Microglia-derived IL-1β Promoted Neuronal Apoptosis Through ER Stress-mediated Signaling Pathway PERK/eIF2α/ATF4/CHOP Upon Arsenic Exposure

Objective:Arsenic is the leading toxicant on the ATSDR list of hazardous environmental chemicals,which is linked with neurotoxicity including cognitive dysfunction,neurodevelopmental alterations and neurodegenerative disorders.It has been suggested that increased and sustained pro-inflammatory response is one of the triggering factors of arsenic-induced neurotoxicity.Microglia,the immune cells in the central nervous system,response to physiological and pathological stress,and release a large array of pro-inflammatory cytokines if activated excessively.Several studies indicated that arsenic was capable of inducing microglia activation,however,the role of the subsequently released pro-inflammatory cytokines in arsenic-induced neurotoxicity remains to be elucidated.Therefore,first,this study intends to establish the mouse model of exposed to NaAsO₂through drinking water,and use minocycline,a recognized inhibitor of microglia activation,to interfere the effect of NaAsO₂on microglia in the hippocampal nerve tissue of mice.To verify the effect of NaAsO₂on microglia in the hippocampus of mice,and the role of activated microglia in inducing apoptosis of mouse hippocampal neurons.Secondly,in this study,N9 microglia cells were exposed to NaAsO₂,and then the HT-22 hippocampal neurons were treated with culture supernatant（N9 CM）for 24 h,observe the effect of activated microglia on HT-22 cells,elucidated the underlying mechanism of microglia-derived IL-1βin the induction of neuronal apoptosis upon arsenic exposure.Methods:21-day-old Kunming mice were choose for this study.Mice of each group were treated correspondingly for 8 weeks as follow:sodium arsenite（NaAsO₂）treated groups（0,20,40,80 mg/L）,minocycline treated group and NaAsO₂（80 mg/L）+minocycline group.At the end of the corresponding treatment,mice were sacrificed to collect samples of brain hippocampus.The protein level of Iba-1,IL-1β,cleaved caspase-3,Bax,Bcl-2,p-PERK,p-eIF2α,ATF4,CHOP were detected by western blotting assay,aim to explain the effect of NaAsO₂on mouse hippocampal tissues.In addition,the intervention effect of minocycline were also measured by western blot.Murine microglial cell line N9 and mouse hippocampal neuronal cell line HT-22were used to study in vitro.N9 cells were treated of NaAsO₂for 24 h,cell viability was detected by Cell Counting Kit-8;The secretion of IL-1βin the supernatant was determined by ELISA;the protein level of Iba-1 and IL-1βwere detected by western blot;“conditioned medium（N9 CM）”was used to culture HT-22 cells,the apoptotic rate of HT-22 cells were detected using an Annexin V/PI double staining and visualized by fluorescence microscope,and apoptosis related protein of cleaved-caspase-3,Bax,Bcl-2 were measured by western blot.In order to verity the effect of microglia in induce HT-22 cell apoptosis,minocycline were choose to intervene the N9 cell.Cell Counting Kit-8 were used to detected the concentration of minocycline.ELISA was choose for determined the effect of minocycline on IL-1β.Western blot were detected the expression of Iba-1,IL-1β.Flow cytometry assay and fluorescence microscope were used to detected the apoptotic rate of HT-22 cells which were treated with N9 CM,cleaved caspase-3,Bax,Bcl-2 and ERS related protein were also detected by western blot.Aim to study the mechanism of microglia activation and whether microglia-derived IL-1βplayed a role in the apoptosis of HT-22 cells through the activation of ER stress-mediated PERK/eIF2α/ATF4/CHOP pathway.The expression of TLR4,p-JNK in N9 cell which were treated by NaAsO₂and/or minocycline were detected by western blot;IL-1ra were used to pre-treated HT-22 cell for 1 h,apoptotic rate of HT-22 cells were determined by flow cytometry assay and fluorescence microscope;western blot were used to measure the effect of IL-1ra on cleaved caspase-3,Bax,Bcl-2 and ERS related proteins.At least,HT-22 cell were exposure to IL-1β（20,40,60 ng/m L）,verify the IL-1βcould induced the HT-22 cell apoptosis,the protein expression of cleaved caspase-3,Bax,Bcl-2 and