本文關(guān)鍵詞：大麻素Ⅱ型受體激動劑改善阿爾茨海默病樣小鼠學(xué)習(xí)記憶能力的神經(jīng)炎癥機(jī)制研究，由筆耕文化傳播整理發(fā)布。

        目的：以大麻素II型受體（cannabinoid2receptor, CB2R）激動劑為研究工具，研究CB2R對β-淀粉樣蛋白（β-amyloid,Aβ）誘導(dǎo)的小鼠學(xué)習(xí)記憶能力損傷的改善作用及其調(diào)節(jié)小膠質(zhì)細(xì)胞功能表型轉(zhuǎn)化的神經(jīng)源性炎癥作用機(jī)制。為確立CB2R作為新的抗神經(jīng)源性炎癥候選藥物靶標(biāo)提供進(jìn)一步的實驗證據(jù)，為治療阿爾茨海默病提供新的藥物治療策略。方法：1CB2R激動劑對AD模型小鼠空間及非空間學(xué)習(xí)記憶能力的影響根據(jù)自發(fā)活動的研究數(shù)據(jù)將10周齡雄性C57/Bl6J小鼠隨機(jī)分為對照組，AD模型組，，CB2R激動劑JWH-015干預(yù)組和JWH-015單藥組。對照組和JWH-015單藥組側(cè)腦室一次性注射生理鹽水8μl。AD模型組和JWH-015干預(yù)組動物側(cè)腦室一次性注射寡聚態(tài)Aβ1-424μg（8μl），以建立AD模型。造模后第2天開始，JWH-015干預(yù)組和JWH-015單藥組小鼠腹腔注射JWH-015（0.5mg/kg/天），對照組和AD模型組小鼠每天腹腔注射等體積含0.1%DMSO生理鹽水，連續(xù)給藥3周。采用Morris水迷宮實驗及新物體識別實驗觀察CB2R激動劑對AD模型小鼠空間及非空間學(xué)習(xí)記憶能力的改善作用。2CB2R激動劑對AD模型小鼠腦內(nèi)膠質(zhì)細(xì)胞功能形態(tài)及數(shù)量的影響通過組織免疫熒光技術(shù)觀察上述不同組別實驗動物腦內(nèi)小膠質(zhì)細(xì)胞標(biāo)志物（CD11b）和星形膠質(zhì)細(xì)胞標(biāo)志物（GFAP）的表達(dá)情況和CB2R激動劑對上述改變的影響。3CB2R激動劑對Aβ誘導(dǎo)的小膠質(zhì)細(xì)胞功能和激活表型的影響采用實時定量PCR技術(shù)，觀察海馬、紋狀體、前額葉等腦區(qū)的CB2R，M1型激活小膠質(zhì)細(xì)胞標(biāo)志物TNF-α、iNOS、IL-6及M2型激活小膠質(zhì)細(xì)胞標(biāo)志物Ym1/2mRNA的表達(dá)情況。4CB2R對LPS誘導(dǎo)的小膠質(zhì)細(xì)胞功能表型的調(diào)節(jié)機(jī)制通過Western Blot的方法觀察CB2R對LPS誘導(dǎo)的激活小膠質(zhì)細(xì)胞內(nèi)絲裂原活化蛋白激酶信號通路ERK、JNK及p38MAPK磷酸化水平的影響，探討CB2R參與小膠質(zhì)細(xì)胞激活功能表型調(diào)節(jié)的信號傳導(dǎo)機(jī)制。結(jié)果：1CB2R激動劑改善AD模型小鼠空間和非空間學(xué)習(xí)記憶能力Morris水迷宮實驗結(jié)果顯示，在定位航行實驗中，AD模型組小鼠逃避潛伏期較對照組小鼠顯著延長（P <0.01）；在空間探索實驗中，與對照組相比，AD模型組小鼠1分鐘穿越平臺次數(shù)顯著減少（P <0.05）；新物體識別實驗結(jié)果顯示，與對照組相比，AD模型組小鼠新物體識別指數(shù)顯著下降（P <0.05），提示AD模型建立成功。與AD模型組小鼠相比，JWH-015干預(yù)組小鼠逃避潛伏期顯著縮短（P <0.05），1分鐘穿越平臺次數(shù)顯著增加（P <0.05）；JWH-015干預(yù)組小鼠新物體識別指數(shù)較AD模型組小鼠相比顯著增加（P <0.05），提示CB2R激動劑能夠改善AD模型小鼠空間及非空間學(xué)習(xí)記憶能力；JWH-015單藥組小鼠與對照組相比，逃避潛伏期、1分鐘穿越平臺次數(shù)及新物體識別指數(shù)均無顯著變化（P>0.05）；此外，各組小鼠體重及自發(fā)活動未見組間差異，提示JWH-015不影響正常小鼠體重及精神活動。2CB2R激動劑抑制腦內(nèi)膠質(zhì)細(xì)胞激活數(shù)量通過免疫組化的方法檢測不同處理組小鼠紋狀體腦區(qū)小膠質(zhì)細(xì)胞和海馬腦區(qū)星型膠質(zhì)細(xì)胞的激活與表達(dá)情況。與對照組相比，AD模型組小鼠紋狀體腦區(qū)小膠質(zhì)細(xì)胞標(biāo)記物CD11b表達(dá)明顯增高，形態(tài)表現(xiàn)為分枝狀（激活狀態(tài)）；JWH-015干預(yù)組小鼠紋狀體腦區(qū)CD11b的表達(dá)與AD模型組相比明顯下降；JWH-015單藥組較對照組未見明顯差異。AD模型組小鼠海馬腦區(qū)星型膠質(zhì)細(xì)胞標(biāo)志物GFAP較對照組表達(dá)顯著增高；JWH-015干預(yù)組小鼠GFAP的表達(dá)水平較AD模型組有減少的趨勢；JWH-015單藥組與對照組相比沒有明顯差異。3CB2R調(diào)節(jié)腦內(nèi)Aβ誘導(dǎo)的小膠質(zhì)細(xì)胞M1/M2激活表型的轉(zhuǎn)化3.1Aβ誘導(dǎo)腦內(nèi)不同腦區(qū)CB2R mRNA表達(dá)水平上調(diào)與對照組相比，AD模型組小鼠海馬、紋狀體和前額葉等腦區(qū)CB2RmRNA表達(dá)均顯著上調(diào)（海馬P <0.01，紋狀體和前額葉P <0.05）。與AD模型組相比，JWH-015干預(yù)組小鼠海馬、紋狀體和前額葉等腦區(qū)CB2RmRNA表達(dá)水平均顯著下調(diào)（海馬P <0.01，紋狀體和前額葉P <0.05）。3.2CB2R激動劑降低M1型激活小膠質(zhì)細(xì)胞標(biāo)記物mRNA表達(dá)水平在海馬腦區(qū)，與對照組小鼠相比，AD模型組小鼠iNOS mRNA表達(dá)水平顯著上調(diào)（P <0.01）。與AD模型組相比，CB2R激動劑干預(yù)組小鼠iNOSmRNA表達(dá)水平顯著下調(diào)（P <0.01）。