• Herbal Formula Science
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• v.10 no.2
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• pp.127-141
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• 2002

Objectives: Haeganjeon(HGJ) has been used for the treatment of liver disease in traditional medicine. The present study was carried out to evaluate the antioxidant and protective effects of HGJ extract on oxidative damage of hepatocytes by tert-butyl hydroperoxide(t-BHP). Methods: In the linoleic acid water-alcohol system, the levels of lipid peroxide(LPO) were determined by TBA method. The scavenging effect of HGJ on ${\alpha},{\alpha}-diphenyl-{\beta}-picrylhydrazyl$(DPPH) radical was determined according to the method of Hatano. In the Fenton system(ferrous ion reaction with hydrogen peroxide), the levels of hydroxyl radical induced LPO in rat liver homogenate were determined according to the method of TBA. Inhibitory effect of HGJ on superoxide generation was measured by xanthine-xanthine oxidase system. In order to evaluate antioxidative activity of HGJ in the liver cell, cultured normal rat liver cells(Ac2F) were prepared and incubated with or without HGJ. After 18hr, cells placed in DMEM medium without serum, and then incubated with 1mM tert-butyl hydroperoxide(t-BHP) for 2hrs. Viable cells were detected by MTT assay. Conclusions: In the linoleic acid autoxidation system, HGJ extract significantly inhibited the time course of the lipid peroxidation. These effects were similar to those of BHA HGJ extracts showed about 70% scavenging effect on DPPH radical. And HGJ extract inhibited the lipid peroxide formation in rat liver homogenate induced by hydroxyl radical derived from Fenton system. In addition, HGJ extract protected the cell death induced by t-BHP and significantly increased cell viability in the normal rat liver cell. These result indicated that HGJ extract might playa protective role against oxidative hepatic cell injury by means of free radical scavenger.

• Animal cells and systems
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• v.1 no.4
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• pp.641-646
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• 1997

Lysophosphatidic acid (LPA; 1-acyl-glycerol-3-phosphate) has been known as an intercellular phospholipid messenger with a wide range of biological activities. In this study, the effect of LPA on both the proliferation and differentiation of rat E63 myoblasts has been investigated. In the serum-free Insulin-Transferrin-Selenium (ITS) media, the proliferation of E63 cells was largely restricted. Addition of LPA into the ITS media strongly promoted the cell proliferation and resulted in two to four fold increase of cell number. Furthermore, it appeared to increase the percent fusion in a dose-dependent manner up to 15 ug/ml. The synthesis of myosin heavy chain (MHC) was increased by LPA as well. These results indicate that LPA is able to promote both cell proliferation and differentiation in rat E63 myoblasts. Suramin, known to have uncoupling activity on growth factor-receptor interaction, was tested for antagonistic activity in myoblast proliferation and differentiation. Myoblasts grown in the ITS medium containing LPA were able to proliferate well even in the presence high concentration of suramin whereas myoblast differentiation was completely blocked by 30 ug/ml of suramin. The inhibitory effect of suramin on the myoblast differentiation was completely reversible by removing the suramin. This result indicates that the intracellular signaling pathway of LPA leading to cell proliferation might be distinct from that leading to cell differentiation on E63 myoblasts. Also, the antagonistic effect of suramin suggests that the differentiation activity elicited by LPA might be mediated by a specific G protein-coupled receptor.

• Journal of Ginseng Research
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• v.29 no.4
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• pp.159-166
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• 2005

