Kyung Soo Nam

Proteomic analysis for inhibitory effect of chitosan oligosaccharides on 3T3-L1 adipocyte differentiation

In the present study, we performed a differential proteomic analysis using 2‐DE combined with MS to clarify the molecular mechanism for the suppressive effect of chitosan oligosaccharides (CO) during differentiation of adipocyte 3T3‐L1. Cell differentiation was significantly inhibited by CO at the concentration of 4 mg/mL. Protein mapping of adipocyte homogenates by 2‐DE revealed that numerous protein spots were differentially altered in response to CO treatment. Out of 50 identified proteins showing significant alterations, six were up‐regulated and 44 were down‐regulated by CO treatment in comparison to control mature adipocytes. Among them, most of the proteins are associated with lipid metabolism, cytoskeleton, and redox regulation, in which the levels of farnesyl diphosphate synthetase (FDS), dedicator of cytokinesis 9 (DOCK9), and chloride intracellular channel 1 (CLIC1) were significantly reduced (>two‐fold) with CO treatment. These results have not previously been examined in the context of adipogenesis, and thus can be used as novel biomarkers. Taken together with immunoblot analysis, it was concluded that the inhibitory effect of CO on adipocyte differentiation was mediated by C/EBPα and PPARγ pathway through significant downregulations of important adipogenic molecules such as fatty acid binding protein and glucose transporter 4.

Inhibitory Effect of Deep-sea Water on Differentiation of 3T3-L1 Adipocytes

Currently, the utilization of deep-sea water (DSW) is receiving much attention due to its high productivity, large quantity, and potential for biological application. The 3T3-L1 cell line is a well-established and commonly used in vitro model to assess adipocyte differentiation. Over the course of several days, confluent 3T3-L1 cells can be converted to adipocytes in the presence of an adipogenic cocktail. In this study, the effects of DSW on differentiation adipocyte 3T3-L1 cells were studied. DSW significantly decreased lipid accumulation, a marker of adipogenesis, in a dose-dependent manner. DSW of hardness 1,000 was the most effective for inhibiting adipocyte differentiation without any cytotoxicity. DSW significantly reduced expression mRNA levels of PPARγ and C/EBPα and protein levels of fatty-acid-binding protein and adiponectin. Our results suggest a potential role for DSW as anti-obesity agents by inhibiting adipocyte differentiation mediated through the down-regulated expression of adipogenic transcription factors and adipocyte-specific proteins.

Age-related changes in matrix metalloproteinase-9 regulation in cultured mouse aortic smooth muscle cells

We previously reported that aortic smooth muscle cells (SMC) from aged mice have an age-related decline in proliferative capacity compared with those derived from young mice. Here we investigated matrix metalloproteinase-9 (MMP-9) regulation in both young and aged SMC. Zymography, immunoblot, and northern blot analysis showed that MMP-9 expression is significantly reduced in response to tumor necrosis factor-alpha stimulation with increasing in vitro age. Mutational analysis, gel shift assays and supershift assays demonstrated that the lower MMP-9 expression in aged SMC is associated with lower activities of NF-kappaB and AP-1. Since mitogen-activated protein kinase ERK1/2 induce MMP-9 expression, we examined whether U0126, an ERK1/2 inhibitor, influenced MMP-9 expression in aged SMC. Treatment with U0126 successfully inhibited MMP-9 expression in both young and aged SMC. Finally, to analyze the causal relationship between replicative senescence and MMP-9 expression, we stably overexpressed the MMP-9 gene in aged SMC and we showed no alteration of the proliferative capacity of the transduced cells. Taken together, these results suggest that down-regulation of MMP-9 expression in SMC may play a role in vascular remodeling during in vitro aging.

