Characterization of recombinant protein ferritin from Pyropia yezoensis (rPyFer) and its biological activities.

Ferritins are iron-binding proteins that are basically participated in iron storage, detoxification, and immune response. In the present study, ferritin gene from the marine red algae Pyropia yezoensis was cloned into a pET21d expression vector. High-efficiency transformation was performed in Escherichia coli BL21, the recombinant protein was expressed by induction with 0.1 mM isopropyl-ß-D-thiogalactoside and purified via ammonium sulfate precipitation, anion exchange and size exclusion chromatography. The purified recombinant ferritin from P. yezoensis (rPyFer) was characterized and analyzed for its antimicrobial activity against both Gram-negative and Gram-positive bacterial cultures and exhibited significant antibacterial activity against Gram-positive cultures. The recombinant protein was also analyzed for its iron-uptake and radical-scavenging activities; rPyFer exhibited significant iron-uptake activity at low concentrations, and its radical-scavenging activity increased in a dose-dependent manner. This research will contribute to the development of new therapeutic proteins from marine algae.

Spirulina protein promotes skin wound repair in a mouse model of full-thickness dermal excisional wound.

The skin protects body from environmental damage. Skin wounds lead to microbial infection and harmful agent injury. Thus, wound repair is crucial for the recovery of the normal function of skin tissue. The present study investigated the promoting effects of spirulina protein (SPCP) in mice on skin wound repair and also aimed to elucidate the potential underlying mechanisms. The results revealed that SPCP promoted the skin wound repair in a mouse model of full­thickness excisional wounds. SPCP induced an increase in the expression level of α­smooth muscle actin (α­SMA). The activities of superoxide dismutase (SOD) and catalase (CAT) were enhanced by SPCP treatment in the granulation tissue. In addition, SPCP decreased the level of malondialdehyde (MDA) in the granulation tissue. Western blot analysis revealed that SPCP enhanced the phosphorylation and activation of protein kinase B (Akt) and extracellular signal­regulated kinase (ERK). Moreover, the expression level of transforming growth factor ß1 (TGF­ß1) was increased in the SPCP­treated groups. The phosphorylation level of Smad2 was also increased by treatment of SPCP. Furthermore, SPCP promoted the expression of collagen in the granulation tissue. Taken together, these findings indicate that SPCP exerts a promoting effect on skin wound repair. The Akt, ERK and TGF­ß1 signaling pathways are involved in this process.

Peptidyl-prolyl isomerase and the biological activities of recombinant protein cyclophilin from Pyropia yezoensis (PyCyp).

Cyclophilins are highly conserved proteins associated with peptidyl-prolyl cis-trans isomerase activity (PPIase). The present study was designed to analyze the biological activity of recombinant cyclophilin from the marine red algae Pyropia yezoensis (PyCyp). The cyclophilin gene from P. yezoensis was cloned into the pPROEX-HTA expression vector. The plasmid was transformed into BL21 Escherichia coli by high efficiency transformation. Recombinant protein was expressed using 0.1 mM IPTG and the fusion protein was purified by affinity column chromatography. The His-tag was removed by TEV protease. The recombinant protein was further purified on a HiPrep Sephacryl S-200 HR column and by reversed-phase high performance liquid chromatography with a Sep-pak plus C18 column. Purified cyclophilin was characterized by a variety of analytical methods and analyzed for its peptidyl-prolyl isomerase activity. Our recombinant PyCyp was shown to catalyze cis-trans isomerization. PyCyp was also evaluated for antimicrobial activity against both Gram-positive and Gram-negative bacteria cultures and showed significant antibacterial activity against tested pathogens. PyCyp was shown to permeabilize bacterial membranes as evidenced by increased fluorescence intensity in SYTOX Green uptake assays with Staphylococcus aureus. The radical scavenging activity of PyCyp increased in a dose-dependent manner, indicating significant antioxidant activity. This study provides information for the development of therapeutic proteins from marine algae.

