Mirim Jin

Gastroprokinetic effects of DA-9701, a new prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber.

To develop a therapeutic for functional dyspepsia (FD), a prokinetic agent, DA-9701 has been newly formulated with Pharbitis Semen and Corydalis Tuber and we evaluated the gastroprokinetic effects of DA-9701 in comparison with conventional prokinetics. Oral administration with DA-9701 not only significantly accelerated gastric emptying in normal rats but also restored the delayed gastric emptying caused by apomorphine and cisplatin up to almost normal levels. For gastrointestinal transit, DA-9701 caused FITC-dextran to travel a significantly longer distance than the control, and in the delayed GI transit models induced by laparotomy and atopine, DA-9701 considerably increased the values of mean geometric center (MGC), while the conventional prokinetics rarely showed significant effects. Furthermore, DA-9701 drastically increased the gastric accommodation in Beagle dogs, shifting the pressure-volume curve toward considerably higher volume compared to the control, which was comparable to that of cisapride. These results indicate that DA-9701 has potential as a safe and effective therapeutic for FD patients with abnormalities in GI motor function.

Activation of selective transcription factors and cytokines by water-soluble extract from Lentinus lepideus.

We isolated a water-soluble extract, PG101, from cultured mycelia of Lentinus lepideus. Treatment of human peripheral blood mononuclear cells (PBMCs) with PG101 increased levels of TNF-α, IL-1β, IL-10, and IL-12 by 100- to 1000-fold, whereas GM-CSF and IL-18 were activated by an order of magnitude. On the contrary, IFN-γ and IL-4 were not affected. The response to PG101 occurred in a dose- and time-dependent manner. From the human PBMCs treated with PG101, TNF-α was a first cytokine to be activated, detectable at 2 hr post-treatment followed by IL-1β at 6 hr post-treatment. IL-12 and IL-10 were the next to follow. GM-CSF and IL-18 both showed significant increases 24 hr after treatment. When PBMCs were sorted into various cell types, monocyte/macrophages, but not T and B cells, were the major target cell type responsive to PG101. Consistent with this result, the profile of cytokine expression upon PG101 treatment was comparable between PBMCs and a human promonocytic cell line (U937), whereas cell lines of T cell and myeloid origins did not respond to PG101. Data from a transient transfection assay involving specific reporter plasmids indicated that cellular transcription factor such as NF-κB, but not AP-1, was highly activated by PG101. Results from a gel retardation assay and the experiment involving a specific NF-κB inhibitor confirmed the involvement of NF-κB. Despite its significant biological effect on various cytokines, PG101 remained nontoxic in both rats and PBMCs even at a biological concentration approximately 20 times greater. PG101 demonstrates great potential as a therapeutic immune modulator.

Inhibition of LPS-induced NO production and NF-κB activation by a sesquiterpene fromSaussurea lappa

To elucidate the molecular mechanisms for the suppression of LPS-induced nitric oxide (NO) production by a dehydrocostus lactone (DL) fromSaussurea lappa, we examined the preventive effect of this compound on NF-κB activation in LPS-treated RAW 264.7 macrophages and U937 human monocytic cells. The results suggest that the suppression of NO production is mediated by the inhibitory action on the i-NOS gene expression through the inactivation of NF-κB and this sesquiterpene lactone can act as a pharmacological inhibitor of the NF-κB activation.

Mycelial protoplast isolation and regeneration of Lentinus lepideus

Generation of fungal protoplast is essential for fusion and transformation systems. Protoplast fusion offers great potential for the improvement of industrially important microorganisms. To establish conditions for the protoplast isolation and regeneration of the mycelia of Lentinus lepideus, various enzymes and osmotic stabilizers were examined. To investigate suitable medium for the culture of L. lepideus, the mycelia were grown in ten different media at 28 degrees C for 10 days. Among them potato dextrose agar (PDA) medium was found to be the best for colony growth. When Novozym 234, cellulase and beta-glucuronidase were added to the mycelia in combination or alone, Novozym 234 alone at the concentration of 10 mg/ml was the most effective for the protoplast yield. Purified spherical protoplasts of the mycelia were osmotically hypersensitive and further incubation of the mycelia with the lytic enzyme resulted in the older parts of the hyphae swollen. When we applied various osmotic stabilizers at the fixed concentration of 0.6 M on the protoplasts, the yields of protoplasts were increased until 4-hr incubation. However application of sucrose or MgSO4 led to further protection of protoplasts after that time and reached a plateau on 5- and 7-hr incubations, respectively. The suitable incubation time and optimal pH with the lytic enzyme for the maximum release of protoplasts were 6 hrs of incubation and pH 5, respectively. When we examined various osmotic stabilizers for the regeneration of the protoplast, the complete medium containing 0.6 M sucrose induced highest hyphal growth with regeneration frequency of 3.28%.

