Itaconate Alleviates Neuroinflammation In Parkinson’s Disease By Inhibiting NLRP3 Inflammasome And Exert The Neuroprotective Effect Of Dopamine

Parkinson’s disease(PD)is a common age-related neurodegenerative motor disorder,mainly caused by the damage of dopaminergic neurons in the substantia nigra.The hallmark clinical symptoms of PD’s motor symptoms,including slowly progressive asymmetric resting tremor,motor retardation,stiffness,and changes in posture and gait.Dyskinesia leads to progressive disability,impairs activities of daily living and reduces quality of life.Various non motor symptoms are common in Parkinson’s disease,including olfactory disorder,sleep disorder,constipation,dementia and depression.The main pathological features of PD are the progressive loss of dopaminergic neurons in the substantia nigra(SN)and striatum,which control the motor system,and intraneuronal protein aggregates in the Lewy body and Lewy neurites.PD is a complex disease caused by the combined influence of environmental and genetic factors.Although the pathophysiological mechanisms involved in PD have been extensively studied,the initial triggers of PD remain unclear.So far,the treatment of PD is still symptomatic,and there is no therapy that can effectively slow down or prevent the progression of PD.Therefore,there is an urgent need to develop an anti PD drug that can not only improve PD but also have neuroprotective effects.Itaconate is an important intermediate metabolite isolated from the TCA cycle,which has received extensive attention in recent years due to its anti-inflammatory function.It was found that itaconate regulates inflammation by inhibiting the production of SDH and ROS,and inhibits NLRP3 inflammasome.Neuroinflammation and oxidative stress play a key role in the pathogenesis of PD.NLRP3 inflammasome is a response to neurotoxicity,misfolded protein and brain autophagy defects.It has become a key neuroinflammatory mechanism driving neurodegeneration and an important therapeutic target for PD.Many immunomodulatory effects of itaconate have revealed its potential as a therapeutic agent for a variety of diseases.However,the effect of itaconate sodium on Parkinson’s disease is rarely studied.In this study,we established an MPTP induced PD mouse model and an MPP+-induced cell model to explore the effect of itaconate on motor deficits in PD mice,and to study the effect of itaconate on dopamine neuron damage,oxidative stress and apoptosis in vivo and in vitro,and to explore its molecular mechanism.This study is mainly divided into two parts:Part Ⅰ itaconate exerts dopamine neuroprotective effect in MPP+-induced SH-SY5Y cells by inhibiting NLRP3 inflammasomePurpose:To study the effect of itaconate on MPP+-induced apoptosis of SH-SY5Y cells,and the inhibitory effect of itaconate on MPP+-induced damage of dopamine neurons and oxidative stress in SH-SY5Y cells,and to explore the molecular mechanism of itaconate inhibiting inflammation and exerting dopamine neuroprotective effect.Methods:The PD cell model in vitro was established by using MPP+.Cell viability was evaluated by MTT assay;TUNEL staining and flow cytometry were used to detect apoptosis;Protein immunoblotting and immunofluorescence were used to detect the expression level of th and evaluate the damage of dopamine neurons;The levels of inflammatory factors were detected by qRT-PCR;The contents of superoxide dismutase(SOD)and reactive oxygen species(ROS)were further determined;At last,the expression of NLRP3 inflammasome related proteins was detected by Western blot and immunofluorescence.Results:MPP+significantly decreased the viability of SH-SY5Y cells,and itaconate significantly increased the viability of SH-SY5Y cells induced by MPP+.The apoptosis of SH-SY5Y cells in MPP+group was significantly increased,and itaconate significantly decreased the apoptosis of SH-SY5Y cells.The expression of TH and Nurrl decreased in MPP+ group,and itaconate alleviated the decrease of th and Nurrl induced by MPP+.In the MPP+group,the ROS level in SH-SY5Y cells increased and the SOD level decreased,which was reversed by itaconate.The mRNA expression of IL-1β、TNF-α、COX-2 and iNOS in SH-SY5Y cells in MPP+ group increased,and itaconate decreased IL-1β、TNF-α、COX-2 and iNOS mRNA expression.The expression of NLRP3,ASC.caspase-1 and IL-1β was significantly increased in SH-SY5Y cells in MPP+ group,and itaconate inhibited the increase of NLRP3 inflammasome related protein.Conclusion:Itaconate increased the viability of MPP+-induced SH-SY5Y cells.inhibited the apoptosis of MPP+-induced SH-SY5Y cells,alleviates the oxidative stress of SH-SY5Y cells,alleviates the inflammatory response of SH-SY5Y cells induced by MPP+,inhibits the activation of NLRP3 inflammasome in PD cells induced by MPP+,and alleviates the dopamine neuron damage of MPP+-induced SH-SY5Y cells.Part Ⅱ:itaconate exerts dopamine neuroprotective effect in MPTP induced PD mice by inhibiting NLRP3 inflammasomePurpose:To study the effect of itaconate on the motor behavior of MPTP induced PD mice,to evaluate the neuroprotective effect of itaconate on MPTP induced PD mice,and to explore the mechanism of itaconate’s neuroprotective effect in the PD mouse model from the behavioral and molecular biological aspects.Methods:MPTP induced PD mouse model was established,and the motor coordination and balance ability of PD mice were evaluated by climbing rod test and rotating rod test.The expression of th in PD mice was detected by immunoblotting and immunofluorescence to evaluate the damage of dopamine neurons.The expression of Iba-1 and apoptosis related proteins in substantia nigra and striatum were detected by immunoblotting,and the level of inflammatory factors was detected by qRT-PCR.The contents of SOD,MDA,GSH and ROS in substantia nigra and striatum were further detected.Finally,the expression of NLRP3 inflammasome related proteins in substantia nigra and striatum was detected by Western blot.Results:In MPTP group,the turnover time and total time increased,and the time of falling from the rotator decreased significantly.Itaconate partially reversed this phenomenon.MPTP resulted in decreased TH expression in substantia nigra and striatum,and itaconate significantly reversed the decrease in th level.The expression of Iba-1 in substantia nigra and striatum was significantly increased in MPTP group,and itaconate inhibited the increase of Iba-1 expression in substantia nigra and striatum of PD mice.IL-1β、TNF-α、COX-2 and iNOS levels in substantia nigra and striatum were significantly increased in MPTP group,and iconic acid reversed the increase of inflammatory factors caused by MPTP.In MPTP group,SOD and GSH levels in substantia nigra and striatum decreased,while MDA levels increased,which was alleviated by itaconate.In MPTP group,cleaved-caspase3 and Bax protein levels were significantly increased in substantia nigra and striatum,and bcl-2 protein levels were significantly decreased.Itaconate partially reversed this phenomenon.In MPTP group,NLRP3,ASC,caspase-1 and IL-1β was significantly increased in substantia nigra and striatum,and itaconate inhibited the increase of NLRP3 inflammasome related protein.Conclusion:itaconate alleviates the motor defects of MPTP induced PD model mice,reduces neuronal damage,and maintains the function of substantia nigra and striatum.Itaconate can inhibit MPTP induced microglial activation and oxidative stress in PD mice,alleviate MPTP induced inflammatory response and neuronal apoptosis in PD mice,and inhibit MPTP induced activation of NLRP3 inflammasome in PD mice.Itaconate could alleviate the neuroinflammation of Parkinson’s disease by inhibiting the activation of microglia and NLRP3 inflammasome and exert dopamine neuroprotective effect.