Objective

The endoplasmic reticulum stress (ERS) and ERS-related neuronal apoptosis contribute to the cerebral ischemia/reperfusion (I/R) injury. (-)-Clausenamide has been reported to be nootropic and improve learning and memory in amnesia animal models. However, whether (-)-Clau could protect neurons from ischemic injury and the possible mechanism needed further study. The present study aimed to explore the effects of (-)-Clau on primary cortical neurons treated with oxygen-glucose deprivation/reoxygenation (OGD/R).

Methods

Rat primary cortical neurons were used to set up an injury model of OGD/R which imitated the clinical I/R injury. Cell viability and apoptosis were measured by CCK-8 assay, LDH detection and TUNEL staining, respectively. The activation of GRP78/eIF2α-ATF4-CHOP signaling pathway, one of the three branches of ERS, and cleaved caspase-3, the apoptotic marker, were assessed by western blotting.

Results

OGD/R induced activation of GRP78/eIF2α-ATF4-CHOP signaling pathway. (-)-Clau significantly attenuated OGD/R-induced decrease in the cellular viability and the activation of GRP78, eIF2α, ATF4 and CHOP. To further confirm the effect of (-)-Clau on OGD/R-induced ERS activation, the ERS inducer Tunicamycin (TM) was applied. TM significantly abolished (-)-Clau’s protective effect against ERS and neuronal apoptosis, indicating that the protective effect of (-)-Clau was dependent on inhibiting ERS.

Conclusions

The present work demonstrated for the first time that (-)-Clau could reverse the activation of GRP78/eIF2α-ATF4-CHOP branch, thus inhibited ERS and the subsequent apoptosis induced by OGD/R and promoted cell viability in vitro. (-)-Clau could serve as a promising therapeutic agent in the treatment for ischemic stroke in the future.

Abbreviations

ATF4: activating transcription factor-4; ATF6: activating transcription factor-6; CHOP: transcriptional induction of CCAAT/enhancer binding protein homologous protein; (-)-Clau: 3-hydroxy-4-phenyl-5a-hydroxybenzylN-methyl-g-lactam; eIF2α: eukaryotic initiation factor 2α; ER: endoplasmic reticulum; ERS: endoplasmic reticulum stress; GRP78: 78-kDa glucose regulated protein; I/R: ischemia/reperfusion; IRE1: inositol requiring enzyme-1; JNK: c-Jun N-terminal kinase; OGD/R: oxygen-glucose deprivation/reoxygenation; PERK: double-stranded RNA-dependent protein kinase-like ER kinase; TM: Tunicamycin; UPR: unfolded protein response

中文翻译：

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（-）-Clausenamide减轻了原代神经元培养物中OGD / R诱导的ER应激和凋亡。

目的

内质网应激（ERS）和ERS相关的神经元凋亡促成脑缺血/再灌注（I / R）损伤。（-）-Clausenamide据报道具有促智作用，可改善健忘症动物模型的学习和记忆能力。然而，（-）-Clau是否能保护神经元免受缺血性损伤，其可能的机制尚待进一步研究。本研究旨在探讨（-）-克劳对氧-葡萄糖剥夺/复氧（OGD / R）治疗的原代皮层神经元的影响。

方法

大鼠原代皮层神经元用于建立模仿临床I / R损伤的OGD / R损伤模型。通过CCK-8测定，LDH检测和TUNEL染色分别测量细胞活力和凋亡。通过蛋白质印迹法评估了ERS的三个分支之一GRP78 /eIF2α-ATF4-CHOP信号通路的激活以及凋亡的标志物caspase-3的裂解。

结果

OGD / R诱导GRP78 /eIF2α-ATF4-CHOP信号通路的激活。（-）-Clau显着减轻了OGD / R诱导的细胞活力下降以及GRP78，eIF2α，ATF4和CHOP的激活。为了进一步证实（-）-Clau对OGD / R诱导的ERS激活的影响，应用了ERS诱导剂Tunicamycin（TM）。TM显着取消了（-）-Clau对ERS和神经元凋亡的保护作用，表明（-）-Clau的保护作用取决于抑制ERS。

结论

本研究首次证明了（-）-Clau可以逆转GRP78 /eIF2α-ATF4-CHOP分支的激活，从而抑制ERS和随后由OGD / R诱导的细胞凋亡，并促进了体外细胞活力。（-）-Clau有望在将来成为缺血性中风的有前途的治疗剂。

缩略语

ATF4：激活转录因子4；ATF6：激活转录因子6；CHOP：CCAAT /增强子结合蛋白同源蛋白的转录诱导；（-）-Clau：3-羟基-4-苯基-5a-羟基苄基N-甲基-g-内酰胺；eIF2α：真核起始因子2α；ER：内质网；ERS：内质网应激；GRP78：78 kDa葡萄糖调节蛋白；I / R：缺血/再灌注；IRE1：需要酶1的肌醇；JNK：c-Jun N-末端激酶；OGD / R：氧葡萄糖剥夺/复氧；PERK：双链RNA依赖性蛋白激酶样ER激酶；TM：衣霉素；UPR：展开的蛋白质反应