Parapyruvate Induces Neurodegeneration in C57BL/6JNarl Mice via Inhibition of the α-Ketoglutarate Dehydrogenase Complex.

Parapyruvate is a substance commonly found in commercial dietary supplements of calcium pyruvate (DSCP) that inhibits the α-ketoglutarate dehydrogenase complex (KGDHC) and has been shown to induce senescence in human Hs68 cells. However, it is unknown whether parapyruvate can induce neurodegeneration. In this study, the parapyruvate content in DSCP was converted to an equivalent dose for mice and administered to the C57BL/6JNarl mice at doses around the equivalent dose for 69 days, including 5, 50, and 500 mg/kg/day. The Morris water maze (MWM) task and the active avoidance test were conducted to assess the learning and memory ability in mice, and then brain tissues were collected for biochemical analyses. The results demonstrated that parapyruvate significantly impaired the learning and memory ability, decreased the KGDHC activity, and promoted the oxidative stress and acetylcholinesterase (AChE) activity in mice in a dose-dependent manner. Additionally, parapyruvate induced Tau and phosphorylated Tau (p-Tau) aggregation at dosages ≥5 mg/kg/day and increased the myelin basic protein (MBP) expression at a dosage of 500 mg/kg/day. These results suggest that the equivalent dose of parapyruvate can induce neurodegeneration in the C57BL/6JNarl mice.

Safety Assessment and Hepatic-Renal Protection of Cajanus cajan (L.) Millsp. Root and Its Soy Isoflavone Contents.

Cajanus cajan (L.) Millsp., also known as pigeon pea, has roots that have exhibited much pharmacological potential. The present study was conducted to assess the safe dose of the ethanolic extract of C. cajan roots (EECR95) and to analyze the main soy isoflavones contents. In vitro, we investigated the mutagenicity and cytotoxic effect of EECR95 on Salmonella typhimurium-TA98 and TA100 (by Ames tests) and RAW 264.7, L-929, and HGF-1 cell lines (by MTT tests) for 24 h of incubation. We found no mutagenic or cytotoxic effects of EECR95. After administration of 0.2 or 1.0 g/kg bw of EECR95 to both male and female Wistar rats for 90 days, there were no significant adverse effects on the behaviors (body weight, water intake, and food intake), organ/tissue weights, or immunohistochemical staining, and the urine and hematological examinations of the rats were within normal ranges. EECR95 potentially decreases renal function markers in serum (serum uric acid, BUN, CRE, and GLU) or liver function markers (cholesterol, triglyceride, and glutamic-pyruvate-transaminase (GPT)). We also found that EECR95 contained five soy isoflavones (genistein, biochanin A, daidzein, genistin, and cajanol), which may be related to its hepatorenal protection. Based on the high dose (1.0 g/kg bw) of EECR95, a safe daily intake of EECR95 for human adults is estimated to be 972 mg/60 kg person/day.

Insulin sensitizer and antihyperlipidemic effects of Cajanus cajan (L.) millsp. root in methylglyoxal-induced diabetic rats.