模型組IL-6，TNF-α mRNA表達(dá)呈上調(diào)趨勢，JWH-015干預(yù)組IL-6，TNF-α mRNA表達(dá)呈下調(diào)趨勢。單藥組與對照組相比，其TNF-α、iNOS、IL-6mRNA表達(dá)未見明顯改變（P>0.05）。在紋狀體腦區(qū)，AD模型組小鼠腦內(nèi)iNOS，IL-6，TNF-α mRNA表達(dá)較對照組均顯著升高（P值分別為P <0.01、P <0.01和P <0.05）。與模型組相比，JWH-015干預(yù)組小鼠腦內(nèi)iNOS，IL-6，TNF-α mRNA的表達(dá)顯著下調(diào)（分別為P <0.01、P <0.01和P <0.05）。與對照組相比，單藥組TNF-α、iNOS、IL-6mRNA表達(dá)無顯著差異（P>0.05）。在前額葉腦區(qū)，與對照組相比，AD模型組小鼠腦內(nèi)iNOS，IL-6mRNA表達(dá)顯著上調(diào)（P值分別為P <0.01，P <0.05）。與AD模型組相比，JWH-015干預(yù)組小鼠腦內(nèi)iNOS，IL-6mRNA表達(dá)顯著下調(diào)（P均<0.01）。TNF-α mRNA表達(dá)各組間未見顯著改變（P>0.05）。上述實驗結(jié)果表明，CB2R激動劑JWH-015通過激活CB2R可下調(diào)AD模型小鼠腦內(nèi)M1型激活小膠質(zhì)細(xì)胞數(shù)量及其促炎癥功能。3.3CB2R激動劑上調(diào)M2型激活小膠質(zhì)細(xì)胞標(biāo)記物mRNA表達(dá)在海馬腦區(qū)，與對照組小鼠相比，AD模型組小鼠腦內(nèi)M2型激活小膠質(zhì)細(xì)胞標(biāo)記物Ym1/2mRNA表達(dá)顯著下調(diào)（P <0.05）。與AD模型組相比，JWH-015干預(yù)組小鼠腦內(nèi)Ym1/2mRNA表達(dá)水平顯著上調(diào)（P <0.05）。在紋狀體腦區(qū)，與對照組小鼠相比，AD模型組小鼠腦內(nèi)Ym1/2mRNA表達(dá)水平顯著下調(diào)（P <0.05），JWH-015干預(yù)組小鼠腦內(nèi)Ym1/2mRNA表達(dá)水平呈升高趨勢，統(tǒng)計學(xué)沒有顯著差異（P>0.05）。在前額葉腦區(qū)，AD模型組小鼠腦內(nèi)Ym1/2mRNA表達(dá)水平較對照組顯著下調(diào)（P <0.05）。與AD模型組相比，JWH-015干預(yù)組小鼠腦內(nèi)Ym1/2mRNA表達(dá)水平顯著上調(diào)（P <0.01）。JWH-015單藥處理組Ym1/2mRNA表達(dá)水平較對照組無顯著差異（P>0.05）。上述實驗結(jié)果表明，CB2R激動劑JWH-015可上調(diào)AD模型小鼠腦內(nèi)M2型激活小膠質(zhì)細(xì)胞表達(dá)及其抗炎作用。4CB2R激動劑顯著抑制LPS誘導(dǎo)的M1型BV-2小膠質(zhì)細(xì)胞ERK1/2和JNK磷酸化水平與對照組相比，LPS處理組BV-2小膠質(zhì)細(xì)胞ERK磷酸化水平顯著增高（P <0.01）；JWH-015低劑量處理組（10nM）與LPS處理組相比，ERK1/2磷酸化水平呈下降趨勢；JWH-015中劑量（100nM）及高劑量組（1μM） BV-2小膠質(zhì)細(xì)胞ERK1/2磷酸化水平顯著下調(diào)（P <0.01）；JWH-015中濃度（100nM）組對LPS誘導(dǎo)的BV-2小膠質(zhì)細(xì)胞ERK1/2磷酸化水平升高的抑制作用可被CB2R拮抗劑SR144528所顯著阻斷（P <0.05）。與對照組相比，LPS處理組BV-2小膠質(zhì)細(xì)胞內(nèi)JNK磷酸化水平顯著增高（P <0.05）； JWH-015能濃度依賴性地抑制LPS誘導(dǎo)BV-2小膠質(zhì)細(xì)胞的JNK磷酸化水平；JWH-015中濃度（100nM）組對LPS誘導(dǎo)的BV-2小膠質(zhì)細(xì)胞JNK磷酸化水平升高的抑制作用可部分被CB2R拮抗劑SR144528阻斷。實驗結(jié)果提示， CB2R激活后，下調(diào)ERK和JNK兩個信號分子的磷酸化水平，因此，阻斷MAPK信號通路過度激活可能是JWH-015抑制M1型激活小膠質(zhì)細(xì)胞表達(dá)和抑制其促炎作用的信號轉(zhuǎn)導(dǎo)機(jī)制之一。結(jié)論：選擇性CB2R激動劑JWH-015對Aβ誘導(dǎo)的AD模型小鼠學(xué)習(xí)記憶能力具有改善作用，其作用機(jī)制可能與其抑制MAPK信號通路過度活化，調(diào)節(jié)小膠質(zhì)細(xì)胞激活數(shù)量及功能表型，進(jìn)一步抑制不同腦區(qū)內(nèi)神經(jīng)炎癥反應(yīng)，減輕神經(jīng)元損傷相關(guān)。

    Objective:To investigate the ameliorative effect of CB2R on learning andmemory dysfunction induced by amyloid-β in C57/Bl6J mice and toclarify its important role involved in microglia phenotypic conversion inmodulating neuroinflammation in CNS using a selective CB2R agonist.This study will provide much credible data that might explain thepromising candidate drug target in treatment of neuroinflammation inAlzhermer’s diseases.Methods:1The effect of CB2R agonist on spatial and nonspatial learning andmemory abilities of AD model miceC57/Bl6J mice of10weeks of age were randomly