The Role Of Endoplasmic Reticulum Stress PERK-autophagy Pathway In The Toxicity Of Dibromoacetonitrile In HT22 Cells

Background and ObjectiveChlorination of drinking water acts as one of the effective public health measures to control water-borne diseases,but produces many disinfection by-products(DBPs)during disinfection,such as haloacetonitriles(HANs),trihalomethanes(THMs),haloacetic acids(HAAs)and so on.Compared with those controlled carbon-containing disinfection byproducts,HANs,one of nitrogenous disinfection byproducts(N-DBPs)has a stronger cytotoxicity and genotoxicity,which has been widely concerned by the public.Studies have shown that chronic exposure to HANs contributes to altered neurodegeneration in the brain tissue or neural cells for reactive oxygen species(ROS)accumulation and mitochondrial dysfunction.The neurotoxicity of HANs should not be ignored,but its specific molecular mechanism has not been elucidated.The central nervous system is very sensitive to redox states.Excessive ROS production has been shown to impair endoplasmic reticulum function,thereby inducing endopasmic reticulum stress(ERS)and autophagy,which is the main mechanisms of apoptotic injury.This article aims to explore the role ofoxidative stress-ERS-autophagy induced with DBAN in mouse hippocampal neuron(HT22)cells injury by regard dibromoacetonitrile(DBAN)as a representative of HANs to establishing intervention targets for the precise prevention of HANs neurotoxicity.Methods1.DBAN inhibited the viability of HT22 cells in a concentration-dependent manner;increased the leakage rate of LDH;changed the normal morphology of HT22 cells,preliminarily suggesting that DBAN induced HT22 cell damage.Compared to the control group,an increase in apoptotic cells was observed under the microscope,and the nucleus was densely thick and small and irregular in size;the results of flow cytometry showed that the apoptosis rate of HT22 cells in the DBAN-treated group was increased;The result of Western blot showed a decreasing ratio of apoptotic protein Bcl-2/Bax,indicating that DBAN induced apoptosis in HT22 cells.2.Investigate the role of ERS-PERK signaling pathway induced by DBAN in HT22 cells:After treating HT22 cells with DBAN,the expression of ERS and PERK signaling pathway related proteins glucose regulated protein(GRP78),phospho-Protein kinase-like ER kinase(p-PERK),activating transcription factor 4(ATF4),C/EBP-homologous protein(CHOP),protein disulfide isomerase(PDI)was detected by Western blot and inmmunofluorescence.HT22 cells were pretreated with the pharmacological inhibitor 4-PBA(1 m M)for 1h and treated with 10 μM DBAN for 24 h.CCK8 and Flow cytometry assay were used to detect the cell viability and apoptosis levels;Western blot was used to detect the expression of the above ERS-PERK signaling pathway-related proteins and apoptotic proteins.3.Investigate the effect of activating the PERK signaling pathway on autophagy induced by DBAN in HT22 cells: HT22 cells were treated with a series of different concentrations of DBAN(0,2.5,5,10 μM)for 24 h.Western blot was to detect the expression of autophagy-related proteins Beclin1 and LC3 B proteins;Acridine orange(AO),monodansylcadaverine(MDC)stainning and immunofluorescence assay were used to observe cells autophagosome formation induced by DBAN.HT22 cells are treated in advance with 3-MA for 1 h before co-treated with DBAN for 24 h and the changes of autophagy-related indexes,cell viability,apoptosis rate and apoptosis-related proteins were observed.CCK8 and Flow cytometry assay were used to detect cells viability and apoptosis levels;Western blot was used to detect the expression of the above autophagy-related proteins and apoptotic proteins.4.Explore the effect of ROS-mediated PERK signaling pathway in DBAN-induced autophagy in HT22 cells: HT22 cells were treated with a series of different concentrations of DBAN(0,2.5,5,10 μM)for 24 h,and DCFH-DA probe was used to detect the levels of intracellular ROS.HT22 cells are treated in advance with NAC(2.5 m M)for 2 h before co-treated with DBAN for 24 h.then the changes of ROS level were observed,and the cell viability changes,toxicity changes,apoptosis levels were detected by CCK8,LDH,and flow cytometry.Western blot was to detect the expression of ERS-PERK signaling pathway and autophagy-related proteins.The DCFH-DA probe was to detect ROS levels after 4-PBA pretreatment in HT22 cells.Results1.DBAN inhibited HT22 cell viability and increased LDH leakage rate in a concentration-dependent manner.TUNEL and Hoechst 33342 staining showed that the apoptotic cells in the DBAN-treated group gradually increased,mostly manifested as single cells,and the nuclei were small and irregular and most of them were dense and thick.Compared with the control group,the results of flow cytometry showed that the apoptosis rate of HT22 cells increased in DBAN-treated group and the flow cytometry results showed a decrease in Bcl-2/Bax.The results showed that DBAN induced apoptosis in HT22 cells.2.Compared with the control group,DBAN induced an increase in the protein levels of p-PERK,GRP78,ATF4,CHOP and PDI in HT22 cells,suggesting that the ERS-PERK signaling pathway was activated.4-PBA pretreatment can effectively reverse DBAN-increased ERS-PERK signaling pathway-related proteins,restore DBAN-induced decreased viability and increased apoptosis of HT22 cells,suggesting that DBAN induced HT22 cell damage by activating the ERS-PERK signaling pathway.3.Compared with the control group,DBAN induced a concentration-dependent increase in the levels of Beclin1 and LC3 B Ⅱ/Ⅰ of HT22 cells;the number of acidic vesicles labeled with AO staining(red fluorescence)and MDC staining(green fluorescence)increased,as well as in fluorescence intensity;in the cellular immunofluorescence experiment,LC3 B with green fluorescence diffused evenly in the control group,and agglomeration and increase occurred in the DBAN treatment group.These results suggested that DBAN induced autophagy in HT22 cells.Pretreatment of 3-MA significantly reduced the level of Beclin1 and LC3 B Ⅱ/Ⅰ,the cell viability inhibition,the apoptosis effect induced by DBAN,and increased the Bcl-2/Bax ratio decreased by DBAN,indicating that DBAN activated autophagy to induce HT22 cell damage.Pretreatment of 4-PBA significantly reversed the Beclin1 and LC3 B Ⅱ/Ⅰ level reduced by DBAN,indicating that inhibition of ERS could reduce autophagy induced by DBAN.4.Compared with the control group,DBAN induced an increase in ROS levels in HT22 cells.NAC pretreatment effectively removed ROS produced by DBAN,and significantly antagonized the cell viability inhibition,increased in the apoptosis rate and decreased in Bcl-2/Bax of HT22 cells induced by DBAN,suggesting that DBAN promoted the increased levels of ROS in HT22 cells.Pretreatment of NAC reduced the levels of GRP78,p-PERK and CHOP proteins,and also lowered the expression of Beclin1 and LC3 B Ⅱ/Ⅰ associated with autophagy increased by DBAN,indicating that reducing ROS levels could inhibit PERK signaling pathways and autophagy.Pretreatment of 4-PBA weakened DBAN-induced ROS levels and autophagy levels,indicating that the inhibition of ERS can effectively reduce ROS levels and autophagy induced by DBAN.ConclusionDBAN reduced HT22 cell viability,increased LDH release rate,changed the normal morphology of cells,and induced apoptosis of HT22 cells leading to HT22 cell damage..DBAN-mediated activation of autophagy by the ERS-PERK signaling pathway induced HT22 cell damage.DBAN induced autophagy by mediating the PERK signaling pathway and ROS interaction,leading to HT22 cell damage.