Cajanus cajan (L.) Millsp., known as pigeon pea, is one of the major grain legume crops of the tropical world. It recognizes as an ethnomedicine to possess various functions, such as helping in healing wound and cancer therapy. We investigated whether 95% ethanol extracts from C. cajan root (EECR) protect against methylglyoxal (MGO)-induced insulin resistance (IR) and hyperlipidemia in male Wistar rats and explored its possible mechanisms. The hypoglycemic potential of EECR was evaluated using α-amylase, α-glucosidase activities, and advanced glycation end products (AGEs) formation. For in vivo study, the rats were divided into six groups and orally supplemented with MGO except for Group 1 (controls). Group 2 was supplemented with MGO only, Group 3: MGO + metformin, Group 4: MGO + Low dose-EECR (L-EECR; 10 mg/kg bw), Group 5: MGO + Middle dose-EECR (M-EECR; 50 mg/kg bw), and Group 6: MGO + High dose-EECR (H-EECR; 100 mg/kg bw). EECR possessed good inhibition of α-glucosidase, α-amylase activities, and AGEs formation (IC50 = 0.12, 0.32, and 0.50 mg/mL), respectively. MGO significantly increased serum levels of blood glucose (GLU), glycosylated hemoglobin, homeostasis model assessment of IR, AGEs, lipid biochemical values, and atherogenic index, whereas EECR decreased these levels in a dose-dependent manner. EECR can also act as an insulin sensitizer, which significantly decreased (47%, P < 0.05) the blood GLU levels after intraperitoneal injection of insulin in the insulin tolerance tests. The hypoglycemic and antihyperlipidemic mechanisms of EECR are likely through several possible pathways including the inhibition of carbohydrate-hydrolyzing enzymes (α-glucosidase and α-amylase) and the enhancement of MGO-trapping effects on inhibition of AGEs formation.

NPS-1034 Induce Cell Death with Suppression of TNFR1/NF-κB Signaling in Testicular Cancer.

Background and objectives: NPS-1034 with a dual inhibitory effect on Met and Axl kinase receptors has exhibited therapeutic potential in previous models. However, no study on treating testicular cancer (TC) cell lines with NPS-1034 has been established. Materials and Methods: In this study, a series of in vitro examinations of the apoptotic effect induced by NPS-1034 in TC cell lines was conducted to clarify the molecular interactions involved. Results: A decrease in cell viability rate was observed following NPS-1034 treatment, as shown in the MTT assay. Induction of the apoptotic effect was observed in TC cells as the sub-G1 and Annexin-PI populations increased in a dose-dependent manner. The involvement of the tumor receptor necrosis factor receptor 1 (TNFR1) pathway was later determined by the proteome array and western blotting. A reduction in TNFR1 and NF-κB downstream protein expressions, an upregulation of cleaved caspase-3 and -7, and a downregulation of survivin and claspin all reassured the underlying mechanism of the TNFR1 involved in the apoptotic pathway induced by NPS-1034. Conclusions: Our findings provide evidence for a potential underlying TNFR1 pathway involved in NPS-1034 treatment. This study should offer new insights into targeted therapy for TC.

Effects of Cajanus cajan (L.) millsp. roots extracts on the antioxidant and anti-inflammatory activities.

Cajanus cajan (L.) Millsp., also named pigeon pea, is widely grown in the tropics and the subtropics. C. cajan roots (CR) and ribs stewed in hot water have been used as a traditional medicine in various cultures to treat diabetes. The purpose of this study was to determine the functional components of hot water (WCR) and 50%, 95% ethanol extracts (EECR50 and EECR95) from CR, then evaluating their antioxidant and anti-inflammatory effects. The results indicated that EECR95 had higher polyphenol, especially the isoflavones (e.x. daidzein, genistein, and cajanol) than those of the other extracts, and it also exhibited the most potent anti-oxidative activities by in vitro antioxidant assay. In the lipopolysaccharide-stimulated RAW 264.7 cells, we found that EECR95 significantly decreased intracellular reactive oxygen species and significantly enhanced the activities of superoxide dismutase and catalase. Mechanism studies showed that EECR95 mainly activated nuclear factor (NF) erythroid 2-related factor 2/antioxidant protein heme oxygenase-1 and inhibited nuclear factor kappa B (NF-κB) signaling pathway, and thus exhibited antioxidant and anti-inflammatory effects. Overall, this study suggests that CR may have the potential to be developed as a biomedical material and that genistein, which has relatively high uptakes (3.44% for the pure compound and 1.73% for endogenous genistein of EECR95) at 24 h of incubation with RAW 264.7 cells, could be the main active component of CR.

Corrigendum to "Protective Effects and Possible Mechanisms of Ergothioneine and Hispidin against Methylglyoxal-Induced Injuries in Rat Pheochromocytoma Cells".

