摘要目的 探讨缺氧加重淀粉样β蛋白(Aβ)对神经细胞毒性作用的通路. 方法 体外培养入神经母细胞瘤细胞(SH-SY5Y细胞),细胞分为对照组、Aβ干预组、缺氧组、Aβ干预十缺氧组,采用实时定量聚合酶链式反应(qRT-PCR)检测细胞中p21激活激酶1(PAK1)、LIM激酶(LIMK1)、cofilin mRNA表达水平,用免疫印迹(Western blot)检测细胞中PAK1、LIMK1、磷酸化LIMK1 (P-LIMK1)、cofilin、磷酸化cofilin(P-cofilin)蛋白表达水平. 结果 Aβ处理后,SH-SY5Y细胞的活力减少;与对照组比较,10μmol/L Aβ干预SH-SY5Y细胞24 h后,PAK1、LIMK1、P-LIMK1、P-cofilin蛋白表达量减少(均P＜0.05),PAK1和LIMK1的mRNA表达减少(P＜0.05),cofilin蛋白和mRNA表达差异无统计学意义.10 μmol/L Aβ+2％氧气干预条件下处理SH-SY5Y细胞24 h后,与Aβ干预组比较,PAK1、LIMK1、P-LIMK1、P-cofilin蛋白表达量减少(均P＜0.05),PAK1和LIMK1的mRNA表达减少,且差异有统计学意义(均P＜0.05),cofilin蛋白和mRNA表达差异无统计学意义. 结论 Aβ可能通过抑制PAK1的活性来减少P-LIMK1,进而导致eofilin磷酸化减少,去磷酸化cofilin形成增多,导致神经元的损伤,缺氧通过该通路加重Aβ对神经细胞的毒性作用.

abstractsObjective To investigate the pathways by which hypoxia aggravates the neurotoxic effects of amyloid-beta protein (Aβ) on neurons.Methods Human neuroblastoma cells (SH-SY5Y cells) were cultured in vitro,and were divided into four groups:the control group,Aβ intervention group,hypoxia group,Aβ intervention plus hypoxia group.Quantitative real time polymerase chain reaction(qRT-PCR) was adopted to detect the mRNA expression levels of p21-activated kinase 1 (PAK1),LIM kinase 1 protein (LIMK1)and cofilin.Western blot was used to measure the protein levels of PAK1,LIMK1,phosphate-LIMK1 (P-LIMK1),cofilin and phosphate-cofilin (P-cofilin).Results After Aβ treatment,the activity of SH-SY5Y cells was decreased.Compared with the control group,the protein levels of PAK1,LIMK1,P-LIMK1,P-cofilin,and the mRNA expression levels of PAK1 and LIMK1 were decreased(all P＜0.05),but the protein and mRNA expression of cofilin had no significant changes after 24 h of treatment with 10μmol/L Aβ.Compared with the Aβ intervention group,the protein levels of PAK 1,LIMK1,P-LIMK 1 and P-cofilin were decreased (all P ＜ 0.05),and the mRNA expression levels of PAK1 and LIMK1 were decreased(both P＜0.05),but the protein and mRNA expression of cofilin had no significant changes after 24 h of treatment of SH-SY5Y cells with 10 μmol/L Aβ plus 2％ oxygen.Conclusions Aβ may reduce P-LIMK1 expression by inhibiting the activity of PAK1,thereby reducing the P-cofilin,increasing the formation of dephosphorylated cofilin,leading to neural cells damage,and hypoxia aggravates the neurotoxicity of Aβ through this pathway.