The Mechanism Of SIRT5 Mediating Anterior Cingulate Cortex Dendritic Remodeling In Neuropathic Pain

BackgroundsNeuropathic pain is a kind of chronic pain caused by somatosensory nervous system injury or disease,which is often accompanied by negative emotions such as anxiety,depression and sleep disorders.The processing dysfunction of related brain nuclei is also one of the causes of neuropathic pain.A large part of the identification of the brain mechanism of neuropathic pain is due to the development of brain magnetic resonance imaging and analysis techniques.Freesurfer magnetic resonance analysis can evaluate the thickness and surface area of gray matter to reflect the morphological characteristics of brain nuclei through local processing of T1-weighted magnetic resonance images.Postherpetic neuralgia is the most common type of neuropathic pain in clinical pain clinic.There are significant individual differences in the therapeutic effect of amitriptyline.Therefore,the use of Freesurfer method to analyze the cortical thickness of brain nuclei in patients with postherpetic neuralgia may be helpful to evaluate the efficacy of amitriptyline.Axonal orientation,diffusion and density imaging（NODDI）is a diffusion magnetic resonance scanning method which can observe the microstructure characteristics of gray matter of brain nuclei.Chronic sciatic nerve compression injury（CCI）is the most widely studied and used animal model of neuropathic pain.Using NODDI scan to observe whether there are gray matter microstructure changes such as dendritic remodeling in the brain nucleus of CCI mice and explore the mechanism may provide a new target for the treatment of neuropathic pain.Dendritic remodeling,which is the microstructure change of gray matter,is the result of the interaction of neuron metabolic state and neuronal microenvironment in the brain nucleus.The change of neuronal mitochondrial biogenetic state can affect dendritic remodeling.Microglia polarization is an important regulator of neuronal microenvironment.After activation,microglia are divided into pro-inflammatory microglia（M1 polarization）and anti-inflammatory microglia（M2 polarization）.M1 polarized microglia produce TNF-αand other cytokines can cause dendritic atrophy and remodeling,while M2 polarized microglia produce IGF-1 and other growth factors can alleviate dendritic atrophy and remodeling.SIRT5 belongs to the sirtuin protein family,which can regulate both energy metabolism and functional state of cells.The purpose of this study is to determine whether SIRT5 can affect the dendritic remodeling of related brain nuclei by regulating neuronal mitochondrial biogenesis and polarization transition of microglia in neuropathic pain.MethodsThe pain visual analogue scale（VAS）and Hamilton depression scale（HAMD）in patients with postherpetic neuralgia before and after treatment with amitriptyline for 8weeks were retrospectively analyzed.Free Surfer method was used to analyze the brain magnetic resonance data of patients before treatment and matched healthy volunteers to evaluate the difference of cortical thickness between the two groups,and to analyze the correlation between the cortical thickness of brain nuclei and the therapeutic effect of amitriptyline.After the establishment of CCI model in male C57/BL6J mice,the changes of mechanical tactile pain and thermal pain hypersensitivity threshold were detected by Von Frey fiber and radiation calorimeter,the anxiety-like behavior was evaluated by opening test and elevated plus maze,and the depression-like behavior was evaluated by forced swimming test and tail suspension test.After the mouse model of neuropathic pain with anxiety-depression behavior was successfully established,NODDI scan was used to analyze and compare the in vivo brain magnetic resonance of mice in CCI group and sham group.The status of dendritic remodeling and activation of microglia in brain nuclei were detected by Golgi-Cox staining and immunohistochemistry respectively.RNA microarray was used to screen differential genes and related signal pathways.Western Blot Analysis,RT-q PCR and immunofluorescence were used to detect the expression of SIRT5,mitochondrial biogenesis related molecules and microglial polarization related molecules in related brain region.Through SIRT5 knockout mice,SIRT5 overexpression of neuronal cell lines,primary neuronal cells and microglia culture and interference,combined with cell experiments and in vivo experiments,to further explore the regulatory mechanism of SIRT5 in dendritic remodeling of related nuclei in neuropathic pain.Results1)The thickness of left anterior cingulate gyrus（ACC）,left posterior cingulate gyrus and dorsolateral cortex of right prefrontal lobe became thinner in patients with postherpetic neuralgia.The thickness of cortex in the left ACC was positively correlated with the reduction rate of VAS score,but there was no correlation between the thickness of each cortex and the reduction rate of HAMD score.It is suggested that the thickness of ACC in patients with postherpetic neuralgia before treatment is related to the therapeutic effect of amitriptyline.2)The pain behavior of mice in CCI group was related to the decrease of NDI index and the increase of ODI index in ACC.Furthermore,Golgi-Cox staining and immunohistochemistry showed the neuron dendritic atrophy remodeling and microglia activation in ACC of CCI group.3)In order to explore the mechanism of ACC dendritic remodeling induced by NP,differential gene screening and expression profile enrichment analysis were carried out in ACC region of CCI group and sham group 21 days after operation.The differential expression of SIRT5 and the change of mitochondrial biogenetic pathway were found respectively.Further observation at each time point after CCI modeling confirmed that the m RNA and protein expression levels of SIRT5 gradually increased in ACC region,and SIRT5 was expressed both in neurons and microglia.And the expression of molecular markers related to mitochondrial biogenesis decreased gradually.In terms of neurons,the overexpression of SIRT5 can also reduce the mitochondrial biogenesis of SH-SY5Y cells.Using Sirt5^-/-mice,it was confirmed that SIRT5 knockout could alleviate the disturbance of mitochondrial biogenesis and poor dendritic remodeling caused by NP on neurons in ACC region,and affect the expression of PGC-1α,a regulator of mitochondrial biogenesis.Furthermore,the morphological observation of primary neurons and the interference of PGC-1αconfirmed that SIRT5 affected the mitochondrial biogenesis and dendritic remodeling of neurons through PGC-1α.4)The expression of SIRT5 protein and m RNA increased in M1-polarized microglia,but not in M2-polarized microglia.Further studies found that SIRT5 knockout could significantly promote the transition from M1-stimulated microglia to M2-polarized microglia.STAT6 is the main regulator of M1/M2 polarization transition in microglia.SIRT5 knockout does not affect the expression of STAT6 protein in primary microglia,but increases the expression of phosphorylated STAT6 in microglia.Co-immunoprecipitation also confirmed the binding of SIRT5 and STAT6 in ACC tissue after CCI modeling.Further use of STAT6 phosphorylation inhibitor AS1517499 confir med that SIRT5knockout mediated the polarization of microglia from M1 to M2 through STAT6,and SIRT5 knockout could indeed alleviate the pain behavior of CCI model mice.Conclusion1)Freesurfer magnetic resonance analysis of patients with postherpetic neuralgia showed that the thinning of ACC cortex was related to the efficacy of amitriptyline;2)NODDI magnetic resonance scanning of CCI model mice showed that dendritic atrophy and remodeling of ACC were related to pain behavior;3)SIRT5 mediated dendritic remodeling of ACC by affecting neuronal mitochondrial biogenesis in neuropathic pain.4)SIRT5 knockout relieves neuropathic pain by promoting M2 polarization of microglia in ACC region.