[This corrects the article DOI: 10.1155/2017/4824371.].

Safety and efficacy of tyrosinase inhibition of Paeonia suffruticosa Andrews extracts on human melanoma cells.

INTRODUCTION: The development of tyrosinase inhibitors is a hot research topic. Recently, the Chinese herb Paeonia suffruticosa Andrews, commonly named as Cortex Moutan (CM), was reported as being capable of reducing melanogenesis. We developed an A2058 human melanoma cell model to test the safety and efficacy of tyrosinase inhibition. The aim was to further clarify the bioactivities of CM extracts and paeonol for the purpose of skin whitening. METHODS: The 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, total polyphenol and flavonoid contents, and in vitro tyrosinase inhibitory effects of water and ethanol CM extracts were determined. Cellular inhibitions of tyrosinase and melanin production were also evaluated. RESULTS: Water and ethanol CM extracts were both shown to have strong DPPH scavenging abilities in a dose-dependent manner. The polyphenol content was higher in the ethanol CM extract compared to the water extract, while the flavanone content was comparable. Kinetic analyses revealed that the ethanol CM extract and paeonol are noncompetitive tyrosinase inhibitors. The cellular melanin content and l-DOPA oxidation assays demonstrated that the ethanol CM extract was an appropriate alternative whitening agent to paeonol and arbutin in ultraviolet-induced A2058 human melanoma cells. CONCLUSION: The results of this study suggest that a human cell model is more suitable for determining tyrosinase activity than mouse cell models for determining cellular tyrosinase activity and melanin production. The ethanol CM extract was also confirmed as a promising ingredient in sun protection and skin whitening cosmetics. Future work should focus on melanogenesis-related gene expressions.

Anti-Fatigue, Antioxidation, and Anti-Inflammatory Effects of Eucalyptus Oil Aromatherapy in Swimming-Exercised Rats.

Eucalyptus globulus possesses important pharmacological activities, including antioxidant and anti-inflammatory effects. We investigated the anti-fatigue, antioxidant, and anti-inflammatory effects of eucalyptus essential oil after swimming exercise using an animal model. Male Sprague­ Dawley rats were administered eucalyptus oil (200 µL/h) daily via inhalation (15 min), and anti-fatigue effects were assessed following eucalyptus essential oil administration for 2 or 4 weeks when forced to swim until exhaustion while carrying ~5% body weight-equivalent. To assess antioxidant and anti-inflammatory effects, control and oil-treated groups were subjected to swimming, which was intensified from 90 min to 120 min daily over 4 weeks, with non-swimming groups included as controls. The 2- and 4-week-treated rats increased their swimming-to-exhaustion time by 46 s and 111 s, respectively. Additionally, lactate (LA), creatine kinase (CK), and lactate dehydrogenase (LDH) activities increased significantly in the non-treated swimming relative to levels observed in the non-swimming groups (P < 0.05); however, no significant differences in these markers were observed between the treated groups. The anti-fatigue effects were related to LA clearance and reduced LDH and CK concentrations. Moreover, compared to the corresponding levels in the non-swimmers, the non-treated swimmers showed markedly elevated levels of liver malondialdehyde (MDA), xanthine oxidase (XO), and other factors, but significantly decreased (P < 0.05) glutathione (GSH) concentrations. However, compared with that of the non-swimmer group, the treated swimming group showed no significant changes in these levels (P > 0.05), suggesting stable XO and MDA production and maintenance of GSH levels. These results suggested that eucalyptus oil aromatherapy increased rat swimming performance and antioxidant capacity and decreased oxidative damage and inflammatory reactions in tissues, indicating good anti-fatigue, antioxidant, and anti-inflammatory effects after high-intensity endurance exercise.

Protective Effects and Possible Mechanisms of Ergothioneine and Hispidin against Methylglyoxal-Induced Injuries in Rat Pheochromocytoma Cells.

