淫羊藿苷對骨母細胞及蝕骨細胞之影響

Effects of Icariin on Osteoblasts and Osteoclasts

謝采蓓(Tsai-Pei Hsieh)

指導教授 ： 許秀蘊

共同指導教授 ： 孫瑞昇(Jui-Sheng Sun)

臺北醫學大學/藥學院/藥學系(博士班)/博士(2010年)

https://doi.org/10.6831/TMU.2010.00089

摘要

淫羊藿是一種中國常用於治療骨質疏鬆症的草藥。淫羊藿中主要活性黃酮配糖體為是淫羊藿苷(Icariin)，有研究顯示可以提高骨癒合及減少骨質疏鬆症的發生。不過，詳細的分子機制仍不清楚。在本研究中探討淫羊藿苷分子機制，主要通過刺激成年小鼠骨母細胞初代培養，抑制蝕骨細胞分化和骨再吸收作用，透過抑制MAPKs / NF-κB活化表現，調控低氧誘導因子-1α (HIF-1α; Hypoxia-Inducible Factor 1α) 和前列腺素E2 (Prostaglandin E2，PGE2)的合成。
本研究中骨母細胞初代培養從八個月Imprinting Control Region (ICR) 成年母鼠取得。研究中發現Icariin會增加成年母鼠骨母細胞的活性，促進骨母細胞生成及分化。從骨母細胞活性分析發現10−8 M icariin表現最佳。在這個濃度下，Icariin會刺激細胞增生，增加鹼性磷酸酵素活性，增加骨母細胞礦化，促進一氧化氮(NO)生成。Icariin會抑制骨母細胞凋亡，藉由抑制caspase-3活化，延長骨母細胞生命週期。Icariin會增加Bone morphogenetic protein-2 (BMP-2) mRNA、SMAD4 mRNA、Core bonding factor 1 (Cbfa1/Runx2) mRNA、Osteoprotegerin（OPG）、Receptor activator of nuclear factor (NF)-kB ligand (RANKL) mRNA的基因表現。利用BMP受體拮抗劑 Noggin及一氧化氮合成酶抑制劑L-NAME來探討相關的機轉，在本研究中發現給予Nogginr及L-NAME後，可以抑制Icariin之細胞增生、ALP之活性增加、NO之生成，也會調控BMP-2 mRNA、SMAD4 mRNA、Cbfa1/Runx2 mRNA、OPG mRNA、RANKL mRNA表現。本研究經體外實驗發現Icariin為一種骨細胞刺激劑，會誘導骨生成作用，藉由BMP-2及NO生成，調控SMAD4 mRNA、Cbfa1/Runx2 mRNA、OPG mRNA、RANKL mRNA表現。
Icariin在蝕骨細胞與骨母細胞共同培養模式下，可以抑制蝕骨細胞的活性，10−8 M Icariin對於抑制蝕骨細胞特有的分化指標:抗酒石酸性磷酸酵素 (tatrate resistances acid phosphatease；TRAP) 效果最佳。Icariin會縮小細菌脂多醣體 (LPS, Lipopolysaccharide) 誘導的蝕骨細胞大小，同時降低TRAP 及酸性磷酸酵素 (acid phosphatease；ACP) 活性，但不會影響蝕骨細胞存活。Icariin也會抑制骨再吸收作用，降低Interleukin-6 (IL-6) 和Tumor necrosis factor-alpha (TNF-α) mRNA及蛋白質表現。Icariin會增強OPG mRNA 基因表現，而減少RANKL mRNA 基因表現。Icariin減少前列腺素E2 (Prostaglandin E2，PGE2)及COX-2之合成。此外，Icariin會在正常氧氣濃度下減少LPS誘導之蝕骨細胞中低氧誘導因子-1α (HIF-1α; Hypoxia-Inducible Factor 1α) 的生成。在蝕骨細胞中，Icariin減少LPS造成的ERK1/2、I-kappa-B-alpha (IκBα)、p38 及JNK活化；而在骨母細胞中，Icariin則會減少LPS造成的ERK1/2及IκBα活化，反而增加p38活化。Icariin經由p38 及 JNK路徑來抑制LPS誘導之蝕骨作用，。
總而言之，本研究發現Icariin會促進體外骨形成作用，藉由BMP-2及NO生成，抑制骨再吸收作用，預防發炎性骨質流失，減少p38 及 JNK路徑活化。由本研究發現Icariin在骨質疏鬆症的治療上深具潛力。

