應用半自動 Bonfire程式分析神經樹突形態評估3,5-二甲基苯胺與其代謝物對於初代皮質神經元毒性之影響以及松杉靈芝對於老化大鼠認知功能障礙之改善效果

第一部分摘要大腦發育歷經神經分化、遷移、排列與網絡形成，這個歷程會受到許多內生性與外源性因子的調控，若此過程受到干擾，會造成大腦發育畸形或功能失常。近年來許多研究發現，合成類化合物以及環境毒物對神經發育有潛在的危險性。單環芳香胺類化合物 (Monocyclic aromatic amines , MAAs) 普遍存在於日常周遭環境中，香菸的煙霧、染劑、橡膠和汽機車燃料中都含有單環芳香胺。單環芳香胺類化合物具有潛在性的致癌危機，特別是其中一類的3,5-二甲基苯胺 (3,5-dimethylanilie , 3,5-DMA) ，文獻指出會高度誘發膀胱癌產生。而單環芳香胺代謝物會和DNA鍵結並誘發活性氧物質 (Reactive oxygen species, ROS) 生成，導致細胞死亡。儘管芳胺族被定義為致癌的危險因素，但是對於中樞神經系統暴露於3,5-DMA的神經毒性之風險鮮為人知。因此本研究假設3,5- DMA和其代謝物可能對皮質神經元造成氧化損傷，並破壞神經樹突之型態發育。本實驗以體外培養模式 (in vitro model) 分別給予3,5-DMA和3,5-DMA與肝臟萃取物 (liver abstract S9) 共培養，以及其代謝物3,5-二甲氨基苯酚 (3,5-dimethylaminophenol , 3,5-DMAP) ，探討皮質細胞與神經元存活率，和細胞體內ROS表現量。實驗結果顯示，加入S9之組別神經毒性高於單獨處理3,5-DMA組別，顯示3,5-DMA經過S9代謝產生具有高毒性之代謝產物。而處理代謝物3,5-DMAP組別和其他組相比則有顯著神經毒性。由於文獻指出3,5-DMA經代謝產生ROS，本實驗中於各組間加入自由基清除劑：N-乙酰半胱胺酸 (N-acetyl cysteine，NAC) ，討探是否可以防止氧化壓力造成神經元的損傷，結果發現經過NAC處理的組別可以有效降低細胞內ROS的產生，進而減少ROS對於神經細胞之傷害，而達到保護之作用。第二部分摘要細胞與組織的老化是內在因子 (如基因) 與外在環境 (如熱、冷、疾病、營養素缺乏等因素) 所造成。隨著年齡增長，細胞與組織的再生能力降低，無法修復身體內損壞或病變的蛋白質，造成異常的蛋白質開始於體內累積，產生氧化壓力與生成自由基，進而攻擊正常蛋白質、細胞與組織，導致疾病發生。氧化壓力所造成的老化現象，特別容易受到影響的是腦部組織，氧化壓力造成的神經損傷包括影響神經再生或造成腦細胞死亡，進而影響到正常腦神經迴路，導致相關疾病病患的學習或行為障礙。舉例來說，於腦神經科學中具有相關成因的常見疾病有失憶症 (Dementia) 、阿茲海默症 (Alzheimer's disease) 、帕金森氏症(Parkinson's) 等。因此，如何抑制氧化壓力的生成為延緩老化的重要研究方向。中國傳統草藥松杉靈芝 (Ganoderma Tsugae) ，自古以來被認為可以延年益壽，而先前研究指出，靈芝具有抗腫瘤、抗發炎、免疫調節、保肝與抗脂質生成的作用。服用靈芝可以促進新陳代謝並維持身體機能運作，可減緩蛋白質汰換速率、抑制細胞凋亡、減輕疼痛。許多研究指出長期暴露於半乳糖D-galactose(D-gal)會導致ROS過度生成引起體內氧化壓力，模擬自然老化大腦。本實驗欲探討Traditional Chinese medicine (TCM) Ganoderma tsugae DMSO extract (GTDE) 是否改善D-galactose (D-gal) 誘導的老化大鼠模型的氧化壓力以及具有保護記憶缺失的潛在作用。將實驗分為4組對照組 (control)、靈芝組 (GTDE)、誘導老化組 (D-gal)、誘導老化靈芝組 (D-gal+ GTDE)，給予大鼠皮下注射D-gal(100 mg/kg/day) 並同時口服Ganoderma tsugae DMSO extract (GTDE) (200 μg/kg/day) 並持續25週。實驗結果發現，GTDE顯著改善D-gal大鼠對於環境的警覺性和空間學習記憶。GTDE顯著降低D-gal大鼠體內AGEs表現。此外，抗氧化相關蛋白 (SOD-1、catalase)以及腦源性神經營養因子 (BDNF) 的表現量在口服GTDE組別有顯著增加。藉由螢光4HNE、NLRP3分別標定脂質過氧化產物以及發炎複合體，可在大腦皮質中發現的4HNE、NLRP3表現量在服用GTDE後有明顯降低。從上述結果表明，GTDE可以藉由調節腦部大腦抗化相關蛋白來降低大腦皮質炎症反應，以及增加BDNF表現量促進突觸可塑性，改善老化所造成的認知功能障礙。

