration of the chamber was significantly promoted, suggesting that the migratory ability was strengthened. We also observed collagen gel contraction and the generation of intracellular ROS in RPE cells. The results showed that Nano-GO induced collagen gel contraction and intracellular ROS production in RPE cells. Moreover, immunofluorescence staining and western blot analysis revealed that Nano-GO significantly regulated key molecules of EMT, including epithelial-cadherin, neural-cadherin, α-smooth muscle actin, vimentin, and matrix metalloproteinases (MMP-2 and MMP-9). Interestingly, Nano-GO-induced RPE cell migration and intracellular ROS production were abrogated in PLD-knockdown RPE cells, indicating that PLD activation played a crucial role in the Nano-GO-induced RPE EMT process. We demonstrate for the first time that Nano-GO promotes RPE cell migration through PLD-mediated ROS production. We provide preliminary evidence to support the hypothesis that Nano-GO has adverse health effects related to RPE damage.

CYTOKININ-RESPONSIVE GROWTH REGULATOR regulates cell expansion and cytokinin-mediated cell cycle progression.

The cytokinin (CK) phytohormones have long been known to activate cell proliferation in plants. However, how CKs regulate cell division and cell expansion remains unclear. Here, we reveal that a basic helix-loop-helix transcription factor, CYTOKININ-RESPONSIVE GROWTH REGULATOR (CKG), mediates CK-dependent regulation of cell expansion and cell cycle progression in Arabidopsis thaliana. The overexpression of CKG increased cell size in a ploidy-independent manner and promoted entry into the S phase of the cell cycle, especially at the seedling stage. Furthermore, CKG enhanced organ growth in a pleiotropic fashion, from embryogenesis to reproductive stages, particularly of cotyledons. In contrast, ckg loss-of-function mutants exhibited smaller cotyledons. CKG mainly regulates the expression of genes involved in the regulation of the cell cycle including WEE1. We propose that CKG provides a regulatory module that connects cell cycle progression and organ growth to CK responses.

Anti-Metastatic Effect of Gold Nanoparticle-Conjugated Maclura tricuspidata Extract on Human Hepatocellular Carcinoma Cells.

PURPOSE: We aimed to study green-synthesized gold nanoparticles (GNPs) from Maclura tricuspidata (MT) root (MTR), stem (MTS), leaf (MTL), and fruit (MTF) extracts and evaluate their anti-metastatic properties in hepatocellular carcinoma cells. Maclura tricuspidata belongs to the Moraceae family and is widely used as a traditional medicinal plant given its biological activities. METHODS: We quantified the phenolic and flavonoid contents, reducing capacity, and antioxidant activity of all four extracts. The facile and optimum synthesis of MT-GNPs was visualized using UV-vis spectra and dynamic light scattering (DLS). Surface morphology, selected area electron diffraction (SAED), and fast Fourier transform (FFT) pattern of MT-GNPs were assessed using high-resolution transmission electron microscopy (HR-TEM). The crystallized gold pattern of MT-GNPs was evaluated using energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD). The functionalizing ligands of MT-extracts and MT-GNPs were determined using Fourier-transform infrared spectroscopy (FT-IR). The photocatalytic capabilities of MT-GNPs were assessed by measuring the reduction of rhodamine B and methylene blue. Cell viability assay was detected using Cell Counting Kit-8 solution. Anti-migratory and anti-invasive effects were assessed using cell migration and invasion assays. Matrix metalloproteinase (MMP)-9 and phospholipase D (PLD) enzymatic activities were measured using gelatin zymography and Amplex Red PLD assay, respectively. Western blotting and luciferase assay were used to detect protein expression. RESULTS: All extracts had high phenolic and flavonoid contents and strong antioxidant and reducing capacities. Results from UV-Vis spectra, DLS, HR-TEM, EDS, XRD, and FT-IR showed the successful formation of MT-GNP with surface morphology, crystallinity, reduction capacity, capsulation, and stabilization. MTR-GNPs and MTS-GNPs had better catalytic activities than MTL-GNPs and MTF-GNPs for reduction of methylene blue and rhodamine B. Moreover, MTS-GNPs and MTR-GNPs exhibited the highest anti-migratory and anti-invasive potential and seemed to be more biologically active than the MTS and MTR extracts. Treatment with MT-GNPs decreased the enzymatic activity, translation levels of MMP-9 and PLD1. Our results showed that MTS-GNPs and MTR-GNPs could dramatically reverse transforming growth factor-ß-induced vimentin and N-cadherin upregulation and E-cadherin downregulation. CONCLUSION: The application of GNPs as a potential treatment approach for hepatocellular carcinoma can improve therapeutic efficiency.

