Effect of different ingredients in traditional Korean medicine for human uterine leiomyoma on normal myometrial and leiomyomal smooth muscle cell proliferation.

Crude water extracts of 13 traditional Korean medicinal ingredients used for leiomyomal treatment were prepared and used to treat human uterine normal myometrial and leiomyomal cell cultures. All the ingredients inhibited proliferation and altered the morphology of both myometrial and leiomyomal cells. Among the 13 ingredients, n-hexane-, chloroform-, and ethylacetate-soluble fractions were extracted from seven ingredients that potently inhibited cell proliferation in their water extract form. Among these, the ethylacetate-fraction of Phlomis umbrosa and Spatholobus suberectus, and the chloroform-fraction of Curcuma zedoaria and S. suberectus inhibited leiomyomal cell proliferation significantly compared to myometrial cell proliferation. Similarly, immunohistochemical analysis showed the inhibition of transforming growth factor-beta receptor 2 in leiomyomal tissue after treatment with the fractions of the ingredients. Moreover, the chloroform-fraction of C. zedoaria was subfractionated by open column chromatography. Two of the eight subfractions (fractions 6 and 7) potently inhibited cell proliferation in leiomyoma compared to myometrium. Further study will be performed with the goal of isolating specific compounds from two effective subfractions of C. zedoaria, ethylacetate-fraction of P. umbrosa, and the ethylacetate and chloroform-fractions of S. suberectus. The present study may be helpful in developing an alternative remedy to leiomyoma with minimal side-effects compared to the current treatments.

Antiproliferative effect of Scutellaria barbata D. Don. on cultured human uterine leiomyoma cells by down-regulation of the expression of Bcl-2 protein.

Scutellaria barbata D. Don (Lamiaceae; SB) inhibited the growth of leiomyomal cells (LM). A time-dependent antiproliferative effect was noted when 10(-5) m buserelin, gonadotrophin-releasing hormone (GnRH) agonist or 20-40 microg/mL SB was added. The inhibition of cell growth decreased with the addition of the PKC activator (12-O-tetradecanoylphorbor-13-acetate; TPA) much as it did with the addition of SB, and the decreases in the viable cells caused by the addition of SB were reversed completely by pretreatment with a protein kinase C (PKC) inhibitor (calphostin C). The findings suggest that SB inhibits cell proliferation in cultured human uterine leiomyoma cells accompanied by PKC activation. Next, the study investigated the effect of SB on fetal development for toxicity. Pregnant Sprague-Dawley rats, from gestation day 6-15, were administered 20 g/L or 50 g/L SB in the drinking water and then killed on day 20. No maternal toxicity was observed, however, embryonic loss in the treatment groups was double that of the controls (p < 0.05). No gross morphologic malformations were seen in the treated fetuses. Fetuses exposed to SB were found to be significantly heavier than the controls, an effect that was greater in female fetuses and was not correlated with increased placental size. The results suggest that the SB had no toxicity and that in utero exposure to SB resulted in increased early embryo loss with increased growth in surviving fetuses. On the other hand, Western blot analyses revealed that Bcl-2 protein of a 26 kDa was abundant in leiomyomal cells, but not in normal myometrial cells. The addition of progesterone (100 ng/mL) resulted in a striking increase in Bcl-2 protein expression in the cultured leiomyoma cells. However, the addition of SB (20 microg/mL) resulted in a significant reduction in Bcl-2 protein expression in the cells. The results indicated that human uterine leiomyomal cells express Bcl-2 protein and progesterone enhances its expression, however, SB reduces the expression of Bcl-2 protein in human uterine leiomyoma cells.

Effect of safflower seeds supplementation on stimulation of the proliferation, differentiation and mineralization of osteoblastic MC3T3-E1 cells.

