| 摘要: | 在之前的研究中我們確定了baicalein會刺激B16F10小鼠黑色素瘤細胞株產生superoxide及hydroxyl radical，且此ROS的產生路徑與12-lipoxygenase (12-LOX)有關。同時也發現baicalein會造成細胞存活率的下降和細胞凋亡；然而baicalein造成細胞死亡到底是因為產生了ROS還是本身抑制12-LOX的關係，原因仍是眾說紛紜。所以本次研究我們利用胞外的ROS清除劑mannitol、catalase及胞內ROS清除劑DMPO、CMH來清除baicalein產生的ROS，結果發現ROS清除劑對於細胞的生長變異影響不大，但在MTT觀察細胞生存率的實驗中可以有效回復baicalein造成的細胞存活率下降，而在研究蛋白表現的western bloting實驗上，ROS清除劑對於細胞凋亡蛋白active caspase-3和內在途徑凋亡蛋白Bax的表現又沒有改變。另外在12-LOX方面，我們使用了12-LOX的下游活性產物 (12-HETE)來抵消baicalein的12-LOX抑制作用，結果發現12-HETE雖然對於細胞生長變異不產生影響，但是可以回復baicalein誘導的B16F10小鼠黑色素瘤細胞存活率下降，其對於baicalein產生的active caspase-3表現增加也有明顯地抑制作用。最終我們也同時給予ROS清除劑和12-HETE共同作用，結果發現兩者同時存在下會將baicalein對小鼠黑色素瘤細胞的傷害降到最低，如此可以說明，baicalein造成的B16F10小鼠黑色素瘤細胞死亡，與ROS產生和抑制12-LOX皆有關係；然而細胞凋亡部分則是因為baicalein抑制了12-LOX所造成。另外我們也用siRNA技術來降低12-LOX的表現，結果發現baicalein造成控制組細胞生存率的下降遠高於12-LOX siRNA的細胞，所以我們推論baicalein造成的細胞死亡必會透過12-LOX，而且12-LOX siRNA與baicalein抑制12-LOX機轉不同，再由之前研究12-LOX siRNA會降低baicalein在B16F10細胞產生的ROS，推論baicalein造成的細胞死亡主因應該是ROS，而此死亡也應為細胞壞死。為了證實以上論點，我們還另外用了流式細胞儀來分析baicalein造成的細胞死亡，結果發現細胞壞死果然是細胞死亡的主要部份，細胞凋亡所佔比率甚小。由以上實驗結果可以推出一個結論，baicalein造成B16F10小鼠黑色素瘤細胞的死亡，主因為其透過12-LOX產生的ROS，所以大部份的死亡是細胞壞死，其中baicalein抑制12-LOX造成之細胞凋亡僅佔細胞死亡的一小部份。
In our previous study, we demonstrated that baicalein induces the formation of superoxide and hydroxyl radicals via 12-lipoxygenase (12-LOX) in B16F10 mouse melanoma cell line; simultaneously we also found that baicalein caused a reduction in cellular viability and induce cell apoptosis. At present, whether baicalein-induced cell death involves the 12-lipoxygenase (12-LOX) suppression or ROS generation is still unclear. In the present investigation, we utilized the extracellular ROS scavengers namely mannitol and catalase, and the intracellular ROS scavengers such as DMPO and CMH to assess their scavenging ability on ROS produced by baicalein. In addition, we also found that the ROS scavengers had no effect on cell growth differentiation, but in the cellular viability (MTT) assay they could effectively reverse the cell viability reduction induced by baicalein. Moreover western blot analysis revealed that the ROS scavengers didn’t respond the cell apoptosis protein (active caspase-3) and the intrinsic apoptosis pathway protein (BAX). In the 12-LOX aspect, we use the 12-LOX downstream product (12-HETE) to counterbalance the 12-LOX inhibitory action of baicalein. We found that the 12-HETE had no difference in cell growth differentiation, but it could reverse the reduction of cellular viability caused by baicalein in B16F10 mouse melanoma cell line effectively, this results are as similar as in ROS scavengers. The 12-HETE also possesses an inhibitory effect on the increase in expression of active caspase-3 caused by baicalein. Finally we pretreated the ROS scavengers and 12-HETE at the same time, the results showed that both drugs minimize the damage caused by baicalein. We inferred that the B16F10 mouse melanoma cell death caused by baicalein is related to both of 12-LOX suppression and ROS generation, but the apoptosis is only because of the 12-LOX suppression by baicalein. In addition, we also used siRNA technology to reduce the performance of 12-LOX; we found the cellular viability reduction of Electro control group is more pronounced than the 12-LOX siRNA group. Therefore, we inferred the cell death caused by baicalein through the 12-LOX; the 12-LOX siRNA and 12-LOX inhibition by baicalein are in different mechanisms. According to our previous study, the 12-LOX siRNA group would decrease the ROS generation caused by baicalein, so we assumed that the main reason of cell death caused by baicalein is the ROS generation and could lead to cell necrosis. In order to confirm the above arguments, we also use the cytometric analysis to examine the cell death caused by baicalein in which we found that the cell necrosis is the major part of cell death, and the apoptosis play a minimum role. The data generated from this study, we can conclude that the B16F10 mouse melanoma cell death caused by baicalein is mainly because of the ROS generation through the 12-LOX. Therefore, the majority of cell death may be due to cell necrosis. The cell apoptosis caused by 12-LOX suppression of baicalein is only a small part. |
