Simvastatin 對人類成骨細胞之增殖及粒線 體功能之影響機制探討

The Effect Mechanism of Simvastatin on Proliferation and Mitochondrial Function of Human Osteoblasts

廖秀蓉(Hsiu-Jung Liao)

指導教授 ： 何美泠

共同指導教授 ： 張瑞根(Je-Ken Chang)

高雄醫學大學/醫學院/醫學研究所/碩士(2007年)

https://doi.org/10.6832/KMU.2007.00112

摘要

Simvastatin，是hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)還原酶抑制劑，可抑制膽固醇的合成作用。近來，許多活體內及活體外研究指出Simvastatin具有促進骨生成的效果。我們之前活體內研究亦發現，Simvastatin 增加了卵巢切除鼠之網狀骨之骨生成蛋白表現如BMP-2、Osteocalcin 及Type I collagen 並且促進了卵巢切除老鼠之骨生成作用。同時，我們也發現Simvastatin 增加了卵巢切除鼠之附著於網狀骨周圍的成骨細胞之數目。因此，本研究擬進一步探討Simvastatin 對活體外培養人類成骨細胞之細胞增殖的影響及其作用之分子機制。我們的實驗結果顯示Simvastatin 能促進活體外培養之人類成骨細胞的細胞增殖。Simvastatin 會促進Cyclin D2 及Bcl-2 之mRNA 表現與Cyclin D2 及Bcl-2，此外；我們亦發現蛋白質之表現。由以上結果顯示，Simvastatin 增加Cyclin D2及Bcl-2 之表現可能是促進成骨細胞增殖之重要機制之一。我們更進一步發現Simvastatin 可促進活體外培養人類成骨細胞粒線體功能及ATP 製造，我們利用了電子傳遞鏈抑制劑進一步研究Simvastatin 對細胞增殖之促進作用與粒線體功能之相關性。結果顯示，粒線體之電子傳遞鏈Complex I 活性的抑制劑Rotenone
會顯著的抑制人類成骨細胞的ATP 製造量及細胞增殖Simvastatin 與Rotenone共同處理能回覆Rotenone 處理之人類成骨細胞之ATP 製造量及細胞增殖，Simvastatin 促進Cyclin D2 及Bcl-2 mRNA 表現之現象並不受Rotenone 之影響。進一步我們利用CompelxⅤ抑制劑Oligomycin 來阻斷電子傳遞鏈之內外膜間之電子經ATP 合成酶所產生的ATP，進而探討粒線體功能與細胞週期之相關性。Oligomycin 能有效抑制人類成骨細胞ATP 製造量及細胞增殖，再與Simvastatin
共同處理能部份回覆人類成骨細胞之ATP 製造量及細胞增殖，顯示ATP 製造量與細胞之增殖具有很大的相關性。此外，我們更進一步發現，以Oligomycin 與Simvastatin 之共同處理下(CompelxⅤ之抑制)，Simvastatin 無法促進Cyclin D2及Bcl-2 之mRNA 表現。根據以上結果，我們推測Simvastatin 可能部份透過促進粒線體功能而影響細胞週期調控因子Cyclin D2 及抗凋亡因子Bcl-2 之表現，
而促進了人類成骨細胞之增殖。統整本研究結果，我們認為Simvastatin 在活體內促進骨生成之作用的機制之一，可能是促進成骨細胞之增殖及粒線體功能所造成。故我們未來將進一步探討Simvastatin 影響細胞週期調控因子之表現對粒線體之電子傳遞鏈功能之相關性，藉此釐清Statins 藥物促進成骨細胞之增殖作
用的機制。

關鍵字

人類成骨細胞 ； simvastatin ； 細胞增殖 ； 粒線體

並列摘要

Simvastatin, a hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, is known to inhibit cholesterol biosynthesis. Recently, several studies indicated that simvastatin had a potent effect on stimulating bone formation in vivo and vitro studies. Our previous study also indicated that simvastatin attenuated ovarietomy-induced (OVX-induced) osteopenia. Furthermore, we also found that osteogenic proteins, BMP-2, osteocalcin and type I collagen, were markedly increased by simvastatin in trabecular bones of OVX rats. Our previous results also
showed that simvastatin increased the number of osteoblastic cells in trabecular bones of OVX rats in comparison to the non-drug treated OVX rats. In this study, we further investigated the molecular mechansim of simvastatin effect on proliferation in cultured human osteoblasts. Our results showed that simvastatin significantly enhanced proliferation of cultured human osteoblasts. We further found that simvastatin elevated the mRNA and protein levels of cyclin D2 and bcl-2 in human
osteoblasts. From these results, we suggest that increases of the expressions of cyclin D2 and bcl-2 may play an important role in the simvastatin stimulated proliferation in osteoblasts. Moreover, we also found that simvastatin increased mitochondrial activity and ATP production of human osteoblasts. Whether the stimulatory effect of
simvastatin on proliferation related to the motichondrial function was also investigated by treating the blockers of respiratory chain. The results showed that complex I blocker, rotenone, decreased ATP production and proliferation of osteoblasts. Furthermore, combined treatment of simvastain and rotenone was also found to rescue the suppression of ATP production and proliferation caused by the blockade of complex I of respiratory chain in mitochondria. We found that the stimulatory effect of simvastatin on the mRNA expressions of cyclin D2 and bcl-2 was not changed by the blockade of complex I of respiratory chain in mitochondria. On the other hand, our data showed that oligomycin suppressed ATP production and proliferation, but combined treatment with simvastain can rescue these suppression effects caused by blockade of complex V of respiratory chain. More importantly, we found that the enhancement of mRNA expressions of cyclin D2 and bcl-2 by simvastatin was eliminated with blockade of complex V of respiratory chain. From these results, we suggest that simvastatin increased proliferation may act, at least partially, through increasing mitochondrial function and the expression of cyclin D2 and bcl-2 in human osteoblasts. Whether the effect of simvastatin on cell cycle regulators related to the function respiratory chain in mitochondria needs to be further investigated.

並列關鍵字

human osteoblasts ； simvastatin ； proliferation ； mitochondria

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