The Study Of Anti-neuroinflammatory Mechanism Of Cannabinoid Type2Receptor Agonist In Improving The Learning And Memory Of Mice In Alzhermer’s Disease Experimental Model

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.