Diabetic encephalopathy (DE) is often a complication in patients with Alzheimer's disease due to high blood sugar induced by diabetic mellitus. Ergothioneine (EGT) and hispidin (HIP) are antioxidants present in Phellinus linteus. Methylglyoxal (MGO), a toxic precursor of advanced glycated end products (AGEs), is responsible for protein glycation. We investigated whether a combination EGT and HIP (EGT + HIP) protects against MGO-induced neuronal cell damage. Rat pheochromocytoma (PC12) cells were preincubated with EGT (2 µM), HIP (2 µM), or EGT + HIP, then challenged with MGO under high-glucose condition (30 µM MGO + 30 mM glucose; GLU + MGO) for 24-96 h. GLU + MGO markedly increased protein carbonyls and reactive oxygen species in PC12 cells; both of these levels were strongly reduced by EGT or HIP with effects comparable to those of 100 nM aminoguanidine (an AGE inhibitor) but stronger than those of 10 µM epalrestat (an aldose reductase inhibitor). GLU + MGO significantly increased the levels of AGE and AGE receptor (RAGE) protein expression of nuclear factor kappa-B (NF-κB) in the cytosol, but treatment with EGT, HIP, or EGT + HIP significantly attenuated these levels. These results suggest that EGT and HIP protect against hyperglycemic damage in PC12 cells by inhibiting the NF-κB transcription pathway through antioxidant activities.

Lycopene Inhibits Metastasis of Human Liver Adenocarcinoma SK-Hep-1 Cells by Downregulation of NADPH Oxidase 4 Protein Expression.

NADPH oxidase 4 (NOX4), with the sole function to produce reactive oxygen species (ROS), can be a molecular target for disrupting cancer metastasis. Several studies have indicated that lycopene exhibited anti-metastatic actions in vitro and in vivo. However, the role of NOX4 in the anti-metastatic action of lycopene remains unknown. Herein, we first confirmed the anti-metastatic effect of lycopene (0.1-5 µM) on human liver adenocarcinoma SK-Hep-1 cells. We showed that lycopene significantly inhibited NOX4 protein expression, with the strongest inhibition of 64.3 ± 10.2% (P < 0.05) at 2.5 µM lycopene. Lycopene also significantly inhibited NOX4 mRNA expression, NOX activity, and intracellular ROS levels in SK-Hep-1 cells. We then determined the effects of lycopene on transforming growth factor ß (TGF-ß)-induced metastasis. We found that TGF-ß (5 ng/mL) significantly increased migration, invasion, and adhesion activity, the intracellular ROS level, matrix metalloproteinase 9 (MMP-9) and MMP-2 activities, the level of NOX4 protein expression, and NOX activity. All these TGF-ß-induced effects were antagonized by the incubation of SK-Hep-1 cells with lycopene (2.5 µM). Using transient transfection of siRNA against NOX4, we found that the downregulation of NOX4 could mimic lycopene by inhibiting cell migration and the activities of MMP-9 and MMP-2 during the incubation with or without TGF-ß on SK-Hep-1 cells. The results demonstrate that the downregulation of NOX4 plays a crucial role in the anti-metastatic action of lycopene in SK-Hep-1 cells.

Antioxidant Properties of Essential Oil Extracted from Pinus morrisonicola Hay Needles by Supercritical Fluid and Identification of Possible Active Compounds by GC/MS.

