Mechanism Of Deacetylase Sirt3 Ameliorating Diabetic Neuropathic Pain By Regulating Glucose Metabolism Of Microglia

Diabetic Neuropathic Pain（DNP）is a common complication of diabetes,primarily affecting the sensory,motor,and autonomic nervous systems of patients.Clinically,DNP is characterized by abnormal pain sensations in the extremities,such as tingling,burning,electric shock-like pain,or numbness,especially pronounced at night.This pain is persistent and/or episodic,significantly impacting the quality of life of patients.Epidemiologically,the incidence of DNP is closely related to the prevalence of diabetes.With the increasing number of diabetic patients globally,the number of individuals with DNP is also growing.It is estimated that about half of diabetic patients will experience varying degrees of neuropathy caused by hyperglycemia,with about 30%-40%of cases accompanied by painful symptoms.Clinical therapies for DNP are limited and often ineffective,mainly due to the unclear pathogenesis of DNP.Therefore,exploring the pathogenesis of DNP and identifying potential molecular targets to counteract it is crucial for improving the quality of life of patients and reducing their physical,mental,and economic burdens.In this study,we constructed a Type I diabetic mouse model for DNP research using a single high-dose intraperitoneal injection of streptozotocin（STZ）,with blood glucose levels above 16.7 mmol/L indicating successful modeling.To explore the molecular mechanisms of DNP,we identified the differential gene Sirt3 as a focus through transcriptome sequencing.We found that differential genes were mainly enriched in insulin signaling pathways,glycolysis/gluconeogenesis,HIF-1,etc.Differential Biological Processes（BP）included carbohydrate metabolic processes,pyruvate metabolic processes,ATP generation,etc.Sirt3is an NAD⁺-dependent deacetylase,primarily located in mitochondria and is the strongest deacetylase among the three sirtuins family proteins in mitochondria.It regulates the activity of many downstream proteins through deacetylation,maintaining mitochondrial function and regulating mitochondrial fission/fusion.Although there are many studies on the relationship between Sirt3 and inflammation and cancer,whether Sirt3 is a key link in the pathogenesis of DNP is not clear.Therefore,this study examined the changes in Sirt3expression over time post-modeling through Western blot and Q-PCR.The results showed that Sirt3 transcription decreased and protein levels lowered over time,suggesting that the downregulation of Sirt3 may be involved in DNP.Thus,we constructed Sirt3 gene knockout mice to explore its role in the pathogenesis of DNP.Twenty-one days after modeling,Sirt3^-/-diabetic mice showed more severe tactile allodynia and thermal hyperalgesia compared to Sirt3^+/+diabetic mice.Notably,Sirt3 deficiency did not affect blood glucose levels in both DNP mice and normal mice.To explore the impact of Sirt3 in DNP,we co-stained Sirt3 with microglia markers（IBA-1）,astrocyte markers（GFAP）,and neuron markers（Neu N）,finding that Sirt3 was mainly located in microglia and neurons.Twenty-one days after modeling,Sirt3 deficiency led to increased IBA-1 expression in the spinal cord,with no effect on GFAP expression,indicating that Sirt3 deficiency increased microglial proliferation and activation.Due to the activation of microglia,the transcription of inflammatory cytokines（IL-6,IL-1β,TNF-α）in the spinal dorsal horn of Sirt3^-/-DNP mice increased significantly compared to Sirt3^+/+DNP mice.Given the established role of NF-κB and MAPK signaling pathways in microglial activation and neuroinflammation,we studied the effect of Sirt3 deficiency on their activation status.Compared to Sirt3^+/+DNP mice,levels of phosphorylation of P65,ERK,JNK,and P38 were elevated in Sirt3^-/-DNP mice.Transmission electron microscopy revealed that in the spinal dorsal horn of Sirt3^+/+diabetic mice,myelin sheaths displayed a concentric arrangement with occasional areas showing slight relaxation and separation.Some individual mitochondria showed mild swelling.In Sirt3 deficient diabetic mice,the structure of the myelin sheath lamellae in the spinal dorsal horn appeared loose,distorted,folded,or even fragmented and disintegrated.In Sirt3 deficient diabetic mice,the demyelination changes were observed,and the mitochondria showed a higher degree of swelling compared to that of control mice.Glycolysis plays a key role in the activation process of microglia.Next,we aimed to explore whether the regulatory effect of Sirt3 on neuroinflammation is related to glycolysis.Western blot analysis confirmed that on day 21post-modeling,the protein