iVObjective Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), are defined by the progressive loss of specific neuronal cell populations and are associated with protein aggregates. Because of its high metabolic rate and relatively reduced capacity for cellular regeneration compared with other organs, the brain is believed to be particularly susceptible to the damaging affects of ROS. The treatment for neurodegenerative diseases is not very good.The Traditional Chinese medicine has its speciality. Exploring the effective Traditional Chinese medicine is meaningful. The purpose of the present study is to determine the protective of 2-methoxy-6-acetyl -7-methyljuglone of Polygonum cuspidatum, to assess its possible protective effects on neurons from oxidative stress, as a preliminary step in the understanding of its mechanism of action. Methods In this study, cell viability assay, morphological assay, flow cytometry detection of cell death and mitochondrial membrane potential assay were used to investigate whether 2-methoxy-6-acetyl-7-methyljuglone exerted neuroprotective effects on H2O2 induced injury in PC12 cells and to study the possible mechanisms. 1. Establish H2O2 induced PC12 cell injury model. 2.Apply MTT asssy to detect the cell viability of different concentrations of ethyl acetate V 澳门大学硕士学位论文 extract(EAPC), petroleum ether extract (PАРС), 95%6 ethanol extract (EEPC) of Polygonum cuspidatum on PC12. 3. The effect of the EAPC and the compounds isolated from EAPC on H2O2 induced PC12 injury by LDH, MTT assay. 4. Effect of 2-Methoxy-6-acetyl-7-methyljuglone on H2O2 induced changes in mitochondrial membrane potential. 5. Adopt Annxin V/PI assay to detect the apoptosis and necrosis rate of e2-Methoxy-6-acetyl-7-methyljuglone on on H2O2 -induced injury. 6. Nuclear staining for assessment of apoptosis was observed using the chromatin dye Hoechst 33324. 7. Effect of 2-Methoxy-6-acetyl-7-methyljuglone on the cell cycle by H₂O2 induced injury in PC12 cells. Results 1.880µM H2O2 induced PC12 injury model was estabilshed. 2.The results showed that the concentration of MAM from 0.3µM to 9.6µM has no cytotoxicity. 3. Following exposure of the cells to 880µM H2O2 for 24h, the significant reduction in cell survival and activities lactate dehydrogenase (LDH) release.The cell survival were increased and the LDH release is reduced by MAM (0.3-9.6µM) plus 880µM H2O2 4.MAM(4. 8µM 1. 2µM 0. 3M) can reduce 880µM H2O2-induced mitochondrial membrane potential change of JC-1 stained mitochondria in PC12 cells at 2H. 5.The apoptotic nature of H2O2-induced cell death was further confirmed by annexin V-FITC labeling of PS exposed on the plasma membrane. MAM(4. 