Pine (Pinus morrisonicola Hay, PM) needles have been used as folk medicine for their antihypertension and lipid-lowering effects. As supercritical fluid extraction (SFE) is considered an ideal technique for the extraction of essential oil from plant materials, the present work investigated the optimal SFE conditions and the protective effects of different resulting fractions of PM needles on lipid peroxidation and foam cell production in macrophages. Nine PM needle extracts (PME1-9) were obtained in 1%-4% yields using different SFE conditions, of which PME1 had the lowest yield (1.1%) and PME3 the highest (3.9%). PME3 exhibited lower cytotoxic effects and stronger inhibition of lipid peroxidation and formation of foam cell in RAW 264.7 macrophages than those of other PME extracts. PME3-1 purified from PME3 by column and thin layer chromatography inhibited LDL oxidation more effectively than did PME3 in a cell-free system oxidized by Cu(2+). PME3-1 dose-dependently (25-100 µg/mL) decreased conjugated diene levels and foam cell formation induced by ox-LDL. GC/MS analyses revealed that 1-docosene, neophytadiene, and methyl abietate were increased 5.2-, 1.7- and 4.3-fold in PME3-1 relative to PME3. A new hydrocarbon compound, cedrane-8,13-diol, was identified in PME3-1. Overall, the present study demonstrates the optimal extraction conditions of SFE of PM and identifies the most potent antioxidant fractions and possible active compounds in PM.

A Nampt inhibitor FK866 mimics vitamin B3 deficiency by causing senescence of human fibroblastic Hs68 cells via attenuation of NAD(+)-SIRT1 signaling.

Vitamin B3 (niacin) deficiency can cause pellagra with symptoms of dermatitis, diarrhea and dementia. However, it is unclear whether the vitamin B3 deficiency causes human aging. FK866 (a Nampt inhibitor) can reduce intracellular NAD(+) level and induce senescence of human Hs68 cells. However, the mechanisms underlying FK866-induced senescence of Hs68 cells are unclear. In this study, we used FK866 to mimic the effects of vitamin B3 deficiency to reduce the NAD(+) level and investigated the mechanisms of FK866-induced senescence of Hs68 cells. We hypothesized that FK866 induced the senescence of Hs68 cells via an attenuation of NAD(+)-silent information regulator T1 (SIRT1) signaling. We found that FK866 induced cell senescence and diminished cellular NAD(+) levels and SIRT1 activity (detected by acetylation of p53), and these effects were dramatically antagonized by co-treatment with nicotinic acid, nicotinamide, or NAD(+). In contrast, the protein expression of SIRT1, AMP-activated protein kinase, mammalian target of rapamycin, and nicotinamide phosphoribosyltransferase (Nampt) was not affected by FK866. In addition, the role of GSH in the FK866-induced cells senescence may be limited, as N-acetylcysteine did not antagonize FK866-induced cell senescence. These results suggest that FK866 induces cell senescence via attenuation of NAD(+)-SIRT1 signaling. The effects of vitamin B3 deficiency on human aging warrant further investigation.

The anti-angiogenic action of 2-deoxyglucose involves attenuation of VEGFR2 signaling and MMP-2 expression in HUVECs.

AIMS: 2-Deoxyglucose (2-DG) is a glucose analogue and has been shown to inhibit angiogenesis in human umbilical vascular endothelial cells (HUVECs) through interference with N-linked glycosylation. However, the anti-angiogenic mechanisms of 2-DG are not fully elucidated. MAIN METHODS: We first employed an ex vivo rat aortic ring model to substantiate the anti-angiogenic action of 2-DG and then used HUVECs to investigate the molecular mechanism underlying such an action. KEY FINDINGS: Results reveal that 2-DG (0.05-1.0mM) significantly inhibited tube formation in both rat aortic rings and HUVECs. 2-DG (0.1-1.0mM) also significantly inhibited cell invasion and migration, as well as the activity and mRNA and protein expression of matrix metalloproteinase (MMP)-2 in HUVECs. In addition, 2-DG (1.0mM) significantly inhibited mRNA and protein expression of vascular endothelial growth receptor 2 (VEGFR2) in a time-dependent manner. 2-DG also significantly inhibited the phosphorylation of the focal adhesion kinase (FAK) and mitogen-activated protein kinase (p38), the downstream molecules of VEGFR2. The effects of 2-DG on tube formation, MMP-2 activity, and VEGFR2 protein expression in HUVECs were reversed by mannose, an N-linked glycosylation precursor. Mannose also reversed 2-DG-induced accumulation of VEGFR2 in the endoplasmic reticulum. SIGNIFICANCE: This ex vivo and in vitro study demonstrates that 2-DG inhibits angiogenesis with an action involving attenuation of VEGFR2 signaling and MMP-2 expression, possibly resulting from interference with N-linked glycosylation of VEGFR2. Further studies are needed to show that 2-DG inhibits VEGF-mediated angiogenesis or that the actual status of N-glycosylation of VEGFR2 is affected by the treatment.

