The Study Of Interleukin-1β Overload And "Double Edge" Effect Instability In Alzheimer’s Disease

Research background:Alzheimer’s disease(AD)is caused by β-amyloid protein(Aβ)excessive deposition,neurofibrillary tangles,extensive neuronal damage,loss and progressive decline of cognitive,memory and behavioral abilities are the main characteristics of a chronic neurodegenerative disease often occurring in the elderly.AD has been reported for more than 100 years.Now,wo don’t know the reason about the AD completely,but some research find CNS may be a impotant reason about the AD.In order to explore the effect of core effector of central inflammation-interleukin-1β(IL-1β)will help to clarify the inflammatory pathology of AD and provide basic theoretical support for clinical intervention treatment of AD.IL-1β is an effector molecule at the core of AD inflammation,which promotes inflammation but also reduces the Aβ deposition of the brain by research report on,and the research group find that the early existence of suitable IL-1β is conducive to improving the repair of injured neurons.We put forward,with the development of AD lesions,IL-1β shows the "double-edged" effect of neuronal protection and injury associated with concentration and time differences.Specifically,low concentrations of IL-1β in the early physiological or pathological stage is conducive to the adaptation of neuron microenvironment and improve the survival of neurons;long term pathological factors(e.g.Aβ insufficient clearance and excessive deposition)induces ultra-high IL-1β accumulation(IL-1β overload),mediated the effect of IL-1β changes from neuroprotection to nerve injury(this paper is referred to the instability of "double edge" effect of IL-1β),and promotes the development of AD lesions.For the assumption of the subject,we need to clarify:①Why is it abnormally high of central IL-1β?What is the cause of overload of central IL-1β?②Why is it shows the "double-edged" effect of central IL-1β?What is the molecular basis of the"double-edged”effect of IL-1β?The study confirmed that the expression of silent information regulator typel/sirtuinl(SIRT1)decreased significantly under the AD pathological condition,while NLRP3,the key signal of shear maturation of IL-1β was significantly activated;it is suggested that SIRT1-NLRP3 is negatively correlated and may be involved in the generation regulation under the AD inflammatory pathology.Studies have also shown that both mature brain-derived neurotrophic factor(mBDNF)and precursor BDNF(proBDNF)perform independent biological functions.However,the role of proBDNF is completely opposite to that of mBDNF.The mBDNF improves neuronal survival,while proBDNF induces neuronal injury.Combined with the experimental report of research group about IL-1β participate in the regulation of BDNF expression.We further speculate that under physiological conditions,SIRT1 is highly expressed,inhibits the overexpression of NLRP3 inflammatory complex and limits microglia IL-1β shear activation to maintain low IL-1β level in the brain,alleviate the expression of proBDNF,maintain the balance of central mBDNF/proBDNF signal,and show neuroprotective effect;and under pathological conditions such as excessive Aβdeposition,the expression of SIRT1 is down-regulated,the inhibition of NLRP3 inflammatory complex is weakened,and the over activation of NLRP3 inflammatory complex leads to the maturation of microglia IL-1β increased release(IL-1βoverload),promote the expression of proBDNF,mBDNF/proBDNF signal imbalance,and show the effect of nerve injury.In order to verify the above hypothesis,this study carried out two parts of experimental work,from SIRT1 signal regulation and IL-1β association,to explore the relationship between IL-1β and BDNF,and to explore the central IL-1β related to AD pathology the molecular involvement mechanism of overload was analyzed by IL-1β“double edge”effect and the execution and transmission of BDNF signal change,so as to verify that“SIRT1-NLRP3 signal drives IL-1β overload,linkage,mBDNF/proBDNF imbalance,mediated the“double-edged”unstable effect of IL-1β and promotes the project hypothesis of AD pathology”.Part I:Experimental study of SIRT1-NLRP3 signal and the overload of IL-1βResearch methods:BV2 microglia and Aβ with incubation for different times,SIRT1 overexpression plasmid was constructed and transfected into cells,which were divided into six groups.