FAM134B介导的自噬在无镁外液致痫海马神经元内质网应激及凋亡中的作用

The role of FAM134B-mediated autophagy in endoplasmic reticulum stress and hippocampal neurons apoptosis induced by Mg^2+-free solution

目的探讨FAM134B介导的自噬在无镁外液致痫海马神经元内质网应激及凋亡中的作用。方法原代培养新生24 h的SD大鼠海马神经元,通过无镁外液诱导体外癫痫模型。将海马神经元随机分为对照组(CON)、无镁诱导组(AE)、阴性对照组(Lenti-pGV)、过表达FAM134B组(Lenti-FAM134B)和低表达FAM134B组(Lenti-FAM134B-shRNA),并进一步采用选择性自噬抑制剂3-MA进行干预;采用LDH法检测细胞活力、TUNEL法检测细胞凋亡、Western blotting法检测自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ、内质网应激相关蛋白GRP78及CHOP的表达。结果与CON组相比,AE组LDH释放率明显升高,海马神经元活力下降,神经元凋亡明显增加,过表达FAM134B显著降低LDH释放率,增加海马神经元活力,减少AE诱导的神经元凋亡;AE组LC3-Ⅱ/LC3-Ⅰ比例增加,过表达FAM134B使LC3-Ⅱ/LC3-Ⅰ比例进一步增加;AE组GRP78和CHOP的表达明显增加,过表达FAM134B显著减少AE诱导的GRP78和CHOP表达的增加,而低表达FAM134B均发挥相反作用。与过表达FAM134B组相比,3-MA消除过表达FAM134B对AE诱导的内质网应激及神经元活力的影响,显著增加GRP78和CHOP的表达,并增加LDH释放率。结论FAM134B可能通过调控自噬途径对内质网应激和神经元凋亡发挥保护作用。

Objective To investigate the role of FAM134B-mediated autophagy in endoplasmic reticulum stress and hippocampal neurons apoptosis induced by Mg2+-free solution.Methods Hippocampal neurons from newborn SD rats were randomly divided into control group,AE group,Lenti-pGV group,Lenti-FAM134B group and Lenti-FAM134B-shRNA group.In addition,the selectively autophagy inhibitor(3-Methyladenine,3-MA)was used.LDH assay was used to assess hippocampal neuron activity.TUNEL assay was used to measure apoptotic neurons.The expression of GRP78,CHOP and LC3-Ⅱ/LC3-Ⅰwere detected by Western blotting.Results Compared with the control group,the release rate of LDH was increased,the hippocampal neuron activity was decreased,and neuronal apoptosis was significantly increased in AE group,while FAM134B Overexpression decreased the release rate of LDH,increased the hippocampal neuron activity and attenuated AE-induced neuronal apoptosis;the ratio of LC3-Ⅱ/LC3-Ⅰwas significantly increased in the AE groups,while FAM134B Overexpression further increased the ratio of LC3-Ⅱ/LC3-Ⅰ;the expression of GRP78 and CHOP were obviously increased in the AE groups,while FAM134B Overexpression decreased the expression of GRP78 and CHOP induced by AE;in addition,FAM134B down-expression all exerted the opposite effect.Compared with Lenti-FAM134B group,3-MA abolished the effects of FAM134B Overexpression on the expression of GRP78 and CHOP and the hippocampal neuron activity.In addition,3-MA significantly increased the expression of GRP78 and CHOP and the release rate of LDH.Conclusion FAM134B-mediated autophagy may play a protective role in endoplasmic reticulum stress and hippocampal neurons apoptosis induced by Mg2+-free solution.