A Role And The Mechanism Of Activated Microgial α₇-nAChR In Attenuating Inflammatory Factor Secretion Inducd By Aβ

With the aging of the word's population,the number of people who have Alzheimer's diseases(AD) that a neurodegenerative disease closely associated with old,rise sharply.Because of the etiology and pathogenesis of AD is not clear, so how to treatment is difficult,as yet there is no cure method.AD involves a progressive disruption of cognition including memory and learing,behavior and social life.its main brain pathology characterizes including:the major constituent of the amuloid plaques(extracellular) is a 42 amion-acidβ-amyloid peptide (Aβ_1-42);whereas the angles(intraneuronal) are composed of modified forms of the microtubule-associated proein,tau;and also by extensive neuronal loss.It has been known that the development and pathogenesis of AD are associated with cerebral chronic inflammation caused by genetic and environmental factors and progressive degeneration of cholinergic system,but about its specific etiopathogenesis is little known.As a result,it is still no effective methods to treatment of AD.AD is a very serious public health care problem that will become of increasing concern in the future,consequently,researchers have devoted an increasing amount of effort to improving our understanding of the relationship between AD and Aβ.The development of AD has been known to be related to central inflammation which is a response secondary to deposition of Aβand also a major factor for the progressive degeneration of neuron.The cells involved in the inflammation are astrocytes,microgliocytes and oligodendrocytes which,once stimulated,can release a variety of cytokines,chemokines,alexins and their activators.The subsequent non-specific inflammatory cell infiltration can lead to chronic inflammation and harm the nervous system.Deposition of Aβnot only produces a direct toxic effect on nerve cells,but also activates gliacytes and oligodendrocytes in the nervous system to release inflammatory neurogens which thus trigger the signal pathway to damage to neurons.It has been found that increase in cholinergic level and nicotinic acetylcholine receptor agonist in the brain may have a therapeutic effect on AD,though the functional mechanism is not clear yet.There has been evidence that the cholinergic neurotransmitters released after peripheral vagus nerve being stimulated can interact withα₇-nAChR receptors in macrophages to inhibit the synthesis and secretion of inflammatory mediators of macrophages so that the peripheral inflammation is weakened.Although there are no peripheral macrophages in the central nervous system,there are microglials with similar function of peripheral macrophages,are involved in the central inflammation induced by Aβ. Consequently,it is natural for us to ask whether microglails containα₇-nAChR or activiation ofα₇-nAChR in microglails may lead to reduced central inflammation induced by Aβ.At present study,is lillte report on how activiation ofα₇-nAChR in microglails may lead to reduced central inflammation induced by Aβ.but great number of inflammation mechanism research demonstrated that Mitogen-activated protein kinase(MAPK) play an important role in the pathogenesis and development of inflammation.Four subfamilies of MAP kinases,i.e.ERK,JNK/SAPK,p38/ RKand ERK5/BMK1,have been identified and cloned in mammaliamcells.These MAP kinases are activated by many proinflammatory stimuli and play an important role in the pathogenesis and development of inflammation,hence we suppose that activiation ofα₇-nAChR in microglails lead to reduced central inflammation induced by Aβweather involve inhibitor MAKP pathway.Based on those researchs,the present study,was designed to conduct the following research:first of all investigate the expression ofα₇-nAChR in the hippocampal microglails in rats,secondly to observe the expression and secretion of Aβ-induced inflammatory mediators from hippocampal microglails,and to explore the effect of activatedα₇-nAChR in hippocampal microglails on Aβ-induced inflammation.Use specific inhibitors for MAPKS,study MAPK pathway on reduce inflammtion Aβ-induced.At last to investigate activation microglia's alpha7 neuromal acetylcholine receptor(α₇-nAChR) whether reduce neuron toxicity.Methods1.Culture of microglia cellSterilized newborn SD rats were used to harvest their cerebrum promptly and aseptically.The hippocampus was resected and digested with trypsin for centrifugalization.The cell suspension was prepared after removal of the supernatant and inoculated into cell culture flasks for culture.After mixed cultivation of the cells for 7-9 days,use lidocaine solution puritied microglia cells,collected for further culture.The microglia cell was detected through the antibody CD11b/c specific to microglia,in order to assess the purity coefficients of microglails.Hoechst33258 used to nucleus staying2.Identification expression ofα₇-nAChR in microglia cell from RNA and protein levelTotal RNA was prepared from rat microglia using Trizol reagent according to manufacturer's protocol.To generate DNA frangment by RT-PCR methods,α₇-nAChR primer:For 5'CAT TGA CGT TCG CTG GTT CC-3' Rev 5'ATG GTG CTG GCG AAG TAT TG -3'.PCR products were analyzed by electrophoresis, stained with ethidium bromide and photographed.Double staining of immunofluorescence was used to identify microglia with-CD11b/c antibodies, and to