June-Ki Kim

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

ABSTRACT Interleukin-1β (IL-1β) regulates several activities of the osteoblast cells derived from mouse calvarial bone explants in vitro. IL-1β 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-1β 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-1β over a similar dose range. Treatment of mouse calvarial bone cells with IL-1β resulted in a dose dependent stimulation of bone resorption and the bone resorption induced by IL-1β was strongly inhibited by calcitonin treatment, indicating osteoclast-mediated bone resorption, suggesting that the bone resorption induced by IL-1β appears to be osteoclast-mediated. This study supports the role of IL-1β in the pathological modulation of bone cell metabolism, with regard to implication of the pathogenesis of osteoporosis by IL-1β.

Ascochlorin suppresses oxLDL-induced MMP-9 expression by inhibiting the MEK/ERK signaling pathway in human THP-1 macrophages.

The critical initiating event in atherogenesis involves the invasion of monocytes through the endothelial walls of arteries and the transformation of monocytes from macrophages into foam cells. Human THP‐1 monocytic cells can be induced to differentiate into macrophages by phorbol myristate acetate (PMA) and can then be converted into foam cells by exposure to oxidized low‐density lipoprotein (oxLDL). Also, during a chronic inflammatory response, monocytes/macrophages produce the 92‐kDa matrix metalloproteinase‐9 (MMP‐9) that may contribute to the extravasation, migration, and tissue remolding capacities of the phagocytic cells. Here, we investigate the effect of ascochlorin (ASC), a prenylphenol antiviral compound from the fungus Ascochyta viciae, on oxLDL‐induced MMP‐9 expression and activity in human THP‐1 macrophages. ASC reduced oxLDL‐induced MMP‐9 expression and activity in a time‐dependent and dose‐dependent manner. Also, an analysis of MMP‐9 activity using pharmacologic inhibitors showed that ASC inhibits MMP‐9 activity via the extracellular signal‐regulated kinase 1 and kinase 2 pathways. Our results suggest that ASC may be useful as a potent clinical antiatherogenic agent, a topic of considerable interest in the biological chemistry of chemotherapeutic agents. J. Cell. Biochem. 102: 506–514, 2007.

Ring-Sp1 decoy oligonucleotide effectively suppresses extracellular matrix gene expression and fibrosis of rat kidney induced by unilateral ureteral obstruction

Tubulointerstitial fibrosis is the consequence of an injury characterized by the accumulation of excess collagen and other extracellular matrix components, resulting in the destruction of the normal kidney architecture and subsequent loss of function. A transcription factor Sp1, originally described as a ubiquitous transcription factor, is involved in the basal expression of extracelluar matrix genes and may, therefore, be important in fibrotic processes. Here, we report on the design of a ring-Sp1 decoy oligonucleotide, containing the consensus Sp1 binding sequence in a single decoy molecule without an open end, to create a novel therapeutic strategy for fibrosis. The ring-Sp1 decoy oligonucleotide is highly resistant to degradation by nucleases or serum compared to the conventional phosphorothioated double-stranded Sp1 decoy oligonucleotide, and effectively suppressed the expression of transforming growth factor-β1 and fibronectin, the binding of Sp1 to the promoter region of these genes, and proliferation in response to serum in normal rat kidney fibroblasts. Moreover, treatment with the ring-Sp1 decoy in vivo significantly attenuates extracellular matrix gene expression in the rat kidney in which a unilateral ureteral obstruction had been induced. These results suggest that the ring-Sp1 decoy oligonucleotide represents promising therapeutic alternative to the conventional treatment of fibrotic disorders.

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

Abstract Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) 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‐1β 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‐1β 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‐1β on days 1 and 2. These results demonstrate that the potency of induction of MMP‐2 by IL‐1β and TNF‐α 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‐1α and IL‐1β.

Immune-stimulating properties of polysaccharides from Phellodendri cortex (Hwangbek)

Heteropolysaccarides were isolated from the Korean medicinal plant, Phellodendri cortex (Hwangbek), by hot water and alkali extractions. The extracted polysaccharides were fractionated into eight fractions and they are mainly composed of D-N-acetylglucosamine, D-galactose, D-mannose, and D-glucose. Among the polysaccharide fractions, Fr.-2 showed a potent B-lymphocyte-stimulating activity in a system using polyclonal antibody forming cells in C57BL/6XC3H mice at dosages of 2–10 mg. On the basis of their solubility in aqueous ethanol, four fractions of Fr.-2-1 to Fr.-2-4 were further obtained from the Fr.-2, and Fr.-2-3 was divided into Fr.-2-3-1, 2, 3, and 4 by DEAE cellulose chromatography. The main activity was found in Fr.-2-3-2, which contained 100% (w/w) of carbohydrates and further purified to Fr.-2-3-2-2 by gel filtration chromatography using TSK Gel HW50S. Fr.-2-3-2-2, having a molecular weight of about 230 kDa, showed the highest B-cell-stimulating activity and the half-maximal concentration for B-lymphocyte-stimulating activity was ca. 2.2 µg/ml.

Prevention of collagen-induced arthritis in mice by Cervus korean TEMMINCK var. mantchuricus Swinhoe

The effect of water extract of deer antler (DAA) prepared from the pilose antler of Cervus korean TEMMINCK var. mantchuricus Swinhoe (Nokyong) on collagen-induced mouse rheumatoid arthritis (RA) model was studied. Identification of common DAA capable of affording protection or modulating the onset and severity of arthritis may have important human health implications. DAA has been shown to possess anti-inflammatory and anti-carcinogenic properties in experimental animals. In this study, we determined the effect of DAA-injection on collagen-induced arthritis in mice. In three independent experiments, mice given DAA in water exhibited significantly reduced incidence of arthritis (30-45%) as compared with mice not given DAA in water (86-98%). The arthritis index also was significantly lower in DAA-injected animals. Western blot analysis showed a marked reduction in the expression of inflammatory mediators such as cyclooxygenase 2, IFN-γ, and tumor necrosis factor α in arthritic joints of DAA-injected mice. The neutral endopeptidase activity was approximately six-fold higher in arthritic joints of non-DAA-injected mice in comparison to non-arthritic joints of unimmunized mice, whereas it was only two-fold higher in the arthritic joints of DAA-injected mice. Additionally, total IgG and type II collagen-specific IgG levels were lower in serum and arthritic joints of DAA-injected mice. Taken together our studies suggest that DAA may be useful in the prevention of onset and severity of arthritis.

