探討KMUP-1抑制由RANKL誘發類蝕骨細胞增生及分化的作用機轉

Study the mechanism of KMUP-1 in attenuating RANKL-induced osteoclast-like cells proliferation and differentiation

徐珮娟(Pei-Chuan Hsu)

指導教授 ： 葉竹來

高雄醫學大學/醫學院/藥理學科研究所/碩士(2010年)

摘要

在骨質汰舊換新的過程中，當骨頭重塑（bone remodeling）的平衡遭到破壞，蝕骨細胞（osteoclasts）的骨溶蝕作用大於造骨母細胞（osteoblasts）的骨形成作用時，將會使骨細胞減少導致骨質的流失或是骨密度的下降，進而造成許多骨頭疾病，例如骨質疏鬆症（osteoporosis）、牙周炎（periodontitis）或發炎性之關節疾病（osteoarthritis）。骨質流失會提高骨折的風險並造成許多嚴重的問題，包含骨頭的畸形、疼痛、死亡率增加以及嚴重的經濟負擔。近年來的研究指出，磷酸二酯酶第四型（phosphodiesterase 4, PDE4）抑制劑在一些引貣骨質流失的實驗模式上具有不錯的治療效果。Receptor activator of nuclear factor-κB ligand（RANKL）是調節蝕骨細胞增生分化及其骨溶蝕活性的關鍵因子，本研究的目的以RANKL誘發RAW264.7巨噬細胞形成類蝕骨細胞的模式當中，探討PDE3、4、5抑制劑KMUP-1對類蝕骨細胞增生及分化的影響，並深入探討KMUP-1抑制類蝕骨細胞增生及分化的作用機轉。實驗的結果發現，KMUP-1於對細胞沒有毒性的劑量下，降低了由RNAKL誘發的類蝕骨細胞增生，降低了蝕骨細胞分化標的酵素－tartrate-resistance acid phosphatase （TRAP）的產量及活性，降低了於蝕骨細胞生成扮演重要角色的促發炎性細胞激素產量和HMGB1的核質轉移，以及降低成
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熟的蝕骨細胞對於骨基質的溶蝕作用，而KMUP-1的這些抑制效果呈劑量相關性。我們發現KMUP-1抑制蝕骨細胞增生分化的機制主要是透過抑制RANKL誘發的MAP kinases、NF-κB及Akt之訊息傳遞路徑，並抑制關鍵轉錄因子c-Fos和NFATc1，以及與蝕骨細胞溶蝕作用相關基因之mRNA的表現。另外，我們於monosodium iodoacetate（MIA）誘發大鼠膝關節炎之動物實驗模式中，證實投與KMUP-1 2.5和5 mg/kg 可以減緩MIA對膝關節軟骨的傷害，減少發炎細胞的浸潤。因此，我們評估於體外試驗KMUP-1具有抑制類蝕骨細胞增生及分化的作用，於體內試驗可預防及治療發炎性之關節疾病，KMUP-1可成為一個預防及治療骨質流失的新製劑。

關鍵字

類蝕骨細胞 ； 骨質流失 ； 膝關節炎

並列摘要

In bone remodeling, an imbalance caused by increased bone resorption over bone formation leads to skeletal diseases such as osteoporosis, periodontitis, and rheumatoid arthritis. Recently, phosphodiesterase (PDE) 4 inhibitors have been shown to have therapeutic effects in different experimental osteopenia models. The receptor activator of nuclear factor-B ligand (RANKL) plays a critical role in the differentiation and bone resorptive activity of osteoclasts. Thus, in this study, we performed to determine whether KMUP-1, a PDE3, 4, 5 inhibitor, can attenuate the differentiation and proliferation of RANKL-induced osteoclast-like cells from RAW264.7 cells. In vitro, we found that KMUP-1 inhibited the RANKL-induced tartrate-resistance acid phosphatase (TRAP, a marker for osteoclast differentiation) activity and the formation of multinucleated osteoclasts in dose-dependent manner without any cytotoxicity. KMUP-1 also attenuated the bone resorption activity of mature osteoclasts. In addition, KMUP-1 inhibited inflammatory cytokines release and HMGB1 translocation which reported to play a major role in osteoclastogenesis. Furthermore, KMUP-1 prevented RANKL-induced activation of signaling molecules (Akt, MAP kinases and NF-κB) and key transcription factors (c-Fos and NFATc1) during early osteoclastogenesis. We also observed that KMUP-1 attenuated the activity and protein expression of matrix metalloproteinase-9 (MMP-9) and MMP-2. In vivo, Osteoarthritis was induced by intraarticular injection of monosodium iodoacetate (MIA) into knee joints of rats, KMUP-1 1, 2.5 and 5 mg/kg were orally
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administered once a day for 7 days before and after MIA injection. We found that KMUP-1 was effective in articular cartilage erosion in MIA-induced osteoarthritis. Taken together, our results demonstrate that KMUP-1 potentially inhibits RANKL-induced osteoclast differentiation and proliferation by attenuating the downstream signaling molecules and transcription factors required for osteoclastogenesis in vitro. KMUP-1 attenuates MIA-induced osteoarthritis in vivo. Thus, we indicate that KMUP-1 may be a new therapeutic treatment for bone loss diseases.

並列關鍵字

RANKL ； RAW264.7 ； osteoclast ； MIA

參考文獻

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