ISRIB对Aβ1-42诱导的SH-SY5Y细胞的神经保护作用

The neuroprotective effect of ISRIB on SH-SY5Y cells induced by Aβ1-42

目的:以β淀粉样蛋白(amyloidβ-protein 1-42,Aβ1-42)诱导的SH-SY5Y细胞为模型,探讨综合应激反应抑制剂(integrated stress response inhibitor,ISRIB)对Aβ1-42诱导的内质网应激和细胞凋亡的影响。方法:SH-SY5Y细胞按不同处理方式分为4组:对照组、Aβ组、ISRIB组、ISRIB+Aβ组。培养48 h后用噻唑蓝比色法检测各组细胞存活率,蛋白免疫印迹试验检测内质网应激相关蛋白的表达,包括葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、蛋白激酶R样内质网激酶(protein kinase R-like ER kinase,PERK)-真核起始因子2α(eukaryotic initiation factor 2α,eIF2α)-活化转录因子4(activation transcription factor 4,ATF4)介导的内质网应激通路,以及下游的CCAAT/增强子结合蛋白同源蛋白(C/EBP homologous protein,CHOP),同时也检测了凋亡相关蛋白的表达,包括Bcl-2、Bax、cleaved-caspase-3。结果:与Aβ组比较,ISRIB能显著提高SH-SY5Y细胞的存活率(P <0.01),显著降低p-PERK(P <0.01)、p-eIF2α(P <0.05)、ATF4(P <0.01)和CHOP的表达(P <0.05),提高Bcl-2/Bax表达(P <0.05),降低cleaved-caspase-3表达(P <0.01)。结论:ISRIB能够抑制Aβ1-42诱导的SH-SY5Y细胞凋亡,其机制与抑制ERS造成的eIF2α通路激活及其相关凋亡信号通路有关。

Objective:This study aims to investigate the effect of integrated stress response inhibitor(ISRIB)in apoptosis and endoplasmic reticulum stress of SH-SY5Y cells induced by amyloid β-amyloid protein1-42(Aβ1-42)and to clarify its mechanism.Methods:SH-SY5Y cells were divided into normal control group,Aβ group,ISRIB group and ISRIB+Aβ group. The survival rate of cells in each group was detected by MTT colorimetric assay. Western blotting was used to examine the expression of endoplasmic reticulum stress marker glucose-regulated protein 78(GRP78)and activation of protein kinase R-like ER kinase(PERK)-eukaryotic initiation factor 2α(eIF2α)-activation transcription factor 4(ATF4)signaling pathway,together with C/EBP homologous protein(CHOP). Meanwhile,apoptosis-related proteins were also detected,including Bcl-2、Bax and cleaved-caspase3. Results:Compared with Aβ group,ISRIB significantly increased the survival rate of SH-SY5Y cells(P < 0.01),significantly decreased the activation of pPERK(P < 0.01),p-eIF2α(P<0.05),ATF4(P < 0.05)and CHOP(P < 0.05),increased Bcl-2/Bax level(P < 0.05)and decreased cleaved-casepase3 expression(P < 0.01). Conclusion:ISRIB could inhibit the apoptosis of SH-SY5Y cells induced by Aβ1-42,and its mechanism is related to the inhibiting the activation of eIF2α pathway and apoptotic signaling pathway induced by ERS.