Ginsenosides, a group of Panax ginseng saponins, exert the lowering effects of plasma cholesterol levels in animals. We had reported earlier that ginsenoside Rb2 upregulate low-density lipoprotein receptor (LDLR) expression via a mechanism that is dependent of the activation of sterol response element binding protein 2 (SREBP-2) expression. This study was conducted to determine the effects of ginsenoside Rb2 on the expression of the hepatic LDLR expression at cellular levels using HepG2 cells, and to evaluate whether the sterol response element binding protein 1 (SREBP-l) was involved in the regulation of LDLR expression. Incubation of HepG2 cells in serum-free medium supplemented with cholesterol $(10{\mu}g/ml)$ for 8 hours decreased the mRNAs of LDLR mRNA by $12\%$ and SREBP-l mRNA by $35\%$. Ginsenoside Rb2 antagonized the repressive effects of cholesterol and increased both LDLR and SREBP-l mRNA expression to 1.5- and 2-fold, respectively. Furthermore, Western blot and confocal microscopic analyses with SREBP-l polyclonal antibody revealed that ginsenoside Rb2 enhanced the maturation of the SREBP-1 from the inactive precursor form in ER membrane to the active transcription factor form in nucleus. These results suggest that ginsenoside Rb2 upregulates LDLR expression via a mechanism that is dependent of the activation of not only SREBP-2 expression, but also SREBP-1 expression and maturation, and also indicate that the pharmacological value of ginsenoside Rb2 may be distinguished from that of lovastatin which is reported that it upregulate LDLR through SREBP-2 only, not through SREBP-1.

• Journal of Life Science
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• v.28 no.10
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• pp.1147-1155
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• 2018

Agaricus blazei mycelial liquid culture extract (ABMLCE) promoted the production of testosterone (TS) in TM-3 mouse Leydig testis cells. Now, we report that ABMLCE containing eritadenine (EA) as a minor constituent (15.3 mg/100 g) reduced $5{\alpha}-reductase$ 2 ($5{\alpha}-R2$) enzyme activity and dihydrotestosterone (DHT) content which are key constituents for the benign prostatic hyperplasia (BPH) inductions. RWPE-1 prostate cells were grown in a Keratinocyte serum-free medium (K-SFM) containing ABMLCE (0~50 ppm), EA (0~10 ppm,), and finasteride (FS $10{\mu}M$: a positive control) in a 24-well plate for 24 hr. Supernatants collected from cell-cultured media were used for the assay of $5{\alpha}-R2$, superoxide dismutase (SOD), catalase (CAT) and cyclooxygenase-2 (COX-2) enzyme activities, and for TS, DHT, tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$) and $interleukin-1{\beta}$ ($IL-1{\beta}$) contents by their assay kits. The $5{\alpha}-R2$ activity and DHT content were proportionally reduced (p<0.05) to concentrations of ABMLCE. The SOD and CAT enzyme activities were significantly (p<0.05) elevated concomitant with ABMLCE concentrations, while COX-2, $TNF-{\alpha}$ and $IL-1{\beta}$ showed reverse results (p<0.05). Similarly, the effects of EA were similar to those of ABMLCE. Efficacies of ABMLCE 50 ppm and EA 10 ppm in $5{\alpha}-R2$ and DHT reduction were similar to those of $10{\mu}M$ FS. These results suggest that ABMLCE and EA reduced $5{\alpha}-R2$ and DHT through their anti-inflammatory and anti-oxidative actions. This implies that ABMLCE containing EA could be a beneficial material in the cure of BPH in humans.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.269-279
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• 2000

The present study was carried out to evaluate the effect of glycosaminoglycans added to the culture medium on the mouse embryo development to the blastocyst stage. In vitro fertilized mouse oocytes were cultured in Ham's F-10 supplemented with 10% FBS either in the absence or presence of 0.1, 0.5, 1.0 mg/$m\ell$ hyaluronic acid, chondroitin sulfate, and dermatan sulfate, respectively. After 4 days in culture, embryos developed to blastocysts were observed in all groups. There was a significant increase in blastocyst yield in the presence of hyaluronic acid and chondroitin sulfate (p<0.05), whereas dermatan sulfate was ineffective. Development to the blastocyst stage was best supported in 0.1, 0.5, 1.0 mg/$m\ell$ hyaluronic acid and 0.5mg/$m\ell$ chondroitin sulfate. It is concluded that hyaluronic acid and chondroitin sulfate support the development of mouse oocyte fertilized in vitro to the blastocyst stage. Furthermore, these results suggest that glycosaminoglycans can be utilized to support embryo development in vitro as a nutrient instead of serum.

