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.

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.

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.

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.

Anti-Cancer Effects of α-Cubebenoate Derived from Schisandra chinensis in CT26 Colon Cancer Cells.

α-Cubebenoate derived from Schisandra chinensis has been reported to possess anti-allergic, anti-obesity, and anti-inflammatory effects and to exhibit anti-septic activity, but its anti-cancer effects have not been investigated. To examine the anti-cancer activity of α-cubebenoate, we investigated its effects on the proliferation, apoptosis, and metastasis of CT26 cells. The viabilities of CT26 cells (a murine colorectal carcinoma cell line) and HCT116 cells (a human colon cancer cell line) were remarkably and dose-dependently diminished by α-cubebenoate, whereas the viability of CCD-18Co cells (a normal human fibroblast cell line) were unaffected. Furthermore, α-cubebenoate treatment increased the number of apoptotic CT26 cells as compared with Vehicle-treated cells and increased Bax, Bcl-2, Cas-3, and Cleaved Cas-3 protein levels by activating the MAP kinase signaling pathway. α-Cubebenoate also suppressed CT26 migration by regulating the PI3K/AKT signaling pathway. Furthermore, similar reductions were observed in the expression levels of some migration-related proteins including VEGFA, MMP2, and MMP9. Furthermore, reduced VEGFA expression was found to be accompanied by the phosphorylations of FAK and MLC in the downstream signaling pathway of adhesion protein. The results of the present study provide novel evidence that α-cubebenoate can stimulate apoptosis and inhibit metastasis by regulating the MAPK, PI3K/AKT, and FAK/MLC signaling pathways.

Laxative Effects of Phlorotannins Derived from Ecklonia cava on Loperamide-Induced Constipation in SD Rats.

This study investigated the laxative effects of phlorotannins (Pt) derived from Ecklonia cava (E. cave) on chronic constipation by evaluating alterations in stool parameters, gastrointestinal motility, histopathological structure, mucin secretion, gastrointestinal hormones, muscarinic cholinergic regulation, and fecal microbiota in SD rats with loperamide (Lop)-induced constipation subjected to Pt treatment. Stool-related parameters (including stool number, weight, and water contents), gastrointestinal motility, and length of intestine were significantly enhanced in the Lop+Pt-treated group as compared to the Lop+Vehicle-treated group. A similar recovery was detected in the histopathological and cytological structure of the mid-colon of Lop+Pt-treated rats, although the level of mucin secretion remained constant. Moreover, rats with Lop-induced constipation subjected to Pt treatment showed significant improvements in water channel expression, gastrointestinal hormone secretions, and expression of muscarinic acetylcholine receptors M2/M3 (mAChRs M2/M3) and their mediators of muscarinic cholinergic regulation. Furthermore, the Lop+Pt-treated group showed a significant recovery of Bifidobacteriaceae, Muribaculaceae, Clostridiaceae, and Eubacteriaceae families in fecal microbiota. Taken together, these results provide the first evidence that exposure of SD rats with Lop-induced constipation to Pt improves the constipation phenotype through the regulation of membrane water channel expression, GI hormones, the mAChR signaling pathway, and fecal microbiota.

S-petasin induces apoptosis and inhibits cell migration through activation of p53 pathway signaling in melanoma B16F10 cells and A375 cells.

Melanoma is a dangerous type of skin cancer that develops from the melanocytes. Activation of p53 in melanoma cells has been validated as a strategy for melanoma therapy. S-Petasin, a dietary sesquiterpene isolated from Petasites japonicus, has been shown to possess multiple biological effects. However, no studies have reported that s-petasin exerted anti-melanoma or inhibited activity in melanoma cells. We investigated the effect of s-petasin in B16F10 cells and A375 cells and the underlying molecular mechanism. S-Petasin exerted a significant anti-proliferation effect on B16F10 cells and A375 cells as measured by the MTT assay and crystal violet staining assay. S-Petasin induced cell apoptosis in B16F10 cells and A375 cells as evidenced by flow cytometry assay and western blot assay. Wound healing assay and transwell cell migration and invasion assay revealed that s-petasin suppressed B16F10 cells and A375 cells migration in vitro. For mechanism study, western blot assay indicated that s-petasin activated the p53 pathway signaling. Furthermore, expression of Bcl-2, Bcl-XL, Bax, MMP-2, MMP-9, p21, CDK4 and cyclin D1 were regulated by s-petasin. Taken together, our data suggest that s-petasin is a novel compound which can induce apoptosis and inhibit cell migration through activation of the p53 pathway signaling in melanoma B16F10 cells and A375 cells.