assigned tocontrol group, Aβ1-42induced group, Aβ1-42induced mice treated withJWH-015, and JWH-015treated group based on locomotion activity. ADmodel group was created with single intraventricular injection of fibrillarAβ1-42（4μg,8μl）and control group was single intraventricular injectionof saline （8μl） accordingly. Aβ1-42induced group and control groupwere treated with or without JWH-015and1%DMSO. Aβ1-42inducedgroup and control group were intrapertoneal treatment with JWH-015according to0.5mg/kg/day for3weeks. Performance in the Morris watermaze and novel objects recognition test to observe the spatial andnonspatial learning and memory abilities of mice in each group.2The effect of CB2R agonist on morphology and amount of activatedmicroglia in brain in each group mice. To apply the immunohistochemistry to determine the effect of CB2Ragonist on CD11b and GFAP expression of microglia and astrocyte ofmice in brain in each group mice.3The effect of CB2R agonist on function and active phenotype ofmicroglia in brain.To detect the mRNA expression of CB2R, M1microglia phenotypemarkers TNF-α, iNOS, and IL-6, as well as M2microglia phenotypemarker Ym1/2in hippocampus, striatum and cortex in each groupthrough the Quantative Real-Time PCR.4The signal transduction mechanism of CB2R agonist in regulatingactivated M1phenotype of LPS-stimulated microglia.Using the Western blot assay to investigate the effect of CB2R agoniston phosphorylation levels of MAPKs（ERK1/2, JNK and p38MAPK）pathway in LPS-stimulated M1phenotype of microglia.Results:1CB2R agonist could improve the learning and memory ability of ADmodel miceResults from place navigation trail of Morris water maze indicatedthat the latency of AD model mice was significantly prolonged than thelatency of control mice （P <0.01）. Compared with the control group,the times across platform of AD model mice showed a significantdecrease trend （P <0.05）. Meanwhile the recognition index of ADmodel mice was significantly reduced compared with the control group.These results indicated that the AD model was successfully established.Moreover the CB2R agonist JWH-015could effectively counteractAβ-induced learning and memory ability impairment shown by thesignificant reduction in the latency and a significant increase in the timesacross platform of AD group as well as the significant improvement inrecognition index. In addition, we didn’t found any impact in body weightand locomotion activity of mice in each group（P>0.05）. These resultsdemonstrated that the CB2R agonist JWH-015is effective in ameliorate the cognition dysfunction induced by Aβ1-42.2SCB2R agonist reduced the amount of the activated microglia in brainCompared with the control