Dae-Yong Han

Korean Scutellaria baicalensis water extract inhibits cell cycle G1/S transition by suppressing cyclin D1 expression and matrix-metalloproteinase-2 activity in human lung cancer cells

AIM OF THE STUDY Scutellaria baicalensis Georgi is a widely used medicinal herb in several Asian countries including Korea. The various medicinal properties attributed to Scutellaria baicalensis include anti-bacterial, anti-viral, anti-inflammatory and anti-cancer effects. The present study investigated the cytotoxicity of Scutellaria baicalensis water extract (SBWE) on A549 non-small-cell-lung cancer cells and the A549 expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and matrix metalloproteinase-2 (MMP-2), and the effects of SBWE on cell cycle progression, especially the G1/S phase, and on cell motility. MATERIALS AND METHODS SBWE cytotoxicity was assessed by a standard colorimetric assay utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and expression of cyclin D1 and CDK4 protein in SBWE-treated A549 cells was assessed by Western blot analysis. Flow cytometry analysis was performed to determine the effect of SBWE on A549 cell cycle progression. A549 cell MMP-2 activity was examined by zymography. Cell motility and migration was assessed by a scratch wound healing assay. RESULTS SBWE was not cytotoxic. The production of Cyclin D1, CDK4 and MMP-2 activity were significantly decreased in a SBWE dose-dependent manner, with maximum inhibition occurring at SBWE concentrations of 250 μg/ml and 500 μg/ml. SBWE inhibited cell cycle progression in the G1/S phase and significantly inhibited the motility of A549 cells. CONCLUSIONS Cyclin D1 protein may be associated with MMP-2 activity and cell motility. Thus, SBWE promotes a strong protective effect against MMP-2 mediated metastasis and cell proliferation through the down-regulation of cyclin D1. SBWE may be a useful chemotherapeutic agent for lung cancer.

Suppressive Effect on Lipopolysaccharide-Induced Proinflammatory Mediators by Citrus aurantium L. in Macrophage RAW 264.7 Cells via NF-κB Signal Pathway

Citrus fruits have been used as an edible fruit and a traditional medicine since ancient times. In particular, the peels of immature citrus fruits are used widely in traditional herbal medicine in Korea, as they are believed to contain bioactive components exerting anti-inflammatory activity. This study examined whether the crude methanol extract of Citrus aurantium L. (CME) has a suppressive effect on inducible enzymes and proinflammatory cytokines by inhibiting the NF-κB pathway in LPS-stimulated macrophage RAW 264.7 cells. The cells were pretreated with the indicated concentrations of CME (5, 10, 20, and 50 μg/mL) and then treated with LPS (1 μg/mL). The results showed that CME (10, 20, and 50 μg/mL) inhibited the LPS- (1 μg/mL) induced mRNA and protein expression of iNOS in macrophage Raw 264.7 cells. In addition, the expression of COX-2 was inhibited at the mRNA and protein levels by CME in a dose-dependent manner. The mRNA expression of proinflammatory cytokines, such as TNF-α and IL-6, were markedly reduced by CME (10, 20, and 50 μg/mL). Moreover, CME clearly suppressed the nuclear translocation of the NF-κB p65 subunits, which was correlated with its inhibitory effect on I-κB phosphorylation. These results suggest that CME has anti-inflammatory properties by modulating the expression of COX-2, iNOS, and proinflammatory cytokines, such as TNF-α and IL-6, in macrophage RAW 264.7 cells via the NF-κB pathway.

Characterization and neutralization of Nemopilema nomurai (Scyphozoa: Rhizostomeae) jellyfish venom using polyclonal antibody.

Jellyfish stings have often caused serious health concerns for sea bathers especially in tropical waters. In the coastal areas of Korea, China and Japan, the blooming and stinging accidents of poisonous jellyfish species have recently increased, including Nemopilema nomurai. We have generated a polyclonal antibody against N. nomurai jellyfish venom (NnV) by the immunization of white rabbits with NnV antigen. In the present study, the antibody has been characterized for its neutralizing effect against NnV. At first, the presence of NnV polyclonal antibody has been confirmed from the immunized rabbit serum by Enzyme linked immunosorbent assay (ELISA). Then, the neutralizing activities of the polyclonal antibody have been investigated using cell-based toxicity test, hemolysis assay, and mice lethality test. When the polyclonal antibody was preincubated with NnV, it shows a high effectiveness in neutralizing the NnV toxicities in a concentration-dependent manner. Moreover, we explored proteomic analyses using 2-D SDS-PAGE and MALDI-TOF mass spectrometry to illustrate the molecular identities of the jellyfish venom. From this, 18 different protein families have been identified as jellyfish venom-derived proteins; the main findings of which are matrix metalloproteinase-14, astacin-like metalloprotease toxin 3 precursor. It is expected that the present results would have contributed to our understandings of the envenomation by N. nomurai, their treatment and some valuable knowledge on the pathological processes of the jellyfish stinging.