Anti-bone resorption properties of the Korean herbal formulation, Gami-Honghwain (HJ), which comprises Carthamus tinctorius L. seed and hominis placenta, were investigated. We demonstrate that the production of PGE2 is inhibited by 20-100 microg/ml HJ in nontransformed osteoblastic cells (MC3T3-E1 cells), indicating that HJ inhibits PGE2 production. The effect of HJ on the proliferation and osteoblastic differentiation in MC3T3-E1 was also studied. HJ dose-dependently increased DNA synthesis (significant at 20-100 microg/ml), and increased alkaline phosphatase (ALP) and prolyl hydroxylase activities of MC3T3-E1 cells (20-100 microg/ml), while anti-estrogen tamoxifen eliminated the stimulation of proliferation and ALP activity of MC3T3-E1 which was induced by HJ. These results indicate that HJ directly stimulates cell proliferation and differentiation of osteoblasts. Also, when we assessed the effects of HJ on osteoblastic differentiation in MC3T3-E1, HJ enhanced ALP activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the HJ was observed at relatively low doses (significant at 20-100 microg/ml and maximal at 100 microg/ml). Northern blot analysis showed that the HJ (60 microg/ml) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. HJ (100 microg/ml) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that HJ has anabolic effect on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases.

Chemoprevention of Scutellaria bardata on human cancer cells and tumorigenesis in skin cancer.

Scutellaria barbata D. Don (Lamiaceae) (SB) is a perennial herb, which is natively distributed throughout Korea and southern China. This herb is known in traditional Chinese medicine as Ban-Zhi-Lian and in traditional Korean medicine as Banjiryun. SB has been used as an antiinflammatory and antitumor agent. The SB showed strong growth-inhibitory activity and cancer chemopreventive activity in assays representing three major stages of carcinogenesis. The SB was found to act as an antimutagen; it mediated antiinflammatory effects; inhibited cyclooxygenase and hydroperoxidase functions (antipromotion activity). In addition, SB inhibited the development of preneoplastic lesions in carcinogen-treated mouse mammary glands in culture and inhibited tumorigenesis in a mouse skin cancer model. On the other hand, an inhibitory effect of SB on the growth of gynecological cancer cell lines such as HeLa cell and human ovary cancer (HOC) was shown. When HOC cells were treated with SB, the expression of cyclooxygenase-2 was inhibited. These data suggest that SB merits investigation as a potential cancer chemopreventive agent in humans, especially in gynecological cancers.

Herbal formulation, Yukmi-jihang-tang-Jahage, regulates bone resorption by inhibition of phosphorylation mediated by tyrosine kinase Src and cyclooxygenase expression.

Anti-bone resorption properties of the Korean herbal medicine, Yukmi-jihang-tang (YJ), which is comprised of seven herbs such as Rehmannia glutinosa Libosch, Dioscorea japonica THUNB, Cornus officinalis SIEB et. ZUCC, Smilax glabra ROXB, Paeonia suffruticosa ANDR, Alisma platago-aquatica var. orientale SAMUELS and Hominis placenta, were investigated. Cyclooxygenase-2 (COX-2) and tyrosine kinase involve on prostaglandin E2 (PGE2) production in mouse calvarial osteoblasts stimulated by cytokine interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and/or interleukin-6 (IL-6). IL-1beta and IL-6 and to a lesser extent TNF-alpha, enhanced COX-2 mRNA levels in calvarial osteoblasts. TGF-beta, YJ (100microg/ml) and their combinations of YJ+TGF-beta reduced the COX-2 mRNA level, PGE2 biosynthesis and bone resorption induced by IL-1beta, TNF-alpha, IL-6 or their combination. Finally, YJ inhibits in vitro and in vivo bone resorption by inhibition of phosphorylation of peptide substrates. The parathyroid hormone-induced bone resorption in mouse fetal long bone cultures was inhibited with an IC(50) of 16microg/ml. YJ dose-dependently reduced the hypercalcemia induced in mice by IL-1beta and partly prevented bone loss and microarchitectural changes in young ovariectomized rats, showing that the protective effect on bone was exerted via the inhibition of bone resorption. These results indicate that the synergy between IL-beta, TNF-alpha, IL-6 on PGE2 production is due to an enhanced gene expression of COX-2 and that tyrosine kinase(s) are involved in the signal transduction of COX-2 in mouse calvarial osteoblasts. Thus, YJ as a possible Src family kinase inhibitor may be useful for the treatment of diseases associated with elevated bone loss. This result also suggested that the YJ extracts is effective for bone resorptive action in bone cells.

Pharmacological activity in growth inhibition and apoptosis of cultured human leiomyomal cells of tropical plant Scutellaria barbata D. Don (Lamiaceae).

