| BackgroundNeuropathic pain(neuropathic pain,NP)is the pain directly caused by the injury or disease of the somatosensory nervous system,and it is a chronic disease caused by abnormal pathological changes caused by tissue damage or inflammation related to the nervous system.NP patients have various clinical manifestations and a long course of disease.It is recognized as one of the most difficult pain syndromes to treat,and its treatment effect is often unsatisfactory.Therefore,clarifying the pathogenesis of NP has important clinical significance and social value.In this study,bioinformatics analysis was performed on spinal cord tissue samples and sequencing data of the mouse SNI model,and the mouse sciatic nerve branch selective injury(SNI)model and the LPS-stimulated mouse spinal cord microglia(BV2)model were used to explore the E3 Ubiquitin ligase MDM2(Transformed 3T3 Cell Double Minute 2)ubiquitination modification activating transcription factor 3(Activating Transcription Factor 3,ATF3)regulates the molecular mechanism of spinal cord microglial inflammatory response mediating neuropathic pain.MethodsUse the GEO database to download the chip data set GSE5425 with SNI mouse model DRG and spinal cord samples and sequencing dataset 123919.The data set was screened for key genes by RStudio software and Comparative Toxicology Database(CTD),and the E3 ubiquitin ligase upstream of the key genes was screened by Ubibrowser online ubiquitination database.Use RStudio software to do downstream functional enrichment analysis,combined with single-cell data sets to analyze the screening target gene ATF3,and verify it through the mouse SNI model and LPS-induced BV2 cell model.In LPS-stimulated BV2 microglial cells,MDM2 was overexpressed and ATF3 was inhibited,the expression level of ATF3 was detected by real-time fluorescent quantitative PCR(qRT-PCR),and the downstream NF-κB,NLRP3 and inflammatory factors were detected by Western blot index.At the same time,the ATF3 knockout mouse spinal cord microglial cells were used to create a SNI model to observe the changes of ATF3 knockout mouse microglial cells on the pain behavior of mice,and the changes in the activation state of microglial cells were detected by immunofluorescence.Finally,the ubiquitination-related experiments were used to verify that MDM2 is the E3 ubiquitin ligase of ATF3.ResultThe differential gene ATF3 was screened out by bioinformatics analysis of GSE5425 dataset and GSE123919 dataset of GEO database.At the same time,Ubibrowser ubiquitination database analysis results showed that MDM2 may be the E3 ubiquitin ligase of ATF3.The study found that in the BV2 cell model,LPS induced the high expression of MDM2 and ATF3 in the microglial activation model,overexpression of MDM2 inhibited the expression of ATF3,overexpression of MDM2 and knockdown of ATF3 significantly reduced NF-κB,NLRP3 and related inflammation.expression of sex factors.And the expression of ATF3 was positively correlated with the expression of NF-κB,NLRP3 and inflammatory factors,and the expression of MDM2 was negatively correlated.In addition,the expressions of ATF3 and MDM2 were significantly increased in the spinal cord of SNI mice.SNI mice knocking out ATF3 in microglia had upregulated mechanical pain threshold and attenuated microglial activation compared with flox mice.Finally,the ubiquitination-related experiments verified that the E3 ubiquitin ligase MDM2 may regulate the expression level of the substrate protein ATF3 through ubiquitination.ConclusionThese results suggest that the E3 ubiquitin ligase MDM2 alleviates the inflammatory response and inhibits microglial activation by regulating ATF3 through ubiquitination,thereby alleviating the development of neuropathic pain. |
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