Deep sea water improves hypercholesterolemia and hepatic lipid accumulation through the regulation of hepatic lipid metabolic gene expression

A high‑fat diet or high‑cholesterol diet (HCD) is a major cause of metabolic diseases, including obesity and diabetes; vascular diseases, including hypertension, stroke and arteriosclerosis; and liver diseases, including hepatic steatosis and cirrhosis. The present study aimed to evaluate the effects of deep sea water (DSW) on rats fed a HCD. DSW decreased HCD‑induced increases in total cholesterol and low‑density lipoprotein (LDL) cholesterol in the blood, and recovered high‑density lipoprotein cholesterol. In addition, DSW decreased levels of liver injury markers, which were increased in response to HCD, including glutamate‑oxaloacetate transaminase, glutamate‑pyruvate transferase and alkaline phosphatase. Lower lipid droplet levels were observed in the livers of rats fed a HCD and treated with DSW at a hardness of 1,500, as compared with those in the HCD only group. Semi‑quantitative reverse transcription‑polymerase chain reaction (RT‑PCR) revealed that mRNA expression levels of fatty acid synthase and sterol regulatory element binding protein‑1c (SREBP‑1c) in rats fed a HCD with DSW were lower compared with the HCD only group. Furthermore, quantitative RT‑PCR revealed that DSW enhanced LDL receptor (LDLR) mRNA expression in a hardness‑dependent manner. Combined, the results of the present study indicated that DSW may reduce HCD‑induced increases in blood and liver lipid levels, indicating that DSW may protect against hypercholesterolemia and non‑alcoholic hepatic steatosis. In addition, the present study demonstrated that DSW‑induced downregulation of lipids in the blood and hepatic lipid accumulation was mediated by enhancement of LDLR expression and suppression of fatty acid synthase and SREBP‑1c.

Effect of Cnidii Rhizoma on nitric oxide production and invasion of human colorectal adenocarcinoma HT-29 cells.

Colorectal adenocarcinoma is the most common type of gastrointestinal cancer. Colon adenocarcinoma is a major health problem worldwide due to the high prevalence and mortality rates associated with the disease. The majority of colorectal carcinomas are adenocarcinomas, which originate from the epithelial cells of the colorectal mucosa. HT-29 cells, which originate from human colon adenocarcinoma, are used as an in vitro model to investigate the effect of malignant transformation on the expression of cellular constituents and functions of the intestinal epithelium. Nitric oxide (NO) is a signaling molecule, which is involved in inflammation and carcinogenesis. It has been reported that enhanced inducible NO synthase (iNOS) activity and the resulting NO concentrations in human colon carcinoma contribute to tumor progression and vascular invasion. The present study investigates the effect of pro-inflammatory cytokine-induced nitric oxide (NO) production and iNOS expression on the invasion of human colorectal adenocarcinoma HT-29 cells, and the effect of extract from Cnidii Rhizoma on NO production and the invasiveness of HT-29 cells. Treatment of HT-29 cells with cytokines, 100 U/ml interferon γ, 10 ng/ml interleukin-1 α and 25 ng/ml tumor necrosis factor α was found to increase NO production. Pretreatment of the cells with Cnidii Rhizoma (0.1–5 mg/ml) resulted in an inhibition of cytokine-induced NO production and iNOS expression. The invasiveness of HT-29 cells through Matrigel was significantly increased by treatment with cytokines. Cnidii Rhizoma inhibited the invasiveness of cytokine-treated HT-29 cells through the Matrigel-coated membrane in a concentration-dependent manner. Matrix metalloproteinase (MMP) activity in HT-29 cells increased following the treatment with cytokines, and pretreatment of the cells with Cnidii Rhizoma inhibited cytokine-induced MMP-2 activity. These results provide sufficient information for the further development of Cnidii Rhizoma as an antitumor metastatic agent for the treatment of colon cancer.