Scutellaria barbata D. Don (Lamiaceae) (SB), which is known in traditional Korean medicine, has been used as an anti-inflammatory and antitumor agent. Since uterine leiomyoma is the most common benign smooth muscle cell tumor of the myometrium, we aimed to determine the growth inhibition and the induction of apoptotic cell death brought about by the herb SB in two different leiomyomal cells, named LM-1 and LM-2, and to clarify the mechanism of this apoptosis. Water-soluble ingredients of SB, and the leiomyomal cell lines of LM-1 and LM-2, were used in vitro. Growth inhibition, induction of cell death, morphological features, the presence of DNA ladders, increases in Caspase 3-like activity, the effects of a Caspase 3 inhibitor on apoptotic cell death, and the release of Cytochrome C by SB were analyzed. SB inhibited the growth and decreased the viability of the leiomyomal cells. The viability of normal myomatrial smooth muscle cells (SMC) in the presence of low concentrations of SB was higher than those of leiomyomal cells. Apoptotic bodies and DNA ladders were observed to be induced in leiomyomal cells of LM-1 and LM-2 by SB. The synthetic tetrapeptide Caspase 3 inhibitor, N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibited the apoptotic cell death of leiomyomal cells induced by SB. The Caspase 3-like activity in leiomyomal cells LM-1 and LM-2 increased after the addition of SB. Cytochrome C was released from mitochondria into the cytosol 8h after the addition of SB, and reached a peak at 16h. The peak of Cytochrome C release was earlier than that of Caspase 3-like activity. We concluded that SB inhibited the growth of the leiomyomal cells and induced apoptosis. The apoptosis of leiomyomal cells induced by SB was associated with the release of Cytochrome C from the mitochondria, followed by an increase in Caspase 3-like activity.

Regulation of IGF-I production and proliferation of human leiomyomal smooth muscle cells by Scutellaria barbata D. Don in vitro: isolation of flavonoids of apigenin and luteolin as acting compounds.

Scutellaria barbata D. Don (Lamiaceae) (SB) is a perennial herb, which is natively distributed throughout Korea and southern China. This herb is known in traditional Chinese Medicine as Ban-Zhi-Lian and traditional Korean medicine as Banjiryun, respectively. SB has been used as an anti-inflammatory and antitumor agent. We aimed to determine the expression of growth factor molecules for growth inhibition after treatment of SB in two different human myometrial smooth muscle cell (SMC)s and leiomyomal SMCs. Water-soluble ingredients of SB, myometrial SMCs, and the leiomyomal cell lines were used in vitro. SB significantly reduced cell numbers in culture and arrested cell proliferation, and also induced apoptosis, indicating that the presence of an intact apoptotic pathway was demonstrated in these cells by SB. Uterine leiomyoma is the most common benign smooth muscle cell tumor of the myometrium. The expression of insulin-like growth factor-I (IGF-I) was measured at the mRNA and protein level in myometrium and leiomyomal cells with and without treatment with a water extract of SB for 3 days. IGF-I mRNA expression was significantly higher in leiomyomal cells than in myometrium cells. The IGF-I protein was more abundant in leiomyomal cells than in myometrium. When SB was treated to the cells, the IGF-I protein concentrations in myometrial and leiomyomal cells from the SB-treated cells were similar. The results indicated that IGF-I expression is probably associated with a proliferation of leiomyomal cells than myometrium. However, SB down-regulated the IGF-I expression where IGF-I contributes to the selective growth of the leiomyoma. Therefore, growth modulation of LMs by SB occurs via mechanisms dependent of apoptosis. The raw materials were extracted and subjected to functional isolation for the active molecules in the present assay systems. The five flavonoids were isolated and the chemical structures of resveratrol, baicalin, berberine, apigenin, and luteolin were determined. The effects of resveratrol, baicalin, and berberine on the above parameters have not been significantly evidenced, whereas apigenin and luteolin were effective. The anti-proliferative compounds apigenin and luteolin belong to the flavones, a class of flavonoids and are characterized as selectively inhibitors of the growth of LM cells. Our findings suggest that flavonoids of apigenin and luteolin are potentially useful for the development of therapeutic treatments of cancer. These data also suggest that SB reduces tumor volume with inducing a concomitant increase in the rate of apoptosis.

The inhibitory effect of a Korean herbal medicine, Zedoariae rhizoma, on growth of cultured human hepatic myofibroblast cells.

