Neuroprotective Effects And Mechanisms Of Cannabinoid Receptor Ⅱ In LPS-induced Mice Model Of Parkinson’s Disease

Parkinson’s disease（PD）is the second most common age-related neurodegenerative disease,characterized by selective loss of Dopamine（DA）neurons in the substantia nigra（SN）.At present,levodopa,the precursor of DA,and DA agonists are widely used in the treatment of PD.However,these drugs can only temporarily improve clinical symptoms,but cannot prevent or slow down the progress of PD.In the autopsy results of patients with PD,it was found that there were a large number of activated glial cells and inflammatory cytokines in SN,which indicated that neuroinflammation was involved in the occurrence and development of PD.In the process of neuroinflammation,glial cells are abnormally activated and produce a large number of inflammatory factors,which induce neuronal degeneration and death.In view of the important role of nerve inflammation in the pathogenesis of PD,the effective inhibition of nerve inflammation will be helpful to the treatment of PD.In recent years,the anti-inflammatory effect of cannabinoid typeⅡreceptor（CB₂ receptor）has attracted wide attention.CB₂ receptor is an important part of endogenous cannabinoid system.CB₂ receptor is mainly expressed in microglia and astrocytes in the central nervous system.CB₂ receptor is encoded by CNR2 gene located on chromosome 1p36.11.Human genome-wide association analysis shows that CNR2 gene is related to PD.Our previous studies found that the activation of CB₂ receptor could inhibit the inflammatory response of astrocytes induced by 1-methyl-4-phenylpyridinium（MPP⁺）.However,there are few studies on CB₂ receptor in the in vivo model of PD.It is not clear whether activating this receptor can inhibit neuroinflammation and protect DA neurons.In this study,C57BL/6 mice were used as the research object,and the PD animal model was established by microinjection of lipopolysaccharide（LPS）into the SN region of the midbrain by stereotaxic technique.CB₂ receptor agonist JWH133 was selected for treatment.The aim of this study was to explore the neuroprotective effect of CB₂ receptor in LPS-induced PD model and its possible mechanism by using behavior,immunoblotting,immunohistochemistry and real-time fluorescence quantitative PCR（q RT-PCR）.The experimental results are as follows:1.Immunofluorescence staining showed that CB₂ receptor was expressed in mice SN region.2.Western blot results showed that compared with the control group,the expression of CB₂ receptor protein in SN region of LPS group was up-regulated（P<0.01）.3.The results of pole climbing showed that the rod climbing time of LPS group was longer than that of control group.The pole climbing time of JWH133 treated mice was significantly lower than that of LPS treated mice（P<0.01）.The results showed that the time of rotarod in LPS group was less than that in control group（P<0.05）.The number of II in JWH133 group was significantly higher than that in LPS group（P<0.05）.Compared with wild type mice LPS group,CB₂ receptor knockout（CB₂-KO）mice spent less time on rods in LPS group（P<0.05）.4.The results of immunofluorescence and immunohistochemical staining showed that compared with the control group,the number of tyrosine hydroxylase（TH）positive neurons in SN region and the density of TH positive fibers in striatum（STR）in LPS group were significantly lower than those in control group（P<0.001）.The number of TH positive neurons in SN region and TH positive fibers in STR region in JWH133 treated group were significantly higher than those in LPS group（P<0.001）.Compared with LPS group,TH positive neurons and TH positive fibers in SN of LPS group of CB₂-KO mice were significantly decreased（P<0.05）.Western blotting also showed that JWH133 treatment inhibited the decrease of TH protein expression induced by LPS（P<0.05）.Compared with wild type mice,TH neurons in SN region of CB₂-KO mice were more vulnerable to LPS damage.5.The results of immunofluorescence staining showed that the number of activated microglia and astrocytes in SN region of LPS group was higher than that of control group（P<0.001）.The number of activated microglia and astrocytes in SN region of JWH133 group was significantly lower than that of LPS group（P<0.001）.Compared with wild-type mice LPS group,the number of activated microglia and astrocytes in SN region of CB₂-KO mice in LPS group was significantly increased（P<0.05,P<0.01）.6.Western blot results showed that the expression of inducible nitric oxide synthase（i NOS）and cyclooxygenase-2（COX-2）in SN region of LPS group was up-regulated as compared with that of control group（P<0.01,P<0.001）.The expression levels of i NOS protein and COX-2 protein in SN region of JWH133 treated mice were significantly lower than those of LPS group（P<0.05）.Compared with wild type mice LPS group,the expression levels of i NOS protein and COX-2 protein in CB₂-KO LPS group were significantly higher than those in wild type mice（P<0.05）.7.The results of q RT-PCR showed that the expressions of i NOS,COX-2,Interleukin-1β（IL-1β）,TNF-αand Interleukin-6（IL-6）m RNA in SN region of LPS group were significantly higher than those of control group（P<0.01,P<0.001）.The expressions of i NOS,COX-2,IL-1βand TNFαm RNA in SN region of JWH133 treated mice were significantly lower than those of LPS group（P<0.05,P<0.01）.The m RNA expression level of these inflammatory cytokines in CB₂-KO LPS group was significantly higher than that in wild type mice LPS group（P<0.05,P<0.01,P<0.001）.8.Western blot results showed that compared with the control group,the expression of Bax protein in SN region of LPS group was up-regulated（P<0.001）and the expression of Bcl-2 protein was decreased（P<0.05）.Compared with LPS group,the expression of Bax protein in SN region of JWH133 treated group was significantly decreased（P<0.05）,while the expression of Bcl-2 protein was significantly increased.Compared with wild-type mice LPS group,the expression of Bax protein in SN region of CB₂-KO mice LPS group was up-regulated and the expression of Bcl-2 protein was decreased.9.Western blot results showed that the expression of p-Akt protein and the ratio of p Akt/Akt in SN region of LPS group were lower than those of control group（P<0.001）.Compared with LPS group,the phosphorylation level of Akt and the ratio of p-Akt/Akt in JWH133 treated group increased significantly（P<0.01）.The ratio of p-Akt/Akt in LPS group of CB₂-KO mice was lower than that of wild type mice（P<0.05）.Conclusion:LPS treatment increases the expression of CB₂receptor in mice SN region.The absence of CB₂ receptor leads to the increased activation of microglia and astrocytes in SN induced by LPS.CB₂ receptor activation can inhibit the activation of microglia and astrocytes in SN induced by LPS,protect DA neurons from injury and death caused by neuroinflammation,and improve motor dysfunction in mice.CB₂ receptor may play a neuroprotective role through PI3K-Akt signal pathway.