過去有研究指出腫瘤壞死因子-alpha（tumor necrosis factor-alpha；TNF-α）與帕金森氏症有關，然而，TNF-α對於神經細胞是保護或是傷害的作用及其機制仍是不明的。本實驗欲探討TNF-α在帕金森氏症疾病中所扮演的角色，因此，我們以1-methyl-4-phenyl-pyridinium（MPP+ ions；MPP+）單獨處理及MPP+ 合併TNF-α作用於PC-12細胞上，作為研究模式。PC-12細胞給予不同濃度的MPP+（600，300，150，75或0μM）或是合併TNF-α（15或30 ng/ml）處理，至24小時後，再以MTT Assay分析存活率。MTT Assay結果顯示MPP+ 處理可以引發細胞死亡，且死亡率隨著劑量增加而提昇。且MPP+ 合併TNF-α處理，可以引發更高的細胞死亡率。在細胞形態上，我們也看到了相似的結果。另外，以DAPI染色的結果證明，此一細胞死亡現象屬細胞凋亡。我們進一步探討此一細胞凋亡現象可能之機制，從西方墨點法的結果得知，MPP+ 處理可以引發caspase-3活化及JNK磷酸化，而且當MPP+ 合併TNF-α處理時可以引發更多的caspase-3活化及JNK磷酸化。但是磷酸化c-Jun（p-c-Jun）的表現量並未有增加的趨勢。我們進一步以kinase assay偵測磷酸化JNK的活性，結果發現MPP+ 合併TNF-α處理引起JNK活性上升。接著，為了探討caspase-3及JNK是否為MPP+合併TNF-α處理導致細胞死亡的主要因子，我 3 們使用caspase-3及JNK專一性抑制劑評估細胞凋亡的情形。由TUNEL實驗結果證實，抑制caspase-3可以有效緩解MPP+ 合併TNF-α處理導致的細胞死亡；然而抑制JNK只能輕微緩解MPP+ 合併TNF-α處理導致的細胞死亡。 從以上實驗結果得知，MPP+ 會經由活化caspase-3而誘導細胞凋亡，而TNF-α加劇MPP+ 之毒性和提昇caspase-3活性有關。而且此一神經毒素引起可經由活化而導致細胞凋亡。MPP+ 合併TNF-α處理也能增加JNK磷酸化，但此一JNK的磷酸化的功能並不全然與細胞凋亡有關，而其詳細功能仍待進一步的實驗調查。; Recent studies indicate tumor necrosis factor-alpha（TNF-α）is associated with Parkinson's disease（PD）. However whether TNF-α plays a neuron protective or neuron destructive role remains to be elucidated. In addition, the mechanism is still not clear. To determine the role of TNF-α in PD, we studied neurotoxic effect on dopaminergic neurons（PC-12 cells）induced by 1-methyl-4-phenyl-pyridinium（MPP+ ions；MPP+）in the presence or absence of TNF-α. PC-12 cells were either treated with MPP+ alone（600, 300, 150, 75 and 0μM） or in combination with TNF-α（15 or 30 ng/ml） for 24 hours and then assayed for cell viability. MTT Assay showed MPP+ treatment increased cell death in a dose-dependent manner. However, death rates were statistically higher in MPP+ plus TNF-α groups than those treated with either MPP+ or TNF-α only. Morphologically, we also observed similar trends. In addition, results from DAPI staining indicated the cell death was apoptotic. We further investigated possible signal molecules involved. Data from western blot analysis showed that caspase-3 and JNK activation increased when treated with MPP+ alone; and the increases were even higher in MPP+ plus TNF-α treated cells. However, the amount of phosphorylated c-Jun was not elevated in all treated 5 groups. We then detected the activation of JNK. Data from kinase assay showed JNK activity increased clearly in MPP+ plus TNF-α treatment cells. We later used Z-VAD （a caspase inhibitor） and a JNK inhibitor （SP600125） to determine whether blockade of caspase-3 and JNK activation can inhibit apoptosis. Data from TUNEL assay demonstrated caspase inhibition prevented the cell death efficiently. However, JNK inhibition only slightly prevented the cell death slightly. Our data suggest that MPP+ caused cell apoptosis through caspase-3 activation, and the enhanced toxicity induced by the addition of TNF-α is aiso related to caspase-3 activation. In addition, JNK activation was involved in the intricate signaling pathways. Nevertheless, JNK activation may partially involved in the induction of apoptosis. Further investigation will be needed to elucidate the role of JNK activation in this apoptosis.
