| 摘要: | 目前已知有許多因素造成神經退化，包括老化、發炎、氧化傷害、錯誤摺疊蛋白的累積，細胞凋亡等。文獻指出當大腦受到氧化傷害時，會將多元不飽和脂肪酸氧化，形成如4-羥基反式2-壬烯 (4-hydroxy-trans-2-nonenal, 4-hydroxynonenal, 4-HNE)、 丙烯醛 (acrolein) 和 丙二醛 (Malondialdehyde, MDA) 等脂質過氧化物。4-HNE已被證實具強氧化能力，能與蛋白質形成共價鍵結產物-胼合物 (protein adduct)，破壞鈣離子恆定，導致細胞死亡。4-HNE也會破壞血腦障壁 (BBB)，造成神經元損傷。巴金森氏症 (Parkinson's disease, PD)、阿茲海默症 (Alzheimer's disease, AD) 、亨丁頓舞蹈症 (Huntington's disease, HD) 及肌萎縮性脊髓側索硬化症 (amyotrophic lateral sclerosis, ALS) 等神經退化性疾病的病理學共同特徵為大腦皆有錯誤摺疊蛋白的累積。目前已知錯誤摺疊蛋白在內質網積累，會引發內質網壓力 (ER stress)，細胞為因應此壓力而改變許多訊息傳遞和基因表現，即稱為未摺疊蛋白質反應（unfolded protein response, UPR）。UPR相關的蛋白質對細胞的影響可分成兩大方向：一為紓解內質網壓力，幫助細胞復原和存活；但是當壓力過度而無法紓解時，則會促進受損細胞的死亡。因此，本論文擬深入探討4-HNE處理PC12類神經細胞引發內質網壓力 (ER stress) 與所涉及的UPR路徑和相關的訊息分子。研究結果發現4- HNE處理未分化或已分化的PC12細胞皆會造成細胞死亡，且其毒性與劑量及處理時間呈正相關。未分化的PC12細胞經4-HNE處理4-8小時，會導致細胞週期停滯於G0/G1；處理16小時後，細胞內鈣離子會大量累積。4-HNE會快速刺激大量的活性氧生成，若添加抗氧化劑N-乙醯基半胱氨酸 (N-Acetylcysteine, NAC) 清除活性氧，則可以保護細胞免於4-HNE的毒害的毒性，因此4-HNE的細胞毒性可能與活性氧有關。4-HNE處理PC12細胞2-4小時即能活化PERK-eIF2α-ATF-4、IRE1α-XBP1及ATF-6三條典型的未摺疊蛋白質反應（unfolded protein response, UPR），隨後並誘導下游的轉錄因子CHOP和伴隨蛋白GRP78的表現，幫助細胞紓解過多的錯誤摺疊蛋白質，並起動細胞凋亡之進程。4-HNE處理4-8小時也能高度誘導促凋亡基因TRB3 (tribbles-related protein 3)、及促進p-eIF2去磷酸化的Growth arrest and DNA damage-inducible protein (GADD34)，使得凋亡蛋白caspase-3活化，導致細胞凋亡。除此之外，4-HNE處理4-8小時會顯著誘導DNA修補基因growth arrest and DNA damage (GADD) 45 mRNA表現，此結果與細胞週期停滯結果相呼應，也暗示4-HNE處理可造成DNA損傷。此外，4-HNE處理4-8小時亦能誘導CHOP的下游的促凋亡基因p53-up-regulated modulator of apoptosis (PUMA) 的轉錄，由此可知4-HNE也能活化p53路徑。4-HNE處理4-8小時也會活化細胞的nuclear factor erythroid 2-related factor 2 (Nrf2)- antioxidant response elements (ARE) 路徑，誘導血紅素加氧酶-1 (Heme oxygenase-1)、麩氨酸半胱氨酸連接酶催化次單元 (catalytic subunit of glutamate cysteine ligase, GCLC) 的表現，前者的誘導倍數尤高。若添加抗氧化劑NAC清除活性氧，HO-1和GCLC的表現皆顯著被抑制。我們進一步利欲Nrf2 siRNA探討UPR與Nrf2-ARE是否有互相交流 (crosstalk)，結果發現降低90%的Nrf2 mRNA後，可抑制50%的HO-1的表現，但對UPR的下游基因如 CHOP或TRB3的表現卻無影響。由此推論UPR與Nrf2-ARE雖然都受活性氧誘導，但各自為獨立之壓力因應系統。4-HNE也會活化絲裂原活化蛋白激酶 (Mitogen-activated protein kinases, MAPKs) 路徑，並大量持久的活化下游的轉錄因子Nuclear factor-kappa B (NF-κB )。此兩者與4-HNE誘導的Nrf2-ARE及UPR 間的相關性，仍有待進一步研究。
The likely mechanisms of neurodegenerative diseases involve oxidative stress, inflammation and neuron cell death. 4-Hydroxy-trans-2-nonenal (4-HNE) is the most common degradation product of 9- and 13-hydroperoxides of n-6 fatty acids such as arachidonic and linoleic acids. 4-HNE is a highly reactive aldehyde that avidly attacks nucleophilic centers in proteins, phospholipids, and nucleotides. There is compelling evidence that 4-HNE is highly elevated in the brains of patients with Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. The endoplasmic reticulum (ER) is involved in the folding and maturation of membrane-bound and secreted proteins. Under ER stress conditions, activation of the unfolded protein response (UPR) reduces unfolded protein load through several prosurvival mechanisms, including the expansion of the ER membrane, the selective synthesis of key components of the protein folding and quality control machinery and the attenuation of the influx of proteins into the ER. When ER stress is not mitigated and homeostasis is not restored, the UPR triggers apoptosis. The exact participation of the UPR in the onset of neurodegeneration is unclear, but there is significant evidence for the alteration of these pathways in the endoplasmic reticulum. Several