| Alzheimer's disease(AD)is the most prevalent neurodegenerative disease in elderly populations,and it is characterized by progressive decline of memory,cognitive function disorder and personality change.In the contemporary society of population aging,AD has become the fourth killer after cardiovascular and cerebrovascular diseases,tumor and stroke,threatening the health of the elderly,reducing the life quality,and increasing the burden on the family and society.However,the pathogenic causes of AD are complex and diverse,and the exact pathogenesis remains unknown.In recent years,a large number of studies have shown that neuroinflammation is one of the major pathogenic causes of AD.Several pieces of evidence indicate the local inflammation in AD,including the increase of activated microglial cells,the presence of elevated levels of proinflammatory cytokines.As an initiation of neuroinflammation,microglia activation currently is one of the hot topics in the study of the nervous system degenerative disease.Modulation of microglia immune function in Alzheimer's disease might open new therapeutic avenues.Activated microglia exerts typical functions including rapid proliferation,phagocytosis,antigen presentation and secretion of proinflammatory cytokines and chemokines to kill and phagocytose foreign pathogens as well as to remove injured cells and debris.However,an uncontrolled inflammatory reaction is a driving force of the chronic progression of neurodegenerative disease.Therefore,strict regulation of MG immune response is the key to prevent neuroinflammation.Acetylcholine(ACh),a key neurotransmitter involved with learning,memory and congnition,binds to two distinct receptor subtypes in the brain: nicotinic(nAChR)and muscarinic(mAChR).In the recent years,studies have indicated a role for nAChRs in modulate microglial-mediated neuronflammation.It is generally accepted that nAChRs are functional expression on microglia,and that the activation of nAChRs generally inhibits the inflammatory response of microglial activation.However,the functional expression of mAChRs of microglia and the possiblemechanism controlling pro or anti-inflammatory microglial response remain to be clarified.Trihexyphenidyl hydrochloride is the most commonly used mAChRs non selective antagonist for the treatment of PD.We previously reported that long-term trihexyphenidyl exposure in SD rats significantly increased microglia proliferation and activation in hippocampal and cerebral cortex.Immunofluorescence assays revealed significant upregulation of M1 muscarinic receptor expression co-localized with CD68 on rat microglia.Our findings suggest that M receptor directly regulates the immunoinflammatory response of microglia.In this study,we firstly confirmed the expression of M receptor in MG,and observed the regulation of M receptor in MG immune response.This study was conducted to explore the relationship between M receptor and neuronflammation.Moreover,the new role of M receptor in the development of AD is opend up to find new target for the treatment of AD.Objective In the current study,we firstly established the primary culture method of rat cortical microglia.Then,we explored the expression of muscarinic acetylcholine receptors on primary microglia cells.Furthermore,to study the involvement of muscarinic acetylcholine receptor in regulation of immune response,the effect of the mAChRs non-selective agonists oxotremorine(OX),antagonists Atropine(Atr),selective M1 receptor inhibitor Muscarinic Toxin 7(MT7)on microglia activation and microglia functions was studied,including antigen processing and presentation and neuroinflammation response Methods(1)Primary microglia cell of rat cortical culture: primary microglia was derived from the brain of postnatal 24 hours SD rat.After culture for 8-9 days,mixed glial cellshave layered obviously,the number of amebic like microglia increased and distributed in the upper layer;combined with mild trypsin digestion method and content temperature shock method to isolate microglia;Iba1 immunofluorescence staining to identify the purity of microglia;(2)Expression characterization of m AChRs on primary microglia enriched cultures:RT-PCR,laser confocal microscopy and western-blot were used to confirm the expression of acetylcholine M receptor in microglia;(3)The effect of mAChRs on microglia antigen processing and presentation fuction:OX(10-5M)treatment of microglia for 72 h,laser confocal microscopy and flow cytometry to detect MHC? expression in microglia;the expression of ?2m,Tap1 andRt1-Aw2 mRNA were detected by quantitative real-time PCR;(4)To identify potential muscarinic receptor-associated changes in MHCI pathway expression: laser confocal microscopy,western-blot and quantitative real-time PCR were used to detect the effect of OX on the expression of M1 receptor and MHCI in microglia;(5)The effect of mAChRs on microglia activation: laser confocal microscopy and western-blot were used to detect the expression of CD68 and CD11 b after OX or M1 receptor blocker MT7 treatment;(6)The effect of m AChRs on secretion of proinflammatory cytokines: the secretion of IL-1?,TNF-? and IL-10 in microglia were detected by luminex technique at 24 h,48h and 72 h after treatment with OX(10-6M,10-4M).Results(1)Modified primary culture of microglia was established successively,and the rate of Iba1 positive cells was about 95%;the rule of microglia growth was observed;mixed microglia was amebic like,microglia isolation and culture three days,microglia was basically branched with single or multi-stage protrusions.(2)Five subtypes mRNA of mAChRs expressed in microglia by RT-PCR analysis;laser confocal microscopy and western-blot showed that the M1 receptor expression and distribution in the microglia cell membrane and cytoplasm.(3)Mcroglia co-incubation with nonselective muscarinic agonist oxotremorine(OX,10-5M)for 72 hours,the expression of MHC? was significantly up-regulated in laser confocal microscopy(P<0.001).(4)Flow cytometry results showed that the positive rate of microglia co-labeled MHC? and ?2m was 8.46±0.47% in normal group,OX group was 51.80±1.48%.Compared with the normal group,the positive rate of OX group significantly increased five times(P<0.001),indicating that OX promotes the expression of MHC?in microglia membrane.(5)OX increased mRNA expression of genes related antigen-presenting ?2m,Tap1 and Rt1-Aw2 in quantitative real-time PCR analysis(P<0.001).(6)Compared with the normal group,OX(10-5M,40min)up-regulated M1 receptor and MHC? expression(P<0.001);M1 receptor selective antagonist MT7(10-8M,5min)partially blocked OX-induced M1 receptor and MHC? expression(P<0.001).The results of laser confocal microscopy indicated that M1 expression was consistent withMHC? and they were co-located.(7)Laser confocal microscopy and western-blot results showed that the M1 receptor expression is up-regulated by OX(10-5M,72h)(P<0.01).The mRNA expression of M1 was decreased by OX(20min),but it is increased after OX(40min,72h)treatment.(8)Up-regulation of CD68 and CD11b(P<0.001)induced by OX(10-5M,72h)further confirmed that mAChRs was directly regulated microglial activation.Compared with OX group,MT7(10-8M,5min)partially blocked OX-induced CD68 and CD11 b expression(P<0.001),suggesting that microglial activation is related to M1 recptor;(9)Microglia and OX(10-4M,10-6M)co-incubation for 24 h can inhibit the secretion of anti-inflammatory factor IL-10(P<0.01)and pro-inflammatory TNF-?(P<0.01),however,OX had no significant effect on IL-1?.Conclusion(1)A modified method for primary microglia culture was established successively.(2)Five subtypes mRNA of mAChRs expressed in rat cortical microglia,and M1 recptor expressed in cell membrane and cytoplasm.(3)Activation of M receptors by OX promoted microglial antigen presentation,microglia activation and regulated the release of inflammatory mediators.(4)The blocking of the M1 receptors inhibited OX-induced microglia activation and MHC? expression,indicating the functional expression of M1 receptor,and M1 receptor may play a major role in regulation of immune response in microglia. |
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