A Comparative Study on Physicochemical, Photocatalytic, and Biological Properties of Silver Nanoparticles Formed Using Extracts of Different Parts of Cudrania tricuspidata.

Green-synthesized silver nanoparticles (SNPs) have great potential for biomedical applications, due to their distinctive optical, chemical, and catalytic properties. In this study, we aimed to develop green-synthesized SNPs from extracts of Cudrania tricuspidata (CT) roots (CTR), stems (CTS), leaves (CTL), and fruit (CTF) and to evaluate their physicochemical, photocatalytic, and biological properties. CTR, CTS, CTL, and CTF extracts were evaluated and compared for their total phenol and flavonoid content, reducing capacity, and antioxidant activity. The results revealed that CTR, CTS, CTL, and CTF extracts have high phenol and flavonoid content, as well as a powerful antioxidant and reducing capacity. CTR and CTS extracts showed the strongest effects. The results from UV-Vis spectra analysis, dynamic light scattering, high-resolution transmission electron microscopy, energy dispersive spectroscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy showed the successful formation of CT-SNPs with surface morphology, crystallinity, reduction capacity, capsulation, and stabilization. Synthesized CT-SNPs successfully photocatalyzed methylene blue, methyl orange, rhodamine B, and Reactive Black 5 within 20 min. The CTR- and CTS-SNPs showed better antibacterial properties against different pathogenic microbes (Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Salmonella enteritidis) than the CTL- and CTF-SNPs. CTS- and CTR-SNPs showed the most effective cytotoxicity and antiapoptosis properties in human hepatocellular carcinoma cells (HepG2 and SK-Hep-1). CT-SNPs also seemed to be more biologically active than the CT extracts. The results of this study provide evidence of the establishment of CT extract SNPs and their physicochemical, photocatalytic, and biological properties.

Petatewalide B alleviates oxygen­glucose deprivation/reoxygenation­induced neuronal injury via activation of the AMPK/Nrf2 signaling pathway.

Neuronal injury is a common, and critical, occurrence in clinical ischemic strokes, and can cause irreversible brain damage. However, the precise pathological mechanisms underlying this condition and effective treatment remain unclear. Increasing evidence shows that the nuclear factor erythroid 2­related factor 2 (Nrf2)/activated protein kinase (AMPK) signaling pathway serves a significant role in neuronal injury and is involved in neuroprotection. The present study demonstrated that petatewalide B, the active constituent of Petasites japonicus, otherwise known as butterbur, can alleviate oxygen­glucose deprivation/reoxygenation (OGD/R)­induced neuronal death via the adenosine monophosphate­AMPK/glycogen synthase kinase (GSK)­3/ß/Nrf2/antioxidant response element (ARE) signaling pathways in human neuroblastoma SH­SY5Y cells. A neuronal injury model was established by depriving SH­SY5Y cells of oxygen and glucose for 8 h, followed by 24 h of reoxygenation (OGD/R). The results indicated that the OGD/R model exhibited reduced cell viability but increased lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) production and apoptosis. These were accompanied by increased levels of cleaved PARP, cleaved caspase­9, cleaved caspase­3, p53, Bax and p21, as well as decreased Bcl­2 levels. Treatment with petatewalide B was able to strengthen cell viability but reduced LDH release, ROS production and the expression levels of apoptosis­related proteins. Additionally, treatment with petatewalide B activated AMPK in the OGD/R­exposed SH­SY5Y cells and upregulated activation of the downstream transcription factor Nrf2, which accompanied heme oxygenase 1 (HO­1) and NAD(P)H quinone dehydrogenase 1 (NQO1) expression. Furthermore, silencing AMPK, Nrf2, HO­1 and NQO1 expression inhibited petatewalide B's protective effect against apoptosis in the OGD/R­exposed SH­SY5Y cells. Therefore, petatewalide B protected human neuroblastoma cells against OGD/R­induced injury by downregulating apoptosis and oxidative stress via upregulation of the AMPK/Nrf2 signaling pathway, suggesting that petatewalide B may be a prospective protector against neuronal injury, having possible therapeutic and medical implications.

