MicroRNA-3473b Regulates The Expression Of TREM2 And Inhibits Autophagy In Inflammatory Pathogenesis Of Parkinson Disease

Objective:To explore the regulation and mechanism of microRNA-3473b(miR-3473b)and triggering receptor expressed on myeloid cells 2(TREM2)on the activation and autophagy of microglia in the pathogenesis of Parkinson’s disease(PD),and then to further clarify whether there is a direct regulatory relationship between miR-3473 b and TREM2,so as to clarify the role of miR-3473 b in the pathogenesis of PD.Methods:BV2(microglial cell line)and C57BL/6 mice were used as research objects.Lipopolysaccharide(LPS)and 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)were used as model drugs.In vitro,BV2 cells were treated with 0.5 μ g / ml and 1 μ g/ ml LPS for 24 hours to obtain the cell model of neuroinflammation,in vivo,the PD mouse model was obtained by intraperitoneal injection of 20mg/kg MPTP into C57BL/6 mice for five consecutive days.In the cell model,real-time quantitative PCR(RT-qPCR)and Western Blot were used to verify that both miR-3473 b and TREM2 were involved in LPS-induced inflammatory activity.The m RNA expression of Tumor necrosis factor alpha(TNF-α)and Interleukin-1 β(IL-1 β)induced by LPS was detected.The changes of autophagy in inflammatory activation of BV2 cells induced by LPS were verified by detecting the protein expression of p-ULK1,ULK1 and LC3B.In order to further verify the role of miR-3473 b,the expression of miR-3473 b in BV2 cells was inhibited through transfecting with miR-3473 b inhibitor,and then treated with 1 μ g / ml LPS for 24 hours.Then the expression of TREM2,p-ULK1,ULK1,LC3 B and the m RNA expression of TNF-α and IL-1 β were detected by Western blotting and RT-qPCR,so as to prove the regulation of miR-3473 b on the activation and autophagy of TREM2 microglia.BV2 cells were further transfected with TREM2 plasmid to increase the expression of TREM2,then the cells were treated with 1μg/ml LPS for 24 hours,then the protein expression of p-ULK1 and ULK1 and the m RNA expression of TNF-α and IL-1 β were detected by Western blotting and RT-qPCR,so as to detect the regulation of TREM2 in microglia activation and autophagy.And verify whether the anti-inflammatory and autophagy-promoting effects after inhibition of miR-3473 b are dependent on TREM2.After transfection of ULK1 plasmid into BV2 cells and treated with LPS,the expression of LC3 B protein was detected to determine the effect of ULK1 overexpression on autophagy and to verify whether the effect of miR-3473 b and TREM2 on autophagy was achieved by regulating the expression of ULK1.Finally,the target genes of miR-3473 b were predicted in Target Scan database,and the direct regulatory relationship between miR-3473 b,TREM2 and ULK1 was verified by double luciferase reporting experiment.In the PD mice,the activation of microglia was verified by immunofluorescence,and then the expression of miR-3473 b,TREM2,p-ULK1,ULK1 and LC3 B was detected by Western blotting and RT-qPCR.In order to further verify the role of miR-3473 b in PD,a cannula was placed in the right ventricle of mice by stereotactic operation,and then miR-3473 b antagomir(20n M,a total of 5μl)was injected through the catheter device every day for five days,and MPTP was injected intraperitoneally on the third day.After the establishment of the model,the expression of microglia was detected by immunofluorescence,and the expression of TREM2,p-ULK1,ULK1 and LC3 B was detected by Western blotting and RT-qPCR to determine the role of miR-3473 b in MPTP-induced PD model.Results:In vitro,the results of Western Blot and RT-qPCR showed that inhibiting the expression of miR-3473 b in BV2 cells significantly decreased the expression of pro-inflammatory factors TNF-α and IL-1 β induced by LPS,significantly promoted the expression of p-ULK1 and LC3 B protein,increased the ratio of LC3II/I,and promoted autophagy.Target Scan database predicted that TREM2 and ULK1 may be the direct targets of miR-3473 b.At the same time,overexpression of miR-3473 b could inhibit the expression of TREM2 and ULK1,while inhibition of miR-3473 b would promote the expression of TREM2 and ULK1.The dual-luciferase reporter confirmed that TREM2 is the direct target gene of miR-3473 b,but ULK1 is not.Overexpression of TREM2 could significantly inhibit the expression of pro-inflammatory cytokines TNF-α and IL-1β,and promote the expression of p-ULK1.Overexpression of ULK1 can significantly increase the ratio of LC3II/I and promote the occurrence of autophagy.In vivo,the results of Western Blot,RT-qPCR and immunofluorescence experiments showed that MPTP significantly induced neuroinflammation and promoted the expression of miR-3473 b,but also inhibited the autophagy of microglia in the midbrain,reduced the expression of p-ULK1 and LC3 B protein,and reduced the ratio of LC3II/I.However,injection of miR-3473 b antagomir into the substantia nigra compact zone of the midbrain can effectively inhibit the expression of miR-3473 b,thus weaken the activation of microglia,promote the expression of TREM2,p-ULK1 and LC3 B proteins,and promote the occurrence of autophagy.Conclusion:Inhibition of miR-3473 b can attenuate LPS-induced inflammatory activation of microglia and promote autophagy;Overexpression of TREM2 in microglia can inhibit LPS-induced inflammatory activation and promote autophagy by promoting the expression of p-ULK1;TREM2 is a direct target gene of miR-3473 b,and miR-3473 b can directly target TREM2 to regulate the activation and autophagy of microglia.Exogenous delivery miR-3473 b antagomir inhibits miR-3473 b in the brain can effectively attenuate MPTP-induced microglial inflammation and enhance autophagy by promoting the expression of TREM2/p-ULK1.