8µM、 1. 2μM 0. ЗµМ) can inhibit the apoptosis. 6.Further evidence for the apoptotic nature of the observed cell death was provided by the nuclear morphology by using Hoechst 33324 under a microscope. H2O2 clearly induced nuclear fragmentation. MAM(4.8µM 1.2µM、 0.3μM) can reduce the nuclear fragmentation. 7.Since apoptosis and the cell cycle are closely linked, EAPC changed the cell cycle exposure to H2O2 for 24h. MAM(4.8µM 1.2µM、 0.3µM) inhibit the S stage. vi 澳门大学硕士学位论文 8.The EAPC and the compounds from EAPC by MTT, LDH assay. The results showed that EAPC, polydatin, Anthraglycoside A and Anthraglycoside B have protective effects. Conclusion The concentrations of MAM at 4. 8µM、 1. 2µM、 0. 3µM can reduce the release of LDH, increase the depolarization of mitochondrial membrane potential (MPP), reduce the apoptosis rate and nuclear fragmentation. Taken together, these results suggest that MAM shows protection against H2O2-induced cell injury. Keywords Polygonum cuspidatum; 2-methoxy-6-acetyl-7-methyljuglone; hydrogen peroxide; PC12 cells; protection

澳门大学硕士学位论文 中文摘要 研究目的 神經退行性疾病如阿爾采默氏病(Alzheimer's disease, AD), 帕金森病(Parkinson's disease,PD), Huntington 舞蹈病(Huntington disease),肌萎縮側索硬化症(Amyotrophic lateral sclerosis, ALS) 及脊髓肌萎縮症(Spinal muscular atrophy) 等和特定的神經細胞減 少和蛋白聚集有關。和其他器官相比,大腦存在著高代謝率和相對較低的細胞再生能力 的問題,因此易受到活性氧自由基(ROS)的攻擊。 我國將步入老齡化社會,神經退行性疾病治療意義重大。為了有效地治療這類疾病,尋找 和鑒定新的藥物靶點非常重要。中藥一般是通過調節機體整體狀況而起作用,藥性較 和,且毒副作用小。開發中藥很有前景和意義。本研究主要集中在研究從虎杖中分離到 的單體2-甲氧基-6-乙醯基-7-甲基胡桃醒,採用細胞活性檢測法、流式細胞儀檢測細胞 凋亡和壞死的方法、線粒體膜電位方法篩選是否對 H2O2誘導 PC12 細胞損傷有保護作用 及存在的機制。 研究方法 1.建立 H2O2誘導的PC12 細胞損傷模型 2.MTT 法檢測不同濃度的虎杖的石油醚、乙酸乙酯、正丁醇萃取部位、95%醇提 部位不同濃度對PC12細胞存活率的影響 3.LDH、MTT 法測定虎杖的乙酸乙酯萃取部位及從中分離的單體對 H2O2 誘導的 PC12 細胞損傷的影響 4.流式細胞儀檢測2-甲氧基-6-乙醯基-7-甲基胡桃配對 H2O2誘導的PC12 細胞線粒 體膜電位的影響 5.Annexin V/PI 法檢測2-甲氧基-6-乙醯基-7-甲基胡桃對H2O2誘導的PC12 細胞 凋亡的影響 6.螢光倒置顯微鏡觀察2-甲氧基-6-乙醯基-7-甲基胡桃配對H2O2誘導的PC12細胞凋 亡的影響 7.流式細胞儀檢測2-甲氧基-6-乙醯基-7-甲基胡桃配對 H2O2誘導的PC12 細胞周期 的影響 研究結果 澳门大学硕士学位论文 1.建立了濃度為880µM的H2O誘導PC12細胞損傷模型。 2. MTT法結果顯示MAM濃度為9.6-0.34M對細胞無毒性。 3. LDH法、MTT法結果顯示MAM在濃度為9.6-0.34M可以增強細胞存活率,降低 LDH的釋放。 4. 濃度為4.84M、1.2µM、0.34M的MAM可以降低PC12細胞線粒體膜電位的去極化。 5. MAM (4.8µM、1.2µM、0.3µM)減少了PS外翻,降低了細胞凋亡。 6. 採用Hochest 33324 染色,用螢光顯微鏡觀察細胞凋亡過程中細胞核的改變的結 果顯示:MAM (4.84M、1.2uM、0.34M)減少了核固縮。 7. MAM (4.84M、1.2uM、0.34M)對細胞S期具有抑制作用。 8.虎杖的乙酸乙酯萃取部位以及從此部位分離的單體,虎杖苷、大黃素甲醚 -8-O-β-D-葡萄糖苷、大黃素-8-O-B-D-葡萄糖苷對880µM的H2O誘導PC12細胞損傷有保 護作用。 研究結論 MAM在濃度為4.84M、1.2µM、0.3M可以降低H2O2誘導的PC12細胞LDH的釋放, 膜電位的下降,減少PS外翻,減少細胞核固縮。通過抑制H2O2誘導的細胞損傷而達到對 PC12細胞的保護作用。 關鍵字:虎杖;2-甲氧基-6-乙醯基-7-甲基胡桃醒;過氧化氫;PC12細胞;保護作用