Mechanism Research Of SIRT3 In Chronic Neuropathic Pain By Ameliorating Neuroinflammation And Regulating Differentiation And Apoptosis Of Microglia

Chronic neuropathic pain is the result of unbalanced homeostasis in the central nervous system,which persists even when the primary causes are removed.Typical neuropathic pain is resulted by peripheral nerve injuries and seriously reduces patients’quality of life,with longer course and difficulty in treatment.Microglia are resident macrophages of the central nervous system.They play the dual roles of "pro-inflammatory" and "anti-inflammatory" by phenotypic differentiation and heterogeneity and maintain the homeostasis of the neuroinflammation.Chronic constriction injury（CCI）of the sciatic nerve is one of the most widely used animal models of neuropathic pain.In this study,gene enrichment analysis screens differential gene sets in the chronic process of CCI,by which the research target is focused.Mitochondrial SIRT3 is a member of the Sirtuins family,which is closely related to many basic biological functions such as cell survival or death,mitochondrial metabolism,immunity/inflammation and circadian rhythm.SIRT3,as class III histone deacetylase,is involved in regulating mitochondrial energy metabolism and protein acetylation.SIRT3protects mitochondria from DNA damage and oxidative stress induced cell death by reducing reactive oxygen species and activating superoxide dismutase and catalase.Basal expression of SIRT3 in mouse cerebral cortex,spinal dorsal horn,and dorsal root ganglion is relatively low,where the expression of SIRT3 increases in the early stage of CCI and then gradually decreases with the development of chronic pain.According to the CCI behavioral tests by measuring the painful threshold,Sirt3 knockout(Sirt3^-/-)mice get more severe symptoms of allodynia and hyperalgesia than the wild-type mice.The painful severity in thermal and tactile hypersensitivity becomes more significant in the later stages of CCI.The transcriptional level of the proinflammatory cytokine TNFαin the brain of Sirt3^-/-mice is significantly higher than that of wild-type mice.The chronic pain induced by CCI causes proliferation and morphological changes of microglia.In pain-related brain regions,the number of microglia in Sirt3^-/-mice is larger than that of wild-type mice.However,the relative extent of proliferation of microglia induced by CCI in Sirt3^-/-mice is less than that of wild-type mice.By flow cytometry analysis,gene silencing of Sirt3 by si RNA is found to inhibit the"anti-inflammatory"phenotype of microglia by reducing the M2 phenotypic marker CD206 and increases the pro-inflammatory M1 phenotypic markers CD45,CD68 and CD86.The differential gene set ratio between brain tissues of Sirt3^-/-mice with CCI chronic pain and brain tissues of wild-type CCI mice is compared with the database of KEGG signaling pathway.Nicotinamide metabolism,tumor necrosis factor signaling pathway,chemokine signaling pathway and JAK/STAT signaling pathway are closely related with the effect of SIRT3 on the outcomes for chronic neuropathic pain.By quantitative analysis of Western blot,the expression of p-STAT,p-STAT3,p-STAT6,and p-P65（NF-κB）in the brain of mice is found to share the same trend with that of SIRT3during the pathogenesis of CCI,namely increases in the acute phase of CCI and decreases in the chronic phase.In microglia of Sirt3^-/-mice,the expression of p-JAK1,p-STAT1,and p-P65 is significantly stimulated by"pro-inflammatory"mediators（INFγ/LPS）or"anti-inflammatory"mediator（IL-4）.After gene silencing of Sirt3 in HMC3 cells,p-JAK1is significantly stimulated by pro-inflammatory mediators.In contrast,p-STAT6 in Sirt3^-/-microglia is significantly reduced under the stimulation of IL-4 while p-STAT3 is neither affected by IL-4 nor IFNγbut significantly decreases under the stimulation of LPS（activator of microglia）.Arg-1（M2 phenotypic marker）is also significantly reduced in Sirt3^-/-microglia.The results of co-immunoprecipitation experiments show that SIRT3directly binds to STAT1 or STAT3,resulting in an enzyme-dependent deacetylation modification.The combination of SIRT3 and STAT1 inhibits the phosphorylation of STAT1 and P65.STAT1 is activated by phosphorylation and then regulates the transcription of its downstream pro-inflammatory genes.In contrast,activated p-STAT3regulates the transcription of anti-inflammatory genes.The combination of SIRT3 and STAT3 increases p STAT3.rt/q PCR is used to quantitatively analyze the cytokines and chemokines associated with different phenotypes of microglia.Pro-inflammatory mediators like TNF-α,i NOS,IL-6,CX3CL1 and CXCL11 get increased transcription while the transcription of anti-inflammatory cytokine IL-10 is decreased in Sirt3^-/-microglia,indicating that SIRT3 inhibits the function of pro-inflammatory M1 microglia and promotes the anti-inflammatory effect of M2 microglia.The Agilent Sure Print G3 Mouse Gene Expression v28x60K Microarray is used to The m RNA expression of two pro-apoptotic protein,XAF1（XIAP-associated factor 1）and GBP2（Guanylate-binding protein 2）,is found to be extremely low in Sirt3^+/+samples but surges in Sirt3^-/-samples by dis-inhibition from SIRT3.Both Xaf1 and Gbp2 transcription are regulated by type I interferon.XAF1 and GBP2 can enhance IFN-β-induced apoptosis.3-TYP specifically inhibits the expression of SIRT3 in HMC3 microglial cells and increases the transcription level of IFN-βstimulated by LPS.LPS also activates the acetylated H4K16,H3K27 and H3K56,and significantly increases cleaved-Caspase3 in Sirt3^-/-microglia.It is indicated that SIRT3 can de-acetylate histones of microglia and inhibit the type I interferon-induced apoptosis under LPS stimulation.Interestingly,STAT1and NF-κB（promoting M1 phenotype）inhibited by SIRT3 are also key components of the pro-apoptotic signaling pathway.To sum up,pain stimulation and chronic pain result in adaptive accommodation of SIRT3 in the central nervous system.SIRT3 relieves allodynia and hyperalgesia in mice with neuropathic pain.On one hand,the protective effect of SIRT3 on microglia contributes to its regulation of phenotypic differentiation by inhibiting the M1 phenotype and promoting the M2 phenotype.On the other hand,SIRT3 inhibits microglial apoptosis thus enhances the tolerance to chronic pain,leading the neuroinflammation towards repairment and restoration homeostasis in the central nervous system.