Treatment with Gold Nanoparticles Using Cudrania tricuspidata Root Extract Induced Downregulation of MMP-2/-9 and PLD1 and Inhibited the Invasiveness of Human U87 Glioblastoma Cells.

In this study, we aimed to elucidate the anti-invasive effects of Cudrania tricuspidata root-gold nanoparticles (CTR-GNPs) using glioblastoma cells. We demonstrated the rapid synthesis of CTR-GNPs using UV-vis spectra. The surface morphology, crystallinity, reduction, capsulation, and stabilization of CTR-GNPs were analyzed using high resolution transmission electron microscopy (HR-TEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FT-IR). Furthermore, CTR-GNPs displayed excellent photocatalytic activity as shown by the photo-degradation of methylene blue and rhodamine B. Cell migration and invasion assays with human glioblastoma cells were performed to investigate the anti-invasive effect of CTR-GNPs on U87 cells that were treated with phorbol 12-myristate 13-acetate. The results show that CTR-GNPs can significantly inhibit both basal and phorbol 12-myristate 13-acetate (PMA)-induced migration and invasion ability. Importantly, treatment with CTR-GNPs significantly decreased the levels of metalloproteinase (MMP)-2/-9 and phospholipase D1 (PLD1) and protein but not PLD2, which is involved in the modulation of migration and the invasion of glioblastoma cells. These results present a novel mechanism showing that CTR-GNPs can attenuate the migration and invasion of glioblastoma cells induced by PMA through transcriptional and translational regulation of MMP-2/-9 and PLD1. Taken together, our results suggest that CTR-GNPs might be an excellent therapeutic alternative for wide range of glioblastomas.

Bioinspired RGD-Engineered Bacteriophage Nanofiber Cues against Oxidative Stress.

Instructive tissue engineering biomaterials provide a vascular niche and protect oxidative stress in injured tissue. In this study, we exploited bioinspired bacteriophage nanofibers, previously recognized by their biochemical and structural cues inducing angiogenesis, as an antioxidant tissue engineering material. We demonstrated that topological cues of Arg-Gly-Asp (RGD)-engineered bacteriophage nanofibers provide angiogenic niches and cytoprotective functions against cellular oxidative stress with increased expression of antioxidant enzymes heme oxygenase-1 (HO-1) and NAD(P)H-quinone oxidoreductase 1 (NQO1) via the extracellular-signal-regulated kinase (ERK)-nuclear factor erythroid 2-related factor2 (Nrf2)-mediated signaling pathway, where a high density of RGD cues on the phage body support efficient interaction of cells with phage cues. These bioinspired RGD-engineered bacteriophage nanofibers can serve as a novel therapeutic platform for curing ischemic diseases.

Anti-neuroinflammatory effects of Ephedra sinica Stapf extract-capped gold nanoparticles in microglia.

Background: Combination therapy remains a promising strategy for treating neurodegenerative diseases, although green synthesis of gold nanoparticles for treating chronic neuroinflammation and studying their efficacy in treating neuroinflammation-mediated neurodegenerative diseases is not well assessed. Results: Here, Ephedra sinica Stapf (ES) extract was used as the reducing, capping, and stabilizing agent for gold nanoparticle synthesis. We developed ES extract-capped gold nanoparticles (ES-GNs) and investigated their anti-neuroinflammatory properties in microglia. ES-GNs displayed maximum absorption at 538 nm in ultraviolet-visible spectroscopy. Dynamic light scattering assessment revealed that ES-GN diameter was 57.6±3.07 nm, with zeta potential value of -24.6±0.84 mV. High resolution-transmission electron microscopy confirmed the spherical shape and average diameter (35.04±4.02 nm) of ES-GNs. Crystalline structure of ES-GNs in optimal conditions was determined by X-ray powder diffraction, and elemental gold presence was confirmed by energy-dispersive X-ray spectroscopy. Fourier transform-infrared spectroscopy confirmed gold nanoparticle synthesis using ES. Anti-neuroinflammatory properties of ES-GNs on production of pro-inflammatory mediators (nitric oxide, prostaglandin E2, and reactive oxygen species) and cytokines (tumor necrosis factor-α, IL-1ß, and IL-6) in lipopolysaccharide (LPS)-stimulated microglia were investigated by ELISA and flow cytometry. ES-GNs significantly attenuated LPS-induced production of pro-inflammatory mediators and cytokines, which was related to suppressed transcription and translation of inducible nitric oxide synthase and cyclooxygenase-2, determined by RT-PCR and western blotting. ES-GNs downregulated upstream signaling pathways (IκB kinase-α/ß, nuclear factor-κB, Janus-activated kinase /signal transducers and activators of transcription, mitogen-activated protein kinase , and phospholipase D) of pro-inflammatory mediators and cytokines in LPS-stimulated microglia. Anti-neuroinflammatory properties of ES-GNs were mediated by ES-GNs-induced AMP-activated protein kinase)-mediated nuclear erythroid 2-related factor 2 /antioxidant response element signaling. Conclusion: Collectively, these findings provide a new insight on the role of ES-GNs in treating chronic neuroinflammation-induced neurodegenerative diseases.