• Journal of the Korean Society of Food Science and Nutrition
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• v.34 no.6
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• pp.743-749
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• 2005

Estrogen is known to play an important role in maintaining bone mass, since the concentration of serum estrogen decrease after menopause and the estrogen deficiency results in bone loss. Phytoestrogens are plant compounds with estrogen-like biological activity, In this study, to investigate the bioactivities of phytoestrogen, which act on bone metabolism, we examined the effect of selected food-borne phytoestrogens (genistein, daidzein and resveratrol) on osteoblast proliferation and IGF-I production using MC3T3-El cells, a mouse calvaria osteoblast-like cell line. Cells were cultured in a serum free medium for 48 hr in the presence of genistein $(10^{-5}\;M)$, daidzein $(10^{-5}\;M)$ and resveratrol $(10^{-5}\;M)$. The effects of genistein, daidzein and resveratrol on the cell proliferation and growth were evaluated by total cell numbers, MTS assay and cell migration assay. Their effect was compared with the $17\beta-estradiol$. Genistein, daidzein and resveratrol exhibited stimulatory effects on the growth of MC3T3-El cells, and the most pronounced effect was shown with daidzein. In addition, these phytoestrogen increased alkaline phosphatase activity of MC3T3-El cells. These effects were similar to that of $17\beta-estradiol$ effects. Moreover, treatment with genistein, daidzein and resveratrol increased production of insulin like growth factor-I (IGF-I) in conditioned media, indicating that the growth promoting effects of these phytoestrogen were related to the changes in production of IGF-I by MC3T3-El cells. These results show that genistein, daidzein and resveratrol have a stimulatory effect on osteoblast function, and that these findings in a cell model may prove relevant to protecting against the loss of bone mass and the development of osteoporosis in human subjects.

• Journal of Life Science
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• v.17 no.5 s.85
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• pp.687-693
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• 2007

Insulin-like growth factor-I(IGF-I) has significant insulin-like anabolic effects which include the stimulation of glucose and amino acid uptake, as well as protein and glycogen synthesis. IGFs exist in serum and other biological fluids as complexes bound to a family of structurally related insulin-like growth factor binding proteins(IGFBPs). Six human IGFBPs can modulate the effects of IGFs on target tissues by several mechanisms, including altering the serum's half-life and the transcapillary transport of IGFs, as well as changing the availability of IGFs to specific cell surface receptors. Human fibroblasts secrete IGFBPs that can modify IGF-I action. Previous to our study using either Northern blotting, and Western blotting have shown that fibroblasts express mRNA IGFBP-3, -4, and -5, and synthesize these proteins. In addition, fibroblast cell lysates revealed that the IGFBP-3 was most abundant. For these reasons, we undertook to gain further insight into the effects of high and low glucose incubation condition on metabolism and IGFBP-3 expression. In results of metabolites and IGFBP-3 expression in GM10 cells cultivated with various glucose concentration, the consumption of glucose and accumulation of triglyceride were increased in condition of high glucose, and total protein level was decreased. in the course of time. After 5 days incubation, levels of free amino acid in medium containing glucose of high concentration glucose were higher than in conditions of low glucose. Although the levels of IGFBP-3 protein and mRNA levels were increased in low glucose, and IGFBP-3 was not affected by any pretense. Taken together, we suggest that the study of growth factors, like IGFs, might be a possible model of diabetes militus in cell, although the results in cell models were not in accord with in vivo.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.155-163
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• 2004

The aims of this study are 1) to test oocytes and embryos collected from in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to Funahashi et al (1994). Glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at $39^{\circ}C$, and 10% fetal bovine serum albumin was added to the culture medium thereafter. Embryos were treated with 7.5 ${\mu}g/ml$ cytochalasin-B for 30 min, centrifuged at 13,000 rpm for 13 min and then exposed sequentially to an ethylene glycol(EG) vitrification solution, aspirated into OPS, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three dornors after AI for control group. Forty-nine embryos were washed 3 times in mPBS + 10% FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients were transferred individually with 100, 100 frozen embryos derived from abattoir and 34 fresh embryos by surgically, and another three recipients were transferred individually with 150, 150 frozen embryos and 100 fresh embryos by nonsurgically, respectively. all recipient sows exhibited delayed returns to estrus. To our knowledge, theses results suggest that required an improved techniques, more vigorous embryos preparation and substitute to gilt with cleaner uterous condition.