Tetrahydroberberine, an Isoquinoline Alkaloid Isolated from Corydalis Tuber, Enhances Gastrointestinal Motor Function

Because delayed gastric emptying and impaired gastric accommodation are regarded as pathophysiological mechanisms underlying functional dyspepsia (FD), prokinetics and fundic relaxants have been suggested as a new treatment for FD. We isolated tetrahydroberberine (THB), an isoquinoline alkaloid (5,8,13,13a-tetrahydro-9,10-dimethoxy-6H-benzo[g]-1,3-benzodioxolo[5,6-a]quinolizine) from Corydalis tuber, and found that it has micromolar affinity for dopamine D2 (pKi = 6.08) and 5-HT1A (pKi = 5.38) receptors but moderate to no affinity for other relevant serotonin receptors (i.e., 5-HT1B, 5-HT1D, 5-HT3, and 5-HT4; pKi < 5.00). Oral administration of THB not only resulted in significantly accelerated gastric emptying of normal rats in a bell-shaped relationship, with a maximal efficacy at a dose of 30 μg/kg, but also restored the delayed gastric emptying caused by apomorphine, which might be mediated by an antidopaminergic effect. Data from electromyography indicated enhanced motor function of the upper gastrointestinal tract by THB, which occurred through strengthening contractility and shortening the contraction interval. Furthermore, in rats stressed by repeated restraint, a significantly higher shift in the pressure-volume curve by THB (10 μg/kg, p < 0.05), which was inhibited by [O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY-100635), a 5-HT1A antagonist, and Nω-nitro-l-arginine methyl ester, a nitric-oxide synthase inhibitor but not a vasoactive intestinal peptide antagonist, was observed. Oral administration of THB resulted in a drastic increase of gastric accommodation in Beagle dogs. Area under the volume versus time curve was increased significantly by THB (30 μg/kg, p < 0.01) and comparable with that of sumatriptan (3 mg/kg), a potent fundic relaxant. Taken together, our data suggested that THB, with D2 receptor antagonist and 5-HT1A receptor agonist properties, has significant potential as a therapeutic for treatment of FD.

Induction of pacemaker currents by DA-9701, a prokinetic agent, in interstitial cells of Cajal from murine small intestine

The interstitial cells of Cajal (ICC) are pacemaking cells required for gastrointestinal motility. The possibility of whether DA-9701, a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, modulates pacemaker activities in the ICC was tested using the whole cell patch clamp technique. DA-9701 produced membrane depolarization and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid, a non-selective cation channel blocker, but not niflumic acid, abolished the generation of pacemaker currents induced by DA-9701. Pretreatment with a Ca2+-free solution and thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum, abolished the generation of pacemaker currents. In addition, the tonic inward currents were inhibited by U-73122, an active phospholipase C inhibitor, but not by GDP-β-S, which permanently binds G-binding proteins. Furthermore, the protein kinase C inhibitors, chelerythrine and calphostin C, did not block the DA-9701-induced pacemaker currents. These results suggest that DA-9701 might affect gastrointestinal motility by the modulation of pacemaker activity in the ICC, and the activation is associated with the non-selective cationic channels via external Ca2+ influx, phospholipase C activation, and Ca2+ release from internal storage in a G protein-independent and protein kinase C-independent manner.

Ergosterol peroxide from Chaga mushroom (Inonotus obliquus) exhibits anti-cancer activity by down-regulation of the β-catenin pathway in colorectal cancer.

AIM OF THE STUDY In this study, we examined the effect of different fractions and components of Chaga mushroom (Inonotus Obliquus) on viability and apoptosis of colon cancer cells. Among them, one component showed the most effective growth inhibition and was identified as ergosterol peroxide by NMR analysis. We investigated the anti-proliferative and apoptosis mechanisms of ergosterol peroxide associated with its anti-cancer activities in human colorectal cancer (CRC) cell lines and tested its anti-tumor effect on colitis-induced CRC developed by Azoxymethane (AOM)/Dextran sulfate sodium (DSS) in a mouse model. MATERIALS AND METHODS We used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, flow cytometry assays, Western blot analysis, colony formation assays, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and AOM/DSS mouse models to study the molecular mechanism of metastatic activities in CRC cells. RESULTS Ergosterol peroxide inhibited cell proliferation and also suppressed clonogenic colony formation in HCT116, HT-29, SW620 and DLD-1 CRC cell lines. The growth inhibition observed in these CRC cell lines was the result of apoptosis, which was confirmed by FACS analysis and Western blotting. Ergosterol peroxide inhibited the nuclear levels of β-catenin, which ultimately resulted in reduced transcription of c-Myc, cyclin D1, and CDK-8. Ergosterol peroxide administration showed a tendency to suppress tumor growth in the colon of AOM/DSS-treated mice, and quantification of the IHC staining showed a dramatic decrease in the Ki67-positive staining and an increase in the TUNEL staining of colonic epithelial cells in AOM/DSS-treated mice by ergosterol peroxide for both prevention and therapy. CONCLUSION Our data suggest that ergosterol peroxide suppresses the proliferation of CRC cell lines and effectively inhibits colitis-associated colon cancer in AOM/DSS-treated mice. Ergosterol peroxide down-regulated β-catenin signaling, which exerted anti-proliferative and pro-apoptotic activities in CRC cells. These properties of ergosterol peroxide advocate its use as a supplement in colon cancer chemoprevention.

