中文摘要

Midazolam是一種在臨床上被廣泛使用在鎮定及痲醉效果的用藥，他同時也是屬於benzodiazepine的藥物之一。在先前的研究發現，midazolam可以藉由調控在中樞神經內的GABAA 受體去達到其鎮定痲醉的效果，而且，先前研究也有發現，比起其它benzodiazepine的藥物而言，midazolam進入體內之後可以迅速的作用及被代謝掉，而在另一方面，也有研究報告指出midazolam它還可以調控腎上腺皮質的類固醇合成。值得注意的是，在一些患有荷爾蒙性質的癌症而言，像是前列腺癌等，在使用midaozlam這一類的痲醉藥物時會不會對其造成怎樣的影響是值得需要被注意的，在我們先前的研究發現，midazolam可以促使正常的小鼠萊氏細胞與MA-10小鼠萊氏細胞癌進行類固醇合成並且是藉由依賴StAR蛋白質所經由蛋白激酶C與A所進行的，特別的是他同時也可以導致MA-10小鼠萊氏細胞癌細胞死亡，有趣的是，這個現像在正常的小鼠萊氏細胞上並不會產生。因此，我們想要更進一步的去探討到底是什麼細胞機制可以促使MA-10小鼠萊氏細胞癌死亡，首先，我們想要先觀察細胞形態及MTT細胞活性檢測，看midazolam是否真的能夠促使MA-10小鼠萊氏細胞癌死亡，結果顯示，midazolam可以促使MA-10小鼠萊氏細胞癌細胞皺縮、飄浮，而在細胞活性檢測上我們也發現其midazolam對MA-10小鼠萊氏細胞癌有時間及劑量依存性的效果存在，此外，我們從流式細胞儀檢測的結果上也發現經由midazolam處理過後的MA-10小鼠萊氏細胞癌其細胞週期有sub-G1週期的增加，因此，我們接下來想要更進一步的證實其midazolam究竟是利用什麼樣的機制去調控MA-10小鼠萊氏細胞癌的死亡，我們由蛋白質電泳結果中發現midazolam可以促進caspae-8, -9與-3的表現量上升，並且去促使PARP這個蛋白質被切割使其失去功用，使細胞走向死亡 (p＜0.05)。但在另一方面對於Bax、cytochrome-c並無進行調控其表現的差異產生 (p＞0.05)，可是卻對Bid這個蛋白質有明顯被切割的現像產生 (p＜0.05)。此外，我們也去觀察了其它可能的凋亡機制路徑的蛋白質，由結果中顯示，我們發現其磷酸化Akt有被明顯抑制的效果產生 (p＜0.05)。但另一方面也可刺激p38、磷酸化ERK及磷酸化JNK蛋白質的表現 (p＜0.05)。更進一步我們也發現說midazolam可以明顯的增加細胞中氧自由基的產生。總括來說，我們發現midazolam可以藉由活化caspae-8, -9及-3並且去抑制磷酸化Akt的表現而使PARP這個蛋白質被去活化，在另一方面，我們也發現midazolam可刺激p38、磷酸化ERK及磷酸化JNK等蛋白質的表現並且伴隨著細胞中氧自由基的增加去促使細胞走向細胞死亡。

Abstract

Midazolam is widely used as a sedative anesthetic induction agent, and it is a derivative from benzodiazepine drugs. In previous study, midazolam could modulate GABAA receptor in central nervous system to achieve sedative effect. Moreover, midazolam could rapidly onset the action with highly metabolic clearance when compared with other benzodiazepine drugs. In fact, it has been reported that benzodiazepine could regulate adrenocortical steroidogenesis, indicating that midazolam might influence the steroidogenesis when it is used as a sedative drug. Particularly in prostate cancer patients, the effect of the midazolam on steroidogenesis may be detrimental to the underlying diseases. In our previous data we found that midazolam induced StAR-dependent steroidogenesis via PKC and PKA signaling transduction pathways in MA-10 mouse Leydig tumor cells and primary Leydig cells. In the present study, our data showed that midazolam would also induce MA-10 Leydig tumor cell rounding-up and membrane blebbing, and then cell death. But the cell apoptotic phenomenon was not found in primary mouse Leydig cells. Thus, we further examined the cell death effect of midazolam on MA-10 cells. First, we exploited MTT assay and flow cytometry to investigate, and results showed that midazolam decreased cells viability with the cell cycle accumulation of sub-G1 phase in time- and dose-dependent manners. We then examined the expression of apoptotic proteins by immunoblotting. We found that the expressions of caspase-8, -9 and -3, and PARP were significantly increased in higher dosages and later time points of midazolam treatment (p＜0.05). Present data illustrated that the expression of Bax and cytochrome-c did not exchange (p＞0.05), and Bid was significantly decreased (p＜0.05). In addition, we detected the expression of possible apoptotic signal pathway proteins by immunoblotting. Results showed that midazolam could significantly reduce pAkt protein expression (p＜0.05). Moreover, midazolam could significantly stimulate p38, pERK and pJNK proteins expression (p＜0.05). Furthermore, we found that midazolam could significantly increase the production of reactive oxygen species (ROS). In conclusion, midazolam could induce MA-10 mouse Leydig tumor cell apoptosis through activating the caspase-8, -9, -3 and PARP pathway with ROS production, and inhibiting pAkt expression plus inducing p38, pERK and pJNK MAPK pathway.

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