Pelargonidin suppresses adipogenesis in 3T3-L1 cells through inhibition of PPAR-γ signaling pathway.

Pelargonidin is a natural compound that exists widely in fruits, and exerts antioxidant, anti-atherosclerotic, anti-inflammatory, anti-hyperglycemic, and anti-diabetic activities. However, there have not been any studies concerning its anti-obesity potential to date. Therefore, we evaluated the anti-obesity potential of pelargonidin via inhibition of adipogenesis in 3T3-L1 cells. The cellular oil droplet content was decreased to 68.14%, 56.75%, and 48.39% and triglyceride accumulation decreased to 74.53%, 61.54%, and 47.86% after incubation with 5 µM, 10 µM, and 20 µM pelargonidin, respectively, when compared with DMSO group. Furthermore, pelargonidin treatment led to decrease in glucose consumption. Western blot assay illustrated that the expression of PPAR-γ was suppressed to 63.25%, 47.52%, and 21.23% after incubation with 5 µM, 10 µM, and 20 µM pelargonidin when compared with DMSO group. Then, we measured the expression of some target proteins of PPAR-γ, and found that pelargonidin decreased the expressions of HMGCR, LPL, Glut4, and A-FABP. Besides, the result of Luciferase Reporter Assay indicated that pelargonidin inhibited PPAR-γ transcription activity. These results indicated that pelargonidin exerts anti-adipogenic activity in 3T3-L1 cells through inhibition of PPAR-γ signaling pathway, and pelargonidin could be used as a potential anti-obesity agent.

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.

Novel Function of α-Cubebenoate Derived from Schisandra chinensis as Lipogenesis Inhibitor, Lipolysis Stimulator and Inflammasome Suppressor.

The efficacy of α-cubebenoate isolated from Schisandra chinensis has been previously studied in three disease areas, namely inflammation, sepsis, and allergy, and its role in other diseases is still being explored. To identify the novel function of α-cubebenoate on lipid metabolism and related inflammatory response, alterations in fat accumulation, lipogenesis, lipolysis, and inflammasome activation were measured in 3T3-L1 preadipocytes and primary adipocytes treated with α-cubebenoate. Lipid accumulation significantly decreased in MDI (3-isobutyl-1-methylxanthine, dexamethasone, and insulin)-stimulated 3T3-L1 adipocytes treated with α-cubebenoate without any significant cytotoxicity. The mRNA levels of peroxisome proliferator-activated receptor (PPAR)γ and CCAAT-enhancer binding protein (C/EBP) α for adipogenesis, as well as adipocyte fatty acid binding protein 2 (aP2) and fatty acid synthetase (FAS) for lipogenesis, were reduced after α-cubebenoate treatment, while cell cycle arrest at G2/M stage was restored in the same group. α-cubebenoate treatment induced glycerol release in primary adipocytes and enhanced expression of lipolytic proteins (HSL, perilipin, and ATGL) expression in MDI-stimulated 3T3-L1 adipocytes. Inflammasome activation and downstream cytokines expression were suppressed with α-cubebenoate treatment, but the expression of insulin receptor signaling factors was remarkably increased by α-cubebenoate treatment in MDI-stimulated 3T3-L1 adipocytes. These results indicate that α-cubebenoate may play a novel role as lipogenesis inhibitor, lipolysis stimulator, and inflammasome suppressor in MDI-stimulated 3T3-L1 adipocytes. Our results provide the possibility that α-cubebenoate can be considered as one of the candidates for obesity management.

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.

Morusin Functions as a Lipogenesis Inhibitor as Well as a Lipolysis Stimulator in Differentiated 3T3-L1 and Primary Adipocytes.

Conflicting results for morusin activity during adipogenic differentiation are reported in 3T3-L1 adipocytes and cancer cells. To elucidate the influence of morusin on fat metabolism, their anti-obesity effects and molecular mechanism were investigated in 3T3-L1 cells and primary adipocytes. Morusin at a dose of less than 20 µM does not induce any significant change in the viability of 3T3-L1 adipocytes. The accumulation of intracellular lipid droplets in 3T3-L1 adipocytes stimulated with 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, 10 µg/mL insulin in DMEM containing 10% FBS (MDI)-significantly reduces in a dose-dependent manner after morusin treatment. The phosphorylation level of members in the MAP kinase signaling pathway under the insulin receptor downstream also decrease significantly in the MDI + morusin-treated group compared to MDI + vehicle-treated group. Also, the expression of adipogenic transcription factors (PPARγ and C/EBPα) and lipogenic proteins (aP2 and FAS) are significantly attenuated by exposure to the compound in MDI-stimulated 3T3-L1 adipocytes. Furthermore, the decrease in the G0/G1 arrest of cell cycle after culturing in MDI medium was dramatically recovered after co-culturing in MDI + 20 µM morusin. Moreover, morusin treatment induces glycerol release in the primary adipocytes of SD rats and enhances lipolytic protein expression (HSL, ATGL, and perilipin) in differentiated 3T3-L1 adipocytes. Overall, the results of the present study provide strong evidence that morusin inhibits adipogenesis by regulating the insulin receptor signaling, cell cycle and adipogenic protein expression as well as stimulating lipolysis by enhancing glycerol release and lipolytic proteins expression.

