The Endoplasmic Reticulum Stress Mediates Ketamine-induced PC12Cell Apoptosis

Objective: Ketamine, a noncompetitive NMDA (N-methyl-D-aspartate)receptor antagonist, produces a dose-related state of unconsciousness andanalgesia commonly referred to as dissociative anesthesia. It is widely used inadults and pediatric anesthesia. However, a growing number of studiesshowed that general anesthetic ketamine can cause nerve damage, causingneurodegenerative diseases and long-term neurocognitive defects. The studyof ketamine neurotoxicity aroused people’s concern.It has been reported that ketamine can cause rodents, zebrafish,non-human primate neuronal apoptosis. Cell apoptosis is an essential event inthe pathophysiological processes that lead to disease. The precise mechanismsof apoptosis are not completely understood. It has become clear that each ofthe main cellular organelles can participate in cell death signaling pathways,including mitochondrial-and death-receptor-mediated apoptotic pathways,and endoplasmic reticulum road. Studies have reported that ketamine caninduce toxicity in human neurons differentiated from embryonic stem cells viamitochondrial apoptosis pathway, but whether ketamine induced apoptosisthrough endoplasmic reticulum stress (ERS) pathway is not clear.Oxygen free radical, stress hormones and cytokine can influence thefunction of endoplasmic reticulum, lead to not/misfolded protein accumulationand markable protein of ERS increased, thus activating the ERS. Moderateendoplasmic reticulum stress can protect cells by enhancing the degradation ofmisfolded proteins. When the stress is prolonged and excessive, it can elitecell damage by inducing apoptosis. ER stress is considered an early or initialresponse of cells under stress or damage and linked to neuronal death invarious neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’sdisease and Huntington disease. GRP78(78-kDa glucose-regulated protein), also known as Bip (immunoglobulin heavy chain binding protein), is theprimary regulator of the unfolded protein response. When calcium overload,oxidative stress and other factors lead to protein aggregation of unfolded in theER lumen, GRP78/Bip release transmembrane protein, thus activating theERS. Therefore, GRP78/Bip is regarded as the marker protein of ERS.Caspase12is a member of the caspase family. It is only produced in theendoplasmic reticulum.Caspase12only can be activated in the endoplasmicreticulum stress, is the key enzyme of endoplasmic reticulum stress mediatedapoptosis pathway.Based on these findings, in this study we investigated the toxicity ofketamine on PC12cell. Furthermore, we also examined the roles ofendoplasmic reticulum stress in ketamine-induced injury. Our study willprovide important views in deeply understanding the mechanism ofneurotoxicity of ketamine.Methods:1PC12cells were treated with different concentration of ketamine, aftervarious time of ketamine treatment, the cell viability was measured by MTTmethod. PC12cell survival rates were tested after different concentration (20,30,40M) Salubrinal (the endoplasmic reticulum stress inhibitor) with andwithout ketamine.2The effects of ketamine on protein levels of ER stress biomarkers,GRP78/Bip and Caspase12, were investigated in PC12cells.The proteinlevels of PC12cell treated with1,1.5,2mM ketamine for24hours weretested by western blotting. The protein levels of PC12cell treated with1.5mMketamine for6,12,24hours were also tested. The effects of Salubrinal withand without ketamine on the protein levels were detected.3To determine whether ketamine cause apoptosis in PC12cells,apoptotic cells of different groups were determined by Annexin V-FITCapoptosis detection kit according to manufacturer’s protocol. The effects ofSalubrinal on the cell apoptosis induced by ketamine on PC12cells weredetected. 4The data were presented as Mean±SD and analyzed with one wayANOVA by SPSS17.0statistical program. A level of p＜0.05was supposed tobe statistically significant.Results：1Exposure PC12cells to0.5mM ketamine for6hours could not reducecell viability. But for12,24hours,0.5mM ketamine produced a significantdecrease in cell viability as compared with untreated controls.1,1.5,2,2.5mM ketamine decreased PC12cell survival rate in a concentration-dependentmanner.20,30,40M Salubrinal alone did not influence the survival rate ofPC12cells.30,40M Salubrinal inhibited ketamine-induced loss of cellviability in PC12cells.2Western blotting indicated that after treated with ketamine, Bip andCaspase12, the biomarkers of ER stress, were elevated in a concentration-andtime-dependent manner, compared with control group. The increases of theseproteins were effectively inhibited by Salubrinal.3Compared with untreated control, the apoptotic rate of PC12cells wassignificantly increased when1.5mM ketamine treatment and decreased whenco-treated with30M Salubrinal. Salubrinal alone did not influence theapoptotic rate of PC12cells.Conclusion:Ketamine can induce PC12cell apoptosis in a concentration-andtime-dependent manner. The endoplasmic reticulum stress pathway wasinvolved in ketamine-induced PC12cell apoptosis.