Abstract

The inflammatory process plays a key role in neurodegenerative disorder. The inflammatory molecule, 5-lipooxygenase (5-LOX), protein is involved in the pathologic phenotype of AD which includes Aβ amyloid deposition and tau hyperphosphorylation. This study aims to identify the mechanistic role in neuroprotection by 5-LOX inhibitor in neurotoxic SH-SY5Y cell line model by evaluating different cell survival pathway. The neurotoxic SH-SY5Y cells were developed by the treatment of Aβ_25–35. The cells were then treated with 5-LOX peptide inhibitor, YWCS to prevent neurotoxicity reported earlier from our lab. The effect of 5-LOX inhibition on cell survival pathways were determined by western blot experiment with different doses of peptide by using polyclonal anti body of p53, anti-Akt and anti-phosphorylated Akt. Immunoprecipitation and mass spectroscopic studies were done to identify the altered proteins appeared on the blot. Over expression of phosphorylated Akt and 3 bands on p53 lane blot other than p53 were observed. Three bands were identified as isoforms of p53 which correspond to p73, Δ133p53 and Δ160p53 in the cells treated only with 80 µM of YWCS compare to untreated cells. However, no alteration of total p53 and Akt were observed in treated cells. The results exposed the novel mechanistic pathway of neuroprotection by 5-LOX inhibition is likely to be mediated by DNA DSB repair through p53 isoforms and PI3K/Akt pathway. Our finding has opened a new window in the therapeutic approach for the prevention of AD.

中文翻译：

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p73，Δ133p53，Δ160p53，pAKT的诱导通过5-LOX抑制通过DNA修复导致神经保护

摘要

炎症过程在神经退行性疾病中起关键作用。炎症分子5-脂氧合酶（5-LOX）蛋白与AD的病理表型有关，包括Aβ淀粉样蛋白沉积和tau过度磷酸化。本研究旨在通过评估不同的细胞存活途径，确定5-LOX抑制剂在神经毒性SH-SY5Y细胞系模型中在神经保护中的机制作用。神经毒性SH-SY5Y细胞由Aβ的治疗开发_25-35。然后将细胞用5-LOX肽抑制剂YWCS处理，以防止我们实验室先前报道的神经毒性。通过使用p53，抗Akt和抗磷酸化Akt的多克隆抗体，通过不同剂量肽段的蛋白质印迹实验，确定了5-LOX抑制对细胞存活途径的影响。进行了免疫沉淀和质谱研究，以鉴定印迹上出现的改变的蛋白质。观察到p53泳道上除p53以外的磷酸化Akt和3条带的过度表达。与未经处理的细胞相比，在仅用80 µM YWCS处理的细胞中，鉴定出三个带为p53的同工型，分别对应于p73，Δ133p53和Δ160p53。然而，在处理过的细胞中未观察到总p53和Akt的改变。结果揭示了通过5-LOX抑制的神经保护的新机制途径可能是通过p53亚型和PI3K / Akt途径的DNA DSB修复介导的。我们的发现为预防AD开辟了治疗方法的新窗口。