PYP1-4 peptide from Pyropia yezoensis protects against acetaminophen-induced hepatotoxicity in HepG2 cells.

Acetaminophen (APAP) is a widely used analgesic and antipyretic. It is safe at normal treatment doses; however, APAP overdose is a major cause of acute liver and kidney failure. A variety of methods to reduce the damage caused by APAP overdose have previously been evaluated. The protein-rich seaweed Pyropia yezoensis has antioxidant, antitumor and anti-inflammatory activities, and protects against cytotoxicity. However, little is known regarding the protective effects of P. yezoensis peptide against APAP-induced hepatotoxicity. The present study investigated the ability of P. yezoensis peptide (PYP1-4) to ameliorate the damage caused by APAP-induced hepatotoxicity using HepG2 as the model cell line in addition to the signaling pathways involved. Briefly, cell viability, nitric oxide, reactive oxygen species and apoptosis assays were performed in conjunction with western blot analysis and reverse transcription-quantitative PCR. First, the present study revealed the minimum toxic concentration of APAP (15 mM) and the resting concentration of PYP1-4 (0-500 ng/ml). Administration of PYP1-4 to APAP-induced cells decreased the nitric oxide and reactive oxygen species levels, and restored the levels of antioxidant-associated proteins (catalase, heme oxygenase 1, superoxide dismutase 2 and quinone oxidoreductase 1). PYP1-4 increased the translocation of nuclear factor, erythroid 2 like 2 to the nucleus and the activities of glycogen synthase kinase-3ß, Akt and AMP-activated protein kinase. In addition, APAP induced apoptosis; however, PYP1-4 inhibited apoptosis by modulating the levels of pro-apoptotic markers (Bad), anti-apoptotic markers (Bcl-2 and BH3 interacting domain death agonist), caspases and poly (ADP-ribose) polymerase 1. Subsequently, the insulin-like growth factor 1 receptor signaling pathway was investigated to determine whether PYP1-4 treatment restored the levels of cell growth-associated factors during APAP-induced hepatotoxicity. PYP1-4 treatment impacted the levels of components of the insulin receptor substrate 1/PI3K/Akt and Ras/Raf/ERK signaling pathways, and promoted cell survival. Therefore, the P. yezoensis peptide PYP1-4 may be useful for preventing APAP-induced hepatotoxicity.

Novel application of an optical inspection system to determine the freshness of Scomber japonicus (mackerel) stored at a low temperature.

This study evaluated the use of an optical inspection system (OIS) to determine the freshness of mackerel (Scomber japonicus). The correlations between the light reflection intensity (LRI) of mackerel eyes (determined using an OIS) and the volatile basic nitrogen content (VBN) and K-value were analyzed. After unloading at the harbor, the mackerel were stored at 4 °C for 9 days and the VBN, K-value, and LRI were determined at 3-day intervals. During storage, the LRI, VBN, and K-value all increased. Furthermore, the LRI was correlated with the K-value and VBN. Therefore, although the LRI cannot be applied as an absolute standard for evaluating freshness, the LRI using an OIS is a suitable nondestructive method for evaluating freshness for quality and risk management in the processing industry when handling large numbers of fish.

Probing Multi-Target Action of Phlorotannins as New Monoamine Oxidase Inhibitors and Dopaminergic Receptor Modulators with the Potential for Treatment of Neuronal Disorders.