studies showed that 4-HNE treatment causes a reduction in glutathione (GSH) pool, an increase in reactive oxygen species (ROS), protein carbonylation, mitochondrial dysfunction and apoptosis in PC12 cells. Although ER stress has also been implicated in the development of neurodegenerative disorders, the relationship between 4-HNE and ER stress has never been studied in PC12 cells.In this research, it was found that 4-HNE caused PC12 cell death dose-and time-dependently. Furthermore, 4-HNE treatment induced intracellular calcium accumulation and cell cycle arrest at G0/G1 phase in PC12 cells. About 100% increase in ROS production was observed 5 min after treatment with 25 μM 4-HNE, indicating a rapid and transient ROS production was triggered. Treatment of PC12 cells with antioxidant NAC (1 mM) completely alleviated PC12 cell death induced by 25 and 35 M 4-HNE. The recovery of cell viability is apparently related to the abrogated 4-HNE-mediated ROS production.Three canonical arms of the UPR: XBP1 splicing, PERK-eIF2-ATF4, and ATF6 were activated after treatment with 4-HNE for 2-4 h. UPR targets, ER chaperone BiP/GRP78 and CHOP, a transcription factor that is homologus to C/EBP (CAAT/enhancer binding protein) and also known as GADD153, were subsequently stimulated. The expressions of target genes of CHOP responsible for ER stress-induced apoptosis, such as carbonic tribbles-related protein 3 (TRB3), and protein phosphase-1 interacting protein GADD34, which enhances protein synthesis through eIF2 dephosphorylation, thus releasing the translational block, were dramatically stimulated by 4-HNE. BH3-only protein PUMA (p53 upregulated modulator of apoptosis) and growth arrest and DNA damage (GADD) 45, a DNA repair gene, were also both induced by 4-HNE significantly. These results indicates that p53 pathway may be activated by 4-HNE as well. Addition of NAC could significantly attenulated 4-HNE-mediated expression of UPR-related genes, indicating 4-HNE-induced UPR signaling in PC12 cells was up-regulated by ROS production.4-HNE induced alteration in the level of active form of caspase-3 after treatment of PC12 cells for 6-8 h, indicating in PC12 cells, 4-HNE induces apoptosis at least in part through the release of cytochrome c from mitochondria and caspase-3 activation.4-HNE also activated nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response elements (ARE) pathway in PC12 cells. Phase II enzymes such as heme oxygenase-1 (HO-1) and catalytic subunit of glutamate cysteine ligase (GCLC) were thus overexpressed, and HO-1 expression was especially high. Addition of NAC could significantly attenulated 4-HNE-mediated expression of HO-1and GCLC, supporting that Nrf2-ARE pathway is ROS-dependent.Nrf2 siRNA was used to further investigate the crosstalk between Nrf2-ARE and UPR. It was found that knockdown of 90% Nrf2 mRNA caused 50% reduction in HO-1 expression, but the expression of CHOP or TRB3 was unchanged. This result indicates that 4-HNE-mediated UPR and Nrf2-ARE are two oxidative stress response pathways without crosstalk.4-HNE also activated mitogen-activated protein kinases (MAPKs) and led to a long-term activation of nuclear factor-kappa B (NF-κB) in PC12 cells. The roles of MAPK signaling and NF-B on 4-HNE-mediated Nrf2-ARE and UPR remain to be studied. |
