The Study Of Mild Endoplasmic Reticulum Stress In Neuroinflammation

Background: Neuroinflammation is a common pathophysiological pathway of many central nervous system(CNS)diseases,which results in synaptic impairment,neuronal death,and the exacerbation of several pathologies within the brain.Neuroinflammation is mainly characterized by over-activation of glial cells,excessive release of pro-inflammatory factors in the brain,and the blood-brain barrier(BBB)damage.Although neuroinflammation-related diseases have different clinical manifestations,but all involve the disturbance of protein proteostasis.The endoplasmic reticulum(ER)plays a role in many essential cellular processes,including the correct synthesis,folding,modification,and transport of proteins.Several physiological and pathological conditions can alter protein folding in the ER,allowing unfolded or misfolded proteins to accumulate in the ER lumen: a cellular state referred to as ER stress(ERS).To recover ER proteostasis,cells activate a complex signal transduction pathway known as the unfolded protein response(UPR).Several reports have linked ERS to various CNS diseases,but the term “mild ERS”has been recently used to describe slight perturbations in ER function that are not fatal to the cell.Mild ERS seems to be characterized by the activation of proximal sensors without the involvement of downstream pro-apoptotic proteins.Therefore,mild ERS has very different pathophysiological effects.Mild ERS has been reported to be successful in reducing pathological features in various CNS diseases,such as cerebral ischemia and Parkinson's disease(PD).These findings indicate the contribution of ERS to CNS diseases is likely complex and non-linear,and its relationship with neuroinflammation deserves further study.Our study aimed to explore the regulation of mild ERS in microglia,astrocytes and BBB during neuroinflammation.This work might provide not only a better understanding of the role of ERS in neuroinflammation but also the basis for a prospective cure for CNS diseases in the future.Part I: The Effects of Mild Endoplasmic Reticulum Stress on Microglia and its Role in Neuroinflammation Objective: In the current study,we investigated the effect of mild ERS on microglia in LPS-induced neuroinflammation.Methods: In experiment 1,the ERS activator tunicamycin(TM)was dissolved in10% dimethyl sulfoxide(DMSO).The dilutions of TM(0.3,3,and 30 ?g)were prepared freshly before the experiment in 0.9% saline containing 10% DMSO,and 2?L of the preparation was administered via the intracerebroventricular(i.c.v.)route to male Sprague–Dawley(SD)rats.Apoptosis and ERS in the hippocampus were measured 24 hours after injection.In experiment 2,the rats received the i.c.v.injection of TM with or without intraperitoneal injection of the ERS stabilizer sodium4-phenylbutyrate(4-PBA;100 mg/kg)1 hour before LPS administration(500 ?g/kg).Apoptosis,proinflammatory cytokine secretion,microglial activation,and ERS were assessed 24 hours after treatment.In addition,the effect of mild ERS on microglia was determined in vitro.In experiment 3,primary cultured microglia were incubated with different concentrations of TM(0.5,5,and 50 ng/m L).The extent of ERS was assessed after 24 hours.In experiment 4,the cells were pre-exposed to 5 ng/m L of TM for 1 hour,with or without 150 ?M of 4-PBA.Subsequently,10 ng/m L of LPS was added for 24 hours.Microglial activation,proinflammatory factor production,and the extent of ERS were then assessed.Results: Low doses of TM(0.3 and 3 ?g)resulted in a mild ER-stress response without inducing tissue toxicity in the hippocampus of rats.LPS induced M1 microglial activation and inflammatory factor production,which initiated the brain inflammatory response and cellular apoptosis.Site-directed pre-injection of the “ERS activator” TM(3 ?g)inhibited this effect,decreasing inflammatory cytokine levels,M1 microglial activation 24 hours after treatment.Our in vitro findings also showed that lower concentrations of TM(0.5 and 5 ng/m L)triggered a benign,mild ER-stress response in primary cultured microglia,without cytotoxicity.Pre-exposure to the non-lethal dose(5 ng/m L)of TM in primary cultured microglia conferred significant protection against LPS-induced proinflammatory cytokine production and M1/2b polarization.However,sodium 4-phenylbutyrate(4-PBA),a compound that inhibits ERS,ablated the TM-induced protective effect,both in vivo and in vitro.Conclusions:(1)Low doses of TM activated a benign,moderate ERS response in the hippocampus of rats,without cellular apoptosis.(2)Low doses of TM caused non-toxic,mild ERS in primary cultured microglia.(3)Mild ERS attenuated LPS-induced neuroinflammation and shifted the primary microglial population from M1/2b to M2 a.Part II: The effects of Mild Endoplasmic Reticulum Stress on Astrocytes and its Role in Neuroinflammation Objective: In the present study,we investigated the effect of mild ERS on astrocytes in LPS-induced neuroinflammation.Methods: Male Sprague–Dawley rats received i.c.v.injections of tunicamycin(TM;3 ?g),with or without intraperitoneal 4-phenylbutyrate(4-PBA;100 mg/kg),before LPS administration(500 ?g/kg).Astrocytic activation,proinflammatory factor secretion,and ERS were measured for 24 hours after treatment.In addition,the effect of mild ERS on astrocytes was determined in vitro.Specifically,primary cultured astrocytes were incubated with different concentrations of TM(0.1,1,and 10 ng/m L),and the extent of ERS was then assessed after 24 hours.In another experiment,the astrocytes were exposed to TM(1 ng/m L),which activates ERS,with or without the ER-stress inhibitor 4-PBA(100 ?M)before LPS treatment(100 ng/m L).Astrocytic activation,proinflammatory factor production,and the extent of ERS were assessed after 24 hours.Results: Our data showed that TM(3 ?g)attenuated LPS-induced astrocytic overactivation in the hippocampus of rats.However,4-PBA diminished this TM-mediated inhibition of astrocytic activation.Our in vitro results showed that lower doses(0.1 and 1 ng/m L)of TM caused a non-cytotoxic,mild ER-stress response in primary cultured astrocytes.Preconditioning with TM(1 ng/m L)ameliorated LPS-induced overactivation and the inflammatory response in astrocytes.Importantly,4-PBA partially blocked this TM-mediated inhibition of astrocytic activation and anti-inflammatory effect.Conclusion:(1)Low concentrations of TM generated non-cytotoxic,mild ERS in primary astrocytes.(2)Mild ERS attenuated LPS-induced astrocytic cytokine production and overactivation.Part III: The Effects of Mild Endoplasmic Reticulum Stress on Blood-Brain Barrier and its Role in Neuroinflammation Objective: In the current study,we aimed to explore whether mild ERS exerted a protective effect against LPS-induced BBB hyperpermeability.Methods: Male Sprague–Dawley rats received intracerebroventricular injections of tunicamycin(TM;3 ?g),with or without intraperitoneal 4-phenylbutyrate(4-PBA;100 mg/kg)before LPS administration(500 ?g/kg).BBB permeability was measured for 24 hours after treatment.Results: LPS significantly increased albumin levels and Evans blue absorbance in the hippocampus of rats,both of which were apparently attenuated by TM treatment.Furthermore,TM negated LPS-induced increases in hippocampal occludin and claudin-5 expression,while LPS significantly increased matrix metalloproteinase(MMP)-2 and MMP-9 expression,which can be partly compromised by pretreatment with TM.Importantly,combined administration of 4-PBA almost completely blocked the TM-mediated protective effect on the BBB.Conclusion: Our results suggest that mild ERS may alleviate LPS-induced BBB disruption.