Mechanism Of Clock Gene Rev-erbα Attenuating Parkinson’s Disease-like Injury By Regulating NLRP3 Inflammasome

Objective:Circadian disruption is the most common non-motor phenomenon in Parkinson’s disease(PD).It usually appears early in the disease and accompanies the disease throughout its course.However,the causal relationship between circadian disruption and PD has not been fully elucidated.Microglia-mediated neuroinflammation is an important pathological feature of PD.In this study,we explored the specific mechanism of circadian disruption involved in the occurrence and development of PD by observing the potential link between Rev-erbαand microglia-mediated neuroinflammation.Methods:(1)The observation of circadian rhythm and neuroinflammation in PD models:BV2 microglia were treated with MPP~+and sonicatedα-Syn pre-formed fbril(α-Syn PFF)to construct two cell models respectively,and the transcription level of Rev-erbαwithin 24 hours was detected by RT-PCR.C57BL/6J mice were used as experimental objects,and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridyl(MPTP)was administered intraperitoneally to establish the animal model.The rest-activity rhythm was evaluated by wheel-running activity,the transcriptional levels of Rev-erbα,Bmal1 and Per2 in the substantia nigra(SN)within one day were measured by RT-PCR,and the protein levels of Rev-erbαat ZT6 and ZT18 were detected by Western blot.In addition,the number,morphology and volume of microglia at ZT6 and ZT18 were detected by immunofluorescence,the transcription levels of NLRP3 and related inflammatory cytokines in the SN were detected by RT-PCR.(2)Exploring the effects of Rev-erbαon motor function and neuropathology in PD animal model:MPTP-induced mice were pretreated with intraperitoneal injection of Rev-erbαagonist SR9009.The mice were weighed at the same time each day using the same electronic scale,and the motor function was evaluated by pole test,rotarod test and balance beam test.The survival number and distribution of dopaminergic neurons were detected by immunohistochemistry and immunofluorescence staining,and the expression levels of Tyrosine hydroxylase(TH)in the SN and striatum ere detected by Western blot.(3)Exploring the effects of Rev-erbαon neuroinflammation in PD animal model:The number,morphology and distribution of glial in nigrostriatal region were observed by immunohistochemistry.The co-localization of IBA1(microglia marker)with i NOS(M1-type marker),Arg-1(M2-type marker)and NLRP3(NLR Family Pyrin Domain Containing 3)in the SN was observed by immunofluorescence.Western blot was used to detect the protein levels of IBA1 and GFAP,phosphorylation of NF-κB p65 and activation of NLRP3 inflammasome in the SN.The transcription levels of TNF-α,IL-6,IL-1βand IL-18 were measured by RT-PCR.(4)Probing potential mechanism of Rev-erbαregulating neuroinflammation:MPP~+-treated BV2 microglia were pretreated with SR9009and SR8278(Rev-erbαinhibitor).JSH-23 was used to inhibit NF-κB and MCC950 was used to inhibit NLRP3 inflammasome.The effects of Rev-erbαon NF-κB/NLRP3inflammasome pathway and microglia polarization were investigated by Western blot.In addition,BV2 microglia treated withα-Syn PFF were pretreated with SR9009 and SR8278.The intracellularα-Syn monomers and oligomers at different time points were detected by Western blot.Results:(1)In the cell model induced by MPP~+andα-Syn PFF,the diurnal transcription of Rev-erbαwas disturbed,and the average transcriptional level was decreased.In the MPTP-induced mice,the rest-activity was fragmented.The transcriptional rhythm of Rev-erbα,Bmal1 and Per2 in the SN was disrupted,and the circadian oscillation was weakened.The protein level of Rev-erbαdecreased at ZT6.The volume of microglia in the SN lost its diurnal variation,and the expression of inflammatory cytokines increased.(2)MPTP-induced mice exhibited significant motor impairment and loss of dopaminergic neurons in nigrostriatal region.Activation of Rev-erbαcould effectively alleviate MPTP-induced motor impairment and pathological damage.(3)The number and volume of glial cells in MPTP-induced mice were increased,as well as microglial M1 markers and inflammatory cytokines,and the NF-κB/NLRP3 inflammasome pathway was also activated.Activation of Rev-erbαcould effectively inhibit MPTP-induced gliosis and the NF-κB/NLRP3 inflammasome pathway,and promote the transformation of microglia from pro-inflammatory M1 type to anti-inflammatory M2 type.(4)SR9009 inhibited MPP~+-induced activation of NF-κB and NLRP3 inflammasome in microglia in a dose-dependent manner.The activation of microglia was more obvious after SR8278 pretreatment.After JSH-23pretreatment,the activation of NF-κB and NLRP3 inflammasome was inhibited,inflammatory cytokines were decreased,and anti-inflammatory cytokines were increased.Pretreatment with MCC950 inhibited the activation of NLRP3 inflammasome,but the expression of p-NF-κB p65 was not affected.In addition,activation of Rev-erbαsignificantly reduced the uptake ofα-Syn PFF by microglia and even slightly promoted the degradation ofα-Syn PFF.Conclusion:The circadian rhythm of PD model was disrupted,especially the diurnal expression of Rev-erbα.Rev-erbαwas involved in the pathological process of PD by regulating the NF-κB/NLRP3 inflammasome pathway,which could effectively alleviate motor dysfunction,dopaminergic neuron injury and neuroinflammation in MPTP-induced mice,and reduce the pathologicalα-Syn load of microglia.Taken together,this study supported that Rev-erbαmay be a potential target for PD treatment.