The aim of this study was to assess the effect of ZR on the growth of cultured human hepatic myofibroblast cells (hMF). The zedoary (Zedoariae Rhizoma) made from the dried rhizome of Curcuma zedoaria Roscoe is an herbal drug used as an aromatic stomachic. The plant is a perennial herb which is natively distributed throughout Korea and is a traditional Korean herbal medicine. Zedoariae rhizoma is a bioactive traditional medicine with anti-tumor, anti-atherosclerosis, anti-inflammation, and growth-regulating properties. During the course of liver fibrogenesis, hMF, mostly derived from hepatic stellate cells, proliferate and synthesize excessive amounts of extracellular matrix components. To evaluate the antiproliferative effect of a traditional herbal medicine, Zedoariae rhizoma water extracts (ZR) was examined on the growth inhibition of hMF since proliferation of hMF is known to be central for the development of fibrosis during liver injury, and factors that may limit their growth are potential antifibrotic agents. The aim of this study was to test the effects of ZR on the proliferation in cultured hMF. hMF were obtained by outgrowth from human liver explants. ZR markedly reduced serum driven cell proliferation, as assessed by nuclear autoradiography experiments and measurement of actual cell growth. Growth inhibition was totally reversed after removal of the ZR. ZR potently inhibited hMF growth (IC50 = 8.5 microg/ml), in a pertussis toxin-insensitive manner. Analysis of the mechanisms involved in growth inhibition revealed that ZR rapidly increased prostaglandin E2 production and in turn cAMP, which inhibited hMF proliferation, did not affect cAMP levels. Production of cAMP by ZR was abolished by NS-398, a selective inhibitor of cycloxygenase (COX)-2. Also, ZR potently induced COX-2 protein expression. Blocking COX-2 by NS-398 blunted the antiproliferative effect of ZR. We conclude that ZR inhibits proliferation of hMF, probably via an intracellular mechanism, through early COX-2-dependent release of prostaglandin E2 and cAMP, and delayed COX-2 induction. Our results indicated a novel role for ZR as a growth inhibitory mediator and pointed out its potential involvement in the negative regulation of liver fibrogenesis. The results that ZR exhibits potent antiproliferative and antifibrogenic effects toward hMF, indicated that ZR might have therapeutic implications in chronic liver disease.

The expression of matrix metalloproteinase-9 in human follicular fluid is associated with in vitro fertilisation pregnancy.

OBJECTIVE: To investigate the role of matrix metalloproteinase-9 (MMP-9) in the pre-ovulatory follicular fluid and culture media during in vitro fertilisation (IVF) cycle and to develop the zymographic pre-diagnosis marker for successful implantation and pregnancy in human IVF. DESIGN: Controlled clinical study. SETTING: IVF Laboratory, Women's Hospital Infertility Clinic and Dongguk University, Korea. SAMPLE: Women undergoing in vitro fertilisation treatment. METHODS: Experiments were designed for controlled clinical study with women undergoing IVF treatment. MMP-9 expressions in follicular fluid and culture media samples that had been collected during transvaginal oocyte retrieval were measured using zymography. MMP-9 activities and expressions were strongly correlated to a higher rate of fertilisation and pregnancy. MAIN OUTCOME MEASURES: Fertilisation rates and ultrasonic evidence of intrauterine pregnancy by four weeks after embryo transfer. RESULT: MMP-9 activity was significantly higher in the pregnant group than in the non-pregnant group (P < 0.01). In contrast, MMP-2 activity was present in the follicular fluid and culture media of all women, and no difference in its expressions was found between the pregnant and non-pregnant groups. No correlation was found between the MMP-9 expression in follicular fluid and culture media and the fertilisation rates. CONCLUSION: The expression of MMP-9 in the follicular fluid and culture media is a prerequisite for successful pregnancy in IVF cycle. The zymography of MMP-9 activity in follicular fluids of human and culture media was developed as a pre-diagnostic method and zymographic diagnosis marker for successful fertilisation, implantation and pregnancy in human IVF.

Inhibitory effects of Scutellaria barbata D. Don. and Euonymus alatus Sieb. on aromatase activity of human leiomyomal cells.