Neuroprotective effect of Dictyopteris divaricata extract-capped gold nanoparticles against oxygen and glucose deprivation/reoxygenation.

Combination therapy remains a promising approach to ameliorate cerebral ischemia injury. Nevertheless, the primary mechanism of the neuroprotective properties of Dictyopteris divaricata extract-capped gold nanoparticles (DD-GNPs) is not completely understood. DD-GNPs displayed maximum absorption at 525 nm and a diameter of 62.6 ± 1.2 nm, with a zeta potential value of -26.1 ± 0.6 mV. High resolution-transmission electron microscopy confirmed the spherical shape and average diameter (28.01 ± 2.03 nm). Crystalline structure and gold nanoparticle synthesis of DD-GNPs were determined by X-ray powder diffraction, and the presence of elemental gold was confirmed by energy-dispersive X-ray spectroscopy and Fourier transform-infrared spectroscopy. We examined the neuroprotective properties of DD-GNPs and explored their potential mechanisms in human SH-SY5Y neuroblastoma cells treated with oxygen and glucose deprivation/reoxygenation (OGD/R). We found that DD-GNPs inhibited OGD/R-induced release of lactate dehydrogenase (LDH), loss of cell viability, and production of reactive oxygen species. This neuroprotection was accompanied by regulation of apoptosis-related proteins, as indicated by decreased levels of cleaved-caspase-3, cleaved-PARP, cleaved-caspase-9, p53, p21, and Bax, as well as an increased level of Bcl-2. Notably, the neuroprotective effects of DD-GNPs were partially abolished by HO-1, NQO1, Nrf2, and AMPK knockdown. Our results established that DD-GNPs effectively attenuated OGD/R-stimulated neuronal injury, as evidenced by reduced neuronal injury. Even though the accumulating evidence has indicated the low toxicity and minimal side effects of GNPs, experimental clinical trials of DD-GNPs are still limited because of the lack of knowledge regarding the effects of DD-GNPs as neuroprotective agents against neurodegenerative diseases.

Lutein protects human retinal pigment epithelial cells from oxidative stress­induced cellular senescence.

Oxidative stress­induced cellular senescence is an important contributor to the pathogenesis of age­related macular degeneration (AMD). Characteristics of premature cellular senescence include a loss of proliferation, change in cell shape, irreversible cell cycle arrest, and elevated senescence­associated ß­galactosidase (SA­ß­gal) activity. It was hypothesized that lutein may have anti­senescence potential and may be useful as a treatment for AMD. In the present study, premature cellular senescence was induced in ARPE­19 cells via treatment with H2O2 and the effects of lutein application were confirmed by observing cell morphology, lysosome contents, reactive oxygen species (ROS) generation and SA­ß­gal activity, and cell cycle progression. The protein expression was also analyzed via western blotting in order to identify the affected signaling pathways. The results revealed that H2O2 treatment induced premature cellular senescence in ARPE­19 cells, as evidenced by an increased production of ROS and SA­ß­gal, altered lysosome contents, changed cellular morphology and arrested cell cycle progression. However, when treated with lutein, ARPE­19 cells were effectively protected from these H2O2­induced effects. Western blot analysis revealed that lutein induced the expression of heme oxygenase­1, NAD(P)H quinone dehydrogenase 1, sirtuin (SIRT)­1, and SIRT3. Together, the results indicated that lutein protects cells from cellular senescence induced by oxidative stress; therefore, it may be able to suppress the progression of AMD. In addition, our increased understanding of the pathways through which lutein acts is useful for the development of novel therapies for the treatment of oxidative stress­associated retinal disease.

