Endoplasmic Reticulum Mechanism In Methamphetamine-induced Blood-brain Barrier

Methamphetamine(METH)commonly known as "ice",is the most popular and rapidly spreading drugs in the world.METH is an amphetaminetype psychostimulant.METH can cause euphoria,excitement,and even hallucinations.Repeated drug use can lead to addiction and have devastating effects on the central nervous system.Long-term METH abuse can induce neurotoxicity,as well as changes in brain structure and function.METH mainly affects the monoamine neurotransmitter system,and induces graymatter atrophy,white-matter hyperintensities and glial cell activation in bain.METH results in hallucinations,delusions,personality disintegration,and cognitive impairment including attention,memory,and executive functions.Hyperthermia,neurotransmitters release,oxidative stress,mitochondrial dysfunction and inflammation are responsible for METH-induced neurotoxicity.Recently,many studies have investigated the effects of METH on blood brain barrier(BBB).In vivo studies have found that both high-dose and prolonged low-dose METH exposure produce a decrease in BBB structural proteins and an increase in BBB permeability.BBB break down leads to increase in BBB permeability to immunoglobulin and large molecules in hippocampus and amygdale,and decreased expression of tight junction proteins such as occludin,claudin-5 and zonula occludens(ZO-1),which may be related to the metalloproteinase-9 increase.The mechanisms responsible for BBB damage mainly focused on oxidative stress.Brain microvascular endothelial cell is the major component of BBB.METH alters BBB function via direct effects on endothelial cells,resulting in increased reactive oxygen species(ROS),decreased tight junction proteins and trans-epithelium electrical resistant.Endoplasmic reticulum(ER)is an important organelle of eukaryotic cells,and critical for the proper folding of secretory and transmembrane proteins.Several disturbances may alter the ER environment and consequently promote the accumulation of unfolded proteins in the ER lumen,a condition known as endoplasmic reticulum stress(ERS).During ERS,chaperone protein,glucose-regulated protein 78(GRP78)is dissociated from the binding sites on the lumen tails of three ER-localized transmembrane signal transducers: protein kinase RNA-like endoplasmic reticulum kinase(PERK),inositol-requiring enzyme(IRE)-1,and activating transcription factor(ATF)-6,that,in unstressed conditions,are maintained in an inactive status by the binding with the GRP78.The initial aim of the ERS is to adapt and restore ER homeostasis by promoting proper protein folding;however,the chronic and prolonged ERS leads to the activation of apoptotic pathways characterized by increased caspase 12,and C/EBP homologous protein(CHOP)expression.The interplay between oxidative stress and ERS exists when the inner and outer cellular environment changes.Oxidative stress induces ERS which is known to activate apoptotic signaling pathways;sustained ERS induces oxidative stress.ROS release has been known to activate CHOP in neurons.This interplay has also been found in thiamine deficiency and neurodegeneration,where oxidative stress disrupts the ER redox state and thereby disrupts proper protein folding in the ER.However,whether ER stress is involved in METH-induced blood-brain barrier damage has yet to be determined,and there are no reports of any such interplay in METH-induced BBB disruption as yet.In the present study,we aimed to elucidate the effects of METH on BBB and the mechanism of METH-induced BBB disruption by studying the role of ERS and the potential oxidative/ER stress interplay.Considering that brain microvascular endothelial cells(BMVECs)are the main component of the BBB,we used immortalized mouse BMVECs(b End.3)cells to model BBB damage in vitro.Moreover,we used C57BL/6J mice to analyze the roles of ERS in METH-induced BBB disruptions in vivo.These observations will provide a novel insight into the mechanism of METH-induced neurotoxicity.Part I Endoplasmic