With ageing populations in most developed countries, the problem of neurodegenerative diseases presents a big challenge and potential economic burden. It is postulated that oxidative stress might induce excessive apoptosis of neuronal cells and cause aged-related neurodegeneration diseases. Here we used tert-butyl hydroperoxide (t-BHP), a commonly used oxidative agent, to induce lethal injury in rat pheochromocytoma PC12 cells as in vitro model for neurodegenerative diseases. The anti-oxidizing activities of several single compounds isolated from the dried rhizome of Polygonum cuspidatum (Chinese named Hu-Zhang) were explored. Interestingly, the potent anti-oxidative property of 2-methoxy-6-acetyl-7-methyljuglone (MAM), was reported for the first time in the present study. Pretreatments (24 h) of MAM (0.5, 1, 2.5, 5 µM) significantly attenuated PC12 cell death induced by t-BHP. MAM were able to reduce cell death induced by t-BHP-oxidative stress at much lower concentrations than resveratrol and lipoid acid, indicating that the protective effect of MAM was much stronger than those of resveratrol and lipoid acid, with cell viability measured by both MTT and LDH assays. viii The protective effects produced by MAM was thought to be through the inhibition of the apoptotic pathways, evidenced by the reduction of the formation of apoptotic bodies inside the cell induced by t-BHP, which was visualized with DNA staining using Hoechst 33342 fluorescent dye. The protective effects of MAM on t-BHPinduced apoptotic cell death was also investigated by TUNEL labeling assay, which measures the level of DNA fragmentation and is a commonly accepted marker of apoptosis. It was shown that MAM could also reduced DNA fragmentation, providing additional evidence of the anti-apoptotic property of MAМ. The underlying mechanisms were further studied and the results suggested that MAM protected PC12 cells against oxidative damage through the inhibition of the MAPK pathways ERK 1/2, JNK and p38 MAPK. In conclusion, MAM is a potential antioxidative and anti-apoptotic agent useful for the prevention and the treatment of agerelated neurodegenerative diseases. Keywords: 2-methoxy-6-acetyl-7-methyljuglone; MAM; anti-oxidative; anti-apoptosis; tert-butyl hydroperoxide; neurodegenerative; MAPK.

摘要 伴隨著人口老齡化問題越來越嚴重,神經退行性疾病的發病率越來越高,大大 加重了社會的醫療、經濟負擔。近年研究表明氧化損傷能够引起神經元細胞的 凋亡,并且同時誘發神經退行性疾病。所以,在抗氧化劑、抗凋亡劑中尋找具 有治療神經退行性疾病功效的潛在藥物,被視為一種成本低,風險小的治療神 經退行性疾病新藥開發策略。 虎杖是被報導的具有抗氧化活性的常用中藥之一,某些成份已報導具有神經保 護作用。為了進一步探究虎杖的抗氧化作用的藥效物質基礎,以及尋找具有神 經保護作用的潛在化合物及探究其作用機制,本研究採用與多巴胺神經元細胞 有很多相似之處腎上腺嗜鉻細胞瘤細胞株 PC12 作為實驗對象,使用叔丁基過氧 化氫(-BHP)誘導其氧化損傷,造成氧化应激模型。利用該模型篩選從虎杖中 分離得到的多種成份的抗氧化活性,我們發現 2-甲氧基-6-乙酰基-7-甲基胡桃 (MAM)展現了很好的抗氧化活性和 PC12 細胞的保護作用,而該成份的抗氧 化活性尚未有報導。 本研究採用 MTT 法以及乳酸脫氫酶(LDH)漏出率分別測試 t-BHP 所致的細 胞存活率下降和細胞膜損傷的程度,以及MAM 的保護作用,並且採用硫辛酸 和白藜蘆醇作為陽性對照。結果表明,MAM 預處理 24 小時可以明顯抗BHP 所致的PC12 細胞存活率下降以及 LDH 的大量釋放,隨著 MAM 濃度增加 保護作用呈增強趨勢。其保護作用强於硫辛酸和白藜蘆醇。在MTT法測試中, 5 µM 濃度的MAM(111.5± 4.8%)可以完全 抗t-BHP 所致的細胞存活率下降 (26.8±13.2%)。而硫辛酸(100µM, 52.1 ±6.6%)與白藜蘆醇(50M, 97.6± 6.4%)也在不同程度上改善了1-BHP 導致的細胞毒作用。LDH 漏出率測 試結果與 MTT 發結果基本一致,5µM 濃度的 MAM(6.2±5.3%, LDH 漏出 率)大大降低了t-BHP 所致的細胞膜損傷(68.8±12.6%),硫辛酸(100µM, 43.0 + 12.1%)與白藜蘆醇(50M, 11.9 12.1%)同樣起到了不同程度的細胞 膜保護作用。 為了考察 MAM 是否具有抗凋亡作用,我們選用 Hoechst 33342 DNA 螢光染料 定性觀察細胞核形態變化,以及 TUNEL 法和流式細胞儀技術定量考察 PC12凋 亡細胞比例。Hoechst 33342 細胞核染色結果顯示,t-BHP 刺激組有大量的細胞 核呈濃染緻密的固縮形態,有凋亡小體產生,並且細胞密度較正常照組低;而 不同濃度的MAM 預處理組改善了 t-BHP 所致的細胞核固縮情況,以及大大降 低了凋亡小體的產生,提示 MAM 具有很好的抗凋亡作用。TUNEL 法流式細胞 儀檢測結果與 Hoechst 33342 細胞核染色結果相近,同樣顯示了-BHP 能夠誘導 大量凋亡細胞產生,而不同濃度的MAM 預處理 后凋亡細胞的比例大大降低,進一步證明 MAM 具有很好的抗凋亡效果。 為了進一步探究 MAM 保護作用的潛在細胞機制,我們觀察了有絲分裂素活化 蛋白酶(MAPK)信號轉導通路的蛋白表達變化。MAPK 級聯途徑是細胞外刺 激信號轉導致細胞核,從而介導細胞產生反應的信息傳遞通路,在細胞凋亡中 起重要作用。MAPK 級聯途徑主要由三條信號通路組成分別為:細胞外調節蛋 白激酶(ERK)通路,c-Jun 氨基末端激酶(JNK)通路、P38 MAP 激酶通路。 蛋白印跡法(western blotting)結果顯示,t-BHP 刺激在15分鐘內磷酸化 ERK、JNK、p38 MAPK 的表達達到最大值;而MAM 預處理很好地抑制了 ERK、JNK、p38 MAPK的激活,呈現濃度依赖性。 總之,本研究初次報導了 MAM 是虎杖中具有抗氧化活性的成份之一,並且通 過下調氧化應激介導的 ERK、JNK,p38 MAPK的激活,發揮很好的抗凋亡作 用。MAM 可能會在氧化損傷造成的神經退行性疾病的預防和治療中起重要作 用。 關鍵詞:2-甲氧基-6-乙酰基-7-甲基胡桃醒;抗氧化;凋亡;神經退行性疾病; MAPK xi