The Research Of Orai1-mediated Ca²⁺ Influx Regulates Neuroinflammation Through PI3K/Akt/NF-κB Pathway In Parkinson’s Disease

Objective:Parkinson’s disease（PD）is a common neurodegenerative disease with unknown etiology and gradual progression,which is more common in the middle-aged and elderly,which greatly damages the quality of life of patients.At present,the pathogenesis is not clear,which may be related to calcium homeostasis imbalance,neuroinflammation,autophagy and other factors.Calcium release-activated calcium modulator 1（Orai1）is an important channel protein in Store-Operated Ca²⁺Entry（SOCE）,which can regulate neuroinflammation through microglial activation,endoplasmic reticulum stress and downstream signal pathways.In the previous study,it was found that Orai1 decreased in PD mice.Phosphatidylinositol 3 kinase/protein kinase B（PI3K/Akt）signal pathway has been shown to affect neuroinflammation by regulating nuclear factor-kappa B（NF-κB）in PD.After blocking Orai1,it was found that PI3K/Akt signal pathway was inhibited.It is not clear whether Orai1 can be used as an upstream protein of PI3K/Akt/NF-κB pathway to regulate neuroinflammation and affect the occurrence and development of PD.In this study,Orai1 pharmacological inhibitors and PI3K/Akt pathway inhibitors were used to study whether Orai1 regulates neuroinflammation and affects PD through PI3K/Akt/NF-κB pathway in the acute PD mouse model.And we aim to explore the protective role of Orai1 in PD.Methods:Groups:control group（Control）,model group（MPTP）,inhibiting Orai1 group（MPTP+GSK）,inhibiting pathway group（MPTP+GSK+LY）.MPTP（20 mg/kg）was given to C57BL/6 mice by intraperitoneal injection by 4 times at intervals of 2 hours,and the acute PD mouse model was made.On this basis,Orai1 inhibitor group and pathway inhibitor group were given GSK7975A and GSK7975A+LY294002 respectively by intraperitoneal injection.When PD mice models were finished,Behavioral testes was performed after 2 hours,such as pole test,suspension test and gait analysis.Western Blot was used to detect the expression of Orai1,p-Akt,Akt,NF-κB and inflammatory factor IL-1βin substantia nigra and striatum of mice.Immunohistochemistry was used to detect the number of TH（+）neurons and the expression of tumor necrosis factorα（TNF-α）in substantia nigra.Results:1.Behavioral tests resultsThe MPTP group showed reduced locomotor performance in all three behavioral tests compared to the control mice.When GSK7975A was used to inhibit Orai1 calcium channel,we found that in the pole test,the time of climbing from the top of the pole to the ground in the MPTP+GSK group was longer than that in the MPTP group.In the suspension test,the falling times of the MPTP+GSK group were increased and the suspension time was shortened compared with the MPTP group.In gait analysis,when orai1 was suppressed,run duration was prolonged,run speed and the swinging speed became slower in MPTP+GSK group（P<0.05）.Inhibiting Orai1 and using LY294002 to inhibit the pathway,compared with the MPTP+GSK group,the time from the pole to the ground was prolonged in the pole test.In gait analysis,run duration was decreased,stride length was shortened,and swing speed was further slowed down（P<0.05）.2.The results of Western BlotCompared with the control group,the expression of Orai1 in striatum and substantia nigra of MPTP group decreased,the expression of p-Akt decreased,Akt expression had no significant difference,but the expression of NF-κB and IL-1βincreased.When we inhibited Orai1,we found that the expression of Orai1 in MPTP+GSK group decreased further,and p-Akt also decreased significantly,while the expression of NF-κB and IL-1βincreased in MPTP+GSK group.In order to verify the relationship between Orai1 and PI3K/Akt/NF-κB further,after using pathway inhibitor,it was found that Orai1expression was no significance compared with the MPTP+GSK group,while the expression of p-Akt was decreased.At the same time,the expression of p-NF-κB and inflammatory cytokines IL-1βincreased in MPTP+GSK+LY group（P<0.05）.3.The results of ImmunohistochemistryIn this study,it was confirmed that the number of TH（+）neurons in PD model mice was less than that in the control group.It was also observed that after inhibition of Orai1,the number of TH（+）neurons decreased further compared with the MPTP group.After the inhibition of PI3K/Akt pathway,the number of TH（+）neurons in MPTP+GSK+LY group further decreased.In this study,it was confirmed that the number of TNF-αin PD model mice was higher than that in control group.It was also observed that after the inhibition of Orai1,TNF-αwas further up-regulated compared with the MPTP group.After the inhibition of PI3K/Akt pathway,TNF-αexpression in MPTP+GSK+LY group further increased（P<0.05）.Conclusion:1.The decreased expression of Orai1 is involved in the occurrence of PD;2.Down-regulation of Orai1 expression impairs the motor ability of PD mice and increases the expression of neuroinflammatory cytokines;3.Down-regulation of Orai1 expression affects neuroinflammation through PI3K/Akt/NF-κB signal pathway,which leads to the decrease of dopaminergic neurons and the development of PD.