ERS related protein were changed along with the treatment of IL-1β.Results:1、NaAsO₂exposure resulted in abnormal behavioral performance,microglia activation and neurocyte apoptosis in miceThe behavioral performance of the animal were assessed by Morris water maze,the results indicated that:the learning and memory abilities NaAsO₂treated groups were decreased in NaAsO₂（0 mg/L）groups,the escape latency time were significantly longer than that of the control group,and demonstrated dose-dependent downward trends in numbers of passing platform,target quadrant time and so on;new object recognition experiment also show that:the new objected recognition index of mice in NaAsO₂treated groups were significantly lower than that of the control group（p<0.05）.The behavioral experiment results indicated that:exposure to NaAsO₂resulted in spatial cognitive deficits in mice.The expression of Iba-1,the hallmark of microglia activation,were significantly highly than that in control group（p<0.05）,and the IL-1β,which is the typical pro-inflammatory cytokines released by microglia after its activation,were up-regulated expression in NaAsO₂exposure groups（p<0.05）.Expression of cleaved caspase-3,Bax were up-regulated expression in NaAsO₂exposure groups,and Bcl-2 were down-regulated expression after exposure to NaAsO₂（p<0.05）,indicated that NaAsO₂could induced hippocampal neurons apoptosis.At the same time,the protein expression of Bip,p-PERK,p-eIF2α,ATF4,CHOP were significantly up-regulated in NaAsO₂exposure groups（p<0.05）,suggested that ER stress-mediated apoptotic pathway PERK/eIF2α/ATF4/CHOP was possibly involved in the induction of neuronal apoptosis.2、Minocycline inhibited microglia activation and attenuated NaAsO₂induced neurocyte apoptosis in miceMorris water maze indicated that:intervention by minocycline diminished NaAsO₂induced in spatial cognitive deficits,the escape latency time were significantly shorter than that of the NaAsO₂group,and demonstrated dose-dependent upward trends in numbers of passing platform,target quadrant time;The new objected recognition index of mice in NaAsO₂treated groups were significantly improved than that of the control group（p<0.05）.This finding indicating that intervention by minocycline could inhibited the effect of spatial cognitive deficits of NaAsO₂,suppression microglia activation could inhibited NaAsO₂induced neurotoxicity.The expression of Iba-1,IL-1βwere significantly decreased by minocycline,compared with NaAsO₂treated groups（p<0.05）,in addition,the protein level of Bip,p-PERK,p-eIF2α,ATF4,CHOP were also decreased in minocycline intervene group（p<0.05）.Indicated that suppressed microglia activated by minocycline may reduced the hippocampal neurons apoptosis through ER stress-mediated apoptotic pathway PERK/eIF2α/ATF4/CHOP.3、NaAsO₂exposure induced N9 microglia activation and up-regulation of IL-1βN9 cells were exposure to NaAsO₂（2,4,6μM）for 24 h,Iba-1 was found to be dose dependently up-regulated in N9 cells upon NaAsO₂exposure.Meanwhile,the protein expression levels of IL-1βwere remarkably increased in the NaAsO₂exposed groups compared with the control group,indicated that:NaAsO₂could induced activated N9 cell.However,minocycline could significantly inhibited the effect of NaAsO₂,the expression of both Iba-1 and IL-1βwere decreased in NaAsO₂（+）/minocycline（-）.4、Conditioned culture leaded to increased apoptosis in hippocampal neuronal HT-22cellsHT-22 cells were directly exposed to NaAsO₂or treated with N9 CM for 24 h.CCK-8 showed that there were no significantly effect on NaAsO₂（0μM）group,however,cell viability of NaAsO₂（2,4,6μM）N9 CM group was significantly lower than that in NaAsO₂（2,4,6μM）direct exposure group（p<0.05）.The apoptosis level of N9 CM culture in the presence of NaAsO₂was significantly higher than that of the direct NaAsO₂exposure within each concentration pair,same findings were also confirmed by fluorescent staining.At the same time,the protein level of cleaved caspase-3、Bax in NaAsO₂（2,4,6μM）N9 CM group were much highly than that in NaAsO₂（2,4,6μM）direct exposure group,but the Bcl-2 were decreased in NaAsO₂（2,4,6μM）N9 CM group.5、N9 microglia activation play a role in the induction of