It is now well documented that a large proportion of breast tumors express their own aromatase. This intratumoral aromatase produces estrogen in situ and therefore may contribute significantly to the amount of estrogen to which the cell is exposed. Thus it is not only important that aromatase inhibitors potently inhibit the peripheral production of estrogen and eliminate the external supply of estrogen to the tumor cell, but that they in addition potently inhibit intratumoral aromatase and prevent the tumor cell from making its own estrogen within the cell. To study the inhibition of intracellular aromatase, we have examined the aromatase-inhibiting potency of the Scutellaria barbata D. Don. (SB) and Euonymus alatus Sieb. (EA) in myometrial and leiomyomal cells which contain aromatase. We have also used human placental tissues. Although SB and EA are approximately equipotent in a cell-free aromatase system (human placental microsomes), EA is consistently 10-30 times more potent than SB in inhibiting intracellular aromatase in myometrial and leiomyomal cells. To provide insights into the effect of SB and EA on aromatase activity in leiomyomal cells, we examined the cell lines, which is induced to differentiate toward the more transformed cell phenotype by 12-tetradecanoylphorbal-13-acetate (TPA) as a protein kinase C activator and transforming growth factor-beta1 (TGF-beta1). Enzyme activity was inhibited in a time-and dose-dependent fashion by SB and EA and by either 1-50 nM TPA or 0.01-0.5 ng/ml TGF-beta1, with maximal responses after 2-3 h exposure.

Differential inhibition of Scutellaria barbata D. Don (Lamiaceae) on HCG-promoted proliferation of cultured uterine leiomyomal and myometrial smooth muscle cells.

Scutellaria barbata D. Don (Lamiaceae)(SB) is a perennial herb which is natively distributed throughout Korea and southern China. This herb is known in traditional Chinese Medicine as Ban-Zhi-Lian and traditional Korean medicine as Banjiryun, respectively. SB has been used as an anti-inflammatory and antitumor agent. We aimed to determine the expressin of cell cycle-related signal molecules for growth inhibition after HCG treatment by the herb SB in two different human myometrial smooth muscle cells (SMCs) and leiomyomal SMCs. Water-soluble ingredients of SB, myometrial SMCs and the leiomyomal cell lines were used in vitro. Uterine myomas often enlarge rapidly during pregnancy, implying that human chorionic gonadotrophin (HCG) may influences cell proliferation in uterine leiomyomata. We investigated the effects of SB on the cell proliferation and the expression of cell cycle-related proteins in these cells. Although HCG/LH receptor was present in both cultured myometrial and leiomyomal cells, as assayed by reverse transcription polymerase chain reaction analysis, treatment with HCG significantly increased cell proliferation in both myometrial and leiomyomal cells. However, SB reduced the proliferative effect of HCG in leiomyoma and myometrial cells, respectively. In HCG-treated leiomyomal cells, the expression of proliferating cell nuclear antigen, cyclin E and cdc2 was significantly reduced by SB treatment. These results suggest that SB reduced the HCG-promoted proliferation of myometrial and leiomyomal cells.

Inhibitory effect of Cho-Deung-San on human aortic smooth muscle cell migration induced by TNF-alpha through inhibition of matrix metalloproteinase-2 and -9 activity.

The migration and matrix metalloproteinases (MMPs) production of vascular smooth muscle cells (VSMC) may play a key role in the development of atherosclerosis. A Korean traditional herbal formulation, Cho-Deung-San (CDS), which is composed of 11 herbal ingredients, has been used to treat vascular diseases for many centuries. In this study, we investigated the inhibitory effect of CDS on tumor necrosis factor-alpha (TNF-alpha)-induced human aortic smooth muscle cells (HASMC) migration and MMP-2 and -9 activity. The cytotoxocity of CDS on HASMC was very low (IC(50)>500 microg/ml) as measured by the XTT assay method. The Matrigel migration assay showed that CDS effectively inhibited the TNF-alpha-induced migration of HASMC as compared with the control group in a dose-dependent manner (IC(50)=85 microg/ml). To explain this inhibitory effect, the extracts prepared from CDS and its herbal ingredients were assayed for gelatin zymography. The results showed that CDS inhibited MMP-2 and -9 activity (IC(50)=180 and 75 microg/ml, respectively). Among the herbal ingredients of CDS, the hooks and stems of Uncaria sinensis (Oliv.) Havil (UR) has shown significant inhibition against MMP-2 and -9 activity. In addition, the inhibitory effect of UR against gelatinolytic activity of MMP-2 and -9 was higher than that of catechin and lower than that of epigallocatechin gallate. These results suggest that CDS could be used as potential antiatherosclerotic agent, and UR is major component of CDS for antimigration in TNF-alpha treated HASMC.