Up-regulation of nicotinamide phosphoribosyltransferase and increase of NAD+ levels by glucose restriction extend replicative lifespan of human fibroblast Hs68 cells.

Calorie restriction (CR) extends lifespan in a remarkable range of organisms. However, the mechanisms of CR related to the longevity effects are not fully elucidated to date. Using human fibroblast Hs68 (Hs68) cells cultured at a lower level of medium glucose (i.e., glucose restriction; GR) to mimic CR, we investigated the crucial role of nicotinamide phosphoribosyltransferase (Nampt), nicotinamide adenine dinucleotide (NAD(+)), and nicotinamide (NAM) in GR-extended replicative lifespan of Hs68 cells. We found that GR extended the lifespan of Hs68 cells, in parallel to significantly increased expression of Nampt, intracellular NAD(+) levels, and SIRT1 activities, and to significantly decreased NAM levels. The lifespan-extending effects of GR were profoundly diminished by FK866 (a noncompetitive inhibitor of Nampt) and blocked by sirtinol (a noncompetitive inhibitor of sirtuins). However, the steady-state intracellular NAM level (averaged 2.5 µM) was much lower than the IC50 of NAM on human SIRT1 (about 50 µM). All these results suggest that up-regulation of Nampt play an important role in GR-extended lifespan of Hs68 cells by increasing the intracellular NAD(+) levels followed by activating SIRT1 activity in Hs68 cells. In contrast, the role of NAM depletion is limited.

Ergothioneine protects against neuronal injury induced by ß-amyloid in mice.

ß-Amyloid peptides (Aß) are neurotoxic and contribute to the development of Alzheimer's disease (AD). Ergothioneine (EGT) has been shown to protect against loss of memory and learning abilities in mice. In this study, mice were orally fed EGT (0.5 or 2 mg/kg body weight) for 16 days before treatment (i.c.v) with a single dose of Aß1-40 in the hippocampus. After resting for 12 days to restore the body weight, the mice were again fed EGT for additional 39 days. Active avoidance tests were conducted on days 37-39 (short-memory avoidance) and on days 37, 44 and 51 (long-memory avoidance). Water maze task was used to evaluate learning and memory abilities by acquisition test and retention test. In both long-memory avoidance and water maze tests, EGT significantly decreased the escape latency and increased the frequency of successful avoidance. Furthermore, EGT significantly prevented Aß accumulation in the hippocampus and brain lipid peroxidation, restored acetylcholinesterase (AChE) activity, maintained glutathione/glutathione disulfide ratio and superoxide dismutase activity in brain tissues of Aß1-40-teated mice. Thus, EGT can protect against Aß-induced loss of memory and learning abilities in mice. Further studies are required to confirm the protective effects of EGT on the development or progression of AD.

S-Adenosylhomocysteine enhances DNA damage through increased ß-amyloid formation and inhibition of the DNA-repair enzyme OGG1b in microglial BV-2 cells.

