| 摘要: | 本研究探討五味子素保護麩胺酸鹽誘發胎鼠腦皮質神經細胞興奮性毒性的研究，並從細胞分子的層次，去探討五味子素的神經保護機制。實驗結果發現經麩胺酸鹽 (10 μM) 誘導24小時後會使胎鼠腦皮質神經細胞之細胞存活率降低，而藉由細胞形態的改變、DAPI螢光染色與DNA斷片的形成結果得知，麩胺酸鹽誘導胎鼠腦皮質神經細胞死亡的方式為細胞凋亡。而前2小時事先給予五味子素 (10 μM與 100 μM)，經由MTT試驗、細胞型態觀察與DNA電泳分析得知，五味子素可減少麩胺酸鹽所誘導的神經細胞毒性，且呈現劑量依存性的關係。而事先給予五味子素會減少細胞內鈣離子 (Ca2+) 的濃度，降低了一連串的凋亡生化特徵，包含了經由提升細胞內抗氧化酵素的含量 (GSH)，降低細胞內的氧化壓力 (ROS)、一氧化氮 (NO) 及脂質過氧化物 (MDA) 的生成，促使粒線體膜電位 (MMP) 下降，使細胞內抑制凋亡蛋白的Bcl-2與Bcl-XL 蛋白表現量增加，而促進凋亡蛋白的Bax與Bak蛋白表現量減少，也因此讓粒線體產物AIF與Nodo G蛋白表現降低，進而減少粒線體內之cytochrome c釋放至細胞質中，阻止caspase 9的活化，並降低下游caspase 3的活化，減少PARP被caspase 3分解的機會。再者，於內質網壓力的部份，五味子素亦會降低caspase-12蛋白表現量，減少過多的內質網壓力存在於細胞中，而來保護腦皮質神經細胞對抗麩胺酸鹽所誘導的細胞凋亡。最後，在MAPK family訊號傳導路徑中，五味子素可改善麩胺酸鹽誘導腦皮質神經細胞凋亡的現象，其作用可能透過降低磷酸化的ERK、JNK與P38的活化來保護因麩胺酸鹽所誘發之細胞凋亡的情形。綜合以上結果顯示，五味子素保護因麩胺酸鹽誘發細胞凋亡之路徑，主要是透過減少凋亡生化特徵與粒線體凋亡路徑去減少細胞凋亡相關的前驅物質被釋放出來，並調控MAPK家族及降低內質網壓力及caspase family的活化。
The neuroprotective effect of schizandrin on the glutamate-induced neuronal excitotoxicity and its potential mechanisms were investigated using primary cultures of rat cortical cells. After exposure of primary cultures of rat cortical cells to 10 μM glutamate for 24 h, cortical cell cultures exhibited remarkable apoptotic death. Pretreatment of the cortical cell cultures with schizandrin for 2 h significantly protected cortical neurons against glutamate-induced excitotoxicity. Schizandrin reduced apoptotic characteristics and the condensed nuclei evidenced by DAPI staining and DNA fragmentation in glutamate-injured cortical cell cultures. In addition, schizandrin diminished the intracellular Ca2+ influx, and inhibited the subsequent overproduction of NO, ROS, cytochrome c and preserved the mitochondrial membrane potential. Furthermore, schizandrin also increased cellular level of GSH and inhibited the membrane lipid peroxidation (MDA). For Western blot, schizandrin increased the expression of Bcl-2 and Bcl-XL protein, whereas decreased the expression of Bax and Bak. Furthermore, schizandrin also decreased the expression of AIF and Nodo G protein. Schizandrin attenuated the protein levels changes of procaspase-9, caspase-9, caspase-3, cleaved PARP and endoplasmic reticulum stress of caspase-12. On the other hand, our results demonstrataed that the protective effect of schizandrin on glutamate-induced apoptosis was inhibited by the activation of phospho-ERK, phospho-JNK and phospho-P38. Taken together, these results indicated that regulation of the MAPK family, mitochondria mediated pathway and Bcl-2 family molecules, decreation of mitochrondrial cytochrome c release, and inactivation of caspase cascade may be the molecular mechanisms of schizandrin protected glutamate-induced apoptosis in the primary cultures of rat cortical cells. |