• Journal of Periodontal and Implant Science
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• v.24 no.2
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• pp.219-237
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• 1994

The ultimate goal of clinical periodontal therapy is to achieve regeneration of a healthy connective tissue reattachment. Conventional therapy including scaling, root planing, gingival curettage, gingivectomy and flap procedures of various types results primarily in repair rather than regeneration of the periodontium. In order for periodontal regeneration to occur, progenitor periodontal ligament cells must migrate to the denuded root surface, attach to it, proliferate and mature into an organized and functional fibrous attachment apparatus. Polypeptide growth factors belong to a class of potent biologic mediators which regulate cell differentiation, proliferation, migration and metabolism. Insulin-like growth factor-I (IGF- I ) of these factors appear to have an important role in periodontal wound healing and bone formation. The purpose of this study is to evaluate the effects of IGF- I on the periodontal ligament cells to use as a regeneration promoting agent of periodontal tissue. Human periodontal ligament cells were obtained from periodontal tissue explants culture of the first premolar tooth extracted for the orthodontic treatment. Cells were cultured in Dulbecco's modified Eagle medium(DMEM) with 10% fetal bovine serum. Fourth to seventh passage cells were plated in 24 well tissue culture plates and medium changed to serum-free medium prior to addition of growth factors. Cell proliferation was measured by the incorporation of $[^3H]-thymidine$ into DNA, Protein synthesis was determined by measurement of $[^3H]-proline$ incorporation into collagenase-digestible protein(CDP) and noncollagenous protein(NCP) according to the method of Peterkofsky and Diegelmann (1971), And alkaline phosphatase activity was measured as one parameter of osteoblastic differentiation. The results were as follows : The DNA synthetic activity was increased in a dose-dependent manner with IGF- I except for 0.1ng/ml concentration of IGF- I At the concentration of 10, 100ng/ml, IGF- I significantly increased the DNA synthetic activity(P<0.05) The total protein, collagen and noncollagen synthesis was increased in a dose-dependent manner with IGF- I except for 0.1ng/ml concentration of IGF- I. At the concentration of 1, 10, 100ng/ml, IGF- I significantly increased the total protein, collagen and noncollagen synthesis activity(P<0.95, P<0.001). The % of collagen was not effected according to the concentration of IGF- I. The alkaline phosphatase activity was increased in a dose-, time-dependent manner with IGF- I (10, 100ng/ml). In conclusions, the present study shows that IGF- I has a potentiality to enhance the DNA synthesis of periodontal ligament cells with including the increase of the total protein and collagen synthetic activity. The use of IGF- I to mediate biological stimulation of periodontal ligament cells shows promise for future therapeutic applications.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.2
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• pp.151-157
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• 2003

Obtaining a sufficient amount of healthy keratinocytes from a small tissue is difficult. However, ORS cells can be a good source of epithelium since they are easily obtainable and patients do not have to suffer from scar formation at donor sites. Accordingly, the current study modified the conventional primary culture technique to overcome the low propagation and easy aging of epithelial cells during culturing. In a conventional primary culture, the average yield of human ORS tells is 2.↑ $\times$ 10$^3$cells/follicle based on direct incubation in a trypsin (0.1%)/EDTA(0.02%) solution for 15 min at 37$^{\circ}C$, however, our modified method was able to obtain about 6.9 $\times$ 10$^3$cel1s/follicle using a two-step enzyme digestion method involving dispase (1.2 U/mL) and a trypsin (0.1%)/EDTA (0.02%) solution. Thus, the yield of primary cultured ORS cells could be increasd three times higher. Furthermore, a total of 2.0 $\times$ 10$^{7}$ cells was obtained in a serum-free medium. while a modified E-medium with mitomycin C-treated feeder tells produced a total of 6.3 $\times$ 10$^{7}$ Cel1s over 17 days When Starting With 7.5 $\times$ 10$^4$cells. Finally, We Confirmed the effectiveness of our ORS tell isolation method by presenting their ability for reconstructing the bioartificial skin epithelium in vitro