reticulum stress in methamphetamine-induced blood-brain barrier break down in miceObjective: To investigate the role of endoplasmic reticulum stress METH-induced blood-brain barrier(BBB)disruption in mice.We investigated METH-induced hyperthermia and stereotyped behavior to evaluate the neurotoxicity of METH.We detected tight junction proteins expression in brain tissue,fluorescein sodium(Na-Fluo)and Evans blue leakage in mice to explore METH-induced structural and functional BBB disruption.We identified effects of ERS inhibitor,4-phenylbutyric acid(PBA)on METHinduced BBB disruption in mice by intraperitoneal injection of PBA before MEETH injection.Methods: 1 Establishment and observation of METH acute administration mice model.We used C57BL/6 mice,and METH(5mg/kg×4,3-h intervals)was injected intraperitoneally.Rectal temperature was detected 30 min before the first injection,1 h after each injection and 24 h after the first injection.Stereotyped behavior score was assessed using Sams-Dodd’s scoring criterion.2 METH-induced BBB damage in mice.Evans Blue and sodiumfluorescein were used to evaluate solute permeability of the BBB.Animals were injected intraperitoneally with 2% Na-Fluo(2.5 ml/kg)or into the tail vein with 2% Evans Blue(4 ml/kg)at 24 h after the first METH injection.The tracers were allowed to circulate for 30 min and 2 h,respectively.Measurements of Na Fluo and Evans Blue fluorescence were then determined.3 Detection of tight junction proteins in brain tissue.The Claudin 5 and Occludin expression in brain tissue were detected at 24 h after the first METH injection.4 Effects of PBA on METH-induced BBB damage in Mice.PBA(50mg/kg)was injected intraperitoneally 30 min before METH injection.At 24 h after the first METH injection,animals were injected intraperitoneally with 2% Na-Fluo or into the tail vein with 2% Evans Blue,respectively.At 30 min and 2 h after the injection of tracer,the determination of brain tissue Na Fluo and Evans blue volume were performed.5 Effects of PBA on METH-induced tight junction proteins decrease.PBA(50mg/kg body weight)was intraperitoneally injected 30 min before METH injection.The rats were sacrificed at 24 h after the first METH injection,and the Claudin 5 and Occludin expression were detected using Western blot.Results: 1 METH(20 mg/kg/d)induced marked stereotyped behavior,including running in circles,repetitive head movements,accompanied by significant hyperthermia(about 2 ° C).2 METH(20 mg/kg/d)increased Na-Fluo and Evans blue leakage in mice,and decreased the Claudin 5 and Occludin expression.3 PBA(200 mg/kg/d)significantly inhibited METH-induced Na-Fluo and Evans blue leakage in mice,and the Claudin 5 and Occludin protein decrease.Summary: METH acute administration induced hyperthermia and stereotyped behavior in C57BL/6 mice,disrupted tight junctions,and increased permeability of the blood-brain barrier.ERS inhibitor,PBA could reverse METH-induced the increase permeability of the blood-brain barrier and tight junctions disruption.These observations indicate that endoplasmic reticulum stress is involved in METH-induced blood-brain barrier damage in mice.Part II The mechanism of endoplasmic reticulum stress in methamphetamine-induced brain microvascular endothelial cell damageObjective: To evaluate the mechanism of endoplasmic reticulum stress in METH-induced blood-brain barrier disruption.We determined the cell viability,apoptosis,trans-epithelial electrical resistance(TEER)and tight junction protein to illustrate that METH induced brain microvascular endothelial cell damage.We evaluated the effects of METH on unfolded protein response(UPR)using mRNA PCR-array and Western blot.We aimed to illustrate the effects of 4-phenylbutyric acid(PBA)and Salubrinal pretreatment on METH-induced the up-regulation CHOP and pro-caspase 12.We performed si RNA CHOP transfection to observe the role of CHOP in METH-induce cell damage.Methods: 1 Detection of METH-induced brain microvascular endothelial cells(b End.3)damage.First,the marker protein CD31 was