- 學位論文
六味地黃丸減少運動神經元存活蛋白缺乏產生之神經損傷
Liuwei Dihuang Wang attenuates neuronal damage induced by survival motor neuron protein deficiency
摘要
六味地黃丸 (Liuwei dihuang wang, LDW) 為傳統的中藥複方，由熟地黃、山茱萸、山藥、澤瀉、茯苓與牡丹皮等六種中草藥組成，使用於治療糖尿病、腎臟疾病與神經系統障礙，其中有些成分已被證實在神經細胞具有對抗氧化壓力、神經發炎與細胞凋亡之特性，而有神經保護效果。因此，本研究的目的為評估，六味地黃丸在運動神經元存活蛋白 (survival motor neuron protein, Smn protein) 表現量下降後對小鼠運動神經元細胞所產生之神經損傷是否具有保護功效，並探討其可能機轉。實驗所使用的細胞株為穩定攜帶Smn-specific shRNA的運動神經元NSC34細胞。投予doxycycline (1 ug/mL) 48小時誘導運動神經元存活蛋白表現量下降後，使用MTT assay與LDH assay分析細胞存活率，透過細胞核染色判斷細胞凋亡伴隨之染色質濃縮現象，並使用西方點漬法觀察細胞凋亡與神經突 (neurite) 生長相關蛋白的表現，搭配倒立型顯微鏡觀察神經突生長情形。實驗結果發現，當運動神經元存活蛋白表現量下降後，將會活化細胞凋亡路徑並抑制神經突生長，造成神經損傷。然而，缺乏運動神經元存活蛋白的細胞給予六味地黃丸 (0.01-10 ug/mL) 後，細胞存活率及運動神經元存活蛋白表現明顯增加。而實驗結果顯示，六味地黃丸減少細胞凋亡作用機轉可能是經由增加Bcl-2的表現量，及減少促凋亡相關蛋白cytochrome c與cleaved-caspase 3在細胞質的含量。我們進一步討論六味地黃丸在運動神經元存活蛋白缺乏下對神經突生長的影響。實驗結果顯示，六味地黃丸可經由抑制神經突生長負向調控路徑RhoA/ROCK/p-LIMK/p-cofilin改善神經突的生長。總結，本研究結果證實六味地黃丸可以減少因運動神經元存活蛋白缺乏所造成的神經細胞損傷，因此，六味地黃丸極具潛力成為神經退化疾病的治療用藥。
並列摘要
Liuwei dihuang wang (LDW), a traditional Chinese herbal formula, consists of Rehmanniae Radix Praeparata (Shu Di Huang), Cornus Fructus (Shan Zhu Yu), Dioscorea Rhizoma (Shan Yao), Alisma Rhizoma (Ze Xie), Poria Cocos (Fu Ling), and Paeonia Cortex (Mu Dan Pi). LDW is traditionally used for treatment of diabetics, renal disorders, and neurosis. Some components of LDW have been confirmed their neuroprotective effects via anti-oxidative, anti-inflammatory, and anti-apoptotic properties. In this study, we aimed to examine the protective effects of LDW on motor neuronal NSC34 cells against survival motor neuron (Smn) protein deficiency. Doxycycline (1 ug/mL) was used to induce Smn deficiency in stable NSC34 cell line carrying Smn-specific shRNA. Cell viability and cytotoxicity were measured by MTT assay and LDH assay, respectively. The condensed chromatin of apoptotic cells were observed by nuclei staining. Protein expressions were determined by western blot analysis. Cell morphology and neurite length were observed by inverted microscope. Our results indicated that Smn-knockdown activated apoptotic pathway, decreased neurite outgrowth and induced neuronal damage. However, LDW (0.01-10 ug/mL) increased cell viability and Smn expression in inducible Smn-knockdown NSC34 cells. Moreover, LDW attenuated apoptotic death via upregulating Bcl-2, reducing cytosolic cytochrome c and decreasing the cleavage of caspase 3. We further investigated the effects of LDW on meurite growth under Smn deficiency. Results indicated LDW promoted neurite outgrowth via inactivation of RhoA/ROCK/p-LIMK/p-cofilin pathway, a negatively regulatory pathway of neurite development. In conclusion, the present results reveal the novel protective effects of LDW against Smn deficiency in NSC34 motor neuronal cells, suggesting the potential therapeutic use of LDW for neurodegerative diseases.