中文摘要：

目的研究中脑星形胶质细胞来源的神经营养因子（mesencephalic astrocyte-derived neurotrophic factor,MANF）对糖氧剥夺/再灌注（oxygen-glucose deprivation/reperfusion,OGD/R）诱导的人神经母细胞瘤细胞（SH-SY5Y）损伤的保护作用。方法体外培养SH-SY5Y细胞,对细胞进行糖氧剥夺（OGD）6 h,再灌注（R）12 h。在再灌注时期,根据是否给予重组人MANF蛋白处理（2μmol·L^-1,12 h）,将细胞分为正常对照组（NC）、NC＋MANF组、OGD/R组和OGD/R＋MANF组。随后光学显微镜下观察SH-SY5Y细胞形态的改变,MTT法检测细胞存活率,PI染色法检测细胞死亡率,免疫印迹法检测内源性MANF蛋白、内质网（ER）应激相关蛋白GRP78/Bi P、p-IRE1、p-e IF2α及促凋亡蛋白cleaved caspase-3和CHOP的表达。结果光学显微镜下可见OGD/R组SH-SY5Y细胞胞体变小、变圆,突起缩短或消失。进一步研究发现,MANF能明显改善OGD/R诱导的SHSY5Y细胞存活率下降和死亡率增加。免疫印迹法检测发现：OGD/R组细胞内源性MANF蛋白表达增高;ER应激相关蛋白GRP78/Bi P、p-IRE1、p-e IF2α表达增高;促凋亡蛋白CHOP及cleaved caspase-3的表达明显高于NC组。给予重组人MANF蛋白可降低OGD/R组GRP78/Bi P、CHOP及cleaved caspase-3的表达水平。结论 OGD/R可诱导凋亡性ER应激;MANF蛋白可通过抑制OGD/R诱导的凋亡性ER应激而对SH-SY5Y细胞具有保护作用。

英文摘要：

Aim To investigate the protective effects of MANF on human neuroblastoma SH-SY5 Y cells suf-fering from oxygen-glucose deprivation/reperfusion （ OGD/R） and the underlying mechanism. Methods SH-SY5Y cells were treated with OGD for 6 h, fol-lowed by reperfusion for 12 h. Meanwhile, the cells were incubated with 2 μmol · L^-1 recombinant human protein MANF for 12 h during reperfusion. The cell morphology was observed under an optical microscope. The cell viability was determined by MTT assay. PI staining was performed to detect the number of dead cells. Western blot was performed to determine the protein levels of endogenous MANF, glucose-related protein 78 （ GRP78/BiP） , phosphorylated inositol re-quiring enzyme 1 （ p-IRE1 ） , phosphorylated eukaryot-ic translation initiator factor 2α （ p-eIF2α） , cleaved caspase-3, and C/EBP-homologous protein （CHOP）. Results The cells exposed to OGD/R became smaller and round, and the neurites of the cells were shortened or disappeared . Recombinant human protein MANF improved the survival rate （ P 〈0. 05 ） and decreased the death rate （ P 〈0. 05 ） of SH-SY5 Y cells treated with by OGD/R. Western blot assay showed that the endoplasmic reticulum （ ER） stress-associated proteins GRP78/BiP, p-IRE1, p-eIF2α, and MANF were in-creased significantly after OGD/R treatment, compared with the untreated controls. However, the increases of secretion levels of apoptosis-associated proteins CHOP and cleaved caspase-3 in SH-SY5 Y cells induced by OGD/R were significantly suppressed by MANF. Con-clusion OGD/R up-regulates the ER stress-associated proteins and causes apoptosis. MANF inhibits OGD/R-induced cell death, which may be related to attenua-ting ER stress-induced apoptosis.