Effect And Mechanism Of Nf-?B Signaling Pathway In Neuroinflammatory Immunity Of Parkinson's Disease

Objective: Based on the neuroinflammatory immunology theory of Parkinson's disease(PD),it included various mechanisms and signaling pathways.To investigate the relationship between NF-?B and Parkinson's disease(PD),the inflammatory factors in the brain,and the damage of dopaminergic(DA)neurons,as well as the possible mechanisms of neuroinflammation injury.The role of NF-?B signaling pathway in the initiation of PD neuroinflammation was explored from the perspective of cellular,animal levels and inflammatory genetics.Methods:(1)PC12 cells was induced into PD inflammatory cell model by Lipopolysaccharide.The optimal concentrations of for LPS and QNZ(NF-?B inhibitor)were detected by CCK8.The expression of TH in the cell model was detected by immunohistochemistry and immunofluorescence in the LPS-PD cell model.The inflammatory factors(TNF-?,IL-1?,IL-6)were detected by ELISA.After QNZ and NF-?B si RNA were used to intervene in LPS-PD cell model,the expression of Caspase3,NF-?Bp65,?-syn,Nurr1 and TH were detected by WB.The co-immunoprecipitation assay was used to investigate the relationship between NF-?B and Nurr1.(2)A rat inflammation model of PD was constructed by LPS through brain stereotaxic technique.Divided into normal control group,PD model group and pramipexole drug treated group(n=20).Animal behavior testing.Immunohistochemistry for the detection of TH.The levels of inflammatory cytokines(TNF-?,IL-1?,IL-6)were detected by ELISA.RT-PCR was used to detect the m RNA expression of IL-6,TNF-?,Nurr1,?-syn and NF-?Bp65.WB was used to detect the proteins of NF-?Bp65,Nurr1 and ?-syn in NF-?B signaling pathway.(3)Choose PD patients(n=135)and normal peoples(n=135),their general data were statistically analyzed.ELISA was used to measure serum TNF-?,IL-1?,and IL-6 levels in all peoples.The polymorphisms of HLArs3129882 related to inflammation were detected and directed sequencing.To analyzed the relationship between inflammatory factors and PD.The association of HLArs3129882 gene polymorphisms with PD and the differences between Uygur and Han nationality.Results:(1)(1)The cellular model of inflammation PD can be induced by LPS through activation of the NF-?B signaling pathway.The optimal concentration of LPS and QNZ was 600ng/m L and 11 n M by CCK8.The content of TH in LPS-PD group was decreased,compared with the normal cell group(139±6 vs 107±4),there was a difference between the two groups(P<0.05).The positive TH cell were show by Immunofluorescence.Inflammatory factor in LPS-PD(TNF-?,IL-1?,IL-6)expression increased,compared with the normal group(P<0.05);The protein of NF-?B p65,?-syn were increased through detection of WB in LPS-PD group,the TH was decreased,There was a statistically significant difference between groups(P < 0.05).(2)After the inhibition of NF-?B signaling pathway by QNZ,the protein levels of NF-?Bp65 and?-syn were decrease,the protein level of TH was increase in QNZ+LPS-PD group.There was a significant difference between the normal cells,LPS-PD,and QNZ+LPS-PD groups(P<0.05).There was no difference between the normal group and the QNZ +LPS-PD group(P>0.05).(3)The function both QNZ and NF-?B si RNA interfered in LPS-PD cells was same,inhibiting caspase 3 and increasing cell survival.In the normal cell group,LPS-PD,QNZ+LPS-PD,and NF-?B si RNA+LPS-PD groups,the protein of?-syn,Nurr1 and TH were detected by WB,there was difference in four groups(P<0.05).Among the QNZ+LPS-PD and NF-?B si RNA+LPS-PD groups,except for the protein of TH,there was no difference between them(P>0.05).(4)The result of co-immunoprecipitation suggested an interaction between Nurr1 and NF-?B.(2)The rat PD models can successfully establish by LPS,the rats behavioral have changed by APO.After treatment with pramipexole for 14 day in PD model group,the number of TH positive cells in the brain were different(37±1 vs 18±2 vs 23±1)in three groups,the normal group,the PD group and the treaty group.There was statistical significance(P<0.05).The levels of inflammatory cytokines(TNF-?,IL-6)in the brain by inhibiting NF-?B by drugs.The m RNA expression of TNF-? and IL-6 in the brain of the treatment group was down-regulated,Nurr1 was up-regulated,and ?-syn and NF-?B p65 were down-regulated.In the normal group,the PD group and the treaty group.there was a difference(P<0.05).The protein contents of Nurr1,?-syn,and NF-?Bp65 also differed among the three groups(P<0.05).(3)The levels of TNF-?,IL-1? and IL-6 in the PD group were significantly higher than those in the normal control group(P<0.05).The HLArs3129882 gene as inflammatory immune-related,the analysis of its polymorphism showed three genotypes: AA,AG and GG.The frequency distributions of AA genotype and A allele are not different in the PD group and the normal group,like both in Uygur and Han nationality(P>0.05).Conclusion:(1)In the inflammatory cell model and inflammatory animal model of PD,the mechanism may be the activation of NF-?B signaling pathway,increased the content of inflammatory factors,increased the expression of ?-syn,decreased the Nurr1,lead to the damage of DA neurons.(2)In the NF-?B signaling pathway,the protective function of Nurr1 were weakened,which increased the expression of ?-syn and further damages DA neurons.(3)In our experiment,the levels of IL-6 and other inflammatory factors in blood of PD patients were increased.The polymorphism of HLArs3129882 was not related to PD.There were not difference between Uygur and Han nationality.(4)Activation of NF-?B signaling pathway may be one of the important signaling pathways in PD inflammatory immune system.