observe the expression ofα₇-nAChR in microglails.The expression ofα₇-nAChR in miroglia cell in vitro was assessed and analyzed in cultured microglia. using immunofluorescence double labeling.3.Secretion of inflammatory mediators effected by activation ofα₇-nAChR in microglia cellsMicroglia cells were isolated and cultured.The supernatant fluids of cultured microglia cell with and withoutα₇-nAChR activated were collected separately.The liquid phase chip technology was employed to investigate the changes of IL-6, TNF-α,MIP-1,MCP-1αand RANTES in the supernatant to determine the role ofα₇-nAChR in microglia in reducing the inflammation caused by Aβ.4.Effect of MAPK pathway on inflammationUse specific inhibitors for P38 and ERK,study MAPK pathway whether involved in reduce inflammtion Aβ-induced.Collecting the supernatant fluids of cultured microglia cells after added P38 and ERK inhibtors 96h separately.The changes of IL-6,TNF-α,MIP-1,MCP-1αand RANTES determined by liquid phase chip technology.5.study of activatedα₇-nAChR on weaken neuron toxicityThe neuron cells were identified by DAB immunocytochemistry and immunofluorescence,in order to assess the purity coefficients of neuron Hoechst 33258 used to nucleus staying after immunostaining with MAP-2 on the tenth day.the tenth day neurons coculture with microglai cells in transwells.The cells randomly divided into four groups:blank control;Aβprotein group;Coculture group; Nicotine pretreated group.Neurons apoptosis model was established by added Abeta(1-42)protein,and the apoptosis rates was analysed by Flow CytoMeter(FCM) and Fluorescence microscopy.Results1.Microglai cell Culture and identificationThe original generation of mixed-cultured cells mainly astrocytes and contains microgial cells,neurons and a small number of oligodendrocytes.After cultured neurons disappear.Lidocaine solution used to purification of microglia cells.Observed under the microscope:at normal microglail cell in resting state,a small cell body,neurite slender,branched.Using immunofluorescence methods labeling by the probe of specific anti-CD11b/c monoclonal antibodies to identification microglia cells ware positive.2.α₇-nAChR expression in microglia cellsTotal RNA extracted from the purified miroglia cells,and using RT-PCR method amplify 450bp band.The PCR product was detected with DNA sequencing, compaired to gene bank indicated thatα₇-nAChR amplified.In addition, immunofluorescence double labeling showed that they were also positive for anti-CD11b/c and anti-α₇-nAChR stainings.3.Relationship between Aβand secretion of inflammatory factors in microgliaAdding 10μmol/L Aβin cultured microglial cells,after 4h,8h,24h,48h,72h, 96h collected cell supernatant separately.Using Liquid chip technology Detect changes of IL-6,TNF-αand MIP-1,MCP-1,RANTES.The result of analysis show that IL-6,MIP-1α,MCP-1,and RANTES change significantly(p＜0.05),with time increase,inflammatory cytokines and chemokines also increased.4.Effect of activatedα₇-nAChR on secretion of inflammatory factors induced by Aβmicroglia cells pre-activated by 10μmol/L nicotine,activatedα₇-nAChR in microglia cells,then adding 10μmol/L Aβin cultured microglia cells,after 96h collected cell supernatant separately.Using Liquid chip technology detect changes of IL-6,MIP-1,MCP-1 and RANTES.The result of analysis shows that activatedα₇-nAChR could significantly reduce the Aβ-induced secretion of inflammatory mediators from microglail cells.The differences before and after activation ofα₇-nAChR were statistically significant.5.Effect of MAPK pathway inhibit by specific inhibitors on inflammationUse specific inhibitors for P38 and ERK,the result of analysis shows that could significantly reduce the Aβ-induced secretion of inflammatory mediators from microglail cells.The changes of IL-6,TNF-α,MIP-1,MCP-1αand RANTES determined by liquid phase chip technology.6.Activation ofα₇-nAChR on weaken neuronal toxicityPrimary cultured neurons,identification by immunohistochemical with neuron specific NSE antibody staining,result show neuronal cytoplasm stained brown.In order to assess the purity coefficients of neuron by Immunofluorescence assay with MAP-2 staining and Hoechst33258 nucleus staying,reached 94.49%±1.22%. neuromal apoptosis rates detected by TUNNEL apoptosis rates reached 13.28%±2.93%,18.65%±2.06%,30.40%±1.14%,20.60%±1.34%;detected by flow cytometry are 16.60%±1.63%,30.90%±4.27%,47.86%±6.70%,28.73%±2.80%. Two methods showed that the rate of apoptosis induced by Aβ_1-42 protein compared the co-culture group with the Aβgroup difference was significant,(P＜0.05); compared nicotine pre-treatment group with coculture group the difference was significant too(P＜0.05).Conclusions 1.Microglia cells can be expressedα₇-nAChR normally in vitro.2.Aβeffect on microglia cells inflammatory cytokines and chemokines IL-6, MCP-1,MIP-1,RANTES secretions increased by time going.Theα₇-nAChR in microglia cells activated can weaken inflammatory cytokines and chemokines such as IL-6,MCP-1,MIP-1,RANTES secretions induced by Aβ.3.P38 and ERK pathway involved in microglia cells secretion cytokine induced by Aβ-mediated,as well as involved in activation ofα₇-nAChR weaken secretion of imflammtory factors induced by Aβ.4.Aβprotein have toxicity on neurons through effect on microglia cells release inflammatory mediators,activated microglia cellsα₇-nAChR can reduce inflammatory mediators release,play neuron protective role.