S-Adenosylhomocysteine (SAH) is a risk factor for neurodegenerative diseases such as Alzheimer's disease, for which ß-Amyliod (Aß) formation is a major risk factor. We recently showed that SAH increases Aß formation in mouse microglial BV2 cells. Here, we show that incubation of BV2 cells with SAH (0-500nM) for 6-24h sequentially increased Aß formation, ROS and DNA damage measured as 8-oxo-deoxyguanosine (8-oxo-dG) levels. Pre-incubation of BV2 cells with 20µM ß-secretase inhibitor IV for 30min followed by incubation with SAH (500nM) markedly decreased Aß formation and 8-oxo-dG levels. Treatment with SAH for 24h concentration-dependently inhibited DNA methyltransferase (DNMT1) activity and inhibited DNMT1 binding to Sp1 site of 8-oxoG-DNA glycosylases I (OGG1) promoter and OGG1 protein and mRNA expression at 24h; the latter effect was attributed to hypomethylation of the OGG1 gene promoter, because pre-incubation of cells with betaine (1.0mM for 30 min) markedly prevented the inhibition of OGG1 protein expression induced by SAH. Overall, we demonstrate that SAH increases DNA damage in BV-2 cells possible by increased Aß formation leading to increased formation of ROS. Furthermore, the DNA damage is enhanced by SAH through inhibition of DNMT1 activity and hypomethylation of OGG1 gene promoter.

Effects of 2-deoxyglucose and dehydroepiandrosterone on intracellular NAD(+) level, SIRT1 activity and replicative lifespan of human Hs68 cells.

2-Deoxy-D-glucose (2-DG) and dehydroepiandrosterone (DHEA) have been hypothesized to extend lifespan via mimicking calorie restriction (CR). Activation of sirtuins has been proposed to contribute to life extension of CR by increasing intercellular levels of NAD(+) in several organisms. However, it is unclear whether 2-DG and DHEA may affect intracellular NAD(+) levels and human sirtuin 1 (SIRT1) activities. Here, using human fibroblast Hs68 cells we showed that 2-DG increased intracellular NAD(+) levels in both time- and concentration-dependent manners. 2-DG also dose-dependently increased SIRT1 activities and the lifespan (measured as the cumulated growth curve of population doubling levels) of Hs68 cells. In contrast, DHEA at non-cytotoxic concentrations (≤50 µM) did not significantly affect NAD(+) levels, SIRT1 activities or the lifespan of Hs68 cells. These results suggest that 2-DG extends the lifespan of Hs68 cells by increased NAD(+) levels and SIRT1 activities, and that 2-DG has a potential as a CR mimetic.

Ellagic acid inhibits oxidized low-density lipoprotein (OxLDL)-induced metalloproteinase (MMP) expression by modulating the protein kinase C-α/extracellular signal-regulated kinase/peroxisome proliferator-activated receptor γ/nuclear factor-κB (PKC-α/ERK/PPAR-γ/NF-κB) signaling pathway in endothelial cells.

Previous studies have shown that vascular endothelium-derived matrix metalloproteinases (MMPs) contribute to the destabilization of atherosclerotic plaques, a key event triggering acute myocardial infarction. In addition, studies have reported that the PKC-MEK-PPARγ signaling pathway is involved in oxidized low-density lipoprotein (oxLDL)-induced expression of MMPs. Ellagic acid, a phenolic compound found in fruits and nuts, has potent antioxidant, anti-inflammatory, and anticancerous properties. However, the molecular mechanisms underlying its antiatherogenic effects remain to be clarified. This study aimed to assess whether the effects of ellagic acid on the fibrotic markers MMP-1 and MMP-3 are modulated by the PKC-ERK-PPAR-γ signaling pathway in human umbilical vein endothelial cells (HUVECs) that have been exposed to oxLDL. It was found that ellagic acid significantly inhibited oxLDL-induced expressions of MMP-1 and MMP-3. Pretreatment with ellagic acid and DPI, a well-known ROS inhibitor, attenuated the oxLDL-induced expression and activity of PKC-α. In addition, ellagic acid as well as pharmacological inhibitors of ROS, calcium, and PKC strongly suppressed the oxLDL-induced phosphorylation of extracellular signal-regulated kinase (ERK) and NF-κB activation. Moreover, ellagic acid ameliorated the oxLDL-induced suppression of PPAR-γ expression. In conclusion, the data suggest that ellagic acid elicits its protective effects by modulating the PKC-α/ERK/PPAR-γ/NF-κB pathway, resulting in the suppression of ROS generation and, ultimately, inhibition of MMP-1 and MMP-3 expression in HUVECs exposed to oxLDL.