Modulation of multiple protein targets with a single compound is essential for the effective treatment of central nervous system disorders. In our previous G protein-coupled receptor (GPCR) cell-based study, a selective human monoamine oxidase (hMAO)-A inhibitor, eckol, stimulated activity of dopamine D3 and D4 receptors. This result led to our interest in marine phlorotannin-mediated modulation of hMAO enzymes and related GPCRs in neuronal disorders. Here, we evaluate the multi-target effects of phloroglucinol, phlorofucofuroeckol-A (PFF-A), and dieckol by screening their modulatory activity against hMAO-A and -B and various neuronal GPCRs. Among the tested phlorotannins, PFF-A showed the strongest inhibitory activity against both hMAO isoforms, with higher selectivity toward hMAO-B than hMAO-A. Enzyme kinetics and docking data revealed that PFF-A noncompetitively acts on hMAOs into the alternative binding pocket of enzymes with allosteric functions. In a functional assay for GPCR screening, dieckol and PFF-A exhibited a multi-target combination of D3R/D4R agonism and D1/5HT1A/NK1 antagonism. In particular, they effectively stimulated D3R and D4R, compared to other GPCRs. Docking analysis confirmed that dieckol and PFF-A successfully docked into the conserved active sites of D3R and D4R and interacted with aspartyl and serine residues in the orthosteric binding pockets of the respective receptors. Based on our experimental and computational data, we established the structure-activity relationship between tested phlorotannins and target proteins, including hMAOs and GPCRs. Our current findings suggest that hMAO inhibitors dieckol and PFF-A, major phlorotannins of edible brown algae with multi-action on GPCRs, are potential agents for treatment of psychological disorders and Parkinson's disease.

Effect of Cyclophilin from Pyropia Yezoensis on the Proliferation of Intestinal Epithelial Cells by Epidermal Growth Factor Receptor/Ras Signaling Pathway.

Cyclophilin (Cyp) is peptidyl-prolyl isomerase (PPIase), and it has many biological functions, including immune response regulation, antioxidants, etc. Cyp from red algae is known for its antioxidant and antifungal activity. However, the other biological effects of Cyp from Pyropia yezoensis are unclear. In this study, we synthesized Cyp from P. yezoensis (pyCyp) and examined its biological activity on IEC-6 cells. First, the MTS assay showed that pyCyp increased cell proliferation in a dose-dependent manner. pyCyp activated the EGFR signaling pathway that regulates cell growth, proliferation, and survival. It induced intracellular signaling pathways, including the Ras signaling pathway. In addition, we observed cell cycle-related proteins. pyCyp increased the expression of cyclin A, cyclin E, and Cdk2, and decreased the expression of p27 and p21 proteins. These results indicate that pyCyp stimulates cell proliferation via the EGFR signaling pathway and promotes cell cycle progression in intestinal epithelial cells. Therefore, we suggest pyCyp as a potential material to promote the proliferation of intestinal epithelial cells.

Protective Effect of Pyropia yezoensis Peptide on Dexamethasone-Induced Myotube Atrophy in C2C12 Myotubes.

Dexamethasone (DEX), a synthetic glucocorticoid, causes skeletal muscle atrophy. This study examined the protective effects of Pyropia yezoensis peptide (PYP15) against DEX-induced myotube atrophy and its association with insulin-like growth factor-I (IGF-I) and the Akt/mammalian target of rapamycin (mTOR)-forkhead box O (FoxO) signaling pathway. To elucidate the molecular mechanisms underlying the effects of PYP15 on DEX-induced myotube atrophy, C2C12 myotubes were treated for 24 h with 100 µM DEX in the presence or absence of 500 ng/mL PYP15. Cell viability assays revealed no PYP15 toxicity in C2C12 myotubes. PYP15 activated the insulin-like growth factor-I receptor (IGF-IR) and Akt-mTORC1 signaling pathway in DEX-induced myotube atrophy. In addition, PYP15 markedly downregulated the nuclear translocation of transcription factors FoxO1 and FoxO3a, and inhibited 20S proteasome activity. Furthermore, PYP15 inhibited the autophagy-lysosomal pathway in DEX-stimulated myotube atrophy. Our findings suggest that PYP15 treatment protected against myotube atrophy by regulating IGF-I and the Akt-mTORC1-FoxO signaling pathway in skeletal muscle. Therefore, PYP15 treatment appears to exert protective effects against skeletal muscle atrophy.