關鍵字

單環芳香胺類化合物；活性氧物質；神經發育； N-乙酰半胱胺酸；半乳糖；松杉靈芝；氧化壓力；認知功能障礙

並列摘要

PartⅠ Abstract The development of the nervous system is a highly complex process, which includes neuron generation, migration, organization and patterning. Disruption of these steps results in malformation of the brain. In recent years, many studies have found that synthetic compounds and environmental toxicants have potential risks to neurodevelopment. Monocyclic aromatic amines (MAAs) are a group of chemicals presence in the environment from multiple sources via tobacco smoke, dyes, rubber, textiles, and gasoline and coal combustion. MAAs are potential carcinogenesis, particularly 3,5-dimethylanilie (3,5-DMA) , which is highly associated with bladder cancers. The possible mechanism of action in MAA-induced mutagenicity is likely to be through redox cycling of bio-metabolized compound, which would generate covalent DNA adducts and induce the production of free radicals, which might lead to cell death. Although some aromatic amines are defined as risk factors for carcinogenesis, little is known about the neurotoxic risk in central nervous system created by 3,5-DMA exposures. We therefore hypothesized that 3,5-DMA and its metabolites may cause the oxidative damage to the cultured cortical neurons and then perturb their dendritic morphogenesis. We therefore investigated the cortical cell and neuron viability, and ROS generation of 3,5-DMA and 3,5-DMA with liver abstract S9, and its main metabolite, 3,5-dimethylaminophenol (3,5-DMAP) in in vitro primary cortical cultures. We found that in the presence of S9, 3,5-DMA showed more toxic comparing to the absence condition, indicating that a combination of 3,5-DMA and its metabolites were produced by the metabolic reaction of S9. Accordingly, the following investigation will focus on the effects of 3,5-DMA plus S9 and its main metabolite, 3,5-DMAP. In comparison with 3,5-DMA with S9, 3,5-DMAP had the highest potency in inducing neuronal toxicity. Since 3,5-DMA and its metabolites induced the production of ROS, we therefore tested whether ROS scavenger, n-acetyl cysteine (NAC) , could prevent the neurons from the oxidative damage. The data show that NAC can effectively reducethe intracellular ROS production and rescue neural death. PartⅡ Abstract The aging of cells and tissues is intrinsic factors (genes) and the external environment (such as heat, cold, disease, lack of nutrients and other factors) causes. Neural aging is a progressive loss of physiological functions and metabolic processes, which is always accompanied by the occurrence of several neurodegenerative diseases, like Dementia, Parkinson’s and Alzheimer’s diseases. Oxidative damage caused by free radical is one of the well-known theories that contributes to the aging process. Brain is highly sensitive to oxidative stress because of high metabolic rate and low antioxidant ability. Ganoderma Tsugae, a mushroom used in traditional Chinese medicine, has been used to promote health and longevity for centuries in the Asia. G. tsugae and its fruiting body, spores, mycelia, have been used to successfully treat hepatitis, immunomodulatory, anti-viral effects and cancers. D -galactose (D-gal) is a reducing sugar that can produce ROS during its metabolism in vivo. D-gal animal model can imitate many characters of nature brain aging. To address whether Traditional Chinese medicine (TCM) Ganoderma tsugae DMSO extract (GTDE) could reduce the aging caused oxidative damage and learning and memory deficits. Rats will be dived into four groups: (1) control group：PBS injection (s.c.)；(2) GTDE group: 200μg/kg GTDE orally；(3) Chemical aging without GTDE group：100 mg/kg D-galactose injection (s.c.)；(4) Chemical aging with GTDE group: 100 mg/kg D-galactose injection (s.c.) and GTDE orally. The total experimental time is 25 weeks. We have found that GTDE have effects degree of awareness and anxiety by open field test; Aging causes the impairment in recognition memory in Novel object and location recognition test; Morris water maze results show that consecutive GTDE significantly improves the spatial memory in D-gal induced aging rats. We found that GTDE decreased the high AGEs level in aging rats brain, indicating that it ameliorated the oxidative damage in the brain induced by D-gal over exposure. Moreover, there is a significantly reduction in antioxidant enzyme (SOD-1、catalase) and BDNF expression in D-gal aged rats, and GTDE is able to increases antioxidant enzyme and BDNF expression. Meanwhile, Meanwhile, GMI modulated the 4HNE and NLRP3 proliferation in response to aging. In summary, GTDE can improve cognitive dysfunction, and regulates oxidative/inflammatory response aging.

並列關鍵字

Monocyclic aromatic amines ； Reactive oxygen species ； Neurodevelopment ； N-acetyl cysteine ； D-galactose ； Ganoderma tsugae ； Oxidative stress ； Cognitive dysfunction

參考文獻

1. Stiles, J. and T.L. Jernigan, The basics of brain development. Neuropsychol Rev, 2010. 20(4): p. 327-48.

2. Moog, N.K., et al., Influence of maternal thyroid hormones during gestation on fetal brain development. Neuroscience, 2015.

3. Beard, J., Iron deficiency alters brain development and functioning. Journal of Nutrition, 2003. 133(5): p. 1468s-1472s.

4. Archer, T., Effects of Exogenous Agents on Brain Development: Stress, Abuse and Therapeutic Compounds. Cns Neuroscience & Therapeutics, 2011. 17(5): p. 470-489.

5. Esmat, A., et al., Dichloroacetonitrile induces oxidative stress and developmental apoptotic imbalance in mouse fetal brain. Environmental Toxicology and Pharmacology, 2012. 33(1): p. 78-84.

國際替代計量

應用半自動 Bonfire程式分析神經樹突形態評估3,5-二甲基苯胺與其代謝物對於初代皮質神經元毒性之影響以及松杉靈芝對於老化大鼠認知功能障礙之改善效果

未授權

主題瀏覽