Enhancement of repopulation and hematopoiesis of bone marrow cells in Irradiated mice by oral administration of PG101, a water-soluble extract from Lentinus lepideus

PG101 is a water-soluble extract from Lentinus lepideus. It is a potential biological response modifier that activates selective cytokines in vitro, mainly by controlling cellular transcription factor NF-κB. Effects of PG101 were tested on bone marrow cells in irradiated mice. Mice were irradiated with a dose of 6 Gy and were given PG101 by gavages daily for 24 days. In PG101-treated mice, the number of colony-forming cells, including colony-forming units (CFU)-granulocytes/macrophages (GM) and erythroid burst-forming units (BFU-E), were increased to almost the levels seen in nonirradiated control as early as 8 days after irradiation. Two-color flow cytometric analysis using antibodies to ER-MP12 and ER-MP20 suggested that in the bone marrow cell population, PG101 increased the number of granulocytes (ER-MP12-20med) and myeloid progenitors (ER-MP12+20+). Analysis of surface c-Kit and Gr-1 proteins in bone marrow cells indicated that PG101 might induce differentiation of progenitor cells to granulocytes and/or proliferation of the committed cells. Lastly, oral administration of PG101 highly increased serum levels of GM-CSF, IL-6, and IL-1β. Interestingly, the level of TNF-α was elevated by irradiation in control mice, but was maintained at the background level in PG101-treated mice, suggesting that PG101 might effectively suppress TNF-α-related pathologic conditions. Our results strongly suggest the great potential of PG101 as an immune enhancer during radiotherapy and/or chemotherapy.

TNF-α increases lipogenesis via JNK and PI3K/Akt pathways in SZ95 human sebocytes

BACKGROUND Tumor necrosis factor-alpha (TNF-α) is an important pathophysiologic factor involved in the development of acne. However, its role is unclear. OBJECTIVE To explore the lipogenic effect by TNF-α and possible molecular mechanisms in sebocyte. METHODS Using SZ95 human sebocytes, lipid formation by TNF-α was assessed by Oil Red O, Nile Red staining and thin layer chromatography (TLC). Expression of lipogenic genes and activation of mitogen-activated protein kinase as well as Akt were examined by real-time polymerase chain reaction and/or Western blot analysis. Activation of peroxisome proliferator-activated receptor (PPAR) was evaluated by luciferase assay using PPAR response element containing reporter plasmids. Involvement of c-Jun N-terminal kinase (JNK) and Akt in TNF-α-induced lipogenesis was investigated by molecule specific small interfering RNA and inhibitors. RESULTS TNF-α treatment significantly increased formation of lipid droplets in accordance with up-regulated expression of FAS and activation of SREBP-1, but not PPARs. Suppression of phosphorylated JNK by the JNK inhibitor SP600125 greatly diminished TNF-α-induced expression of FAS and SREBP-1. TNF-α could not induce both expression of lipogenic proteins and lipid synthesis when Akt expression was attenuated with siRNA. CONCLUSIONS TNF-α induces lipogenesis in SZ95 human sebocytes through the JNK and phosphoinositide-3-kinase/Akt pathways. These results will be valuable in developing therapeutic strategies for control of seborrhea and acne.

Blockade of Atopic Dermatitis-Like Skin Lesions by DA-9102, a Natural Medicine Isolated from Actinidia arguta, in the Mg-Deficiency Induced Dermatitis Model of Hairless Rats

DA-9102 isolated from Actinidia arguta is a candidate of natural medicine currently under Phase II clinical trial for atopic dermatitis in Korea. In this study, spontaneous dermatitis was induced by magnesium deficiency in hairless rats and this system was applied to assess the suppressive effects of DA-9102 on atopic dermatitis-like skin disease. Oral administration of DA-9102 at a dose of 100 mg/kg for 16 days substantially suppressed the occurrence of spontaneous dermatitis. Eczematous skin lesions, water loss and scratching behavior were significantly decreased by DA-9102 in a dose-dependent manner. Infiltration of inflammatory cells into the skin and pathologic remodeling of the epidermis and dermis were much less than the Mg-def. group. Results from flow cytometry analysis of peripheral blood mononuclear cells indicated that DA-9102 suppressed activation of leukocytes. The decrease in the number of CD45RA+ cells was accompanied by a lower level of IgE in DA-9102 treated rats, and the reduction in the number of CD11b+ cells by DA-9102 in both periphery and skin was significant. Further, DA-9102 not only suppressed the mRNA expression of TH2 cytokines including IL-4 and IL-10 in the lymph node but it also decreased the levels of inflammatory mediators such as nitric oxide and leukotriene B4 (LTB4) in the serum. Taken together, these results suggest that DA-9102 is an orally applicable potent immune modulator capable of controlling the occurrence of atopic dermatitis-like skin disease.