Spirulina Crude Protein Promotes the Migration and Proliferation in IEC-6 Cells by Activating EGFR/MAPK Signaling Pathway.

Spirulina is a type of filamentous blue-green microalgae known to be rich in nutrients and to have pharmacological effects, but the effect of spirulina on the small intestine epithelium is not well understood. Therefore, this study aims to investigate the proliferative effects of spirulina crude protein (SPCP) on a rat intestinal epithelial cells IEC-6 to elucidate the mechanisms underlying its effect. First, the results of wound-healing and cell viability assays demonstrated that SPCP promoted migration and proliferation in a dose-dependent manner. Subsequently, when the mechanisms of migration and proliferation promotion by SPCP were confirmed, we found that the epidermal growth factor receptor (EGFR) and mitogen-activated protein (MAPK) signaling pathways were activated by phosphorylation. Cell cycle progression from G0/G1 to S phase was also promoted by SPCP through upregulation of the expression levels of cyclins and cyclin-dependent kinases (Cdks), which regulate cell cycle progression to the S phase. Meanwhile, the expression of cyclin-dependent kinase inhibitors (CKIs), such as p21 and p27, decreased with SPCP. In conclusion, our results indicate that activation of EGFR and its downstream signaling pathway by SPCP treatment regulates cell cycle progression. Therefore, these results contribute to the research on the molecular mechanism for SPCP promoting the migration and proliferation of rat intestinal epithelial cells.

Wound Healing Potential of Spirulina Protein on CCD-986sk Cells.

Wound healing is a dynamic and complex process. The proliferation and migration of dermal fibroblasts are crucial for wound healing. Recent studies have indicated that the extracts from Spirulina platensis have a positive potential for wound healing. However, its underlying mechanism is not fully understood. Our previous study showed that spirulina crude protein (SPCP) promoted the viability of human dermal fibroblast cell line (CCD-986sk cells). In this study, we further investigated the wound healing effect and corresponding mechanisms of SPCP on CCD-986sk cells. Bromodeoxyuridine (BrdU) assay showed that SPCP promoted the proliferation of CCD-986sk cells. The wound healing assay showed that SPCP promoted the migration of CCD-986sk cells. Furthermore, cell cycle analysis demonstrated that SPCP promoted CCD-986sk cells to enter S and G2/M phases from G0/G1 phase. Western blot results showed that SPCP significantly upregulated the expression of cyclin D1, cyclin E, cyclin-dependent kinase 2 (Cdk2), cyclin-dependent kinase 4 (Cdk4), and cyclin-dependent kinase 6 (Cdk6), as well as inhibited the expression of CDK inhibitors p21 and p27 in CCD-986sk cells. In the meanwhile, SPCP promoted the phosphorylation and activation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt). However, the phosphorylation of Akt was significantly blocked by PI3K inhibitor (LY294002), which in turn reduced the SPCP-induced proliferation and migration of CCD-986sk cells. Therefore, the results presenting in this study suggested that SPCP can promote the proliferation and migration of CCD-986sk cells; the PI3K/Akt signaling pathway play a positive and important role in these processes.

Crude protein from spirulina increases the viability of CCD­986sk cells via the EGFR/MAPK signaling pathway.

Spirulina, an edible blue­green alga, has great potential for various applications in human health, possibly including reduced skin aging. The mechanisms by which spirulina crude protein (SPCP) may influence human skin fibroblast viability are not yet understood; therefore, a human dermal fibroblast cell line (CCD­986sk) was used as a cell model system to study the influence of SPCP on human skin fibroblast viability. An enzyme­linked immunosorbent assay showed that collagen formation improved in SPCP­treated cells in a dose­dependent manner, while elastase activity was decreased. In addition, western blot analysis showed a dose­dependent decrease in the expression of the aging­associated gene matrix metalloproteinase­8, a collagen­degradative enzyme. It was also shown that SPCP upregulated epidermal growth factor receptor (EGFR) activity, leading to activation of the mitogen­activated protein kinase (MAPK)/extracellular signal­regulated kinase (ERK) signaling pathway. Together, these results demonstrated that SPCP increases human fibroblast viability by activation of the EGFR/MAPK signaling pathway. This contribution sheds light on the molecular mechanism for SPCP increasing the viability of human skin cell and provides a potential efficient cosmeceutical for protecting human skin.