Quercetin promotes gastrointestinal motility and mucin secretion in loperamide-induced constipation of SD rats through regulation of the mAChRs downstream signal.

CONTEXT: Quercetin (QCT) has been known as a potential therapeutic strategy for gastrointestinal diseases because it contributes to the stabilization of mast cells, the prevention of histamine release and modulation of CaCC chloride channel. OBJECTIVE: We investigated the laxative effect and action mechanism of QCT in Lop-induced constipation model. MATERIALS AND METHODS: Constipation of SD rats was induced by subcutaneous injection of loperamide (Lop) (4 mg/kg weight) in 0.5% Tween 20 twice a day for three days. After 24 h, the constipation group was further treated with 1× PBS (Lop + Vehicle treated group), 10 mg/kg of QCT (Lop + LQCT treated group), 20 mg/kg of QCT (Lop + MQCT treated group) or 40 mg/kg QCT (Lop + HQCT treated group) at once. At 24 h after QCT treatment, the constipation phenotypes were measured and the transverse colon was collected from SD rats. RESULTS: The gastrointestinal motility, the number of stools and histological structures were significantly recovered in Lop + QCT treated group compared with the Lop + Vehicle treated group. Also, above activity of epithelial cells and smooth muscle cells were regulated by the mRNA expression of the muscarinic acetylcholine receptors M2 and M3 (mAChR M2 and M3) and some mediators of their downstream signalling pathway. Finally, laxative effects of QCT on mAChR signalling pathway were significantly inhibited by the treatment of mAChR antagonist in primary smooth muscle of rat intestine cells (pRISMCs). CONCLUSIONS: This study provides the first strong evidence that QCT can be considered an important candidate for improving chronic constipation induced by Lop treatment in animal models.

α-Isocubebene modulates vascular tone by inhibiting myosin light chain phosphorylation in murine thoracic aorta.

α-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis (SC), a well-known medicinal herb that ameliorates cardiovascular symptoms, but the mechanism responsible for this activity has not been determined. To determine the role played by ICB on the regulation of vascular tone, we investigated the inhibitory effects of ICB on vascular contractile responses by adrenergic α-receptor agonists. In addition, we investigated the role on myosin light chain (MLC) phosphorylation and cytosolic calcium concentration in vascular smooth muscle cells (VSMC). In aortic rings isolated from C57BL/6J mice, ICB significantly attenuated the contraction induced by phenylephrine (PE) and norepinephrine (NE), whereas ICB had no effects on KCl (60 mM)-induced contraction. In vasculatures precontracted with PE, ICB caused marked relaxation of aortic rings with or without endothelium, suggesting a direct effect on VSMC. In cultured rat VSMC, PE or NE increased MLC phosphorylation and increased cytosolic calcium levels. Both of these effects were significantly suppressed by ICB. In conclusion, our results showed that ICB regulated vascular tone by inhibiting MLC phosphorylation and calcium flux into VSMC, and suggest that ICB has anti-hypertensive properties and therapeutic potential for cardiovascular disorders related to vascular hypertension.

Anti-neuroinflammatory Effect of Emodin in LPS-Stimulated Microglia: Involvement of AMPK/Nrf2 Activation.

