中草藥抑制細胞興奮性毒殺模式以治療第十七型脊髓小腦共濟失調症

Abstract

神經退化性疾病 (neurodegeneration diseases) 當中存在著由麩醯胺酸活化的興奮性毒殺 (excitotoxicity) ，如多麩醯胺酸疾病 (polyglutamine diseases) 、阿茲海默氏症疾病 (Alzheimer’s diseases) 和柏金森氏症疾病 (Parkinson’s disease) ，會導致過多的鈣離子流入、粒線體內細胞色素 C 的釋放、導致神經細胞凋亡的蛋白質活化，最後使細胞存活率的下降。許多研究報導指出，中草藥的使用，可能是一個治療退化性疾病新方法。因此，我們研究在人類神經母細胞 (neuroblastoma) SH-SY5Y 細胞以麩醯胺酸 (glutamate) 誘導的興奮性毒殺以及使用多西環素 (doxycycline) 誘導出小腦脊髓萎縮症第十七型 (Spinocerebellar ataxia, SCA17) nTBP/Q79-EGFP 細胞中 TBP/79Q-EGFP 融合蛋白的表現後，探討中草藥是否有保護神經細胞的作用。加入麩醯胺酸後導致神經細胞生存率下降，另一項實驗結果顯示NH018和其純化物NH018-1可能為有效的藥物可以增加神經細胞的存活率、NH018-1可以抑制神經細胞的凋亡 (apoptosis) 、降低乳酸脫氫酶 (LDH) 的釋放以及自由基的生成，並且降低鈣離子所造成的Calpain-2 及 α-spectrin breakdown products (SBDPs) 的表現，降低從粒線體釋放出的細胞色素C、增加細胞存活相關蛋白質：Bcl-2、Bax及降低凋亡相關：蛋白質 cleaved-caspase-9、cleaved-caspase-3和cleaved-PARP等的表現。在誘導TBP/79Q-EGFP 融合蛋白表現的細胞上，NH018-1為有效的藥物可以增加細胞的存活率以及降低cleaved-caspase-9、cleaved-caspase-3和cleaved-PARP等的表現。而施打NH018-1藥物在SCA17的基因轉殖鼠上，能夠有效地延長其在滾輪 (rotarod) 上面跑的時間、步行實驗 (footprint) 上的好轉，在其運動失調的症狀上發揮療效，以及降低 TATA box-binding protein (TBP) 、 Calpain-2 和 cleaved-caspase-3的表現。總合以上，實驗結果顯示出NH018和NH018-1藥物在加入麩氨酸以及誘導出小腦脊髓萎縮症第十七型細胞TBP/79Q-EGFP 融合蛋白的表現後，藉由抑制細胞的凋亡可以增加 SH-SY5Y 細胞以及小腦脊髓萎縮症第十七型細胞的存活率，因此NH018和NH018-1可能為有潛力治療小腦脊髓萎縮症第十七型疾病的藥物。

Excitotoxicity induced neurodegeneration, and polyglutamine (polyQ) diseases, such as Alzheimer’s disease, and Parkinson’s disease, via glutamatergic activation, resulting in excessive calcium influx, cytochrome C release from mytochondria, pro-apoptotic protein activation, and finally leads to decrease the cell viability. It was believed that Chinese herbal medicines (CHMs) might be one of new approaches to treat neurodegenerative diseases. Therefore, we investigated whether CHMs could protect neurons from monosodium glutamate (MSG)-induced excitotoxicity in human neuroblastoma SH-SY5Y cells and doxycycline (DOX) induced-nTBP-EGFP expression in spinocerebellar ataxia type 17 (SCA17) nTBP/Q79-EGFP cells. Our study showed that NH018 and its active compound NH018-1 were effective against MSG induced neurotoxicity by increasing the cell viability measured by MTT assay, and decreased the LDH release and the production of reactive oxygen species, and deduced cell apoptosis after MSG treatment in SH-SY5Y cells, DOX induced SCA17 nTBP/Q79-EGFP cells. NH018-1 also showed the remarkably protective activity against the neuronal cell death as demonstrated by : (1) reducing the Annexin V-FITC and Propidium Iodide (PI) staining, (2) the decrease of cytochrome C released from mitochondria, (3) the reduction of apoptosis-related proteins such as m-calpain (calpain-2), cleaved-caspase-9, cleaved-caspase-3 and cleaved-PARP expression, (4) the decrease of survival-related proteins such as Bcl-2, and (5) the improving of SCA17 mice performance in rotarod and footprint experiments. In conclusion, present studies showed that NH018 and NH018-1 protect cell viability after MSG treatment in SH-SY5Y cells, and DOX treatment in SCA17 nTBP/Q79-EGFP cells by inhibition of cell apoptosis. Furthermore, NH018-1 of NH018 could improve the SCA17 mice performance in rotarod test and footprint analysis as well as decrease the expression of TATA box-binding protein (TBP), calpain-2, and cleaved-caspase-3. Thus, NH018 and NH018-1 could be potential CHMs for the treatment neurodegenerative diseases.