Pyropia yezoensis Protein Prevents Dexamethasone-Induced Myotube Atrophy in C2C12 Myotubes.

Glucocorticoids (GCs), which are endocrine hormones released under stress conditions, can cause skeletal muscle atrophy. This study investigated whether Pyropia yezoensis crude protein (PYCP) inhibits synthetic GCs dexamethasone (DEX)-induced myotube atrophy associated with proteolytic systems. Mouse skeletal muscle C2C12 myotubes were treated with DEX in the presence or absence of PYCP. DEX exposure (100 µM) for 24 h significantly decreased myotube diameter and myogenin expression, which were all increased by treatment with 20 and 40 µg/mL PYCP. Additionally, PYCP significantly reduced the nuclear expression of the forkhead box transcription factors, FoxO1 and FoxO3a, and ubiquitin-proteasome pathway activation. Further mechanistic research revealed that PYCP inhibited the autophagy-lysosome pathway in DEX-induced C2C12 myotubes. These findings indicate that PYCP prevents DEX-induced myotube atrophy through the regulation of FoxO transcription factors, followed by the inhibition of the ubiquitin-proteasome and autophagy-lysosome pathways. Therefore, we suggest that inhibiting these two proteolytic processes with FoxO transcription factors is a promising strategy for preventing DEX-related myotube atrophy.

Pyropia yezoensis Protein Supplementation Prevents Dexamethasone-Induced Muscle Atrophy in C57BL/6 Mice.

We investigated the protective effects of Pyropia yezoensis crude protein (PYCP) against dexamethasone (DEX)-induced myotube atrophy and its underlying mechanisms. DEX (3 mg/kg body weight, intraperitoneal injection) and PYCP (150 and 300 mg/kg body weight, oral) were administrated to mice for 18 days, and the effects of PYCP on DEX-induced muscle atrophy were evaluated. Body weight, calf thickness, and gastrocnemius and tibialis anterior muscle weight were significantly decreased by DEX administration (p < 0.05), while PYCP supplementation effectively prevented the DEX-induced decrease in body weight, calf thickness, and muscle weight. PYCP supplementation also attenuated the DEX-induced increase in serum glucose, creatine kinase, and lactate dehydrogenase levels. Additionally, PYCP supplementation reversed DEX-induced muscle atrophy via the regulation of the insulin-like growth factor-I/protein kinase B/rapamycin-sensitive mTOR complex I/forkhead box O signaling pathway. The mechanistic investigation revealed that PYCP inhibited the ubiquitin-proteasome and autophagy-lysosome pathways in DEX-administrated C57BL/6 mice. These findings demonstrated that PYCP increased protein synthesis and decreased protein breakdown to prevent muscle atrophy. Therefore, PYCP supplementation appears to be useful for preventing muscle atrophy.

Protective effect of Pyropia yezoensis glycoprotein on chronic ethanol consumption-induced hepatotoxicity in rats.