關鍵字

淫羊藿苷骨母細胞 ； 蝕骨細胞

並列摘要

Epimedii herba is one of the most frequently used herbs in formulas prescribed for the treatment of osteoporosis in China. The main active flavonoid glucoside extracted from Epimedium pubescens Maxim. is icariin, which has been reported to enhance bone healing and reduce osteoporosis occurrence. However, the detailed molecular mechanisms remain unclear. In this present study, we examine the molecular mechanisms of icariin by using primary osteoblast cell cultures obtained from adult mice; and icariin can inhibit osteoclast differentiation and bone resorption by suppressing MAPKs/NF-κB regulated hypoxia inducible factor-1α (HIF-1α) and prostaglandin E2 (PGE2) synthesis.
The osteoblast cells were harvested from 8-month old female Imprinting Control Region (ICR) mice. The effects of icariin stimulation on the proliferation, differentiation and maturation of osteoblasts were examined. The viability of the osteoblasts reached its maximum at 10−8 M icariin. At this concentration, icariin increased the proliferation and matrix mineralization of osteoblasts and promoted nitric oxide (NO) synthesis. Icariin decresed osteoblasts apotosis by inhibiting caspase-3 activation to promote cell life cycle. With icariin treatment, the BMP-2, SMAD4, Cbfa1/Runx2, and OPG gene expressions were up-regulated; the RANKL gene expression was however down-regulated. Concurrent treatment involving the BMP antagonist (Noggin) or the NOS inhibitor (L-NAME) diminished the icariin-induced cell proliferation, ALP activity, NO production, as well as the BMP-2, SMAD4, Cbfa1/Runx2, OPG, RANKL gene expressions. In this study, we demonstrate that in vitro icariin is a bone anabolic agent that may exert its osteogenic effects through the induction of BMP-2 and NO synthesis, subsequently regulating Cbfa1/Runx2, OPG, and RANKL gene expressions.
After treatment with icariin, osteoclast co-culture cells were decreased tatrate resistances acid phosphatease (TRAP) activity significantly at the concentration of 10-8 M. Icariin reduced the size of LPS-induced osteoclasts formation, and diminished their TRAP and acid phosphatease (ACP) activity without inhibition of cell viability. Icariin also inhibited LPS-induced bone resorption and interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) mRNA and protein expression. The gene expression of OPG was up-regulated, while RANKL was down-regulated. Icariin also inhibited the synthesis of cyclo-oxygenase type-2 (COX-2) and PGE2. In addition, icariin had a dominant repression effect on LPS-induced HIF-1α expression of osteoclasts. On osteoclasts, icariin suppresses LPS-mediated activation of the ERK1/2, I-kappa-B-alpha (IκBα), p38 and JNK; while on the osteoblasts, icariin reduced the LPS-induced activation of ERK1/2 and IκBα, but increased the activation of p38. Icariin inhibited LPS-induced osteoclastogenesis program by suppressing the p38 and JNK pathway.

In conclusion, we demonstrated that icariin in vitro has bone formation activity through the induction of BMP-2 and NO synthesis, and inhibitory effects on bone resorption that can prevent inflammatory bone loss by suppressing the p38 and JNK pathway. This study was found that icariin should be a potential compound used for osteoporosis treatment.

並列關鍵字

Icariin ； oteoblast ； osteoclast

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