豨薟草萃取物對經RANKL活化蝕骨細胞分化之影響及其作用機制探討

Effects of Siegesbeckia orientalis extract on differentiation of RANKL-mediated osteoclastogenesis and its signaling pathways

蔡泓諺(Hung-Yen Tsai)

指導教授 ： 洪哲穎

義守大學/理工學院/化學工程學系暨生物技術與化學工程研究所/碩士(2014年)

摘要

骨組織的破壞與許多疾病有關，包括骨質疏鬆、類風濕性關節炎及骨癌的轉移等，並會導致關節疼痛及骨頭畸形。脊椎動物的骨骼發育是由成骨細胞和蝕骨細胞等兩個因子的協調平衡。Receptor activator of nuclear factor-κB ligand (RANKL)已被證實為分化及活化蝕骨細胞的重要因子。透過RANKL影響骨質再吸收細胞之作用，且對蝕骨細胞的活化其影響和骨質再吸收相關訊息傳遞系統之瞭解，有可能發展出有效方法來治療像骨質疏鬆症方面的疾病。豨薟草(Siegesbeckia orientalis)傳統上被使用於輔助治療類風濕性關節炎及高血壓等方面疾病。本研究針對豨薟草乙醇萃取物(S. orientalis ethanol extract, SE)以RANKL誘導RAW264.7細胞分化之抑制作用及蝕骨細胞凋亡之誘導作用進行探討。實驗結果顯示，SE能在未造成細胞毒性情形下有效抑制由RANKL誘發之蝕骨細胞形成，且在TRAP活性測試中也呈現出劑量依存性。由TRAP染色法可看出，在RAW264.7細胞加入SE培養五天後，成熟蝕骨細胞的生成數量隨著SE劑量的上升而逐漸減少。而在蝕骨細胞溶蝕作用中，利用Osteo Assay Surface 檢測套組來測試SE對抑制骨頭侵蝕的效果，顯示SE對於成熟蝕骨細胞在骨頭切片的侵蝕能力呈現劑量依存性的抑制作用。進一步以Western blot方法分析，顯示SE可藉由向下調控TRAF6、p38、NF-κB、JNK、ERK、MMP-9及下游NFATc1蛋白量來抑制蝕骨細胞之生成。此外，SE亦能藉由向下調節NF-κB、p38、JNK、ERK及NFATc1等蛋白質表現量以促進成熟蝕骨細胞之死亡。因此，SE具有應用於抑制蝕骨細胞形成與骨質方面相關疾病輔助治療之潛力，值得更進一步評估。

關鍵字

蝕骨細胞 ； 骨質疏鬆 ； 豨薟草 ； 細胞分化 ； 細胞凋亡

並列摘要

Bone tissue destruction is the features of many symbolic diseases, including osteoporosis, rheumatoid arthritis and bone metastasis. It is the result of joint pain and bone deformities. Bone development in vertebrate animals is maintained by two coordinated actions of osteoblast and osteoclast. Receptor activator of nuclear factor-κB ligand (RANKL) has been identified as osteoclast differentiation and activation of an important factor. As such, the down-regulation of RANKL expression or its downstream signals may be a valuable approach to the treatment of pathological bone loss. Siegesbeckia orientalis Linne has been used in traditional rheumatoid arthritis and adjuvant treatment of high blood pressure. This study investigated the inhibitory effects of S. orientalis ethanol extract (SE) on the RANKL-induced osteoclastogenesis of RAW264.7 cell and the survival of mature osteoclast cells. Experimental results showed that SE could effectively inhibit the RANKL-induced formation of osteoclasts and exhibited a dose-dependency on the TRAP activity. It can be seen from the TRAP-staining, after five-day cultivation of RAW264.7 cells with SE, the number of mature osteoclast cells decreased as the SE concentration increased. Additionally, SE inhibited pit formation on Osteo Assay Surface system in a dose-dependent manner that indicated its ability to inhibit the bone resorptive activity of mature osteoclasts in a RAW264.7 cells model. Western blot analysis demonstrated that SE could down-regulated the expression of TRAF6, p38, NF-κB, JNK, ERK, MMP-9 and downstream NFATc1 proteins. In addition, SE could induce the cell death of mature osteoclasts via the down-regulation of NF-κB, p38, JNK, ERK and NFATc1. Taken together, our findings clearly show that SE can attenuate RANKL-induced differentiation and formation of osteoclasts. Therefore, SE has a potential to become complementary agents for treating various bone diseases associated with excessive osteoclast formation and bone destruction. It is worth for further evaluation.

並列關鍵字

Osteoclasts ； Osteoporosis ； Siegesbeckia orientalis ； differentiation ； apoptosis

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