Endoplasmic Reticulum Stress Mediates Methamphetamine-induced Microglial Activition

Objective: Drug abuse has caused a widely public attention worldwide,which not only damages to personal physical and mental health,also affects the family and society.Methamphetamine(METH),represented by amphetamine-type stimulant(ATS),commonly known as“ice”,is a highly addictive drug that is abused throughout the world.METH can pass through the blood-brain barrier and increase the excitability of the central nervous system.METH abuse leads to adverse neuropsychiatric consequences that include addiction,cognitive impairments and neuronal apoptosis in humans.Inflammatory is one of the main mechanisms of long-term abuse of METH causing nerve injury.Increasing evidence suggests that activated glial cells have an important role in releasing inflammatory factors and cytotoxic substances,which contribute to dopamine neuronal damage or even death by trigging inflammation.However,the precise mechanisms responsible for METH-induced microglia activation are still being elucidated.Altered endoplasmic reticulum(ER)homeostasis lead to the accumulation of unfolded or misfolded proteins in the ER lumen,known as ER stress(ERS),which activates the unfolded protein response(UPR).UPR activation can promote cell survival or cell death if ER stress is chronic or severe.Previous studies have focused on the role of ERS associated with inflammatory.However,its involvement in METH-induced inflammatory remains largely unexplored.Based on the above viewpoints,the present study aimed to evaluate the effects of ERS on METH-induced microglial activation and investigate the mechanisms of inflammatory signaling.The data would provide new ideas for the treatment of METH abuse and neurotoxic injury.1 Western blot and RT-PCR were used to observe the GRP78 protein and mRNA after 1 mM METH treatment for 5 min,15 min,30 min,60 min and 90 min in N9 cells.2 Effects of METH and 4-PBA on the expression of GRP78 protein and mRNA in N9 cells were observed by Western Blot and RT-PCR after treated with 1 mM METH and 6 mM ERS suppressor(4-PBA,pretreatment with 60 min)for 30 min.3 After pretreatment with 6 mM 4-PBA for 60 min,Western blot was performed to evaluate the expression of phospho-NF-κB p65,NF-κB p65 after 1 mM METH and 6 mM 4-PBA treatment for 30 min in N9 microglia cells.Flow Cytometry were performed to test the expression of Iba1 and RT-PCR was performed to estimate the effect of 1 mM METH and 6 mM 4-PBA for 3 h on expression of IL-6 and TNF-α in N9 microglia cells.4 With Western blot,we observed the protein levels of IRE1α,p-IRE1α,TRAF2,PERK,p-PERK and ATF6 after 1 mM METH treatment for 5 min,15 min,30 min,60 min and 90 min in N9 cells.The aim was to screen the signaling mechanisms involved in the inflammatory.5 The relationship between IRE1α and TRAF2 was studied after 1 mM METH and 40 μM IRE1α inhibitor STF-083010 treatment for 30 min in the N9 cells by Co-Immunoprecipitation method.Western blot was performed to evaluate the expression of phospho-NF-κB p65.RT-PCR was performed to estimate the effect of METH and STF-083010 for 3 h on expression of IL-6 and TNF-α.6 Transfection with RNAi was used to knock down IRE1α on N9 cells.we observed the expression of p-IRE1α,TRAF2 and phospho-NF-κB p65 by Western blot after 1 mM METH treatment for 30 min,and the expression of IL-6 and TNF-α by RT-PCR after 1 mM METH treatment for 3 h.7 The data were presented as Means±S.E.M and analyzed with one way ANOVA by SPSS21.0 statistical program,Student-Newman-Keuls did the comparison between groups.A level of P < 0.05 was supposed to be statistically significant.Methods:Results:1 The role of endoplasmic reticulum stress in the activation of N9 cells by METH:(1)The level of GRP78 protein and mRNA was significantly increased after 1 mM METH treatment for 30 min,compared with the normal control group.After 4-PBA administration,the above effects were partially reversed.It is suggested that the METH could induced endoplasmic reticulum stress.(2)The expression of Iba1、phospho-NF-κB p65、IL-6 and TNF-α increased after METH treatment,while 4-PBA could reduce the increase.The results showed that ERS could mediate METH-induced microglial activation and the release of inflammation cytokines,such as IL-6 and TNF-α.2 Based on above results,we further investigated which endoplasmic reticulum stress signaling mediates METH-induced N9 microglial cells activation.(1)The effects of p-IRE1α/TRAF2、p-PERK and ATF6 after 1 mM METH treatment for 30 min on the N9 cells were observed.Three signal pathways were activated,which p-IRE1α/TRAF2 change was the most significant.Therefore,we chose this pathway for subsequent study.(2)The result of Co-Immunoprecipitation showed that the interaction between IRE1α and TRAF2 was increased after METH treatment,while STF-083010 could reduce the increase.(3)The expression of phospho-NF-κB,IL-6 and TNF-α increased after METH treatment,while STF-083010 could reduce the phospho-NF-κB,IL-6 increase.But the expression of TNF-α had no change.(4)The transfection of si-IRE1α was found to attenuate the level of p-IRE1α,TRAF2,phospho-NF-κB,IL-6 and TNF-α increased by METH treatment.It is suggested that METH could mediate the activation of microglia through endoplasmic reticulum stress by IRE1α/NF-κB signaling.Conclusions:1 METH can mediate the endoplasmic reticulum stress in microglia.2 METH can mediate the activation of microglia and the release of inflammative cytokines,such as IL-6 and TNF-α by endoplasmic reticulum stress.3 METH-induced microglial activition is related to IRE1α/NF-κB signaling.