group, the expression of CD11b wassignificantly increased in the striatum of AD model mice by detectingimmunofluorescence intensity. Meanwhile we found the morphology ofthe microglia is dendroid. JWH-015decreased the expression of CD11binduced by Aβ1-42. We also found a significant increase in the expressionof GFAP in the hippocampus of AD model mice and the lower expressionof GFAP in JWH-015intervention group. There was a similar expressionof CD11b and GFAP in single saline injected group treated withJWH-015compared with the control group.3CB2R is involved in modulating the M1/M2microglia phenotypeconversion induced by Aβ3.1Aβ induced an increase in mRNA expression of CB2R in differentbrain region.Compared with the control group, the mRNA expression of CB2R instriatum, hippocampus and cortex were significantly increased（P <0.01and P <0.05）. JWH-015could signifantly counteract the up-regulatedexpression of CB2R induced by Aβ1-42in striatum, hippocampus andcortex（P <0.01and P <0.05）. This means the therapeutic effect ofJWH-015on the recognition dysfunction of AD model mice is related tothe decreased expression of CB2R.3.2CB2R agonist reduced the mRNA expression levels of the M1microglia phenotype markers.In the hippocampus, the mRNA expression of iNOS was significantlyincreased in the AD model mice compared with the control mice. Yet theexpression of iNOS of AD model mice was markedly decreased byJWH-015（P <0.01）. Otherwise the expression of IL-6and TNF-αshowed an increased trend in AD model mice. The AD mice treated withJWH-015showed a reduced expression of these two cytokines. JWH-015did not alter the expression of these M1microglia phenotype markers observed in control mice（P>0.05）.In the striatum, a marked expression of iNOS，IL-6and TNF-α wasfound in AD model mice（P <0.01, P <0.01and P <0.05）. Comparedwith the control group, the expression of iNOS，IL-6and TNF-α wassignificantly decreased in JWH-015intervention group（P <0.01, P <0.01and P <0.05）. JWH-015did not affect the expression of these M1microglia phenotype markers in control mice（P>0.05）.In the cortex, we found a significant increase in the expression ofiNOS and IL-6compared with the control group （P <0.01, P <0.05）.However, the expression of these two cytokine was dramatically reducedin the JWH-015intervention group（P <0.01）. The change of theexpression of TNF-α was not observed in each group（P>0.05）.These results indicated that CB2R agonist JWH-015was effective inreducing Aβ-induced increase in the expression of M1microgliaphenotype markers.3.3SCB2R agonist up-regulated the mRNA expression levels of the M2microglia phenotype markerIn the hippocampus, AD model mice showed a significantly decreasedexpression of