The present study investigated the protective effect of Pyropia yezoensis glycoprotein (PYGP) against chronic ethanol consumption­mediated hepatotoxicity in rats. Male Sprague-Dawley rats (n=20; 6 weeks old) were randomly divided into four groups. The rats in each group were treated for 30 days with the following: i) CON group, distilled water only; ii) EtOH group, 20% ethanol 3.7 g/kg/BW; iii) EtOH+150 group, 20% ethanol 3.7 g/kg/BW+PYGP 150 mg/kg/BW; iv) EtOH+300 group, 20% ethanol 3.7 g/kg/BW+PYGP 300 mg/kg/BW. EtOH, PYGP and water were orally administered. The rats were sacrificed after 30 days, and blood and liver samples were collected for analysis. Treatment with ethanol caused significant elevation of serum levels of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT). Furthermore, inhibition of the antioxidant defense system in the liver, including glutathione (GSH), glutathione peroxidase (GSH­px) and catalase (CAT) was observed. However, co­administration with PYGP recovered the antioxidant defense system, and the serum levels of GOT and GPT. PYGP was shown to attenuate ethanol toxicity via the inactivation of mitogen­activated protein kinases (MAKPs). PYGP suppressed the overexpression of cytochrome P450 2E1 (CYP2E1), inducible nitric oxide synthase and cyclooxygenase­2. These results suggested that the protective effect of PYGP was associated with antioxidant activities, MAPKs and the CYP2E1 signaling pathway.

Anti-obesity effects of boiled tuna extract in mice with obesity induced by a high-fat diet.

The aim of this study was to examine the anti-obesity effects of boiled tuna extract in C57BL/6N mice with obesity induced by a high-fat diet (HFD). We determined the anti-obesity effects of boiled tuna extract (100, 200, or 400 mg/kg) on the progression of HFD-induced obesity for 10 weeks. The mice were divided into 5 groups as follows: the normal diet (ND) group (n=10); the HFD group (n=10); the mice fed HFD and 100 mg/kg boiled tuna extract group (n=10); those fed a HFD and 200 mg/kg boiled tuna extract group (n=10); and those fed a HFD and 400 mg/kg boiled tuna extract group (n=10). Changes in body weight, fat content, serum lipid levels and lipogenic enzyme levels were measured. The consumption of boiled tuna extract lowered epididymal tissue weight and exerted anti-obesity effects, as reflected by the serum glucose, triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL­C), low-density lipoprotein cholesterol (LDL-C), insulin and leptin levels. In addition, we demonstrated changes in liver adipogenic- and lipogenic-related protein expression by western blot analysis. Boiled tuna extract downregulated the levels of the CCAAT/enhancer-binding protein α, ß and δ (C/EBPα, ß, Î´), and peroxisome proliferator-activated receptor-γ (PPAR-γ) adipocyte marker genes. Boiled tuna extract also attenuated adipogenic and lipogenic gene expression, namely the levels of fatty acid synthase (FAS), lipoprotein lipase (LPL), acetyl-CoA carboxylase (ACC), glucose transporter type 4 (Glut4) and phosphorylated adenosine monophosphate-activated protein kinase α and ß (AMPKα, ß) in a dose-dependent manner. Moreover, the consumption of boiled tuna extract restored the levels of superoxide dismutase (SOD), catalase (CAT), glutamic oxaloacetic transaminase (GOT), glutamic-pyruvate transaminase (GPT), aspartate transaminase (AST) and alanine transaminase (ALT) to those of the control group. These results suggest that boiled tuna extract attenuates the progression of obesity by stimulating fatty acid oxidation through the upregulation of AMPK genes, as well as by inhibiting the synthesis of adipogenic and lipogenic enzymes. These characteristics of boiled tuna extract highlight its potential anti-obesity effects.

Pyropia yezoensis glycoprotein promotes the M1 to M2 macrophage phenotypic switch via the STAT3 and STAT6 transcription factors.

