咖啡因誘導人類骨母細胞產生氧化壓力及細胞毒性

Caffeine induces oxidative stress generation and cytotoxicity in human osteoblasts

陸品蓁(Pin-Zhen Lu)

指導教授 ： 詹文雄

中原大學/理學院/化學研究所/碩士(2006年)

https://doi.org/10.6840/cycu200600317

摘要

咖啡因的攝取會增加骨質疏鬆的風險，過去研究顯示停經後的婦女骨質疏鬆與喝咖啡有關。近來研究證明，細胞處理咖啡因之後，會誘導細胞停在細胞週期G2/M 和細胞凋亡。咖啡因同時會引起數種細胞凋亡生物化學變化，包括活化 p53 和蛋白質分解酶-3 (caspase-3)，增加粒線體調控細胞凋亡路徑蛋白質Bax表現以及抑制Akt 活性。無論如何，咖啡因對細胞影響明確的分子機制並不清楚。在本研究，我們的結果顯示，使用MTT分析人類骨母細胞處理咖啡因之後，細胞的存活率會依劑量的增加及處理時間的加長而減少。西方點墨分析細胞凋亡蛋白質標記，包括caspase-9，caspase-3，PARP，PAK2，Bcl-2和Bax 等蛋白質，顯示在細胞處理咖啡因之後，與細胞凋亡有關的蛋白質都會隨著咖啡因的劑量增加表現增加。另外，使用lactate dehydrogenase (LDH) release分析，同樣在處理咖啡因劑量的增加及處理時間的加長情況下，培養液中LDH的百分比與控制組比較，培養液中的LDH愈多，表示咖啡因會引發細胞壞死。使用可滲透細胞染劑 2’,7’-dichlorofluorescin diacetate (DCF-DA) 測量 reactive oxygen species (ROS) 產生，我們更進一步發現咖啡因處理細胞後在早期的階段就會直接增加細胞內的氧化壓力。 根據我們的發現，ROS可能是咖啡因處理人類骨母細胞後，調節細胞凋亡與細胞壞死的重要因子。同時，咖啡因對人類骨母細胞所引發的損害同時透過細胞死亡的兩種機制調控。

關鍵字

咖啡因 ； 骨母細胞 ； 細胞壞死 ； 細胞凋亡 ； 氧化壓力

並列摘要

Caffeine consumption is a risk factor for osteoporosis. The previous reports showed that the postmenopausal woman’s bone loss is related to this risk factor. Further study demonstrated that treatment of cells with caffeine could induce G2/M arrest and apoptosis. The caffeine induces various apoptotic biochemical changes, including activation of p53 and caspase-3, increase of mitochondrial-mediated pathway of apoptosis Bax protein level, and inhibition of Akt activity. However, the precise regulatory molecular mechanisms are unknown. Our results revealed that cell viability decreased dose-dependently in osteoblasts treated with caffeine in MTT assay. Immunoblotting analysis of apoptotic markers including caspase9, caspase3, PARP, PAK2, Bcl-2 and Bax activation revealed that caffeine increased the apoptosis-associated parameter in time- and dose-dependent manner. At the same time, increasing activity of LDH released from cells into culture medium also reflected the necrotic cell death process by a lactate dehydrogenase (LDH) release assay. LDH activity in medium of caffeine groups markedly increased time- and dose-dependently versus normal control groups. Using the cell permeable dye 2’, 7’-dichlorofluorescin diacetate (DCF-DA) as an indicator of reactive oxygen species (ROS) generation, we showed that caffeine treatment directly increased intracellular oxidative stress during early period. Our findings suggest that ROS is likely to be important regulator of the apoptotic and necrotic cell death pathways in caffeine-treated osteoblasts. Our data provide evidence that caffeine has impact on bone loss also the apoptotic and necrotic effects on osteoblasts.

並列關鍵字

Osteoblasts ； Apoptosis ； Oxidative stress ； Necrosis ； Caffeine