levels of key glycolysis enzymes（HK2,PKM,LDHA）in the spinal dorsal horn of DNP mice increased significantly,and their expression further increased in the spinal dorsal horn of Sirt3^-/-DNP mice.Additionally,Sirt3 deficiency further increased the levels of glycolysis products lactate and pyruvate in the spinal dorsal horn of diabetic mice.Therefore,we used the glycolysis inhibitor 2-DG to explore the impact of glycolysis on the pain phenotype and neuroinflammation of DNP mice.Behavioral test showed that intraperitoneal injection of 2-DG significantly relieved the tactile allodynia and thermal hyperalgesia in DNP mice;transmission electron microscopy showed that intraperitoneal injection of 2-DG relieved the demyelination and mitochondrial swelling in the spinal dorsal horn of DNP mice,suggesting that Sirt3 deficiency may exacerbate neuroinflammation and diabetic neuropathic pain by increasing spinal cord tissue glycolysis.To confirm the role of Sirt3 in microglial aerobic glycolysis and inflammation,we isolated and cultured primary microglia from Sirt3^-/-and Sirt3^+/+mice.Western blot showed that at high glucose environment,IBA-1 protein levels in Sirt3^-/-microglia were higher than in Sirt3^+/+counterparts,and the phosphorylation levels of inflammation-related signaling pathways including P65,P38,JNK,and ERK were elevated.Q-PCR results suggested that the transcription of inflammatory factors in Sirt3^-/-microglia was significantly increased compared to that of Sirt3^+/+microglia at high glucose environment.To determine the role of Sirt3 in metabolic reprogramming induced by high glucose,we examined the expression of glycolysis-related enzymes and the production of lactate and pyruvate in primary microglia:Sirt3 deficiency further enhanced the upregulation of HK2,PKM2,and LDHA expression caused by high glucose stimulation,accompanied by increased levels of pyruvate and lactate production.In addition,we also assessed the extracellular acidification rate（ECAR）and oxygen consumption rate（OCR）of primary microglia:under high glucose conditions,the intensity of aerobic glycolysis in microglia increased,and Sirt3^-/-microglia showed a further increase in aerobic glycolysis intensity.In contrast,compared to Sirt3^+/+microglia,the OCR of Sirt3^-/-microglia under high glucose conditions decreased.We then overexpressed Sirt3in the BV-2 cell line using lentiviral vectors.Western blot results showed that overexpression of Sirt3 can alleviate the upregulation of IBA-1 and the activation of NF-κB and MAPK signaling pathways caused by high glucose stimulation.Q-PCR results suggested that overexpression of Sirt3 can significantly reduce the expression of inflammatory factors caused by high glucose stimulation.Overexpression of Sirt3 could also alleviate the upregulation of key glycolysis enzymes and the increase in glycolysis metabolite production caused by high glucose stimulation.We also assessed the ECAR and OCR of the BV-2 cell line:overexpression of Sirt3 could alleviate the increase in aerobic glycolysis intensity and the decrease in oxygen consumption rate caused by high glucose stimulation.Many studies have focused on the downstream mechanisms by which Sirt3 maintains mitochondrial homeostasis and cellular oxidative phosphorylation,such as the accumulation of ROS and stabilization of HIF-1αcaused by the absence of Sirt3.However,there are few studies focus on the upstream mechanisms of Sirt3 expression and degradation,i.e.,how high glucose stimulation regulates Sirt3 protein levels.Therefore,we conducted a protein-protein interaction network analysis and found that Fox O1 may be the key molecule regulating Sirt3 expression.The GEPIA database also suggested a positive correlation between Fox O1 and Sirt3 expression in human spinal cord tissues.We then searched the promoter sequence of the mouse Sirt3 gene and used dual-luciferase reporter assays to explore whether the transcription factor Fox O1 affects the Sirt3 promoter.The results showed that exogenously expressed Fox O1 protein could activate Sirt3 luciferase activity in293T cells;after point mutation of the potential Fox O1 binding site in the Sirt3 promoter,the luciferase activity induced by exogenously expressed Fox O1 protein was significantly reduced,confirming that Fox O1 positively regulates Sirt3 expression.Ch IP-q PCR results also suggested that Fox O1 could bind to the Sirt3 promoter,and high glucose stimulation could reduce their binding.Phosphorylation of Fox O1 is usually accompanied by translocation out of the nucleus and loss of transcriptional activity.We analyzed the subcellular localization of Fox O1 in microglia through Western blot and