Macrophage polarization has been well documented. Macrophages can aquire two phenotypes, the pro-inflammatory M1 phenotype, and the anti-inflammatory and wound healing M2 phenotype. The M1 macrophage phenotype has been linked to metabolic disease and is also associated with cancer-related inflammation. Of note, macrophage polarization can be influenced by the extracellular environment. In the current study, we examined the effects of Pyropia yezoensis glycoprotein (PYGP) on M1 to M2 macrophage polarization in lipopolysaccharide (LPS)-stimulated macrophages. RAW 264.7 macrophages stimulated with LPS exhibited an upregulated expression of pro-inflammatory mediators, namely of the M1 markers, nitric oxide (NO), reactive oxygen species (ROS), interleukin (IL)-6, IL-1ß, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and nitric oxide synthase­2 (NOS-2). Treatment with PYGP inhibited the production of M1 markers and increased arginase 1 (ARG1), chitinase-like 3 (Chil3; also known as Ym1), resistin like beta (RETNLB; also known as FIZZ1), IL-10, CD163, CD206, peroxisome proliferator-activated receptor Î³ (PPARγ) and Krüppel-like factor 4 (KLF4) M2 marker gene expression. The signal transducer and activator of transcription (STAT)3 and STAT6 transcription factors were phosphorylated following treatment with PYGP. However, the silencing of STAT3 and STAT6 using siRNA in the macrophages decreased ARG1, Ym1 and FIZZ1 M2 marker gene expression in spite of treatment of PYGP. These findings suggest that PYGP exerts anti-inflammatory effects by regulating the M1 to M2 phenotypic switch through STAT3 and STAT6. Thus, PYGP may have potential for use as a natural remedy for inflammatory diseases.

Pyropia yezoensis glycoprotein regulates antioxidant status and prevents hepatotoxicity in a rat model of D-galactosamine/lipopolysaccharide-induced acute liver failure.

The present study aimed to investigate the effects of Pyropia yezoensis glycoprotein (PYGP) on hepatic antioxidative enzyme activity and mitogen-activated protein kinase (MAPK) phosphorylation in a rat model of D-galactosamine/lipopolysaccharide (D-GalN/LPS)-induced hepatotoxicity. Glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) were measured to determine the severity of hepatotoxicity. Treatment with D­GalN/LPS significantly increased the GOT, GPT and lipid peroxidation levels, and decreased the antioxidant capacity of the rats. Treatment with PYGP (150 and 300 mg/kg/body weight) decreased the levels of GOT, GPT and lipid peroxidation levels. The activities of antioxidative enzymes, including catalase, glutathione S­transferase and glutathione were upregulated following PYGP treatment. Furthermore, D­GalN/LPS­induced MAPK phosphorylation, and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression were downregulated by PYGP. These results indicated that PYGP may exert hepatoprotective effects via the upregulation of antioxidative enzymes, and the downregulation of the MAPK signaling pathway and iNOS and COX-2 expression.

The anti-obesity effects of a tuna peptide on 3T3-L1 adipocytes are mediated by the inhibition of the expression of lipogenic and adipogenic genes and by the activation of the Wnt/ß-catenin signaling pathway.

The differentiation of 3T3-L1 cells into adipocytes involves the activation of an organized system of obesity-related genes, of which those encoding CCAAT/enhancer-binding proteins (C/EBPs) and the Wnt-10b protein may play integral roles. In a previous study of ours, we found that a specific peptide found in tuna (sequence D-I-V-D-K-I-E-I; termed TP-D) inhibited 3T3-L1 cell differentiation. In the present study, we observed that the expression of expression of C/EBPs and Wnt-10b was associated with obesity. The initial step of 3T3-L1 cell differentiation involved the upregulation of C/EBP-α expression, which in turn activated various subfactors. An upstream effector of glycogen synthase kinase-3ß (GSK-3ß) inhibited Wnt-10b expression in 3T3-L1 adipocytes. In a previous study of ours, we sequenced the tuna peptide via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quadrupole time-of-flight mass spectrometry (Q-TOF MS/MS) and confirmed the anti-obesity effects thereof in 3T3-L1 adipocytes. In the present study, we demonstrate that TP-D inhibits C/EBP and promotes Wnt-10b mRNA expression, thus activating the Wnt pathway. The inhibition of lipid accumulation was measured using a glucose and triglyceride (TG) assay. Our results confirmed that TP-D altered the expression levels of C/EBP-related genes in a dose-dependent manner and activated the Wnt signaling pathway. In addition, we confirmed that total adiponectin and high-molecular weight (HMW) adiponectin levels were reduced by treatment with TP-D. These data indicate that TP-D inhibits adipocyte differentiation through the inhibition of C/EBP genes and the subsequent activation of the Wnt/ß-catenin signaling pathway.