Neochlorogenic acid inhibits against LPS-activated inflammatory responses through up-regulation of Nrf2/HO-1 and involving AMPK pathway.

Acute and chronic inflammatory diseases are associated with excessive inflammation due to the accumulation of pro-inflammatory mediators and cytokines produced by macrophages. In the present study, we investigated the anti-inflammatory properties of neochlorogenic acid (nCGA) from Lonicera japonica on lipopolysaccharide (LPS)-activated inflammation in macrophages and participation of the AMPK/Nrf2 pathway. nCGA pretreatment significantly reduced the production of nitric oxide, prostaglandin E2, TNF-α, reactive oxygen species, IL-1ß, and IL-6 by LPS-activated macrophages. Moreover, both transcript and protein levels of inducible nitric oxide synthase and cyclooxygenase-2 were reduced by nCGA in LPS-activated macrophages. nCGA inhibited NF-κB activation by attenuating IKKα/ß and IκBα phosphorylation in LPS-stimulated macrophages. Moreover, nCGA attenuated LPS-elevated JAK-1, STAT-1, and MAPK phosphorylation. We further evaluated the possible role of nCGA in the induction of AMPK/Nrf2 signal pathways required for the protein expression of HO-1 and NQO-1. nCGA induced AMPK activation via phosphorylation of LKB1 and CaMKII and by the inhibitory phosphorylation of GSK3ß. It stimulated the overexpression of Nrf2/ARE-regulated downstream proteins, such as NQO-1 and HO-1. Furthermore, the anti-inflammatory effects of nCGA were attenuated in macrophages subjected to siRNAs specific for HO-1, NQO-1, Nrf2, and AMPK. Accordingly, these results indicate that nCGA, as an AMPK/Nrf2 signal activator, prevents excessive macrophage-mediated responses associated with acute and chronic inflammatory disorders.

Nrf2-mediated neuroprotection against oxygen-glucose deprivation/reperfusion injury by emodin via AMPK-dependent inhibition of GSK-3ß.

OBJECTIVES: Our study verified the neuroprotective properties of emodin against oxygen-glucose deprivation/reoxygenation (OGD/R) and demonstrated its mechanism. METHODS: Human neuronal SH-SY5Y cells were investigated by analysing cell viability, lactate dehydrogenase levels, expression of molecules related to apoptotic cell death, and using biochemical techniques, flow cytometry and Western blot assays. KEY FINDINGS: Emodin reduced OGD/R-lead to neurotoxicity in SH-SY5Y cells. OGD/R significantly increased levels of cleaved poly ADP ribose polymerase, cleaved caspase-3, cleaved caspase-9, p53, p21 and Bax protein. However, emodin treatment effectively inhibited these OGD/R-induced changes. Emodin treatment also increased HO-1 and NQO1 expression in a concentration- and time-dependent manner and caused antioxidant response element (ARE) transcription activity and nuclear Nrf2 accumulation. Emodin phosphorylated AMPK and GSK3ß, and pretreatment of cells with an AMPK inhibitor suppressed emodin-induced nuclear Nrf2 accumulation and HO-1 and NQO1 expression. AMPK inhibitor treatment decreased GSK3ß phosphorylation, suggesting that AMPK is upstream of GSK3ß, Nrf2, HO-1 and NQO1. Emodin's neuroprotective effect was completely blocked by HO-1, NQO1 and Nrf2 knock-down and an AMPK inhibitor, indicating the action of AMPK/GSK3ß/Nrf2/ARE in the neuroprotective effect of emodin subjected to OGD/R. CONCLUSIONS: Emodin treatment protected against OGD/R-lead to neurotoxicity by potentiating Nrf2/ARE-regulated neuroprotection through the AMPK/GSK3ß pathway, indicating that emodin may be useful for treating neurodegenerative disorders.

AMPK/Nrf2 signaling is involved in the anti-neuroinflammatory action of Petatewalide B from Petasites japonicus against lipopolysaccharides in microglia.