①blank control group;②transfection reagent control group;③SIRT1 overexpression plasmid control group;④Aβ experimental group;⑤Aβ+transfection reagent experimental group;⑥Aβ+SIRT1 overexpression plasmid experimental group.After 48h of Aβ treatment,cells and culture supernatants were collected,and NLRP3 inflammatory complex(NLRP3,ASC,caspase-1),SIRT1 and upstream regulatory signal AMPK,M1 and M2 microglia activation markers CCL3 and Argl were detected by Western Blot;the content of IL-1β in cell culture supernatant was determined by ELISA;the activation markers CCL3 and Argl of M1 and M2 microglia were detected again by cellular immunofluorescence.Experimental results:1.Aβ correlation analysis with SIRT1-NLRP3 signalCompared with the control group,in Aβ group,the expressions of NLRP3,Caspase1 and ASC were significantly up-regulated,while the expression of SIRT1 was significantly down regulated;However,the high expression of SIRT1 plasmid transfection was significantly resistant to the expression changes of NLRP3 induced by Aβ,suggest that SIRT1 signal negatively regulates the activation of AD related NLRP3 signal,showing a potential release effect of reduction of microglia IL-1β;2.SIRT1 signal and microglia activationCompared with the control group,in Aβ group,Arg1’s expression down stream,at the same time,the number of Argl positive cells became little;the expression of CCL2 protein increased,and the number of CCL3 positive cells also increased;However,SIRT1 high expression plasmid transfection significantly reversed the above changes induced by Aβ,suggesting that SIRT1 signal interferes with microglia activation and may reduce the release of IL-1β by reducing microglia M1 activation and reduce IL-1β overload;3.SIRT1 signal and IL-1β releaseCompared with the control group,in Aβ group,the content cell IL-1β increased significantly;SIRT1 high expression plasmid transfection significantly reduced IL-1βrelease induced by Aβ,suggesting that SIRT1 alleviates AD related IL-1β overload;4.Aβ intervention SIRT1 signal pathway analysisCompared with the control group,in Aβ group,while the expression of SIRT1 was down regulated,the expression of phosphorylated AMPK decreased significantly,suggesting that the intervention of AMPK activation may be Aβ signal participation pathways that limit SIRT1 expression.Conclusion:1.SIRT1 signal inhibits NLRP3 activation and reduces AD related central IL-1βoverload;2.SIRT1 signal regulates the activation and differentiation of microglia and reduces the activation of AD related M1 microglia,which may be the cellular basis of central IL-1β overload;3.Inhibition of AMPK signal phosphorylation may be due to the loss of SIRT1 expression and expression in AD pathology,upstream participation mechanism of IL-1β overload;Part II:mBDNF/proBDNF signal balance and experimental study on the"double-edged " effect of neuron protection/injury of IL-1βResearch methods:HT22 mouse hippocampal neurons and IL-1β with different concentration/time incubation,divided into seven groups.①blank control group:②0.02ng/ml IL-1βexperimental group;0.05ng/ml IL-1β experimental group;④0.1ng/ml IL-1βexperimental group;⑤1ng/ml IL-1β experimental group;⑥1Ong/ml IL-1βexperimental group;⑦20ng/ml IL-1β experimental group.The seven groups were treated for 6h,12h,24h,48h,72h and 96h respectively.Cells were collected at each time point;we decide to use the way of CCK8 and western blot,then testing mBDNF,proBDNF and their receptors include p75NTR,the proBDNF shear maturation related factors of Furin and PC 1/3;Real-time PCR was used to detect BDNF related signal pathways ERK,MEK and CREB.Experimental results:1.The analysis of“double edge”effect of IL-1βCCK8 experimental results showed that compared with the control group,the low concentration of IL-1β in the 12-72 hours,it showed obvious cell proliferation promoting effect;with the extension of action time,high concentration of IL-1β cell injury effect gradually appeared.At the action time points of 48 and 72 hours,the high concentration of IL-1β both induced significant cell damage,suggesting IL-1β have a“double-edged”effect oneuronal protection/injury associated with the action of concentration and time;2.IL-1β correlation analysis between“double edge”effect and mBDNF/proBDNF signalWe employ the way of Western Blot and the consequence reveal the IL-1β’s time and content enlargement,the mBDNF to cut loss,while proBDNF has raised a lot;in addition,with high concentration of IL-1β,the receptor of mBDNF to cut loss,at the same time,the receptor of proBDNF has raised a lot,suggesting that IL-1β may induce the imbalance of mBDNF/proBDNF ratio,interfere with the execution of BDNF signaling,and participate in the concentration related IL-1β neuroprotection-the conversion of“double-edged”effect of injury;3.IL-1β and BDNF signal regulation analysisWe employ the way of Western Blot and the consequence reveal the high proportion IL-1β,the production of shear mature proteins furin and PC 1/3 of proBDNF decreased significantly;We employ the way of real-time PCR,and we find that compared with the blank group,high concentration of IL-1β restrain the expression of mRNA of MEK,ERK,CREB,the low concentration of IL-1β promote the expression of BDNF related signal molecules,such as MEK,ERK,but have no obvious effect on the expression of CREB;suggesting the IL-1β may be interfere with the BDNF expression,regulate BDNF shear mature,influence signal balance of mBDNF/proBDNF and finally participate in the production of“double-edged”effect.Conclusion:1.Correlation with action concentration and time,IL-1β has the "double-edged"effect of neuroprotection/injury;2.The“double-edged”effect of neuroprotection/injury of IL-1β may be mediated by the imbalance of differential expression ratio of mBDNF/proBDNF signals;3.The regulation of BDNF matures maybe a key pathway in the balance of mBDNF/proBDNF signals mediated by IL-1β.