Protective Effects Of Cannabinoid Type Ⅱ Receptor On MPP~+-induced Neurotoxicity In Primary Cultured Ventral Mesencephalon Neurons

Parkinson’s disease（PD）is a neurodegenerative disease characterized by the loss of dopaminergic（DA）neurons in the substantia nigra pars compacta and the presence of intracellular Lewy bodies.PD patients present with movement disorders such as tremor and rigidity,as well as non-motor symptoms such as dementia,anxiety,lethargy,urinary symptoms,attention deficit,and hypoglycemia.The pathogenesis of PD is unclear,neuroinflammation,oxidative stress is considered to play an important role in the pathogenesis of PD.Growing evidences indicate that the abnormal accumulation of iron in the substantia nigra（SN）can lead to the loss of DA neurons.Excessive iron reacts with H₂O₂ to promote the production of free radicals to damage cells and cause the death of DA neurons.Iron concentrations correlated with severity of PD.It is known that levodopa（L-DOPA）is effective in the early stage of PD treatment,however the complications caused by long-term use will greatly limit the drug intervention of L-DOPA.It is important to find the effective strategies to protect DA neurons to slow down the progression of the disease.Recent studies revealed that the endogenous cannabinoid system（ECS）plays an important role in the pathogenesis of PD.So far,two cannabinoid receptors have been cloned,namely cannabinoid type 1 receptor（CB1R）and cannabinoid type 2 receptor（CB2R）.It was reported that participates in the pathogenesis of PD.Activation of CB2R by cannabinoid-related compounds may maintain neuronal homeostasis in the neurodegenerative diseases including but not limited to PD.An elevated CB2R protein levels were observed in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP）induced PD model by western blot and immunofluorescence staining in the ventral mesencephalon（VM）.Treatment with JWH133 or AM1241,the Selective CB2R agonists,may attenuate MPTP-induced degeneration of DA neurons and axon terminals.AM1241 also effectively reversed MPTP-induced motor deficits in CB1R-knockout mice,and improved the survival of DA neurons.Furthermore,GW842166x,another selective CB2R agonist,also protects 6-hydroxydopamine（6-OHDA）-induced loss of DA neurons and related motor defects in mice.However,until now,the function of these CB2Rs and their role in the pathogenesis of PD are unclear.The purpose of this study was to explore the protective effect of CB2Rs activation on 1-methyl-4-phenylpyridinium（MPP⁺）induced neurotoxicity in the primary cultured VM neurons and the possible mechanisms involved.The VM neurons were pretreated with specific CB2R agonist JWH133 and/or specific CB2R antagonist AM630 for 30 min,respectively,and then co-treated with MPP⁺for 24 h.The changes in mitochondrial transmembrane potential（ΔΨm）and reactive oxygen species（ROS）were detected by flow cytometry in the VM neurons.The expressions of tyrosine hydroxylase（TH）,cleaved caspase-3,Bcl-2,Bax,inducible nitric oxide synthase（i NOS）,cyclooxygenase-2（COX-2）,divalent metal transporter1（DMT1）and ferroportin 1（FPN1）were detected by western blots in each group.The results are as follows:1.The TH protein expression in the VM neurons was markedly decreased in the MPP⁺treatment group（P<0.01,compared with the control）.JWH133 pre-treatment significantly inhibits the decrease of TH protein expression（P<0.01,compared with MPP⁺treatment group）.This effect was blocked by AM630（P<0.01,compared with JWH133 treatment group）.2.TheΔΨm in the VM neurons was significantly decreased in the MPP⁺treatment group compared with the control（P<0.001）.JWH133 pre-treatment significantly inhibits the decrease ofΔΨm（P<0.05,compared with MPP⁺treatment group）.This effect was inhibited by AM630 co-treatment（P<0.05,compared with JWH133 treatment group）.3.Compared with the control,the ROS production was obviously increased in VM neurons in the MPP⁺treatment group（P<0.001）.JWH133 pre-treatment significantly inhibits the increase of ROS generation compared with the MPP⁺treatment group（P<0.05）.The effect of JWH133 was blocked by AM630 co-treatment（P<0.001,compared with JWH133 treatment group）.4.The Bcl-2/Bax protein expression in VM neurons was decreased in the MPP⁺treatment group（P<0.001,compared with the control）.JWH133 pre-treatment significantly inhibits the decrease of Bcl-2/Bax protein expression（P<0.001,compared with MPP⁺treatment group）.This effect was blocked by AM630（P<0.01,compared with JWH133 treatment group）.5.The cleaved caspase-3 protein expression in VM neurons in the MPP⁺treatment group was up-regulated（P<0.01,compared with the control）.This effect was partly reversed by JWH133（P<0.01,compared with MPP⁺treatment group）.Co-treatment with AM630 reverses the effect of JWH133 pre-treatment on cleaved caspase-3（P<0.01,compared with JWH133treatment group）.6.The i NOS protein expression in VM neurons in the MPP⁺treatment group was up-regulated（P<0.01,compared with the control）.JWH133 inhibits the MPP⁺-induced up-regulation of i NOS（P<0.05,compared with MPP⁺treatment group）.This effect was blocked by AM630（P<0.05,compared with JWH133 treatment group）.7.The expression of COX-2 protein was increased in the MPP⁺-treated VM neurons compared with the control（P<0.05）.JWH133 inhibits the MPP⁺-induced up-regulation of COX-2（P<0.05,compared with MPP⁺treatment group）.AM630 attenuates the effect of JWH133 on COX-2（P<0.01,compared with JWH133 treatment group）.8.The DMT1 protein expression was increased in the MPP⁺treatment group compared with the control in the VM neurons（P<0.01）.JWH133 pre-treatment inhibits MPP⁺-induced up-regulation of DMT1 protein expression（P<0.05）.Co-treatment with AM630 reverses the effect of JWH133 pre-treatment on DMT1 protein expression（P<0.05,compared with JWH133 treatment group）.9.The protein expression of FPN1 in VM neurons was down-regulated in the MPP⁺treatment group（P<0.05,compared with the control）.This effect was alleviated by JWH133（P<0.01,compared with MPP⁺treatment group）.Co-treatment with AM630 reverses the effect of JWH133 on FPN1（P<0.05,compared with JWH133 treatment group）.In summary,these results suggested that JWH133 protects VM neurons from MPP⁺-induced injury.JWH133 inhibits the MPP⁺-induced ROS production andΔΨm decrease,and prevents the decrease in Bcl-2/Bax ratio and the increase in cleaved caspase-3 protein expression.Furthermore,JWH133 also antagonizes the up-regulation of DMT1,the down-regulation of FPN1 and the up-regulation of COX-2 and i NOS induced by MPP⁺.These results indicate that activation of CB2R may play a protective role by reducing oxidative stress and iron accumulation.The protective effect of JWH133 activating CB2R may also be related to anti-apoptosis and anti-inflammation.Our study provides a new experimental basis for further understanding of the protective role of CB2R.