| Objective: To study the mechanism on activation of P2Y6 receptor on microglia and its effect on dopaminergic neurons.To investigate whether P2Y6 receptor could be used as a diagnostic biomarker for Parkinson disease by detecting IL-8 levels released from peripheral blood mononuclear cells of patients with Parkinson disease in case of inflammation.Methods: Measure the expression level of P2Y6 receptor on microglia after LPS treatment as well as that of MIP-2 m RNA and i NOS,COX-2 protein induced by UDP and LPS through RT-PCR and western blot.To confirm the influence of P2Y6 receptor on the apoptosis and vitality of SH-SY5 Y cells by western blot and MTT asssay.Not only the phosphorylated level of MAPK signal transduction pathway after incubation of BV-2 cells with LPS or UDP but also the change of NF-κB of BV-2 cells induced by UDP was tested by Western blot.Expression level of i NOS and COX-2 protein following LPS stimulation after inhibition of the MAPK signal transduction pathway was analyzed,The levels of IL-8 in the supernatant of peripheral blood momonuclear cells(PBMC)from patients with Parkinson’s disease and healthy controls before and after LPS stimulation was analyzed by ELISA methods.Results: LPS can upregulate the expression of P2Y6 on BV-2 microglia.LPS alone but not UDP can induce microglia to release MIP-2,i NOS and COX-2,however UDP can downregulate the expression of i NOS induced by LPS.Knocking down P2Y6 receptor can reduced the apoptosis and toxicity of SH-SY5 Y cells added by conditional medium of microglia.P2Y6 receptor activate microglia partially depended on activation of ERK and NF-κB pathway.The PBMC can express and release more IL-8 after LPS stimulation in both PD patients and healthy controls.The PBMC of PD patients can express and release more IL-8 before and after LPS stimulation than controls,as well as the increasing rate of IL-8,although these was not significantly.Conclusion: P2Y6 receptor can partially participate in the activation of microglia and production of pro-inflammatory factors induced by LPS through ERK and NF-κB pathway,causing apoptosis and dopaminergic neuron death.P2Y6 receptor might play an important role in the pathogenesis of Parkinson’s disease.The level of IL-8 released by PBMC of PD patients is higher than that of healthy controls before and after LPS treatment,combined with our previous results(expression P2Y6 m RNA in PD patients is 3.7 times than that of healthy controls)indicate that P2Y6 receptor could be used as a reliable biomarker in diagnosis of Parkinson disease. |
