| Parkinson's disease(PD)is a common neurodegenerative disease.Its pathological characteristics are the degeneration of dopaminergic(DA)neurons in substantia nigra par compacta(SNpc)and the formation of Lewy body.Growing evidences showed that neuroinflammation plays an important role in the pathogenesis of PD.Astrocytes and microglia were activated to produce a large number of inflammatory factors to cause DA neuron degeneration.The inflammatory factors produced by astrocytes further activate microglia to enlarge the inflammatory state and accelerate the occurrence of PD.In addition,abnormal brain iron accumulation is associated with degeneration of DA neurons.Studies have shown that the iron content of SN in PD patients' brains increases and that divalent metal transporter 1(DMT1)is overexpressed.The accumulation of iron in specific regions will inevitably exacerbate the activation of astrocytes.It was reported that cannabinoid type 2 receptor(CB2 receptor)plays an important role in the neuropathological basis of PD.Activation of CB2 receptors on glial cells protect DA neurons by inhibiting the release of inflammatory factors.CB2 receptors also mediate serine phosphorylation of DMT 1,thereby regulating iron transport.Previous studies in our laboratory also showed that activation of CB2 receptors inhibited MPP+-induced iron influx and up-regulation of DMT 1 protein expression,thereby reducing the damage to SH-SY5Y cells.Therefore,we hypothesized that activation of CB2 receptor may inhibit MPP+-induced iron influx and DMT1 protein up-regulation,thereby exerting an anti-inflammatory effect in astrocytes.In the present study,by using western blots,real-time PCR(RT-PCR)and enzyme-linked immunosorbent assay(ELISA),we investigated effects of CB2 receptors on the inflammatory factors levels and iron transport on the 1-methyl-4-phenylpyridinium(MPP+)-treated primary cultured rat astrocytes.Astrocytes were pretreated with specific CB2 receptor agonist JWH-133 and CB2 receptor antagonist AM630 for 40 min,and then stimulated with MPP+.the iNOS,COX-2,TNF-? and IL-1? protein and mRNA expressions were detected.The phosphorylated DMT1 and its total protein expression in each group were also detected.Iron influx was detected by laser scanning confocal microscopy.The results are as follows:1.The purity of primary astrocytes were detected by GFAP and Hoechst double staining.Astrocytes purity were?95%based on immunofluorescence staining with the specific astrocytes marker(GFAP).2.The protein expressions of CB2 receptor in astrocytes were detected by western blot at different time points(2,4,8,and 24 hours)after MPP+treatment.Compared with the control,the protein expression of CB2 receptors in astrocytes in the MPP+group was significantly increased.The peak of its protein expression appeared after MPP+treated for 4 h(P<0.001),and its protein expression also increased at 8 h and 24 h after MPP+treatment(P<0.05).3.The protein expressions of iNOS and COX-2 in astrocytes were detected by western blots at 4h and 24h.The protein expressions of iNOS and COX-2 in the MPP+group were increased compared with the control(P?0.05).The protein expressions of iNOS and COX-2 were decreased after JWH-133 pre-treatment compared with MPP+group(P<0.05),AM630 and JWH-133 co-treatment may blockthe effect of JWH133.The protein expressions of iNOS and COX-2 were increased compared with the JWH133/MPP+group(P<0.05).4.The mRNA expressions of iNOS,COX-2,TNF-? and IL-1? in astrocytes were detected by RT-PCR at 4h and 24h.The mRNA expressions of iNOS,COX-2,TNF-? and IL-? in the MPP+group were increased compared with the control(P<0.05).Pre-treatment with JWH-133 inhibited MPP+-induced up-regulation of iNOS,COX-2,TNF-? and IL-1? mRNA expression(P<0.05),AM630 and JWH133 co-treatment may block the effect of JWH133(P<0.05,compared with the JWH133/MPP+group).5.The protein contents of TNF-? and IL-1? in different treatment groups at 4h and 24h were detected by ELISA.Pre-treatment with JWH133 inhibited MPP+-induced production of TNF-? and IL-1?(P<0.05).These effects were abolished by co-treatment with AM630(P<0.05,compared with the JWH133/MPP+group).6.The expressions of P-DMT1 protein were detected by western blots.The expression of P-DMT1 in the MPP+treatment group increased compared with the control(P?0.05).Pre-treatment with JWH133 inhibited P-DMT1 expression(P?0.05).Co-treatment with AM630 inhibited JWH133-induced down-regulation of P-DMT1(P?0.05,compared with the JWH133/MPP+group).Total DMT1 protein expressions in the MPP+group were also increased(P?0.05),JWH133 inhibited this increase(P?0.05),and AM630 and JWH-133 co-treatment may block the effect of JWH133(P?0.05,compared with the JWH133/MPP+group)7.Iron influx was examined by laser scanning confocal microscopy.The primary cultured astrocytes were pre-treated with 200 ?mol/L MPP+,and then perfused with 1 mmol/L FeSO4.Calcein was used as a fluorescence indicator.The weaker the fluorescence in the cell,the stronger the ability of iron influx.Compared with the control,the fluorescence intensity in the MPP+-treated astrocytes significantly decreased,indicating that iron influx increased.Compared with MPP+group,the decrease in the fluorescence intensity was partly inhibited by JWH133,indicating that iron influx in the JWH133/MPP+group decreased.AM630 blocks the effect of JWH133 and accelerate the fluorescence quenching.In summary,the above results showed that activation of CB2 receptor by JWH133 inhibits MPP+-induced inflammatory response in astrocytes,confirming the anti-inflammatory role of CB2 receptors in astrocytes.Activation of CB2 receptor by JWH133 also inhibit DMT1 phosphorylation and DMT1 up-regulation induced by MPP+,thereby inhibiting MPP+-induced iron influx. |
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