apoptosis in HT-22 cellsN9 microglia was pretreated in the presence or absence of minocycline（5μM）,followed by NaAsO₂exposure at the concentration of 4μM for 24 h.N9 CM were then collected for the subsequent experiments.HT-22 cells were directly exposed to NaAsO₂at the concentration of 4μM or treated with N9 CM for 24 h.The results showed that,treatment by minocycline could suppression the effect of NaAsO₂on induce the apoptosis of HT-22 cells.Compared with NaAsO₂（+）/minocycline（-）/N9 CM（+）group,the apoptosis level was decreased in NaAsO₂（+）/minocycline（+）/N9 CM（+）group.The same results were present in western blot,intervention by minocycline could decreased the protein level of cleaved caspase-3 and Bax,but increased the expression of Bcl-2.These findings provided evidences that inhibition of N9 microglia activation by minocycline could diminish NaAsO₂induced apoptosis of HT-22 cells.6、ER stress-mediated apoptotic pathway was involved in N9 microglia activation-induced HT-22 cell apoptosisThe expressions of Bip,p-PERK,p-eIF2α,ATF4 and CHOP were significantly increased in both of the direct NaAsO₂exposure and indirect NaAsO₂exposure through N9 CM when compared with its relative control.But,the expression of them were much higher in NaAsO₂（+）/minocycline（-）/N9 CM（+）group.However,intervention by minocycline significantly attenuated the activation of PERK/eIF2α/ATF4/CHOP pathway,which were demonstrated by the sharply decreased expressions of Bip,p-PERK,p-eIF2α,ATF4 and CHOP in NaAsO₂（+）/minocycline（+）group compared with those of the NaAsO₂（+）/minocycline（-）group under N9 CM culture.These findings suggested that NaAsO₂induced N9 microglia activation also contributed to the activation of ER stress-mediated apoptotic pathway PERK/eIF2α/ATF4/CHOP apart from the direct action of NaAsO₂.7、NaAsO₂could activation the TLR4/JNK signaling pathway in N9 cellN9 cell were exposure to NaAsO₂（2,4,6μM）for 24 h,compared with NaAsO₂（0μM）group,the protein level of TLR4、p-JNK were significantly increased（p<0.05）,however,minocycline could suppression the effect of NaAsO₂,the protein level of TLR4,p-JNK could significantly decreased compared with NaAsO₂（4μM）group（p<0.05）.This finding indicated that,NaAsO₂could activation the TLR4/JNK signaling pathway,but minocycline may inhibited the activation of N9 cell by suppression of TLR4/JNK signaling pathway.8、IL-1βplayed a role in the induction of ER stress-mediated HT-22 cell apoptosisHT-22 cells were pretreated in the presence or absence of IL-1 receptor antagonist IL-1ra,followed by direct NaAsO₂exposure at the concentration of 4μM or N9 CM treatment for 24 h.Both flow cytometry and fluorescent staining indicated that:compared with NaAsO₂（+）/N9 CM（+）/IL-1ra（-）group,treatment of IL-1ra remarkably attenuated apoptosis level in the N9 CM treated HT-22 cells;at the same time,under the treatment of IL-1ra,the expression of cleaved-caspase-3、Bax were significantly decreased in NaAsO₂（+）/N9 CM（+）/IL-1ra（+）group,but the level of Bcl-2 was significantly increased（p<0.05）.And then,The expression level of Bip,p-PERK,p-eIF2α,ATF4 and CHOP of NaAsO₂（+）/N9 CM（+）/IL-1ra（+）group were remarkably decreased than those of NaAsO₂（+）/N9 CM（+）/IL-1ra（-）group（p<0.05）,which indicated that microglia-derived IL-1βpromoted HT-22 cells apoptosis through ER stress-mediated PERK/eIF2α/ATF4/CHOP pathway.HT-22 cell were exposure to IL-1β,compared with IL-1β（0 ng/m L）group,the protein expression of cleaved-caspase-3、Bax in IL-1β（20,40,60 ng/m L）group were significantly increased,but the expression of Bcl-2 were significantly decreased（p<0.05）;at the same time,IL-1βcould activate the PERK/eIF2α/ATF4/CHOP in HT-22 cell,the expesssion of Bip,p-PERK,p-eIF2α,ATF4 and CHOP were in a does-dependent manner in IL-1β（20,40,60 ng/m L）group（p<0.05）.These findings suggested that IL-1β,possibly derived from NaAsO₂induced N9 microglia activation,was involved in the induction of apoptosis in HT-22 cellsConclusion:1、Arsenic resulted in microglia activation accompanied by release of IL-1β;2、Microglia-derived IL-1βmay promoted neuronal apoptosis through ER stress-mediated pathway;3、Microglia activation was involved in arsenic-induced cognitive dysfunction.