Activation Of Cannabinoid TypeⅡ Receptors Promotes The Conversion Of MPP~+-Treated Microglia From M1 To M2

Parkinson’s disease（PD）is a neurodegenerative disorder caused by progressive degeneration of dopamine neurons in the substantia nigra and the formation of Lewy bodies.Neuroinflammation is an important factor inducing the development of PD.Studies have shown that microglia overactivation during abnormalα-synuclein accumulation and neuronal degeneration leads to an imbalance in the dynamic phenotype of microglia,further exacerbating neuroinflammation and promoting PD progression.Therefore,effective regulation of the phenotypic state of microglia improves the progression of PD.Recent studies have reported that the endogenous cannabinoid system plays a neuroprotective role in neurodegenerative diseases.Among them,the cannabinoid type 2receptor receptor（CB2R）is mainly expressed on microglia.Activation of CB2R inhibits microglia overactivation and reduces the release of inflammatory factors,thereby alleviating neuroinflammation in PD.In studies of hemorrhagic encephalopathy,activation of CB2R attenuates brain damage precisely by regulating M1/M2 polarization in microglia.It has also been shown that the inhibitory effect of CB2R on neuroinflammation is associated with phosphatidylinositol-3-kinase（PI3K）/protein kinase B（Akt）and nuclear factor E2-related factor 2（Nrf2）signaling pathways.In addition,it was confirmed that Nrf2is also one of the important factors involved in regulating the transformation of microglia phenotype.Previous studies in our laboratory demonstrated that activation of CB2R inhibits1-methyl-4-phenylpyridinium（MPP⁺）-induced neuroinflammation in astrocytes.Therefore,we hypothesized that activation of CB2R may promote MPP⁺-induced microglia conversion from M1 to M2 phenotype and this effect may be associated with activation of PI3K/Akt/Nrf2 pathway.In this study,primary rat microglia were pretreated with CB2R agonist JWH133 and CB2R antagonist AM630,followed by the addition of MPP⁺treatment.Protein expression of inducible nitric oxide synthase（i NOS）,interleukin 6（IL-6）,Arginase（Arg-1）and interleukin 10（IL-10）,markers of different phenotypes of microglia were detected by western blots in each group.The surface markers CD86 and CD206 in each group of microglia with different phenotypes were detected by flow cytometry.The phosphorylation levels and total protein expression of PI3K and Akt as well as Nrf2 protein expression in each group of microglia were detected by western blots.Microglia were pretreated with related pathway protein inhibitors,and the expression of phosphorylated and total Akt proteins,Nrf2 protein,i NOS and Arg-1 protein in each group of cells was detected by western blots.The results are as follows:1.Immunofluorescence was applied to detect the purity of primary microglia using CD11b and Hochest double staining.The positive rate of CD11b in primary microglia was93%on average.2.We examined the experssion of i NOS and IL-6,the markers of M1 microglia,in different groups by western blots.Compared with the control,i NOS and IL-6 protein expression were significantly increased in the MPP^+-treated microglia（P<0.0001,P<0.001）.Compared with MPP⁺group,i NOS and IL-6 protein expression decreased in the JWH133pretreatment group（P<0.001）.Compared with JWH133 pretreatment group,i NOS and IL-6 protein expression were elevated in the AM630 and JWH133 co-treatment group（P<0.05）.3.We examined the expression of Arg-1 and IL-10,the markers of M2 microglia,in different groups by western blots.Arg-1 and IL-10 protein expression were elevated in the JWH133 pretreatment group compared with MPP⁺group（P<0.001）.The protein expression of Arg-1 and IL-10 were reduced in the AM630 and JWH133 co-treatment group compared with JWH133 pretreatment group（P<0.05）.4.We examined the expression of M1 surface marker CD86 and M2 surface marker CD206 in microglia in different treatment groups by flow cytometry.Compared with control,the expression of CD86 in MPP⁺-treated microglia was markedly increased（P<0.001）.Compared with MPP⁺group,JWH133 pretreatment significantly inhibited the expression of CD86（P<0.001）.Co-treatment of JWH133 and AM630 enhanced the expression of CD86（P<0.05,compared with JWH133 pretreatment group）.JWH133pretreatment promoted the expression of CD206 compared with the MPP⁺group（P<0.001）.Co-treatment with AM630 reversed the effect of JWH133（P<0.05,compared with JWH133 pretreatment group）.5.We examined the levels of phosphorylated and total proteins of PI3K and Akt as well as Nrf2 protein expression in different groups by western blots.Phosphorylated proteins of PI3K and Akt and Nrf2 protein expression were reduced in the MPP⁺treated group compared with control（P<0.05）.While phosphorylated proteins of PI3K and Akt and Nrf2protein expression were elevated in the JWH133 pretreatment group compared with MPP⁺group（P<0.001）,which could be blocked by AM630（P<0.05,compared with JWH133pretreatment group）.Microglia was pretreated with PI3K inhibitor,and the expression of phosphoprotein and total protein of Akt and Nrf2 protein were detected by western blots in different groups.Co-administration of JWH133 and PI3K inhibitor inhibited Akt phosphorylation and Nrf2 protein expression compared with JWH133 pretreatment group（P<0.05）.6.We detected the expression of i NOS and Arg-1 in cells pretreated with Nrf2 inhibitors by western blots.Compared with the JWH133 pretreatment group,the i NOS protein expression was elevated in the group co-treated with Nrf2 inhibitor（P<0.05）.In addition,Nrf2 inhibitor blocked the upregulation of Arg-1 protein promoted by JWH133（P<0.05）.These results demonstrated that JWH133 inhibits MPP⁺-induced expression of M1-type microglia markers,including i NOS,IL-6 and CD86,and promotes the expression of M2-type microglia markers,including Arg-1,IL-10 and CD206,suggesting that activation of CB2R by JWH133 promotes MPP⁺-induced microglia conversion from M1 to M2phenotype.The regulation of microglia phenotype by activation of CB2R is associated with activation of PI3K/Akt/Nrf2 pathway.