大麻素Ⅱ型受体激动剂改善阿尔茨海默病样小鼠学习记忆能力的神经炎症机制研究

  本文关键词：大麻素Ⅱ型受体激动剂改善阿尔茨海默病样小鼠学习记忆能力的神经炎症机制研究，由笔耕文化传播整理发布。

        目的：以大麻素II型受体（cannabinoid2receptor, CB2R）激动剂为研究工具，研究CB2R对β-淀粉样蛋白（β-amyloid,Aβ）诱导的小鼠学习记忆能力损伤的改善作用及其调节小胶质细胞功能表型转化的神经源性炎症作用机制。为确立CB2R作为新的抗神经源性炎症候选药物靶标提供进一步的实验证据，为治疗阿尔茨海默病提供新的药物治疗策略。方法：1CB2R激动剂对AD模型小鼠空间及非空间学习记忆能力的影响根据自发活动的研究数据将10周龄雄性C57/Bl6J小鼠随机分为对照组，AD模型组，，CB2R激动剂JWH-015干预组和JWH-015单药组。对照组和JWH-015单药组侧脑室一次性注射生理盐水8μl。AD模型组和JWH-015干预组动物侧脑室一次性注射寡聚态Aβ1-424μg（8μl），以建立AD模型。造模后第2天开始，JWH-015干预组和JWH-015单药组小鼠腹腔注射JWH-015（0.5mg/kg/天），对照组和AD模型组小鼠每天腹腔注射等体积含0.1%DMSO生理盐水，连续给药3周。采用Morris水迷宫实验及新物体识别实验观察CB2R激动剂对AD模型小鼠空间及非空间学习记忆能力的改善作用。2CB2R激动剂对AD模型小鼠脑内胶质细胞功能形态及数量的影响通过组织免疫荧光技术观察上述不同组别实验动物脑内小胶质细胞标志物（CD11b）和星形胶质细胞标志物（GFAP）的表达情况和CB2R激动剂对上述改变的影响。3CB2R激动剂对Aβ诱导的小胶质细胞功能和激活表型的影响采用实时定量PCR技术，观察海马、纹状体、前额叶等脑区的CB2R，M1型激活小胶质细胞标志物TNF-α、iNOS、IL-6及M2型激活小胶质细胞标志物Ym1/2mRNA的表达情况。4CB2R对LPS诱导的小胶质细胞功能表型的调节机制通过Western Blot的方法观察CB2R对LPS诱导的激活小胶质细胞内丝裂原活化蛋白激酶信号通路ERK、JNK及p38MAPK磷酸化水平的影响，探讨CB2R参与小胶质细胞激活功能表型调节的信号传导机制。结果：1CB2R激动剂改善AD模型小鼠空间和非空间学习记忆能力Morris水迷宫实验结果显示，在定位航行实验中，AD模型组小鼠逃避潜伏期较对照组小鼠显著延长（P <0.01）；在空间探索实验中，与对照组相比，AD模型组小鼠1分钟穿越平台次数显著减少（P <0.05）；新物体识别实验结果显示，与对照组相比，AD模型组小鼠新物体识别指数显著下降（P <0.05），提示AD模型建立成功。与AD模型组小鼠相比，JWH-015干预组小鼠逃避潜伏期显著缩短（P <0.05），1分钟穿越平台次数显著增加（P <0.05）；JWH-015干预组小鼠新物体识别指数较AD模型组小鼠相比显著增加（P <0.05），提示CB2R激动剂能够改善AD模型小鼠空间及非空间学习记忆能力；JWH-015单药组小鼠与对照组相比，逃避潜伏期、1分钟穿越平台次数及新物体识别指数均无显著变化（P>0.05）；此外，各组小鼠体重及自发活动未见组间差异，提示JWH-015不影响正常小鼠体重及精神活动。2CB2R激动剂抑制脑内胶质细胞激活数量通过免疫组化的方法检测不同处理组小鼠纹状体脑区小胶质细胞和海马脑区星型胶质细胞的激活与表达情况。与对照组相比，AD模型组小鼠纹状体脑区小胶质细胞标记物CD11b表达明显增高，形态表现为分枝状（激活状态）；JWH-015干预组小鼠纹状体脑区CD11b的表达与AD模型组相比明显下降；JWH-015单药组较对照组未见明显差异。AD模型组小鼠海马脑区星型胶质细胞标志物GFAP较对照组表达显著增高；JWH-015干预组小鼠GFAP的表达水平较AD模型组有减少的趋势；JWH-015单药组与对照组相比没有明显差异。3CB2R调节脑内Aβ诱导的小胶质细胞M1/M2激活表型的转化3.1Aβ诱导脑内不同脑区CB2R mRNA表达水平上调与对照组相比，AD模型组小鼠海马、纹状体和前额叶等脑区CB2RmRNA表达均显著上调（海马P <0.01，纹状体和前额叶P <0.05）。与AD模型组相比，JWH-015干预组小鼠海马、纹状体和前额叶等脑区CB2RmRNA表达水平均显著下调（海马P <0.01，纹状体和前额叶P <0.05）。3.2CB2R激动剂降低M1型激活小胶质细胞标记物mRNA表达水平在海马脑区，与对照组小鼠相比，AD模型组小鼠iNOS mRNA表达水平显著上调（P <0.01）。