identified using immunofluorescence.MTT was performed to observe the time-dependent and dose-dependent effects of METH on b End.3 cell viability.Flow cytometry and TUNEL were performed to evaluate apoptotic rate at indicated time(24 h,48 h,72 h)after 1m M METH exposure in b End.3 cells.TUNEL was performed to evaluate apoptotic rate at indicated time(24 h)after 1m M METH exposure in b End.3 cells.Trans-endothelial electrical resistance(TEER)assay was performed at indicated time(24 h,48 h,72 h)after 1m M METH exposure in b End.3 cells.Western blot was performed to measure the Claudin 5 and Occludin expression.2 Effects of METH on the unfolded protein response signal pathway.The m RNA PCR-array method was performed to detect unfolded protein response(UPR)signal pathway related 84 genes at 24 h after 1m M METH exposure in b End.3 cells.Western blot was performed to evaluate GRP78 / Bip,p-IRE1α,p-PERK and ATF6 proteins at indicated time(0,0.5,1,3,6,12 and 24 h)after 1m M METH exposure in b End.3 cells.3 Effects of METH on endoplasmic reticulum stress-related CHOP and pro-caspase12 proteins.Western blot was performed to detect CHOP and pro-caspase 12 expressions at indicated time(0,1,3,6,12 and 24 h)after 1m M METH exposure in b End.3,and evaluate effects of endoplasmic reticulum stress inhibitors,50 μM PBA and 5 m M Salubrinal on CHOP and pro-caspase12 expression.4 Role of CHOP in METH-induced cell damage.The si RNA CHOP transfection was performed before the METH exposure in b End.3 cellls.Western blot was performed to verify the efficiency of si RNA CHOP transfection.MTT was performed to evaluate the cell viability,flow cytometry and TUNEL were performed to evaluate the apoptotic rate,TEER assay was performed,Western blot was performed to evaluate Claudin 5 and Occludin expression.Results: 1 Laser confocal microscopy demonstrated that the cytoplasm and membrane of b End.3 cells were green,nuclei were stained blue using DAPI,endothelial cell marker CD31 was strongly positive in b End.3 cells,suggesting that b End.3 cells have endothelial cell characteristics and can be used for subsequent study.2 Cell viability was significantly decreased after 1 m M METH treatment for 24 h.Cell viability was significantly decreased,apoptosis was significantly increased,TEER,Claudin 5 and Occludin expression was significantly decreased after 1 m M METH treatment for 24 h,48h and 72 h.3 The m RNA PCR-array demonstrated that Hspa5(GRP78 / Bip),XBP-1(p-IRE1α downstream signal),ATF6,Ddit3(CHOP),ATF4(p-PERK downstream signal)and e IF2α(p-PERK downstream signal)in the unfolded protein response(UPR)signals were increased after 1m M METH exposure to b End.3 cells for 24 h.Western blot demonstrated that Bip was increased after 1m M METH exposure to b End.3 cells for 3h in time-dependent manner;p-IREα significantly increased from 3h,reached to peak at 12 h,then decreased;p-PERK significantly increased from 1h,reached to peak at 6 h,and then decreased;the increase of p-IREα and p-PERK sustained to 24h;ATF6 significantly increased from 0.5 h,reached to peak at 3 h,which sustained to 24 h.4 Western blot demonstrated that CHOP and pro-caspase 12 were increased from 12 h to 24 h after 1m M METH exposure to b End.3 cells.Endoplasmic reticulum stress inhibitor,50 μM PBA and 5 m M Salubrinal significantly inhibited METH-induced increase of CHOP and pro-caspase 12.5 Western blot demonstrated that CHOP si RNA significantly inhibited CHOP expression.MTT illustrated that CHOP si RNA significantly inhibited METH-induced decrease of cell viability.Flow cytometry and TUNEL demonstrated that CHOP si RNA significantly inhibited METH-induce apoptosis.CHOP si RNA significantly inhibited METH-induced down-regulation of TEER,Claudin 5 and Occludin expression.Summary: METH could decrease brain microvascular endothelial cell viability,increase apoptosis and down-regulate tight junction protein in time-dependent manner.METH could induce sustain increase of GRP78 / Bip,p-PERK,p-IRE1α,ATF6,CHOP and pro-caspase12 expression.Inhibition of endoplasmic