OBJECTIVES: Abnormal microglia secrete neuroinflammatory factors that play a pivotal role in neurodegenerative-disorder development. Thus, regulating abnormal microglia-activation could be a promising therapeutic strategy. The purposes of this study included investigating the effect of Petatewalide B on lipopolysaccharide- (LPS-) stimulated microglia and exploring the role of the AMPK/Nrf2- (adenosine monophosphate-activated protein kinase/nuclear factor erythroid 2-related factor 2) signaling pathway in the anti-neuroinflammatory function of Petatewalide B. METHODS: We divided the microglia into four groups: a control group, a Petatewalide B-treated group, an LPS-treated group, and an LPS and Petatewalide B-treated group. The four groups of microglia were experimented with, using the NO, ELISA, and promoter assays, and western blotting was conducted to determine LPS-stimulated neuroinflammatory responses. RESULTS: We found that pretreatment with Petatewalide B strongly alleviates interleukin- (IL-) 1ß, IL-6, and tumor-necrosis-factor-α (TNF-α) production, and suppresses iNOS and nitric oxide (NO) overexpression in LPS-stimulated microglia. The AMPK/Nrf2-signaling pathway is important for inducing anti-neuroinflammatory responses. Mechanistic studies report that Petatewalide B increases nuclear-Nrf2 translocation, and heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) expression in a dose-dependent manner. Furthermore, Petatewalide B significantly up-regulates HO-1 and NQO1 by specifically improving antioxidant-response-elements-transcription activity. We then investigated whether Nrf2/HO-1/NQO1 contribute to the anti-neuroinflammatory properties of Petatewalide B. Nrf2, HO-1, and NQO1 small-integrating-ribonucleic-acids (siRNAs) significantly blocked Petatewalide B-attenuated iNOS-promoter-activity in LPS-stimulated microglia. Furthermore, Petatewalide B also up-regulated AMPK-phosphorylation in a dose-dependent manner. We next evaluated whether blocking AMPK-phosphorylation using an inhibitor (compound C) would critically affect anti-neuroinflammatory responses. We found that the AMPK-phosphorylation is associated with nuclear-Nrf2 translocation and elevated HO-1 and NQO1 expression levels. Our data also showed that AMPK-inhibitor pretreatment significantly reverses Petatewalide B-attenuated iNOS-promoter-activity in LPS-stimulated microglia. CONCLUSIONS: Our findings provide the possible mechanism of the anti-neuroinflammatory properties of Petatewalide B that result from beneficial responses in the AMPK/Nrf2-signaling pathway.

Petasites japonicus bakkenolide B inhibits lipopolysaccharide­induced pro­inflammatory cytokines via AMPK/Nrf2 induction in microglia.

Abnormal neuroinflammatory responses have diverse roles in neuronal death, oxidative stress and neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. Microglia regulate these responses via molecular signaling cascades that involve inflammatory cytokines and complement proteins. Bakkenolide B from Petasites japonicus exhibits significant anti­inflammatory and anti­allergic bioactivities. The present study investigated the anti­neuroinflammatory effects and underlying molecular mechanisms of bakkenolide B on the lipopolysaccharide (LPS)­mediated neuroinflammatory response in microglia. The results indicated that bakkenolide B pretreatment significantly reduced microglial production of interleukin (IL)­1ß, IL­6, IL­12, and tumor necrosis factor (TNF)­α. Furthermore, this effect was associated with reduced production of reactive oxygen species. The role of bakkenolide B was then evaluated in the upregulation of nuclear factor erythroid 2­related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathways. The results suggested that bakkenolide B significantly upregulated Nrf2/ARE pathway­related downstream factors, such as NADPH dehydrogenase quinone­1 (NQO­1) and heme oxygenase­1 (HO­1). Silencing of Nrf2, HO­1 and NQO­1 diminished the anti­neuroinflammatory properties of bakkenolide B. AMP­activated protein kinase (AMPK) activates the Nrf2/ARE signaling pathway, and the results of the present study demonstrated that bakkenolide B increased AMPK phosphorylation in microglia. In addition, an AMPK inhibitor abolished the bakkenolide B­induced increase in nuclear Nrf2, NQO­1 and HO­1 protein expression. Finally, an AMPK inhibitor diminished the bakkenolide B­mediated inhibition of LPS­stimulated TNF­α production. Taken together, the present results demonstrate that bakkenolide B may be an effective and therapeutically relevant AMPK/Nrf2 pathway activator for suppressing abnormal neuro-inflammation in neurodegenerative diseases.