Protective effects of deep sea water against doxorubicin‑induced cardiotoxicity in H9c2 cardiac muscle cells

Doxorubicin (DOX) is one of the most effective chemotherapeutic agents in the treatment of a variety of tumors. However, its clinical use has been compromised by the risk of cardiotoxicity. Thus, many efforts have been focused on exploring new strategies to prevent or reverse DOX-induced cardiotoxicity. Recently, deep sea water (DSW) has drawn much scientific interest for therapeutic intervention due to its enrichment in nutrients and minerals. In this study, we investigated whether DSW has protective effects against DOX-induced cardiotoxicity. Pre-treatment with DSW significantly increased the viability of DOX-treated rat H9c2 cardiac muscle cells. This protective effect of DSW appears to be mediated through the inhibition of DNA damage rather than suppression of reactive oxygen species (ROS) production in DOX‑treated H9c2 cardiac muscle cells. The inhibitory effect of DSW on DOX-induced DNA damage subsequently attenuated apoptotic signaling such as activation of cysteine-aspartic acid protease-3 (caspase-3) and fragmentation of poly(ADP-ribose) polymerase (PARP), whereas the expression of anti-apoptotic protein B-cell lymphoma-extra large (Bcl-xL) was increased. Moreover, DSW treatment rescued the activation of protein kinase B (Akt) to protect cells from DOX-triggered apoptosis. Taken together, our data showed that DSW has protective effects against DOX-induced cardiotoxicity, suggesting that DSW has some promise as a novel protective supplement for promoting the successful use of DOX in clinical regimen.

Deep Seawater Increases Dendritic Branches of Cultured Rat Hippocampal Neurons

Deep seawater (DSW; deep ocean water) is pure, rich in inorganic materials which have attracted attention for various applications. In this study we investigated the effects of the DSW upwelled from the East Sea, offshore Yang Yang (Korea) on the morphological differentiation of cultured rat hippocampal neurons, which were grown in the minimal essential medium containing 10% (v/v) fetal bo-vine serum and 25% (v/v) DSW with various hardness. DSW had no effect on initial morphological differentiation (17 hr post-plating). When observed on DIV3, 7, 14, and 17, low hardness (0 and 200) DSW reduced dendritic branching. However, dendritic branches within 80 μm diameter from the center of soma nearly doubled in neurons grown in hardness 1,000 DSW-containing media. DSW with hardness 600 was more or less same as control groups. These results indicate that DSW with appropriate hardness ameliorates neuronal health.

Promotion of Synaptic Maturation by Deep Seawater in Cultured Rat Hippocampal Neurons

Deep seawater (DSW) refers to water extracted from the ocean, usually at depths of 200 meters or more, which is rich in inorganic materials and has attracted attention for various applications. We investigated the effects of the DSW on the synaptic maturation of cultured rat hippocampal neurons. Immunocytochemical examination of DIV21 showed that PSD-95, αCaMKII, and synGAPα1 clusters were strengthened and coupling rates of SV2 and NR2B were significantly increased in neurons grown in the presence of H-800 and H-1000 DSW. Our results indicate that DSW promotes the formation of excitatory postsynaptic signal transduction complexes NRC/MASC and functional synapses.

Effects of ATP on the stability of enzymes against heat treatment in 6-aminonicotinamide treated quail.

The stabilities of liver and pectoral muscle enzymes in 6-aminonicotinamide (6-AN) treated quail against heat treatment in the presence and absence of added ATP were investigated. Only ATP level in the brain and pectoral muscle of 6-AN treated group was significantly reduced compared to the control group whereas ADP and AMP levels were not affected. In the thermal stability (55 degrees C) of liver enzymes, the activity of acetylcholinesterase (AChE) was not affected whereas the activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) were significantly lowered (P<0.01). The addition of 1mM ATP to liver enzyme extracts of 6-AN group afforded 4- and 1.7-fold more protection for GAPDH and LDH, respectively (P<0.01). In liver, LDH appeared to be more protected by ATP than GAPDH. In muscle, however, GAPDH and AChE activity were significantly affected but not LDH. The addition of 1mM ATP to muscle enzyme extracts of 6-AN group afforded 1.7-fold more protection for GAPDH (P<0.01) but rather inactivated AChE. A marked reduction in ATP levels in muscle did not affect specifically muscle enzyme activities only since liver enzyme activities were also affected to the same degree as muscle.