Inhibitory effects of Scutellaria barbata D. Don on human uterine leiomyomal smooth muscle cell proliferation through cell cycle analysis.

Scutellaria barbata D. Don (SB) is one of the herbs belonging to perennial plants, which is known in traditional Korean medicine as 'Ban-Ji-Ryun,' and has been used as an anti-inflammatory and anti-tumor agents against human uterine leiomyoma, mammalian and ovarian cancers. Although the difference between uterine smooth muscle cell (SMC) and leiomyomal SMCs has not been clearly established, the action of SB water extract was investigated using SMCs from normal myometrium and leiomyoma. The proliferation of cultured myometrial and leiomyomal SMC was inhibited by SB treatment. Flow cytometric analysis showed that the population in the G1 phase of the cell cycle increased under SB treatment. Western blotting analysis showed that markers of SMC differentiation such as alpha-smooth muscle actin (alpha-SMA), calponin h1 and cyclin-dependent kinase inhibitor p27 were induced by treatment with SB in myometrial and leiomyomal SMCs. In contrast, cell-cycle-related gene products from the G1 phase of the cell cycle, such as cyclin E and cdk2, were not affected. Taken together, these results indicate that SB inhibits the proliferation of myometrial and leiomyomals SMC through the induction of alpha-SMA, calponin h1 and p27. It is suggested that SB may induce differentiation in uterine SMC and may influence tissue remodeling and reconstruction during physiological and pathophysiological events.

IL-1beta induces and TGF-beta reduces vitamin D3-induced bone resorption in mouse calvarial bone cells.

Bone cells produce multiple growth factors and cytokines that have effects on bone metabolism and can be incorporated into the bone matrix. The present study was designed to extend these observations by examining the interactions between transforming growth factor-beta (TGF-beta) or interleukin-1beta (IL-1beta) and bone cells in a rat long bone culture model. IL-1beta regulates several activities of the osteoblast cells derived from rat long bone explants in vitro. IL-1beta stimulated cellular proliferation and the synthesis of prostaglandin E2 and plasminogen activator activity in the cultured cells in a dose-dependent manner. TGF-B is present in the bone matrix and potentially can be released during bone resorption. TGF-beta reduced basal bone resorption and inhibited vitamin D3 [1,25(OH)2D3]-induced bone resorption in rat long bone cells. These studies support the role of IL-1beta in the pathological modulation of bone cell metabolism, with regard to implication in the pathogenesis of osteoporosis by IL-1beta, and that TGF-beta is positively inhibiting the bone resorption.

Interleukin-1 and tumor necrosis factor-alpha induce collagenolysis and bone resorption by regulation of matrix metalloproteinase-2 in mouse calvarial bone cells.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) greatly induces osteoclast formation and stimulates bone resorption of mouse calvaria in culture. We examined the effects of the two cytokines on the collagenolysis and bone resorption by induction of matrix metalloproteinases (MMPs). The cells were analyzed using zymographic analysis. It was shown that the mouse calvarial osteoblasts constitutively synthesize progelatinase-A (MMP-2). Interleukin-1beta markedly enhanced the messenger RNAs (mRNAs) expression of MMP-2 (gelatinase A), but slightly MMP-9 (gelatinase B), which associated with increases in bone matrix degradation. Both pro- and active-forms of MMP-2 were detected in the conditioned medium collected from calvarial cultures, and IL-1beta markedly stimulated both pro- and active-forms of the MMP-2. The expression of MMP-2 mRNAs could be detected, and they were markedly enhanced by IL-1beta on days 1 and 2. These results demonstrate that the potency of induction of MMP-2 by IL-1beta and TNF-alpha is closely linked to the respective bone-resorbing activity, suggesting that MMP-2-dependent degradation of bone matrix plays a key role in bone resorption induced by these cytokines. On the other hand, when the mouse osteoblasts were stimulated with parathyroid hormone, 1,25(OH)2D3, mononuclear cell conditioned medium (MCM) and IL-1 as bone resorption agents, collagenolysis was increased by producing the active gelatinase. Interleukin-1 in stimulating bone resorption was examined using fetal mouse long bone organ culture. Interleukin-1 stimulated bone resorption and produced marked resorption when present simultaneously. Furthermore, treatment of indomethacin and dexamethasone clearly abolished the responses of IL-1alpha and IL-1beta.