Kalopanacis Cortex extract-capped gold nanoparticles activate NRF2 signaling and ameliorate damage in human neuronal SH-SY5Y cells exposed to oxygen-glucose deprivation and reoxygenation.

Recently, environment-friendly synthesis of gold nanoparticles (GNPs) has been extensively explored by biologists and chemists. However, significant research is still required to determine whether "eco-friendly" GNPs are beneficial to human health and to elucidate the molecular mechanisms of their effects on human cells. We used human neuronal SH-SY5Y cells to show that treatment with Kalopanacis Cortex extract-capped GNPs (KC-GNs), prepared via an eco-friendly, fast, one-pot synthetic route, protected neuronal cells against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced damage. To prepare GNPs, Kalopanacis Cortex was used without any chemical reducing and stabilizing agents. Ultraviolet-visible spectroscopy showed maximum absorbance at 526 nm owing to KC-GN surface plasmon resonance. Hydrodynamic size (54.02±2.19 nm) and zeta potential (-20.3±0.04 mV) were determined by dynamic light scattering. The average diameter (41.07±3.05 nm) was determined by high-resolution transmission electron microscopy. Energy-dispersive X-ray diffraction spectroscopy and X-ray diffraction confirmed the presence of assembled GNPs. Fourier transform infrared analysis suggested that functional groups such as O-H, C-C, and C-N participated in KC-GN formation. Cell viability assays indicated that KC-GNs restored the viability of OGD/R-treated SH-SY5Y cells. Flow cytometry demonstrated that KC-GNs inhibited the OGD/R-induced reactive oxygen species production and mitochondrial membrane potential disruption. KC-GNs also inhibited the apoptosis of OGD/R-exposed cells. Western blot analysis indicated that the OGD/R-induced cellular apoptosis and simultaneous increases in the expression of cleaved caspase-3, p53, p21, and B-cell lymphoma 2-associated X protein were reversed by KC-GNs. The KC-GN-mediated protection against OGD/R-induced neurotoxicity was diminished by NRF2 and heme oxygenase-1 gene knockdowns. Collectively, these results suggested that KC-GNs exerted strong neuroprotective effects on human neuronal cells, which might be attributed to the attenuation of OGD/R-induced neuronal cell injury through the NRF2 signaling pathway.

Anti-inflammatory effects of novel polygonum multiflorum compound via inhibiting NF-κB/MAPK and upregulating the Nrf2 pathways in LPS-stimulated microglia.

The incorporation of Polygonum multiflorum into the diet can result in anti-aging effects owing to its wide range of biological and pharmaceutical properties. We investigated the anti-neuroinflammatory properties of CRPE56IGIH isolated from P. multiflorum by focusing on its role in the induction of phase II antioxidant enzymes and the modulation of upstream signaling pathways. In microglia, CRPE56IGIH significantly inhibited lipopolysaccharide (LPS)-stimulated nitric oxide and prostaglandin E2 production with nonspecific cytotoxicity. CRPE56IGIH also markedly inhibited LPS-inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 protein and mRNA expression in the same manner as it inhibited nitric oxide and prostaglandin E2 production. In the control cells, NF-κB transactivation and nuclear translocation occurred at a baseline level, which was significantly increased in response to LPS. However, pretreatment with CRPE56IGIH concentration-dependently inhibited the LPS-induced NF-κB transactivation and nuclear translocation. The phosphorylation of Janus kinase-signal transducers and activators of transcription and mitogen-activated protein kinases was markedly upregulated by LPS, but considerably and dose-dependently inhibited by pretreatment with CRPE56IGIH. Furthermore, CRPE56IGIH induced the expression of phase II antioxidant enzymes, including heme oxygenase-1 (HO-1) and NADPH dehydrogenase quinone-1 (NQO-1). The activation of upstream signaling pathways, such as the Nrf2 pathway, was significantly increased following CRPE56IGIH treatment. Furthermore, the anti-neuroinflammatory effect of CRPE56IGIH was reversed by transfection of Nrf2, HO-1, and NQO-1 siRNA. Our results indicated that CRPE56IGIH isolated from P. multiflorum could be used as a natural anti-neuroinflammatory agent that induces phase II antioxidant enzymes via Nrf2 signaling.