The Role And Mechanisms Of MIF Meditates NLRP3 Inflammasome Of Microglia In MPP+/MPTP Model

Objective:Parkinson’s disease is a common degenerative disease,which is related with neuroinflammation induced by excessive activation of microglia and the exact mechanisms remains unknown.To investigate the mechanisms of MIF in 1-methyl-4-phenylpyridinium iodide(MPP~+)-induced activation of Nod-like receptor protein 3(NLRP3)inflammasome in microglia and its effect on substantia nigra dopaminergic neurons and microglia cells activation in MPTP model mouse.Method:The Bv-2 microglia cells were treated with different concentrations of MPP~+to activate NLRP3 inflammasome and evaluated the expression of MIF and NLRP3.MIF sh RNA lentivirus and negative control lentivirus were applied to infect Bv-2 cell to knockdown MIF and act as negative control.Quantitative real-time PCR and Western blot were used to detect the m RNA and protein level of MIF.According to treatments the Bv-2cells were divided into control group(no treatment),MPP~+group(treated with MPP~+),LV-con group(infected by negative control lentivirus and treated with MPP~+)and MIF-sh RNA group(infected by MIF-sh RNA lentivirus and treated with MPP~+).In groups,Western blot was used to detect the total protein level of NLRP3,cleaved caspase-1 and p65.Also,ELISA was used to detect the protein of IL-1βand IL-18 in plasma of treated condition.Further,cell immunofluorescence and Western blot were used to detect the p65 in nuclear as well as plasma.The protein level of TH was detected in MN9D dopaminergic neuronal cells with conditional treated plasma of Bv-2 cells.C57BL/6 mouse were treated with Adreno-Associated Virus(AAV)and MPTP and were divided into control group(25mg/kg body weight physiological saline),MPTP group(25mg/kg body weight MPTP),PBS group(25mg/kg body weight MPTP and PBS through stereotactic injection),AAV-con group(25mg/kg body weight MPTP and AAV negative control through stereotactic injection)and AAV-MIF-sh RNA(25mg/kg body weight MPTP and AAV MIF sh RNA through stereotactic injection).We conducted the open field test,pole test and traction test comprehensive statistical analysis.TH and Iba-1 immunohistochemical staining were performed to observe the substantia nigra neurons and microglia cells activation.Results:The significant activation of NLRP3 inflammasome is Bv-2 treated with 0.2m M MPP~+.After affection by MIF sh RNA lentivirus,the protein level of NLRP3,cleaved caspase-1 and plasma protein level of IL-1βand IL-18 were lower than control group but the total protein level of p65 was not statistically different from MPP~+group.Further,we found the protein level of p65 in nuclear is less than MPP~+group and higher in plasma.In Bv-2treated by conditioned medium,MN9D expressed more TH in MIF sh RNA condition than MPP~+.In AAV-MIF-sh RNA MPTP model mouse,substantia nigra TH-positive neurons increased and Iba-1 microglia cells activation decreased compared with MPTP group.Conclusion:MIF sh RNA can reduce the expression of NLRP3 inflammasome caused by MPP~+intervention in Bv-2 microglia cell,relieve the damage of substantia nigra dopaminergic neurons and microglia cell activation in vivo,and play a role in development of neuroinflammation in Parkinson’s disease.