AMPK/Nrf2 signaling regulates multiple antioxidative factors and exerts neuroprotective effects. Emodin is one of the main bioactive components extracted from Polygonum multiflorum, a plant possessing important activities for human health and for treating a variety of diseases. This study examined whether emodin can activate AMPK/Nrf2 signaling and induce the expression of genes targeted by this pathway. In addition, the anti-neuroinflammatory properties of emodin in lipopolysaccharide (LPS)-stimulated microglia were examined. In microglia, the emodin treatment increased the levels of LKB1, CaMKII, and AMPK phosphorylation. Emodin increased the translocation and transactivity of Nrf2 and enhanced the levels of HO-1 and NQO1. In addition, the emodin-mediated expression of HO-1 and NQO1 was attenuated completely by an AMPK inhibitor (compound C). Moreover, emodin decreased dramatically the LPS-induced production of NO and PGE2 as well as the protein expression and promoter activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In addition, emodin effectively inhibited the production of pro-inflammatory cytokines, TNF-α and IL-6, and reduced the level of IκBα phosphorylation, leading to the suppression of the nuclear translocation, phosphorylation, and transactivity of NF-κB. Emodin also suppressed the LPS-stimulated activation of STATs, JNK, and p38 MAPK. The anti-inflammatory effects of emodin were reversed by transfection with Nrf-2 and HO-1 siRNA and by a co-treatment with an AMPK inhibitor. These results suggest that emodin isolated from P. multiflorum can be used as a natural anti-neuroinflammatory agent that exerts its effects by inducing HO-1 and NQO1 via AMPK/Nrf2 signaling in microglia.

Upregulation of Human ST8Sia VI (α2,8-Sialyltransferase) Gene Expression by Physcion in SK-N-BE(2)-C Human Neuroblastoma Cells.

In this research, we firstly demonstrated that physcion, an anthraquinone derivative, specifically increased the expression of the human α2,8-sialyltransferase (hST8Sia VI) gene in SK-N-BE(2)-C human neuroblastoma cells. To establish the mechanism responsible for the up-regulation of hST8Sia VI gene expression in physcion-treated SK-N-BE(2)-C cells, the putative promoter region of the hST8Sia VI gene was functionally characterized. Promoter analysis with serially truncated fragments of the 5'-flanking region showed that the region between -320 and -240 is crucial for physcion-induced transcription of hST8Sia VI in SK-N-BE(2)-C cells. Putative binding sites for transcription factors Pax-5 and NF-Y are located at this region. The Pax-5 binding site at -262 to -256 was essential for the expression of the hST8Sia VI gene by physcion in SK-N-BE(2)-C cells. Moreover, the transcription of hST8Sia VI induced by physcion in SK-N-BE(2)-C cells was inhibited by extracellular signal-regulated protein kinase (ERK) inhibitor U0126 and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, but not c-Jun N-terminal kinase (JNK) inhibitor SP600125. These results suggest that physcion upregulates hST8Sia VI gene expression via ERK and p38 MAPK pathways in SK-N-BE(2)-C cells.

A novel natural compound from garlic (Allium sativum L.) with therapeutic effects against experimental polymicrobial sepsis.

Sepsis is a serious, life-threatening, infectious disease. In this study, we demonstrate that sucrose methyl 3-formyl-4-methylpentanoate (SMFM), a novel natural compound isolated from garlic (Allium sativum L.), markedly enhances survival rates by inhibiting lung inflammation in a cecal ligation and puncture (CLP) experimental polymicrobial sepsis model. SMFM strongly reduced bacterial colony units from peritoneal fluid in CLP mice by stimulating the generation of reactive oxygen species. Lymphocyte apoptosis in spleens from CLP mice was also markedly decreased by SMFM administration. SMFM also significantly inhibited the production of proinflammatory cytokines, such as TNF-α, interleukin-1ß (IL-1ß) and IL-6, in CLP mice. Lipopolysaccharide-stimulated production of TNF-α and IL-6 were also strongly inhibited by SMFM in mouse bone marrow-derived macrophages. Taken together, our results indicate that SMFM has therapeutic effects against polymicrobial sepsis that are mediated by enhanced microbial killing and blockage of cytokine storm.

Partially purified components of Uncaria sinensis attenuate blood brain barrier disruption after ischemic brain injury in mice.

BACKGROUND: Uncaria sinensis (US) has long been used in traditional Korean medicine to relieve various nervous-related symptoms and cardiovascular disease. We recently showed the neuroprotective and cerebrovascular protective effects of US on cerebral ischemia; however, its effects on the blood-brain barrier (BBB) are poorly understood. In this study, the effects of partially purified components of US (PPUS) on BBB disruption were investigated in mice subjected to ischemic brain injury. METHODS: Focal cerebral ischemia was induced in C57BL/6J mice by photothrombotic cortical ischemia. PPUS was injected intraperitoneally 30 min before ischemic insults. Infarct volume, neurological score, wire-grip test, Evans blue leakage and brain water content were then examined 24 h after ischemic brain injury. RESULTS: Infarct volume was significantly reduced and neurological deficit and motor deficit were greatly improved in PPUS-pretreated mice relative to those treated with vehicle following photothrombotic cortical ischemia. Brain edema-induced change of Evans blue extravasation and water content in the ipsilateral hemisphere were alleviated by treatment with PPUS. In addition, PPUS significantly reduced ischemic brain injury-induced degradation of tight junction proteins and elevation of matrix metalloproteinase-9 (MMP-9). CONCLUSIONS: PPUS prevents cerebral ischemic damage by BBB protection, and these effects were associated with inhibition of tight junction degradation and MMP-9 induction.

