4-苯基丁酸钠通过抑制内质网应激减轻皮层神经元氧糖剥夺/再灌注损伤

4-Phenylbutyric Acid Reduces Oxygen-glucose Deprivation/Reoxygenation-induced Injury in Cortical Neurons by Inhibiting Endoplasmic Reticulum Stress

4-苯基丁酸钠(4-phenylbutyric acid,4-PBA)是协助内质网中蛋白质转录后修饰和折叠的分子伴侣,故可减轻非折叠蛋白反应(unfolded protein response,UPR)及其介导的细胞凋亡。既往研究表明,4-PBA可以减轻脑组织的缺血性损伤,但采用原代皮层神经元构建氧糖剥夺/再灌注(oxygen-glucose deprivation/reoxygenation,OGD/R)损伤模型,来研究4-PBA对神经元损伤的保护作用及其机制尚未见报道。本文采用原代培养的皮层神经元OGD/R损伤模型,同时给予4-PBA处理,探讨4-PBA对OGD/R诱导的神经元内质网应激(endoplasmic reticulum stress,ERS)的作用及其机制。分别采用MTT、LDH和Hoechst 33342染色法检测神经元存活率、细胞膜完整性和细胞凋亡情况。Western印迹检测ERS标志物葡萄糖调节蛋白78(glucose regulated protein 78,GRP78),以及肌醇必需酶1(inositol-requiring enzyme 1,IRE1)通路相关蛋白质的表达。Western印迹结果显示,在OGD/R后0~48 h,GRP78的表达较对照组明显升高。MTT、LDH漏出率和Hoechst 33342染色法检测显示,4-PBA显著改善OGD/R所导致的神经元存活率下降、LDH漏出率升高和细胞凋亡增加,且具有明显的剂量依赖性。通过Western印迹检测发现,4-PBA显著逆转OGD/R所致GRP78蛋白表达水平的上调。此外,对肌醇必需酶1通路相关蛋白质的检测显示,4-PBA下调氧糖剥夺/再灌注组神经元p-IRE1和p-JNK的表达,增加抗凋亡蛋白Bcl-2表达。上述研究结果表明,4-PBA在氧糖剥夺/再灌注情况下对神经元具有保护作用,该保护作用可能是通过抑制肌醇必需酶1信号通路介导的非折叠蛋白反应和内质网应激实现的。

4-Phenylbutyric acid （4-PBA） , a chemical chaperone that contributes to post-transcriptional modification and folding of proteins in endoplasmic reticulum （ ER ）, could inhibit unfolded protein response （UPR） and UPR-induced apoptosis. Although previous studies show that 4-PBA could alleviate the ischemic brain damage, the effect and mechanism of 4-PBA on the injury of primary cortical neurons exposed to oxygen-glucose deprivation/reoxygenation （OGD/R） has not been reported. To investigate the effect of 4-PBA on OGD/R-induced endoplasmic reticulum stress （ERS） and its mechanisms, primary cortical neurons were subjected to OGD/R and treated simultaneously with 4-PBA in the present study. The cell viability, cell membrane integrity, and apoptosis were detected by MTT assay, LDH test, and Hoechst 33342 staining respectively. Moreover, the expression levels of glucose regulated protein 78 （GRP78）, an ERS marker protein, and the inositol essential enzyme 1 （IRE1） pathway-associated proteins were detected by Western blotting. Our results revealed that the expression of GRP78 was significantly increased after OGD/R （0 - 48 h）. 4-PBA could remarkably ameliorate the decrease of neurons viability and the increase of LDH leakage and neuronal apoptosis. In addition, Western blotting analysis showed that 4-PBA down-regulated the elevated expression of GRP78, p-IRE! and p-JNK, and up-regulated the suppressed anti-apoptotic protein Bcl-2 expression in OGD/R neurons. The results confirm that 4-PBA has a protective effect on neuron against OGD/R induced injury, by which the effect may be achieved by inhibiting IRE1 signaling-mediated UPR and ERS.