Effects on Lipid Peroxidation and Antioxidative Enzymes of Euonymus alatus in Cultured Rat Hepatocytes

The Euonymus alatus (Thunb.) Sieb. has long been used as a crude drug. In this paper, we investigate the effects of E. alatus on cultured hepatocyte cell system and lipid peroxidation in hydrogen peroxide (H(2)O(2)) treatment conditions. The study covers the physiological activity (the antioxidative activity and the nitrite-scavenging effect) of E. alatus. H(2)O(2) that can produce intracellular free radical was used for inducer of the peroxidation of cellular lipids. Treatment of E. alatus attenuated in cell killing enhanced by increasing concentrations of H(2)O(2). The increased malondialdehyde level induced by H(2)O(2) treatment was reduced by pre-treatment of E. alatus. Furthermore, addition of E. alatus in cell culture medium significantly reduced cell killing and content of intracellular antioxidants. Changes in nitrite-scavenging effect of E. alatus at various concentrations (5-25 mg/ml) and various pH levels (pH 1.2, 4.2 and 6.0) were also observed. The present study was also done to investigate the effects of E. alatus on cultured hepatocyte cell system, H(2)O(2)-induced cytotoxicity and antioxidative enzyme activities, including catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase in H(2)O(2 )treatment conditions. E. alatus treatment had significant protective or elevating activities on these antioxidative enzyme activities compared to a normal group. The results indicate that E. alatus provides a strong antioxidant protection of cells against H(2)O(2)-induced oxidative stress.

Inhibitory effect of Uncaria sinensis on human aortic smooth muscle cell migration is based on matrix metalloproteinase-9 inhibitory activity.

Medicinal extracts of Cho-Deung-san and Uncaria sinensis Havil. (UR) have previously been shown to have inhibitory effects on migration of vascular smooth muscle cells (VSMC) and matrix metalloproteinase (MMP)-2/9 production, which play key roles in the development of atherosclerosis. In this study, we have more extensively investigated the inhibitory effect of UR on MMP-9 activity and TNF-α induced human aortic smooth muscle cells (HASMC) migration. The result from gelatin zymography showed that UR inhibited MMP-9 activity in a dose-dependent manner (IC(50)=55μg/ml). In addition, UR strongly inhibited the migration of HASMC induced by TNF-α treatment (IC(50)=125μg/ml), although it has very low cytotoxic effect on HASMC (IC(50)>500μg/ml). These results suggest that UR is a potential anti-atherosclerotic agent through inhibition of MMP-9 activity and VSMC migration.

Inhibition of Ulmus davidiana Planch (Ulmaceae) on bone resorption mediated by processing of Cathepsin K in cultured mouse osteoclasts

Ulmus davidiana Planch (Ulmaceae) (UD) has long been known to be antiinflammatory in traditional Korean medicine. This experiment investigated the effects of UD on bone resorption using bone cell culture. Different concentrations of crude extract of UD were added to mouse bone cell culture. The mitochondrial activity of the bone cells after exposure of UD was determined by colorimetric 3‐(4,5‐dimethylthiazolyl‐2)‐2,5‐diphenyltetrazolium bromide (MTT). It was demonstrated that UD has potential effects on bone cell culture without cytotoxicity. The most effective concentration of UD in bone cells was 100 µg/mL. Cathepsin K (Cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. When mouse long bone cells including osteoclasts and osteoblasts were treated with UD, UD prevented the osteoclast‐mediated intracellular processing of Cat K, suggesting that UD may disrupt the intracellular transport of pro Cat K. Since secreted proenzymes have the potential to reenter the cell via the mannose‐6‐phosphate (M6P) receptor, to prevent this possibility, UD was tested in the absence or presence of M6P. Inhibition of Cat K processing by UD was observed in a dose‐dependent manner. Furthermore, the addition of M6P resulted in enhanced potency of UD. UD dose‐dependently inhibited in vitro bone resorption with a potency similar to that observed for inhibition of Cat K processing. Copyright

Effect of the Traditional Korean Immunomodulating Formulation, Gamguntang (GGT), on Experimental Thyroiditis Model

The crude herbal formulation, Gamgungtang (GGT), is an immunomodulator showing marked down-regulation of several experimental autoimmune diseases. In this study, its effect on different experimental models of thyroid disease was investigated. Although very effective at preventing thyroid infiltrates in mice immunized with mouse deglycosylated thyroglobulin and complete Freunds adjuvant and in spontaneous models of thyroiditis, it completely failed to modify experimental autoimmune thyroiditis (EAT) induced in mice immunized with mouse thyroglobulin and lipopolysaccharide. There was no significant shift in the observed isotypes of anti-mouse thyroglobulin antibodies and only anti-mouse thyroglobulin antibodies in the spontaneous model were completely down-modulated by the GGT. One surprising fact to emerge was that GGT-treated donor mice, although protected from thyroid lesions themselves, were still able to transfer EAT showing that they must have been effectively primed while being treated with GGT. It is possible that the drug down modulated EAT by interfering with the trafficking of primed effector cells.