Proteomic Analysis of Effects on Natural Herb Additive Containing Immunoglobulin Yolksac (IgY) in Pigs

Thirty male pigs were infected orally with E. coli and Salmonella typhimurium, and divided into a control group and two additive groups to determine the effect of an additive mixture on the changes in protein expression. The pigs were given a food supplemented with a natural herbal additive containing immunoglobulin yolksac (IgY) at concentrations of 0.5% or 1%. On the 1st day and after eight weeks of feeding, the body weight gain, food intake and serum GOT/GPT levels were examined. The GOT/GPT levels on the 1st day were similar in the three groups. However, after eight weeks of feeding, the GOT level was significantly lower in the additive treatment groups (0.5% and 1.0%). In addition, the changes in the spleen proteome as a response to the herbal additive were examined using two-dimensional polyacrylamide gel electrophoresis. A total of 31 differentially expressed protein spots were identified by comparing the protein profiles of the control and additive treated porcine spleens. Finally, 19 proteins were detected by MALDI-TOF/MS. Overall, the proteins detected are involved in a range of biological process, such as metabolic processes, biological processes, transport, carbohydrate metabolic processes, generation of precursors and energy. In conclusion, these results support of the hypothesis that a natural herbal additive containing IgY can affect the immune regulation system and reduce the stress of microbial infections.

Proteome Analysis of the Anti-inflammatory Response of Flavonoids Isolated from Korean Citrus aurantium L. in Lipopolysaccharide- Induced L6 Rat Skeletal Muscle Cells

Skeletal muscle is an important organ in our body and a dynamic composite of proteins. Citrus aurantium L. has been widely used in oriental medicine in Eastern Asia for a long time. It contains over 100 bioactive compounds and flavonoids that regulate the inflammatory response and tumorigenesis, through various mechanisms. In the present study, we investigated changes in the protein pattern using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectroscopy (MALDI-TOF/MS) to assess the anti-inflammatory effect of flavonoids isolated from Korean C. aurantium L. in lipopolysaccharide (LPS)-induced L6 cells. L6 skeletal muscle cells were pretreated with flavonoids for 1 h and stimulated with LPS for 24 h. Proteins from the L6 cells of the control, LPS treated and flavonoid treated groups were extracted and resolved by 2-DE using pH 4-7 IPG strips loaded with 150 μg of protein. Forty-one differentially expressed protein spots were identified (more than two-fold was considered significant, p < 0.05), and 18 were detected by MALDI-TOF/MS. These results suggest that proteomics can be used to identify changes in the expression of marker proteins and the anti-inflammatory effect of flavonoids isolated from Korean C. aurantium L.

Hypothyroidism induced by polychlorinated biphenyls and up-regulation of transthyretin.

Polychlorinated biphenyls are environmental pollutants that are toxic to many biological systems. This study examined whether or not PCB126 and PCB114 have adverse effects on the serum thyroxine level and the serum proteome in rats. The results showed a lower serum total thyroxine level in the PCB126 and PCB114-treated groups than the control. Western blotting showed that the levels of transthyretin expression were significantly higher in the PCB-treated group than the control group. These results suggest that the PCB-mediated hypothyroidism is caused by the displacement of thyroxine from transthyretin.

Physiological Function of Insoluble Dietary Fiber Prepared from Exploded Oak Wood (Quercus mongolica)

This study investigated the production of insoluble dietary fiber using exploded and chemically treated oak wood (Quercus mongolica) and the physiological functions of prepared insoluble dietary fiber in laboratory animals. To produce high quality insoluble dietary fiber, the steam explosion treatment was performed at 25 kgf/cm2 pressure for 6 minutes. In the chemical analysis of insoluble dietary fiber, exploded oak wood was pretreated by 1% sodium hydroxide solution. The insoluble dietary fiber contained 7.6% residual lignin and 61.7% of alpha-cellulose. In order to compare the physiological functions of prepared insoluble dietary fiber with those of commercial insoluble dietary fiber, Sprague-Dawley male rats weighing 100 +/- 10 g were randomly assigned to one normal diet and five high cholesterol diets, containing 1% cholesterol. The high cholesterol diet groups were classified as the fiber-free diet (FF group), 5% commercial alpha-cellulose diet group (5C group), 10% commercial alpha-cellulose group (10C group), 5% insoluble dietary fiber group (5M group) and 10% insoluble dietary fiber group (10M group). Food intake, weight gain and food efficiency ratio in high cholesterol groups were significantly higher than those of the normal group, but there were no significant differences among the high cholesterol diet groups. In addition, there were no significant differences in the weights of liver, kidney and small intestine in insoluble dietary fiber-supplemented groups. Cecum weights in all insoluble dietary fiber groups were significantly higher than those of the FF group. There were no significant differences in the activities of the glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) among the insoluble dietary fiber-supplemented groups. In conclusion, the prepared insoluble dietary fiber and the commercially available insoluble fiber showed the same physiological effects. Moreover, the preparation method for the insoluble dietary fiber from the exploded oak wood was successful.