Ergothioneine protects against neuronal injury induced by cisplatin both in vitro and in vivo.

The neuroprotective effects of ergothioneine (EGT) against cisplatin toxicity were investigated both in vitro and in vivo. For in vitro study, two types of neuronal cells, primary cortical neuron (PCN) cells and rat pheochromocytoma (PC12) cells, were incubated with EGT (0.1-10.0 µM) for 2 h followed by incubation with 0.5 µM cisplatin for 72 h. Results show that cisplatin markedly decreased the proliferation of PC12 cells and strongly inhibited the growth of axon and dendrite of PCN cells, but these effects were significantly prevented by EGT. For in vivo study, CBA mice were orally administered with 2 or 8 mg EGT/kg body weight for 58 consecutive days and were injected i.p. with 5mg cisplatin/kg body weight on days 7, 9 and 11. We found that EGT significantly restored the learning and memory deficits in mice treated with cisplatin evaluated by active and passive avoidance tests. EGT also significantly prevented brain lipid peroxidation, restored acetylcholinesterase (AChE) activity and maintained glutathione/glutathione disulfide ratio in brain tissues of mice treated with cisplatin. These results demonstrate that EGT protects against cisplatin-induced neuronal injury and enhances cognition, possibly through the inhibition of oxidative stress and restoration of AChE activity in neuronal cells.

EGCG protects against oxidized LDL-induced endothelial dysfunction by inhibiting LOX-1-mediated signaling.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), originally identified as the major receptor for oxidized low-density lipoprotein (oxLDL) in endothelial cells, plays a major role in the pathology of vascular diseases. Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. In the present study, we hypothesized that the most abundant polyphenolic compound in tea, epigallocatechin-3-gallate (EGCG), can downregulate parameters of endothelial dysfunction by modulating LOX-1-regulated cell signaling. In cultured human umbilical vein endothelial cells (HUVECs), exposure to oxLDL (130 microg/ml), which led to an increase in LOX-1 expression at the RNA and protein levels, was abrogated by addition of EGCG or DPI, a well-known inhibitor of flavoproteins, suggesting the involvement of NADPH oxidase. Furthermore, oxLDL rapidly activated the membrane translocation of Rac-1 and p47phox and the subsequent induction of ROS generation, which was suppressed markedly by pretreatment with EGCG or anti-LOX-1 monoclonal antibody. OxLDL also increased p38 MAPK phosphorylation and decreased phosphorylation of the amino-terminal region of Akt, with maximal induction at about 30 min, and NF-kappaB phosphorylation within 1 h, resulting in redox-sensitive signaling. In addition, oxLDL diminished the expression of endothelial nitric oxide synthase (eNOS), enhanced the expression of endothelin-1 and adhesion molecules (ICAM, E-selectin, and monocyte chemoattractant protein-1), and increased the adherence of monocytic THP-1 cells to HUVECs. Pretreatment with EGCG, however, exerted significant cytoprotective effects in all events. These data suggest that EGCG inhibits the oxLDL-induced LOX-1-mediated signaling pathway, at least in part, by inhibiting NADPH oxidase and consequent ROS-enhanced LOX-1 expression, which contributes to further ROS generation and the subsequent activation of NF-kappaB via the p38 MAPK pathway. Results from this study may provide insight into a possible molecular mechanism by which EGCG suppresses oxLDL-mediated vascular endothelial dysfunction.