Ym1/2compared with control group（P <0.05）. TheJWH-015was effective in decreasing the expression of Ym1/2in ADmodel mice（P <0.05）.In the striatum, compared with control group, the mRNA expressionof Ym1/2of AD mice was markedly reduced. Unexpectedly, JWH-015intervention did not significantly increase the expression of Ym1/2of ADmice but showed an increased tendency.In the cortex, the expression of Ym1/2of AD mice was significantlyincreased compared with control mice. But compared with the AD group,JWH-015intervention group showed a significantly increase in theexpression of Ym1/2.These results indicated that CB2R agonist JWH-015could promotethe expression of M2actived phenotype micrglia. 4SCB2R agonist could counteract the phosphorylation of ERK1/2andJNK in BV-2microglia cells induced by LPS1μg/ml LPS induced a significantly increase phosphorylation level ofERK1/2in BV-2microglia（P <0.01）.10nM JWH-015showed a mildefficacy on inhibiting the phosphorylation of ERK1/2induced by LPS.However, JWH-015showed a marked counteraction on the LPS-inducedphosphorylation of ERK1/2in the dose of100nM. and1μM （P <0.01）.Meanwhile the CB2R antagonist SR144528could significantly block thedephosphorylation effect of ERK1/2treated with JWH-015（P <0.05）.The phosphorylation level of JNK was significantly increased inLPS-induced BV-2microglia (P <0.01). JWH-015could concentrationdependently inhibited the phosphorylation levels of JNK induced by LPS.CB2R atagnist SR144528lead to partly inverte the effect of JWH-015oninhibition of phosphorylation of JNK.BV-2microglia treated by LPS resulted in the significantly increasedphosphorylation levels of p38MAPK （P <0.05）. The JWH-015did notcounteract the elevated phosphorylation level of p38MAPK induced byLPS（P>0.05）.These results indicated that the effect of JWH-015on inhibiting theexpression of M1microglia phenotype markers was partly obtained fromdepressing the MAPK signal pathway activation by regulating thephosphorylation level of ERK1/2and JNK.Conclusions:Meditation of the amount and functional phenotype of the microgliaand neuroinflammation in different brain region is a potential mechanismof the ameliorative effect of CB2R agonist JWH-015on improving thedysfunction of learning and memory of AD mice induced by Aβ. CB2Ragonist could regulate the actived phenotype of the microglia by partlyinhibiting the activation of MAPK signal pathway.

大麻素Ⅱ型受體激動劑改善阿爾茨海默病樣小鼠學(xué)習(xí)記憶能力的神經(jīng)炎癥機(jī)制研究

中文摘要4-8ABSTRACT8-12英文縮寫13-14前言14-16材料與方法16-26結(jié)果26-29附圖29-36附表36-37討論37-45結(jié)論45-46參考文獻(xiàn)46-52綜述 小膠質(zhì)細(xì)胞激活功能表型與阿爾茨海默氏病52-68    參考文獻(xiàn)61-68致謝68-70個人簡歷70

  本文關(guān)鍵詞：大麻素Ⅱ型受體激動劑改善阿爾茨海默病樣小鼠學(xué)習(xí)記憶能力的神經(jīng)炎癥機(jī)制研究，由筆耕文化傳播整理發(fā)布。