NaAsO₂ Induces Apoptosis Of HT-22 Cells Through ROS Activation Of DAPK1 And Inhibition Of ERK/CREB/Bcl-2 Pathway

Objective:arsenic is easy to cross the blood-brain barrier and accumulate in the central nervous system（CNS）,resulting in the impairment of intelligence and neurobehavioral function,such as the decline of learning ability,the impairment of recent memory and attention concentration.Although the mechanism remains to be elucidated,apoptosis of nerve cells is considered to be one of the potential mechanisms of arsenic induced neurotoxicity.So far,the specific mechanism of neuronal apoptosis induced by as exposure needs to be explored.Most studies have shown that the production of reactive oxygen species（ROS）is an important toxic mechanism of arsenic.ROS is an important signal molecule in inducing apoptosis and plays a crucial role.Death associated protein kinase 1（Dapk1）is the protein kinase with the largest molecular weight in DAPK family.It is a calcium/calmodulin(Ca²⁺/CAM)-dependent serine/threonine（Ser/Thr）protein kinase,which can activate death signal and regulate apoptotic neuronal cell death,and can be activated by a large number of ROS.No studies have shown that arsenic can activate Dapk1 in neuronal cells.In hippocampal neurons,ERK regulates apoptosis by regulating survival pathway.However,there is no research to elucidate its regulatory pathway.The aim of this study was to investigate the effect of As on Dapk1 activation,nerve injury and apoptosis induced by oxidative stress.And under the influence of oxidative stress,the survival pathway is blocked,further promoting cell apoptosis.In order to elucidate the specific mechanism of learning and memory impairment caused by as exposure,and to further explore the mechanism of ROS induced neuronal apoptosis.Methods:HT-22 cells were cultured in MEM medium with double antibody and 10%bovine serum,and cell counting kit-8（CCK-8）kit was used to detect cell viability.Then HT-22 cells were randomly divided into 5 groups,and cultured in As of 0,4.0,6.0,8.0 and10.0μmol/L for 12 hours.The levels of ROS,Ca²⁺and apoptosis rate were detected by flow cytometry.Western blot was used to detect the protein contents of Dapk1,p-Dapk1,NR2B,p-nr2b,nuclear p-ERK,plasma p-ERK,p-CREB,Bcl-2,p-JNK,Bimel and cleaved caspase-3.HT-22 cells were divided into four groups:con group,as group,NAC group and As+NAC group.NAC group and As+NAC group were treated with NAC for 30minutes,and then treated with 10.0μmol/L As for 12 hours.The levels of ROS,Ca²⁺and apoptosis rate were detected by flow cytometry.Western blot was used to detect the protein contents of Dapk1,p-Dapk1,NR2B,p-NR2B,nuclear p-ERK,plasma p-ERK,p-CREB,Bcl-2,p-JNK,Bimel and cleaved caspase-3.Finally,HT-22 cells were divided into four groups:sicrl group,si Dapk1 group,siCtrl+As group and si Dapk1+As group.After24 hours of transfection,HT-22 cells were exposed to 10.0μmol/L As and cultured.The protein content of p-JNK was detected by Western blot.Results:1.The results of HT-22 cells exposed to different concentrations of sodium arsenite showed that the cell viability decreased with the increase of exposure concentration.Flow cytometry showed that the apoptosis rate of HT-22 cells increased in a dose-dependent manner.There was a significant difference between the 10.0μmol/L As group and the control group.The levels of ROS and Ca²⁺increased with the increase of arsenic dose.Western blot showed that the protein content of cleaved caspase-3increased with the increase of arsenic dose;the protein content of Dapk1 and p-NR2B increased with the increase of arsenic dose;the protein content of p-Dapk1 and NR2B decreased with the increase of arsenic dose;the protein content of nuclear p-ERK increased with the increase of arsenic dose The protein content of p-CREB and Bcl-2 decreased with the increase of arsenic dose,the protein content of plasma p-ERK increased with the increase of arsenic dose,and the protein content of p-JNK and Bimel increased with the increase of arsenic dose（P<0.05）.Flow cytometry showed that the expression of ROS in HT-22 cells in As+NAC group was significantly lower than that in As group,and the level of apoptosis was significantly lower than that in As group.Western blot showed that the protein expression of cleaved caspase-3 in As+NAC group was significantly lower than that in As group;the protein expression of Dapk1 and p-NR2B in As+NAC group was significantly lower than that in As group;the protein expression of p-Dapk1 and NR2B in As+NAC group was significantly higher than that in As group;nuclear p-ERK in As+NAC group were significantly lower than that in As group The protein expression of p-CREB and Bcl-2 in As+NAC group was significantly higher than that in As group,the protein expression of plasma p-ERK in As+NAC group was significantly lower than that in As group,and the protein expression of p-JNK and Bimel in As+NAC group was significantly lower than that in As group（P<0.05）.The protein expression of p-JNK in si Dapk1+As group was significantly lower than that in siCtrl+Asgroup（P<0.05）.Conclusion:1.As can increase and activate DAPK1 expression in HT-22 cells through ROS,activate downstream signaling pathway and induce HT-22 cell apoptosis.Moreover,As can induce HT-22 cells to reduce NR2B receptor through ROS,leading to nerve injury2.As can induce p-ERK nuclear metastasis block,inhibit CREB activation,inhibit the activation of downstream survival signal Bcl-2 and promote apoptosis of HT-22 cells through ROS.