IL-1beta regulates cellular proliferation, prostaglandin E2 synthesis, plasminogen activator activity, osteocalcin production, and bone resorptive activity of the mouse calvarial bone cells.

Interleukin-1beta (IL-1beta) regulates several activities of the osteoblast cells derived from mouse calvarial bone explants in vitro. IL-1beta stimulated cellular proliferation and the synthesis of prostaglandin E2 in the cultured cells in a dose-dependent manner. Furthermore, plasminogen activator activity of the mouse osteoblast was positively affected by IL-1beta in a dose-dependent manner over the dosage range of 0.01 ng-2 ng/mL with a maximal effect being observed at 2 ng/mL. However, the induction of osteocalcin synthesis and alkaline phosphatase activity in response to vitamin D, two characteristics of the osteoblast phenotype, were significantly antagonized by IL-1beta over a similar dose range. Treatment of mouse calvarial bone cells with IL-1beta resulted in a dose dependent stimulation of bone resorption and the bone resorption induced by IL-1beta was strongly inhibited by calcitonin treatment, indicating osteoclast-mediated bone resorption, suggesting that the bone resorption induced by IL-1beta appears to be osteoclast-mediated. This study supports the role of IL-1beta in the pathological modulation of bone cell metabolism, with regard to implication of the pathogenesis of osteoporosis by IL-1beta.

Inhibitory effect of Carthamus tinctorius L. seed extracts on bone resorption mediated by tyrosine kinase, COX-2 (cyclooxygenase) and PG (prostaglandin) E2.

Anti-bone resorption properties of the Korean herbal formulation, Honghwain (HHI; Carthamus tinctorius L. seed) was biochemically investigated. On processing bone metabolism, PGE2 accelerated production of IL-1beta in fetal mouse osteoblast and stimulated physiological activation substance, IL-1beta. The novel class of Src tyrosine kinase inhibitors, Herbimycin A (HERB) and HHI reduced COX-2 mRNA levels as well as PGE2 production induced by IL-1beta, TNF-alpha and IL-6. HHI inhibited in vitro and in vivo bone resorption by inhibition of phosphorylation of peptide substrates. HHI dose-dependently reduced the hypercalcemia induced in mice by IL-1beta and partly prevented bone loss and microarchitectural changes in young ovariectomized rats, showing that the protective effect on bone was exerted via the inhibition of bone resorption. These results indicate that the synergy between IL-beta, TNF-alpha, IL-6 on PGE2 production is due to an enhanced gene expression of COX-2 and that tyrosine kinase (s) are involved in the signal transduction of COX-2 in mouse calvarial osteoblasts. Thus, HHI as a possible Src family kinase inhibitor may be useful for the treatment of diseases associated with elevated bone loss.

Inhibitory effect of a Korean traditional medicine, Honghwain-Jahage (water extracts of Carthamus tinctorius L. seed and Hominis placenta) on interleukin-1-mediated bone resorption.

A Korean herbal formulation, Honghwain-Jahage (HJ), which is comprised of a herb of Carthamus tinctorius L. seed and Hominis Placenta, was investigated for inhibiting effects on IL-1 beta-stimulated bone resorption in the fetal mouse bone culture system. Results of in vitro cytotoxicities showed that HJ extracts have no any cytotoxicities in concentrations of 1-200 microg/ml on the cultured osteoblast cells derived from mouse calvarial bone explants. Cell viability was not significantly affected by treatment with the indicated concentration of the extracts. The HJ extracts were shown to have inhibitory effects against the synthesis of PGE(2). We also examined the effect of the pretreatment with various concentrations of the HJ extracts then treated by the PGE(2)-induction agents. Pretreatment of the HJ extracts for 1 h, which by itself had little effect on cell survival, reduced the synthesis of PGE(2). Furthermore, the HJ extracts were shown to have protective effects against plasminogen dependent fibrinolysis induced by IL-1 beta. Pretreatment of the HJ extracts for 1 h did not enhance the plasminogen dependent fibrinolysis. Finally, co-treatment of HJ with calcitonin showed significant inhibitory activity on the IL-1 beta-stimulated bone resorption. From these results, it was found that HJ extracts inhibited IL-1 beta-induced bone resorption.