The Effect And Mechanism Of Escitalopram On Tunicamycin-induced Endoplasmic Reticulum Stress And Oxidative Stress

Background:The early research evidences from our team have showed that endoplasmic reticulum stress(ERS)may be the one of causes of the major depressive disorder(MDD).AttenuatingERS by regulating molecular chaperone levels may be one of the effective approaches to treat MDD in UPR and relative apoptotic pathway.The treatment of ERS may have positive significance in reducing neurocognitive dysfunction and comorbidity in MDD.ERS and oxidative stress(OS)are highly correlated biological processes,and they regulate multiple cellular signaling pathways in normal physiology and disease states.ERS combined OS(ERS/OS)may be the pathogenesis of MDD,which will become a new pathogenic model of severe mental diseases。ERS/OS as a possible pathogenic model of severe mental illness,may be associated with the onset of MDD,and the treatment of ERS/OS may have positive implications for the reduction of cognitive dysfunction and prevention of comorbidities in MDD patients.Objectives:To investigate the effects of ESC on ERS/OS-related pathways in brain microvascular endothelial cells induced by tunicamycin(Tm),so as to expand the mechanism of the antidepressant effect of ESC and provide a basis for ERS/OS as a new therapeutic target of MDD.Methods:According to the research purpose,bEnd.3 cell is divided into the following four groups:Control group(normal control group),TM(5 ug/ml Tm for 24 h),ESC+TM group(20μM ESC for 1h,then 5μg/ml Tm for 24h),ESC group(20μM ESC for 24 h).Then,the cell viability of each group were detected by the CCK-8.The cell apoptosis rate was determined by FITC-Annexin V/PI assay.The mean fluorescence intensity of GRP78 was detected by immunofluorescence.The protein relative expression of GRP78,PERK,XBP1,ATF6,CHOP,caspase-12,JNK,p-eNOS,CaMK Ⅱ,p-gp,MMP9 was detected by Western-Blot.The mRNA relative expression of GRP78,PERK,XBP1,ATF6,CHOP,caspase-12,JNK,p-eNOS,CaMK Ⅱ,p-gp,MMP9 was detected by RT-qPCR.The fluorescence intensity expression of Ca2+,NO,ROS in each group were detected by immunofluorescence.The activity of SOD and the concentration of MDA were detected by colorimetry.Results:Compared with the Control group,the cell apoptosisrate increased and the viability rate decreased in the TM group.Compared with the TM group,the cell apoptosis rate decreased and the viability rate increased in the ESC+TM group.The apoptosis rate and viability rate of ESC group were not significantly changed compared with Control group.Compared with the Control group,the protein and mRNA relative expressions of GRP78,PERK,XBP1,ATF6,CHOP,JNK,CaMKII,MMP9 were up-regulated,and the expressions of P-gp,P-eNOS decreased in the TM group.Compared with the TM group,in the ESC+TM group,the proteins and mRNA relative expressions of GRP78,PERK,XBP1,CHOP,CaMKII,MMP9 decreased,andthe expressions of p-gp and p-eNOS increased,the expressions of ATF6 and JNK showed no significant changes.While the allproteins and mRNA relative expressions in the ESC group were not significant different compared with the Control group.However,there was no significant difference in the proteins and mRNA expressions of caspase-12 in different groups.The immunofluorescence expression of GRP78 in each group was consistent with the protein expression trend.Compared with the Control group,the mean fluorescence intensity of intracellular Ca2+ and ROS increased and NO decreased in the TM group.Compared with the TM group,the mean fluorescence intensity of Ca2+,ROS decreased and NO increased in the ESC+TM group.Compared with the Control group,the SOD activity decrease and MDA concentration increase in TM group.Compared with the TM group,the activity of SOD increase and the concentration of MDA decreased in the ESC+TM group.Conclusion:Tm could activate the GRP78,then the ERS is induced by starting the IRE1-XBP1,PERK-ATF4-CHOP,IRE1-JNK-CHOP,ATF6-CHOP pathways in bEnd.3 cells,induced cell apoptosis and death.24h 5ug/ml Tm could induce ERS model of brain microvascular endothelial cells.ESC could activate IRE1-XBP1 PERK-ATF4CHOP pathways,by inhibited the protein and mRNA relative expression of GRP78,PERK,XBP1,CHOP,could attenuate ERS in bend.3 cell induced by Tm,and could reduce cell apoptosis rate and reduce cell death,which may be one of the new mechanisms of ESC antidepressant therapy.Tm could produce ERS/OS in bend.3 cells,resulting in increased cell permeability.The mechanism may be related to activation of CaMKⅡ pathway and eNOS decoupling.ESC could inhibit ERS/OS and reduce ERS related BBB permeability by inhibiting the CaMKⅡ pathway and eNOS coupling,which may be one of the new mechanisms of ESC in the treatment of MDD.