法舒地尔通过抑制NogoA/NgR信号通路减轻H_(2)O_(2)诱导的SH-SY5Y人神经母细胞瘤细胞凋亡并增强突触可塑性

Fasudil reduces apoptosis of SH-SY5Y cells induced by H2O2 and promotes synaptic plasticity by inhibiting neurite outgrowth inhibitor A and its receptor (NogoA/NgR) signaling pathway

目的探讨法舒地尔(Fasudil)对H(2)O_(2)诱导的SY5Y人神经母细胞瘤细胞凋亡和突触可塑性的影响及其机制。方法细胞分为PBS对照组、250μmol/L H(2)O_(2)处理组和250μmol/L H(2)O_(2)联合15μg/mL Fasudil处理组。噻唑蓝(MTT)法检测细胞活性,原位末端转移酶标记技术(TUNEL)检测细胞凋亡,免疫荧光细胞化学染色检测神经突生长抑制因子A(NogoA)、NogoA受体(NgR)和突触小泡蛋白(Syn)的表达,Western blot法检测NogoA、NgR、p75神经营养因子受体(p75NTR)和含富含亮氨酸重复序列免疫球蛋白结构域的Nogo相互作用蛋白1(LINGO-1)、Syn和突触后致密区蛋白95(PSD-95)的表达。结果与PBS组比较,H(2)O_(2)组细胞活性降低,细胞凋亡率升高,Fasudil处理可显著提高细胞活性,降低细胞凋亡率。与H(2)O_(2)模型组比较,Fasudil处理能使Syn和PSD-95的表达增加。与PBS组比较,H(2)O_(2)组NogoA及其受体复合物NgR/p75NTR/LINGO-1的表达明显增加,而Fasudil处理可以抑制NogoA及其受体复合物NgR/p75NTR/LINGO-1的表达。结论Fasudil可能通过抑制NogoA/NgR信号通路的激活,从而减轻H(2)O_(2)诱导的SH-SY5Y细胞凋亡以及增强突触的可塑性。

Objective To investigate the effect of Fasudil on H(2)O_(2)-induced apoptosis and synaptic plasticity in human neuroblastoma SY5Y cells and its mechanism.Methods The cells were divided into three groups:PBS control group,H(2)O_(2)model group(250μmol/L H(2)O_(2)treatment)and Fasudil intervention group(250μmol/L H(2)O_(2)combined with 15μg/mL Fasudil treatment).MTT assay was applied to detect cell activity and TUNEL was performed to detect cell apoptosis respectively.Immunofluorescence cytochemical staining was used to determine the expression of neurite outgrowth inhibitor A(NogoA),Nogo receptor(NgR)and synaptophysin(Syn).Western blotting was then conducted to detect the expression of NogoA,NgR,p75 neurotrophin receptor(p75NTR),leucine-rich repeat Ig domain-containing Nogo-interacting protein 1(LINGO-1),Syn and postsynaptic density protein-95(PSD-95).Results Compared with the PBS group,the H(2)O_(2)group showed decreased cell viability and increased apoptosis rate while Fasudil treatment significantly increased the cell viability and reduced the apoptosis rate.Compared with the H(2)O_(2)model group,Fasudil intervention increased expressions of Syn and PSD-95.Compared with the PBS group,the expression of NogoA and its receptor complex NgR/p75NTR/LINGO-1 grew significantly in the H(2)O_(2)group,suggesting Fasudil treatment could inhibit the expression of NogoA and its receptor complex NgR/p75NTR/LINGO-1.ConclusionFasudil may inhibit the activation of the NogoA/NgR signaling pathway,therefore reducing the apoptosis induced by H(2)O_(2)in SH-SY5Y cells and enhancing the plasticity of the synapses.