外源性葡萄糖皮質素誘發骨鬆之細胞與分子機轉

Cellular and molecular mechanism of exogenous glucocorticoid-induced osteoporosis

陳俞旭(Yu-Hsu Chen)

指導教授 ： 吳信志

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2019年)

https://doi.org/10.6342/NTU201901508

摘要

葡萄糖皮質素所引起的骨質流失，是常見的後天骨鬆的類型，也是造成年輕人骨鬆的重要成因。在骨骼新陳代謝中，高劑量葡萄糖皮質素會造成成骨細胞與骨細胞凋亡而引發骨質流失。根據先前的研究，成骨細胞與骨細胞的凋亡可能為氧化壓力所導致，但是其中的機制並不完全清楚。且葡萄糖皮質素是否也會對蝕骨細胞造成氧化壓力也還沒有定論。在本論文的第一部分研究中，我們觀察到隨著葡萄糖皮質素劑量增加和培養時間增加，氧化壓力也隨之上升，造成成骨細胞凋亡，粒線體的功能也受到影響。相對的，相同劑量的葡萄糖皮質素卻沒有造成蝕骨細胞的粒線體功能顯著的改變，氧化壓力也沒有上升，反而呈現下降趨勢。如此相反的現象，可能可以藉由一氧化氮合酶nos2基因表現量來說明，在蝕骨細胞中一氧化氮合酶表現是被抑制的，在成骨細胞中則是上升的。除了氧化壓力的探討，我們也繼續探討葡萄糖皮質素對粒線體的影響與氧化壓力的關係，實驗結果呈現，由葡萄糖皮質素所引起的質子洩漏與非粒線體的呼吸作用可能是造成細胞氧化壓力上升的主因。並且長時間的葡萄糖皮質素刺激，累積了上述的細胞變化，導致粒線體崩解和cytochrome C釋放，進而導致成骨細胞的凋亡。延續這樣的觀點，我們繼續的研究葡萄糖皮質素如何調控粒線體的功能。葡萄糖皮質素的受器在與葡萄糖皮質素結合後，會進入細胞核中調控基因的表現。所以從粒線體的型態以及粒線體調控基因的表現做探討，發現短時間的葡萄糖皮質素刺激，會促進粒線體生成，但是隨後粒線體開始分裂成較小的型態，然後細胞開始走向凋亡。綜合整個研究，從葡萄糖皮質素刺激成骨細胞後, 造成氧化壓力的上升，引起質子洩漏，同時調控粒線體基因促使粒線體分裂，最終導致成骨細胞凋亡，實驗結果前後呼應。藉此研究了解，需要使用高劑量或長期使用葡萄糖皮質素的患者治療計畫很重要的一環，調整輔助治療以減少氧化壓力來降低骨鬆之副作用。

關鍵字

外源性葡萄糖皮質素 ； 骨鬆

並列摘要

Glucocorticoid-induced bone loss is the most common form of secondary osteoporosis and the important cause of osteoporosis in the young adults. Among the different types of bone cells, high dose of exogenous glucocorticoids triggers apoptosis in the osteocytes and osteoblasts. Previous literature suggest that death of osteoblasts and osteocytes are associated with oxidative stress, although the mechanisms are not fully understood. Also, it is not clear whether glucocorticoids generate oxidative stress to osteoclasts. First part of this study, we demonstrated the effects of exogenous glucocorticoids on osteoblastic mitochondrial functions and elevated intracellular oxidative stress in a dose- and time-dependent manner. In contrast, the same dose of glucocorticoids did not induce mitochondria dysfunctions and oxidative stress in osteoclasts. Also, the production of reactive oxygen/nitrogen species was decreased in osteoclasts. The opposite effects may be explained by the nitrogen oxide synthase-nos2 expression level which is suppressed in osteoclasts but elevated in osteoblasts. We further revealed that glucocorticoids affect mitochondrial functions. The glucocorticoids induced increase of proton leak and non-mitochondria respiration suggest a potential source of glucocorticoids induced oxidative stress. Long term incubation of glucocorticoids accumulates these detrimental changes and result in cytochrome C release and mitochondria breakdown, consequently, lead to apoptosis in osteoblasts. In this regard, we also visualized the morphological alterations of mitochondria by staining with mitotracker and detect gene expression of mitochondrial biogenesis, resulting that mitochondrial biogenesis were transiently upregulated after treated with dexamethasone (DEX). But mitochondria fission also increased, and apoptosis became prominent. Our results indicated that the primary effects of glucocorticoids on mitochondria is promoting their functions and biogenesis. Mitochondria breakdown and the activation of the apoptotic pathways appeared to be the secondary effect after long term treatment. In our study, we know that we have to make an adjuvant therapy strategy for long-term or high dose steroid demanded patients to prevent bone loss.

並列關鍵字

exogenous glucocorticoid ； osteoporosis

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