reticulum stress could partially reverse METH-induced increase of CHOP and pro-caspase12.CHOP knockdown could reverse METHinduced down-regulation of cell viability,increase of apoptosis,decrease of TEER and tight junction proteins,suggesting that METH induce microvascular endothelial cells damage via endoplasmic reticulum stress.Part III The interplay between endoplasmic reticulum stress and oxidative stress in methamphetamine-induced brain microvascular endothelial cell damageObjective: To observe whether METH could induce oxidative stress in brain microvascular endothelial cells,and explore METH-induced crosstalk between endoplasmic reticulum stress and oxidative stress in microvascular endothelial cells.Methods: 1 Effects of METH on oxidative stress-related signals in b End.3 cells.Reactive oxygen species(ROS)was detected using confocal microscopy and fluorescence spectrophotometer at indicated time(0,0.5,1,3,6,12 and 24 h)after 1 m M METH exposure to b End.3 cells;Western blot was performed to evaluate Bcl2 / Bax expression;mitochondrial membrane potential(MMP)was detected using fluorescence spectrophotometer;Western blot was performed to detect cytochrome c protein in cytoplasm and mitochondria.Immunofluorescence was performed to indentify cytochrome c after 1 m MMETH exposure in b End.3 cells for 24 h.2 Effects of ROS on METH-induced up-regulation of endoplasmic reticulum stress related signals in b End.3 cells.200 μM Apocynin(ROS inhibitor)and 100 μM N-tert-butyl-α-phenylnitrone(NBP)(ROS scavenger)were administered at 30 min before 1 m M METH treatment,and Western blot was performed to evaluate GRP78 / Bip,p-IRE1α,p-PERK and ATF6 proteins.3 Effects CHOP knockdown on METH-induced release of cytochrome c from mitochondria to cytoplasm.Cells were transfected with CHOP si RNA before 1 m M METH treatment,and western blot was performed to evaluate cytochrome c in cytoplasm and mitochondria.Results: 1 ROS was increased from 1h,reached peak at 3 h,declined at 6 h,and came to normal at 24 h.2 Bcl2 / Bax protein was significantly decreased at 3 h after 1m M METH exposure in b End.3 cells in a time-dependent manner.3 MMP was significantly decreased at 3 h after 1m M METH exposure in b End.3 cells in a time-dependent manner.4 Immunofluorescence demonstrated that cytochrome c protein was significantly increased in cytoplasm at 24 h after MEETH exposure in b End.3 cells.Western blot demonstrated that cytochrome c in cytoplasma was up-regulated and cytochrome c in mitochondria was down-regulated at 6 h in a time-dependent manner.5 Apocynin and NBP significantly inhibited METH-induced increase of GRP78/Bip,p-IRE1α,p-PERK and ATF6 in b End.3 cells.6 CHOP si RNA significantly reversed METH-induced down-regulation of cytochrome c in mitochondria and up-regulation of cytochrome c in cytoplasma in b End.3 cells.Summary: METH could induce oxidative stress and apoptosis,inhibition of oxidative stress could reverse the activation of the endoplasmic reticulum stress pathway,in turn,inhibition of CHOP pathway in endoplasmic reticulum stress could reverse METH-induced oxidative stress,suggesting the interplay between oxidative stress and endoplasmic reticulum stress is triggered in METH-induced brain microvascular endotheli-al cell damage.Conclusions: 1 METH induces the increase of blood-brain barrier permeability,disruption of tight junction and abnormal behavior,which is reversed by endoplasmic reticulum stress inhibitor,suggesting that endoplasmic reticulum stress is involved in METH-induced blood-brain barrier disruption.2 Inhibition of endoplasmic reticulum stress can partially reverse METH-induced brain microvascular endothelial cell viability decrease,apoptosis,and tight junction disruption,which is related to endoplasmic reticulum stress molecule chaperone GRP78 / Bip,receptor protein p-PERK,p-IRE1α,ATF6,and apoptotic signal CHOP and pro-caspase12.3 The interplay between oxidative stress and endoplasmic reticulum stress is involved in METH-induced brain microvascular endothelial cell damge.