| Objective:To explore the effect and mechanism of glycolysis on dopaminergic neuron apoptosis,α-Synuclein deposition and peripheral Th17/Treg axis changes in PD.Methods:The PD mouse model was established by intraperitoneal injection of MPTP,and the model was evaluated by behavior test,immunofluorescence staining and Western blot.Glycolysis was inhibited by injection of hexokinase 2(HK2)inhibitor,and the apoptosis of dopaminergic neurons,α-Syn deposition,and changes in Th17/Treg axis were assessed in PD mice.The expression of HK2 and LDHA and levels of pyruvate and lactate in Substantia nigra pars compacta(SNpc)of PD mice were detected to analyze the changes of glycolysis.The effect of lactate on apoptosis of human neuroblastoma SH-SY5Y cell line was evaluated,and the changes of AMPK/AKT/m TOR pathway were detected by Western blot to reveal the effect of glycolysis on dopaminergic neurons andα-Syn deposition.The level of Th17/Treg axis andα-Syn in peripheral blood of PD patients were detected by flow cytometry and Elisa,and the correlation between them was analyzed.The changes of peripheral Th17/Treg axis in MPTP-injected mice and A53T mice were detected by flow cytometry.Transgenic reporter mice carrying Foxp3 lineage reporter gene were employed and injected with MPTP,and the Treg stability in PD mouse model was detected.CD4~+T cells were sorted by MACS from A53 transgenicα-Syn overexpressing(A53T)mice and C57BL6 control mice.Differently expressed genes in CD4~+T cells were screened by RNA-Seq,and verified in vivo and in vitro.The gene overexpressed plasmid and Luciferase reporter activity were used to explore the effect ofα-Syn on the transcription of target gene.The effect of target gene in PD animal model was verified.Results:By inhibiting glycolysis,the apoptosis of dopaminergic neurons andα-Syn deposition in SNpc of PD mice were alleviated;flow cytometry and immunofluorescence staining showed that the activation of immune cells was alleviated,mainly manifested as reduced microglia activation,and improved peripheral Th17/Treg axis transition.The expression of HK2 and LDHA were significantly up-regulated in SNpc of PD mice.The level of pyruvate was decreased,and which of lactate was increased,suggesting that the level of glycolysis in SNpc of PD mice was increased.In addition,lactate induced the activation of the AMPK/AKT/m TOR pathway and promote cell apoptosis in SH-SY5Y cells,while inhibition of glycolysis alleviated the accumulation of lactate and the activation of the AMPK/AKT/m TOR pathway.In the peripheral blood of PD patients,the proportion of Th17was increased and which of Treg was decreased.The level ofα-Syn was significantly elevated in the plasma of PD patients,which was positively correlated with the elevation of Th17 and the reduction of Treg.In the MPTP-induced PD mouse model and A53T mice,the proportion of Th17 increased while Treg decreased in the spleen.Furthermore,the stability of Tregs was impaired in MPTP-injected transgenic reporter mice of the Foxp3 lineage,and the proportion of CD4~+IL-17A~+Foxp3~+Treg increased,indicating IL-17A~+acquisition in Tregs.Transcriptomic results indicated that RORC gene was significantly up-regulated in CD4~+T cells of A53T mice.At the same time,RORC-encoded RORγt was also significantly up-regulated in Th17 and Treg of MPTP-injected mice and inα-Syn specific Tregs,na?ve T cells;the dual-luciferase reporter gene results suggested thatα-Syn promoted the expression of RORC promoter fragment in 293T cells.And RORγt inhibitor treatment in MPTP-injected mice alleviated neurodegeneration,Th17 cell infiltration in SNpc and peripheral Th17/Treg imbalance in spleen.Conclusion:Inhibition of glycolysis alleviated the activation of AMPK/AKT/m TOR pathway induced by lactate in PD,and then rescued the apoptosis of dopaminergic neuron and deposition ofα-Syn.α-Syn deposition induced the Th17 acquisition in Th17/Treg axis by promoting the transcription of RORC.Therefore,inhibition of glycolysis may regulate Th17/Treg axis by alleviatingα-Syn deposition. |
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