The proliferative effects of Pyropia yezoensis peptide on IEC-6 cells are mediated through the epidermal growth factor receptor signaling pathway.

For a number of years, seaweed has been used as a functional food in Asian countries, particularly in Korea, Japan and China. Pyropia yezoensis is a marine red alga that has potentially beneficial biological activities. In this study, we examined the mechanisms through which a Pyropia yezoensis peptide [PYP1 (1-20)] induces the proliferation of IEC-6 cells, a rat intestinal epithelial cell line, and the involvement of the epidermal growth factor receptor (EGFR) signaling pathway. First, cell viability assay revealed that PYP1 (1-20) induced cell proliferation in a concentration-dependent manner. Subsequently, we examined the mechanisms responsible for this induction of proliferation induced by PYP1 (1-20). EGFR is widely expressed in mammalian epithelial tissues, and the binding of this ligand affects a variety of cell physiological parameters, such as cell growth and proliferation. PYP1 (1-20) increased the expression of EGFR, Shc, growth factor receptor-bound protein 2 (Grb2) and son of sevenless (SOS). EGFR also induced the activation of the Ras signaling pathway through Raf, MEK and extracellular signal-regulated kinase (ERK) phosphorylation. In addition, cell cycle analysis revealed the expression of cell cycle-related proteins. The results demonstrated an increased number of cells in the G1 phase and an enhanced cell proliferation. In addition, the upregulation of cyclin D, cyclin E, Cdk2, Cdk4 and Cdk6 was observed accompanied by a decreased in p21 and p27 expression. These findings suggest that PYP1 (1-20) stimulates the proliferation of rat IEC-6 cells by activating the EGFR signaling pathway. Therefore, PYP1 (1-20) may be a potential source for the development of bio-functional foods which promotes the proliferation of intestinal epithelial cells.

Protective effect of Porphyra yezoensis glycoprotein on D-galactosamine­induced cytotoxicity in Hepa 1c1c7 cells.

The present study aimed to examine the signaling pathways and enzyme activity associated with the protective effect of Porphyra yezoensis glycoprotein (PYGP) on D­galactosamine (D­GaIN)­induced cytotoxicity in Hepa 1c1c7 cells. D­GaIN is commonly used to induce hepatic injury models in vivo as well as in vitro. PYGP was extracted from Porphyra yezoensis, a red algae distributed along the coasts of Republic of Korea, China and Japan. In the present study, Hepa 1c1c7 cells were pre­treated with PYGP (20 and 40 µg/ml) for 24 h and then the media was replaced with D­GaIN (20 mM) and PYGP (20 and 40 µg/ml). The results demonstrated that D­GaIN induced Hepa 1c1c7 cell death and pretreatment with PYGP was found to attenuate D­GaIN toxicity. In addition, D­GaIN decreased the antioxidant activity and increased lipid peroxidation processes; however, pre­treatment with PYGP reduced the generation of lipid peroxidation products, such as thiobarbituric acid reactive substances, as well as increased the activity of antioxidant enzymes, including superoxide dismutase, catalase and glutathione­s­transferase (GST). PYGP was shown to suppress the overexpression of extracellular signal­regulated kinase, c­jun N­terminal kinase and p38 mitogen­activated protein kinase (MAPK) phosphorylation induced by D­GaIN. Furthermore, PYGP increased the protein expression of nuclear factor erythroid 2­related factor 2 (Nrf2), quinine oxidoreductase 1, GST and heme oxygenase 1 protein expression. These results suggested that PYGP had cytoprotective effects against D­GaIN­induced cell damage, which may be associated with MAPKs and the Nrf2 signaling pathway.