WSS25 is a sulfated polysaccharide extracted from the rhizome of Gastrodia elata BI, which has been found to bind to bone morphogenetic protein 2 (BMP-2) in hepatocellular cancer cells. Since BMP-2 may regulate both osteoclasts and osteoblasts, here we investigated the effects of WSS25 on osteoclastogenesis in vitro and bone loss in ovariectomized mice.

METHODS:

RAW264.7 cells or mouse bone marrow macrophages (BMMs) were treated with RANKL to induce osteoclastogenesis, which was assessed using TRAP staining, actin ring formation and pit formation assays, as well as bone resorption assay. Cell viability was detected with MTT assay. The mRNA levels of osteoclastogenesis-related genetic markers (TRAP, NFATc1, MMP-9 and cathepsin K) were detected using RT-PCR, while the protein levels of p-Smad1/5/8 and Id1 were measure with Western blotting. WSS25 was administered to ovariectomized mice (100 mg·kg(-1)·d(-1), po) for 3 months. After the mice were euthanized, total bone mineral density and cortical bone density were measured.

RESULTS:

In RAW264.7 cells and BMMs, WSS25 (2.5, 5, 10 μg/mL) did not affect the cell viability, but dose-dependently inhibited RANKL-induced osteoclastogenesis. Furthermore, WSS25 potently suppressed RANKL-induced expression of TRAP, NFATc1, MMP-9 and cathepsin K in RAW264.7 cells. Treatment of RAW264.7 cells with RANKL increased BMP-2 expression, Smad1/5/8 phosphorylation and Id1 expression, which triggered osteoclast differentiation, whereas co-treatment with WSS25 or the endogenous BMP-2 antagonist noggin suppressed the BMP-2/Smad/Id1 signaling pathway. In RAW264.7 cells, knockdown of Id1 attenuated RANKL-induced osteoclast differentiation, which was partially rescued by Id1 overexpression. In conformity to the in vitro experiments, chronic administration of WSS25 significantly reduced the bone loss in ovariectomized mice.

CONCLUSION:

WSS25 inhibits RANKL-induced osteoclast formation in RAW264.7 cells and BMMs by blocking the BMP-2/Smad/Id1 signaling pathway. WSS25 administration reduces bone loss in ovariectomized mice, suggesting that it may be a promising therapeutic agent for osteoporosis.

2013-05-01·Glycobiology3区 · 生物学

WSS25 inhibits Dicer, downregulating microRNA-210, which targets Ephrin-A3, to suppress human microvascular endothelial cell (HMEC-1) tube formation

3区 · 生物学

Article

作者: Fei Xiao ; Kan Ding ; Ling Zhou ; Liping Yang ; Xiaokun Shen ; Hong Qiu

WSS25 is a sulfated polysaccharide that inhibits angiogenesis. However, the mechanism underlying the regulation of angiogenesis by WSS25 is not well understood. Using microRNA (miRNA) microarray analysis, a total of 25 miRNAs were found to be upregulated and 12 (including miR-210) downregulated by WSS25 in human microvascular endothelial cells (HMEC-1). Interestingly, Dicer, a key enzyme for miRNA biosynthesis, was downregulated by WSS25 in HMEC-1 cells. Further studies indicated that HMEC-1 cell tube formation and miR-210 expression were suppressed while Ephrin-A3 expression was enhanced by the silencing of Dicer. In contrast, HMEC-1 cell tube formation and miR-210 expression were induced while Ephrin-A3 expression was suppressed by Dicer overexpression. Moreover, miR-210 was downregulated while Ephrin-A3 was upregulated by WSS25 in HMEC-1 cells. HMEC-1 cell migration and tube formation were arrested, while Ephrin-A3 expression was augmented by anti-miR-210. In addition, HMEC-1 cell tube formation was significantly attenuated or augmented when Ephrin-A3 was overexpressed or silenced, respectively. Nevertheless, the tube formation blocked by WSS25 could be partially rescued by manipulation of Dicer, miR-210 and Ephrin-A3. These results suggest a new pathway whereby WSS25 inhibits angiogenesis via suppression of Dicer, leading to downregulation of miR-210 and upregulation of Ephrin-A3.

2012-08-01·Glycoconjugate journal4区 · 生物学

Structure-activity relationship study of WSS25 derivatives with anti-angiogenesis effects

4区 · 生物学

Article

作者: Fei Xiao ; Ying Wang ; Jianping Fang ; Xia Chen ; Kan Ding

WGEW, an α(1-4) linked glucan with an α(1-4) linked branch attached to C-6, was isolated from the rhizoma of Gastrodia elata Bl. WSS25, a sulfated derivative of WGEW, was reported to inhibit angiogenesis by disrupting BMP2/Smad/Id1 signaling pathway. However, the structure-activity relationship (SAR) for WSS25 is not known. To study the SAR, seven sulfated saccharides derived from WGEW degradation products, six sulfated polysaccharides with varying degrees of substitution, and four aminopropylated, carboxymethylated, phosphorylated, and acetylated derivatives of WGEW were prepared. A sulfated, unbranched product of polysaccharide was also obtained. The structural features of these derivatives were characterized by infrared spectroscopy and nuclear magnetic resonance spectroscopy. An HMEC-1 cell tube formation assay was employed to measure the antiangiogenic effect of the derivatives. The results indicated that only sulfated polysaccharides with molecular weights of more than 41,000 Da could inhibit HMEC-1 cell tube formation. The inhibition effect was dependent on the presence of a sulfate group, since the tube formation was not blocked by aminopropylated, carboxymethylated, phosphorylated, or acetylated WGEW. A higher degree of sulfate substitution on the polysaccharide led to a stronger inhibitory effect, and the degree of sulfate substitution between 0.173 and 0.194 was found to be optimal. Interestingly, the WGEW side chain was not required for anti-tube formation activity. All these preliminary results may provide a clue for further modification of the core structure of WSS25 to discover polysaccharide derivatives as novel anti-angiogenic inhibitors.

100 项与 WSS-25 相关的药物交易

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