Neuroprotection and spatial memory enhancement of four herbal mixture extract in HT22 hippocampal cells and a mouse model of focal cerebral ischemia.

BACKGROUND: Four traditional Korean medicinal herbs which act in retarding the aging process, Polygonum multiflorum Thunb., Rehmannia glutinosa (Gaertn) Libosch., Polygala tenuifolia Willd., and Acorus gramineus Soland., were prepared by systematic investigation of Dongeuibogam (Treasured Mirror of Eastern Medicine), published in the early 17th century in Korea. This study was performed to evaluate beneficial effects of four herbal mixture extract (PMC-12) on hippocampal neuron and spatial memory. METHODS: High performance liquid chromatography (HPLC) analysis was performed for standardization of PMC-12. Cell viability, lactate dehydrogenase, flow cytometry, reactive oxygen species (ROS), and Western blot assays were performed in HT22 hippocampal cells and immunohistochemistry and behavioral tests were performed in a mouse model of focal cerebral ischemia in order to observe alterations of hippocampal cell survival and subsequent memory function. RESULTS: In the HPLC analysis, PMC-12 was standardized to contain 3.09% 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside, 0.35% 3',6-disinapoyl sucrose, and 0.79% catalpol. In HT22 cells, pretreatment with PMC-12 resulted in significantly reduced glutamate-induced apoptotic cell death. Pretreatment with PMC-12 also resulted in suppression of ROS accumulation in connection with cellular Ca(2+) level after exposure to glutamate. Expression levels of phosphorylated p38 mitogen-activated protein kinases (MAPK) and dephosphorylated phosphatidylinositol-3 kinase (PI3K) by glutamate exposure were recovered by pretreatment with either PMC-12 or anti-oxidant N-acetyl-L-cysteine (NAC). Expression levels of mature brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP response element binding protein (CREB) were significantly enhanced by treatment with either PMC-12 or NAC. Combination treatment with PMC-12, NAC, and intracellular Ca(2+) inhibitor BAPTA showed similar expression levels. In a mouse model of focal cerebral ischemia, we observed higher expression of mature BDNF and phosphorylation of CREB in the hippocampus and further confirmed improved spatial memory by treatment with PMC-12. CONCLUSIONS: Our results suggest that PMC-12 mainly exerted protective effects on hippocampal neurons through suppression of Ca(2+)-related ROS accumulation and regulation of signaling pathways of p38 MAPK and PI3K associated with mature BDNF expression and CREB phosphorylation and subsequently enhanced spatial memory.

Schisandrae Fructus Inhibits IL-1ß-Induced Matrix Metalloproteinases and Inflammatory Mediators Production in SW1353 Human Chondrocytes by Suppressing NF-κB and MAPK Activation.

Proinflammatory cytokine interleukin-1 beta (IL-1ß) plays a crucial role in the pathogenesis of osteoarthritis (OA) by stimulating several mediators that contribute to cartilage degradation. Schisandrae Fructus (SF), the dried fruit of Schisandra chinensis (Turcz.) Baill. (Magnoliaceae), is widely used in traditional medicine for the treatment of a number of chronic inflammatory diseases. This study investigated the antiosteoarthritis properties of an ethanol extract of SF on IL-1ß-stimulated SW1353 chondrocytes. SF attenuated IL-1ß-induced expression and activity of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 and also reduced the elevated levels of cyclooxygenase-2 and inducible nitric oxide synthase associated with the inhibition of prostaglandin E2 and nitric oxide production in IL-1ß-stimulated SW1353 chondrocytes. In addition, SF markedly suppressed the nuclear translocation of nuclear factor-kappa B (NF-κB) by blocking inhibitor κB-alpha degradation and inhibited the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). These results indicate that the inhibitory effect of SF on IL-1ß-stimulated expression of MMPs and inflammatory mediators production in SW1353 cells were associated with the suppression of the NF-κB and JNK/p38 MAPK signaling pathways. The results from this study indicate that SF may have therapeutic potential for the treatment of OA due to its anti-inflammatory and chondroprotective features.