observed that with increasing glucose concentration in the environment,the cytoplasmic localization of Fox O1increased while its nuclear localization decreased.Additionally,overexpression of Fox O1was able to weaken aerobic glycolysis in microglia induced by high glucose,as reflected by the reduced protein levels of key enzymes including HK2,PKM2,and LDHA.This effect was not observed in Sirt3^-/-microglia,indicating that the regulatory role of Fox O1 on aerobic glycolysis depends on Sirt3.In microglia treated with the Fox O1 inhibitor AS1842856 under normal culture medium glucose concentrations,Sirt3 m RNA and protein levels significantly decreased in a concentration-dependent manner.These results suggest that the downregulation of Sirt3 induced by high glucose is dependent on Fox O1.Numerous studies have confirmed the important role of Akt in regulating intracellular metabolic pathways and its regulatory effects on the Fox O family of proteins.Therefore,we explored whether the effect of high glucose on Fox O1 is related to Akt activity.Western blot results showed that phosphorylation of Akt at Ser-473 significantly increased in the spinal dorsal horn of diabetic neuropathy（DNP）mice,and similar results were observed in microglia cultured in high glucose.Subsequently,we used the pan-Akt inhibitor GSK690693,and immunofluorescence results suggested that inhibiting Akt activity could reverse the translocation of Fox O1 from the nucleus induced by high glucose.Similarly,these findings were verified by Western blot,indicating that GSK690693 could alleviate the phosphorylation of Fox O1 and the downstream reduction in Sirt3 expression induced by high glucose stimulation.Based on these findings,we further confirmed the regulatory effect of GSK690693 on Sirt3 expression at the transcriptional level using Q-PCR.The autophagy-lysosomal pathway（ALP）and ubiquitin-proteasome system（UPS）are the two main mechanisms for intracellular protein degradation.To further study the mode of Sirt3 degradation under high glucose stimulation,we treated microglia in a high glucose environment with the ALP inhibitor chloroquine（CQ）and the UPS inhibitor MG132.Western blot results showed that the downregulation of Sirt3expression could be blocked by CQ but not MG132,indicating that the reduction in Sirt3 is caused by the Akt-Fox O1 signaling pathway and the autophagy-lysosomal pathway（ALP）under high glucose stimulation.Furthermore,the addition of CQ to microglia simultaneously treated with high glucose and GSK690693 did not further increase Sirt3 protein levels.Metformin is a commonly used oral medication for the treatment of type II diabetes,and its molecular mechanism involves activating adenosine monophosphate-activated protein kinase（AMPK）,which may affect the Akt signaling pathway.Therefore,we wanted to know whether metformin has the same effect as the Akt inhibitor GSK690693.We have already demonstrated that in primary microglia,metformin effectively alleviated the reduction of Sirt3 expression caused by high glucose,both at the m RNA and protein levels.Finally,we explored the potential of metformin in treating DNP through daily intraperitoneal injections.Since our study used a type I diabetes model,metformin showed limited ability to reduce blood glucose.However,metformin treatment significantly alleviated tactile allodynia and thermal hyperalgesia in DNP mice.Additionally,we observed that intraperitoneal injection of metformin rescued the downregulation of Sirt3 at both m RNA and protein levels caused by high blood glucose.Considering the established association between Sirt3 and microglial inflammatory activation,we further explored the effect of metformin on IBA-1 expression in the dorsal horn of the spinal cord.Immunofluorescence analysis showed that metformin reduced the increase in IBA-1 expression induced by high blood glucose,which was further confirmed by Western blot.Q-PCR results suggested that metformin significantly alleviated the production of inflammatory factors caused by high blood glucose.These results indicate that metformin can alleviate neuroinflammation and DNP,an effect that depends on its regulation of Sirt3 protein levels.In summary,high blood glucose reduces Sirt3 expression through the Akt-Fox O1signaling pathway on one hand,and on the other hand,activates the ALP pathway to enhance the degradation of Sirt3.The combined effect of these mechanisms leads to the downregulation of Sirt3,causing shift in microglial metabolism towards glycolysis,thereby promoting the neuroinflammation and DNP.Targeting Sirt3 directly or indirectly may be a viable strategy to control neuroinflammation and alleviate DNP.