Midazolam被廣泛使用作為鎮靜麻醉誘導劑，它是一種衍生於苯二氮卓類的藥物。在之前的研究，我們發現midazolam是藉由通過PKA和PKC途徑，再經由PBR和StAR proteins的影響，引起MA-10小鼠Leydig cell類固醇的產量增加。此外，在較高劑量的midazolam則會引起的MA-10細胞的膜出泡，引發在細胞的死亡的現象。之前研究我們已經發現，midazolam能刺激凋亡蛋白酶(caspase)和抑制絲裂原活化蛋白激酶(MAPK)；而midazolam也會抑制Akt的途徑誘導MA-10細胞凋亡。在本研究中，我們想進一步調查midazolam詳細機制，我們也推測，midazolam誘導MA-10細胞凋亡藉由通過粒線體途徑。我們觀察到內生性淍亡途徑蛋白，促使MA-10細胞死亡，我們將重點放在細胞凋亡、細胞自噬的途徑、內質網壓力途徑和PKC路徑相關蛋白的表達。結果顯示，midazolam可誘導內生性淍亡途徑活化，包括增加Bax和細胞色素C蛋白的表達及LC3II蛋白，但降低Bid蛋白的表達。Midazolam處理課磷酸化的Akt和磷酸化的mTOR蛋白質的表達皆下降。這些數據表示，midazolam處理MA-10細胞可以激活內生性淍亡途徑與促進細胞自噬現象。此外，midazolam處理後誘導裂解caspase-7, -12和增加磷酸化的PKC表現, 但卻降低p53蛋白的表達。這些結果說明，midazolam處理MA-10細胞後，可能激活內質網壓力和PKC途徑誘導凋亡。
其他自噬相關蛋，ER壓力途徑蛋白; ATF6,XPB1和eIF2α，以及不同的PKC亞型蛋白表達改變受midazolam處理。總結來說是midazolam可以活化凋亡途徑，造成內質網壓力上升，和細胞自噬的現象與抑制PKC路徑繼而去誘導MA-10細胞凋亡。

Midazolam is widely used as sedative anesthetic induction agent, and it is a derivative from benzodiazepine drug. In previous study, we have found that midazolam could induce steroidogenesis in MA-10 mouse Leydig cells via PKA and PKC pathways along with the expression of PBR and StAR proteins1. In addition, midazolam at higher dosages induced rounding-up, membrane blebbing, and then cell death phenomenon in MA-10 cells. In fact, we have found that midazolam could stimulate caspase and MAPK, but inhibit Akt, pathways to induce apoptosis in MA-10 cells. In the present study, we would like to further investigate the detail mechanism activated by midazolam in MA-10 cells, and will focus on the apopototic pathway-, autophage pathway-, ER strees pathway-, and PKC pathway-related protein expressions. The results showed that midazolam induced the expression of Bax, cytochrome C and Light Chain 3 II (LC3II) proteins, but decreased the expression of Bid, phosphor-Akt and phosphor-mTOR proteins. These data illustrated that midazolam could activate intrinsic apopototic pathway plus autophagy in MA-10 cells. Moreover, midazolam treatment induced the cleavage of caspase-7,-12 and the phosphorylations of PKC, but decreased the expression of p53. These results demonstrated that midazolam might activate ER stress and PKC pathways to induce MA-10 cell death. We also hypothesized that midazolam could induce apoptosis via the mitochondrial pathway in MA-10 cell. We observated that the expressions of intrinsic apoptotic pathway proteins, other autophagy-related proteins, ER stress pathway-related proteins; activating transcription factor 6 (ATF6), X box-binding protein 1 (XBP1), eukaryotic translation initiator factor 2α (eIF2α), and different isoforms of PKC proteins were changed by treatment of midazolam. In conclusion, midazolam could activate apoptotic pathway, ER stress pathway, PKC pathway with autophage involvement to induce MA-10 cell apoptosis.

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