与AD模型组相比，CB2R激动剂干预组小鼠iNOSmRNA表达水平显著下调（P <0.01）。模型组IL-6，TNF-α mRNA表达呈上调趋势，JWH-015干预组IL-6，TNF-α mRNA表达呈下调趋势。单药组与对照组相比，其TNF-α、iNOS、IL-6mRNA表达未见明显改变（P>0.05）。在纹状体脑区，AD模型组小鼠脑内iNOS，IL-6，TNF-α mRNA表达较对照组均显著升高（P值分别为P <0.01、P <0.01和P <0.05）。与模型组相比，JWH-015干预组小鼠脑内iNOS，IL-6，TNF-α mRNA的表达显著下调（分别为P <0.01、P <0.01和P <0.05）。与对照组相比，单药组TNF-α、iNOS、IL-6mRNA表达无显著差异（P>0.05）。在前额叶脑区，与对照组相比，AD模型组小鼠脑内iNOS，IL-6mRNA表达显著上调（P值分别为P <0.01，P <0.05）。与AD模型组相比，JWH-015干预组小鼠脑内iNOS，IL-6mRNA表达显著下调（P均<0.01）。TNF-α mRNA表达各组间未见显著改变（P>0.05）。上述实验结果表明，CB2R激动剂JWH-015通过激活CB2R可下调AD模型小鼠脑内M1型激活小胶质细胞数量及其促炎症功能。3.3CB2R激动剂上调M2型激活小胶质细胞标记物mRNA表达在海马脑区，与对照组小鼠相比，AD模型组小鼠脑内M2型激活小胶质细胞标记物Ym1/2mRNA表达显著下调（P <0.05）。与AD模型组相比，JWH-015干预组小鼠脑内Ym1/2mRNA表达水平显著上调（P <0.05）。在纹状体脑区，与对照组小鼠相比，AD模型组小鼠脑内Ym1/2mRNA表达水平显著下调（P <0.05），JWH-015干预组小鼠脑内Ym1/2mRNA表达水平呈升高趋势，统计学没有显著差异（P>0.05）。在前额叶脑区，AD模型组小鼠脑内Ym1/2mRNA表达水平较对照组显著下调（P <0.05）。与AD模型组相比，JWH-015干预组小鼠脑内Ym1/2mRNA表达水平显著上调（P <0.01）。JWH-015单药处理组Ym1/2mRNA表达水平较对照组无显著差异（P>0.05）。上述实验结果表明，CB2R激动剂JWH-015可上调AD模型小鼠脑内M2型激活小胶质细胞表达及其抗炎作用。4CB2R激动剂显著抑制LPS诱导的M1型BV-2小胶质细胞ERK1/2和JNK磷酸化水平与对照组相比，LPS处理组BV-2小胶质细胞ERK磷酸化水平显著增高（P <0.01）；JWH-015低剂量处理组（10nM）与LPS处理组相比，ERK1/2磷酸化水平呈下降趋势；JWH-015中剂量（100nM）及高剂量组（1μM） BV-2小胶质细胞ERK1/2磷酸化水平显著下调（P <0.01）；JWH-015中浓度（100nM）组对LPS诱导的BV-2小胶质细胞ERK1/2磷酸化水平升高的抑制作用可被CB2R拮抗剂SR144528所显著阻断（P <0.05）。与对照组相比，LPS处理组BV-2小胶质细胞内JNK磷酸化水平显著增高（P <0.05）； JWH-015能浓度依赖性地抑制LPS诱导BV-2小胶质细胞的JNK磷酸化水平；JWH-015中浓度（100nM）组对LPS诱导的BV-2小胶质细胞JNK磷酸化水平升高的抑制作用可部分被CB2R拮抗剂SR144528阻断。实验结果提示， CB2R激活后，下调ERK和JNK两个信号分子的磷酸化水平，因此，阻断MAPK信号通路过度激活可能是JWH-015抑制M1型激活小胶质细胞表达和抑制其促炎作用的信号转导机制之一。结论：选择性CB2R激动剂JWH-015对Aβ诱导的AD模型小鼠学习记忆能力具有改善作用，其作用机制可能与其抑制MAPK信号通路过度活化，调节小胶质细胞激活数量及功能表型，进一步抑制不同脑区内神经炎症反应，减轻神经元损伤相关。

    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型受体激动剂改善阿尔茨海默病样小鼠学习记忆能力的神经炎症机制研究

中文摘要4-8ABSTRACT8-12英文缩写13-14前言14-16材料与方法16-26结果26-29附图29-36附表36-37讨论37-45结论45-46参考文献46-52综述 小胶质细胞激活功能表型与阿尔茨海默氏病52-68    参考文献61-68致谢68-70个人简历70

  本文关键词：大麻素Ⅱ型受体激动剂改善阿尔茨海默病样小鼠学习记忆能力的神经炎症机制研究，由笔耕文化传播整理发布。