The Function And Mechanism Of Sirt3 In Regulating Microglial Activation During Vascular Cognitive Impairment

ObjectiveVascular Cognitive Impairment（VCI）refers to a broad category of diseases ranging from mild cognitive impairment to dementia caused by cerebrovascular diseases and their risk factors,imposing a great burden on society and families.White Matter Lesions（WMLs）are important pathological changes in VCI,and oligodendrocytes are the main target cells attacked during the process of white matter damage in VCI.Previous studies and preliminary studies by our research group have found that microglial activation and its related neuroinflammatory response play a key role in the occurrence and development of white matter damage and cognitive impairment in VCI.Inhibiting microglia activation can significantly reduce white matter damage and improve cognitive impairment in VCI mice.These results suggest that regulating neuroinflammation mediated by microglia may be an effective therapeutic target for VCI.Sirt3 is a mitochondrial deacetylase primarily expressed in mitochondria,which can deacetylate various mitochondrial proteins and participate in multiple functions of mitochondria,including anti-inflammatory,antioxidant,energy metabolism,and apoptosis.Studies have shown that Sirt3 has an inhibitory effect on neuroinflammation in various central nervous system diseases,including depression and postoperative cognitive dysfunction.Currently,there is no research on the role and mechanism of Sirt3 in VCI,while neuroinflammation is an important pathological process in white matter damage in VCI.This study aims to explore the role of Sirt3 in neuroinflammatory responses in VCI,providing experimental data and theoretical basis for further elucidating the pathogenesis of VCI and exploring new therapeutic targets.MethodsThis study consists of four parts:Part Ⅰ:Weighted Gene Co-expression Network Analysis（WGCNA）,pathway enrichment,and other bioinformatics methods,are used to analyze VCI-related dataset GSE122063 in the GEO database,screening key gene expression modules and performing functional enrichment.After constructing a Bilateral Carotid Artery Stenosis（BCAS）model,single-cell transcriptome sequencing is performed on mouse hippocampal tissue,drawing a single-cell transcriptomic map of the hippocampal tissue in the VCI mouse model,and specifically analyzing the gene expression characteristics of microglia therein.The correlation between Sirt3 expression levels and VCI in the GSE122063 dataset is analyzed.Part Ⅱ:The BCAS mouse model mimicking VCI is constructed,and changes in cerebral blood flow in mice 30 days after BCAS modeling are detected using a laser speckle blood flow detection system.Cognitive functions of the two groups of mice are evaluated through Y-maze,New Object Recognition（NOR）,and Morris water maze.Luxol Fast Blue（LFB）staining,Immunofluorescence（IF）staining,and Western Blotting（WB）are used to assess white matter damage in the corpus callosum and hippocampus 30 days after modeling.Changes in the expression level of Sirt3 in primary microglia and BV2microglia after BCAS modeling and Oxygen Glucose Deprivation/Re-oxygenation（OGD/R）are verified by WB and Real-Time Quantitative Polymerase Chain Reaction（RT-q PCR）.Part Ⅲ:Cognitive functions of Sirt3^+/-,Sirt3^-/-,Sirt3^+/-BCAS,and Sirt3^-/-BCAS mice are evaluated through Y-maze,NOR,and Morris water maze experiments.Iba-1 IF staining is used to label microglia,and Enzyme-linked Immunosorbent Assay（ELISA）is used to detect the expression levels of IL-1β,IL-6,TNF-α,and IL-4 in hippocampal tissue from different groups of mice.LFB staining,MBP IF staining,and electron microscopy are used to examine white matter conditions in the corpus callosum of the four groups of mice.After constructing a Sirt3 interfering lentivirus and transfecting BV2 microglia,the transfection efficiency is verified by WB;Ed U staining is used to detect cell proliferation in sh-Ctrl,sh-Sirt3,sh-Ctrl OGD,and sh-Sirt3 OGD groups;ELISA is used to detect the expression levels of IL-1β,IL-6,TNF-α,and IL-4 in supernatants from different cell groups.Part Ⅳ:Single-cell transcriptome sequencing is performed on hippocampal tissue from Sirt3^+/-BCAS and Sirt3^-/-BCAS mice,and differential expressed genes in microglia from the two groups are enriched through various databases.WB is used to detect the expression of glycolysis-related proteins PKM2,HK2,LDHa,and HIF-1αin hippocampal tissue from the four groups of mice and in BV2 microglia after knocking down and overexpressing Sirt3;colorimetric assay kits are used to detect the expression levels of pyruvate and lactate in tissues and cells.ResultsPart Ⅰ:WGCNA identified the gene expression module most related to VCI.Pathway enrichment revealed that this gene module is primarily enriched in inflammatory responses,microglial activation,NF-κB signaling pathway,and cytokine production,suggesting that neuroinflammation plays a significant role in the pathogenesis of VCI.Differential gene enrichment analysis of microglia in single-cell transcriptomics suggested significant activation of microglial migration,inflammation,mitochondria,NF-κB,and histone acetylation modification in the BCAS group.Sirt3 expression was found to be decreased in brain tissue of VCI patients in the GSE122063 dataset.Part Ⅱ:After BCAS modeling,the spontaneous alternation rate and new arm index in the Y-maze,as well as the new object recognition index in the NOR experiment,were lower in the BCAS group than in the Sham group.In the water maze experiment,the escape latency of the BCAS group began to significantly increase from day 4,and the number of platform crossings and the percentage of time spent in the target quadrant significantly decreased,indicating significant cognitive impairment in the BCAS group mice.LFB staining results showed that 30 days after BCAS modeling,myelin fibers in the corpus callosum became loose,indicating significant white matter damage.WB and IF staining results suggested a significant decrease in MBP expression in the hippocampal region,indicating white matter damage in the corpus callosum and hippocampus of VCI model mice induced by BCAS.WB and PCR experiments showed that the expression of Sirt3 in mouse hippocampal tissue after BCAS modeling,primary microglia and BV2microglia after OGD/R significantly decreased.Part Ⅲ:After BCAS modeling in Sirt3^+/-mice and Sirt3^-/-mice,the spontaneous alternation rate and new arm index in the Y-maze,as well as the new object recognition index in the NOR experiment,were significantly lower in Sirt3^-/-BCAS mice than in Sirt3^+/-BCAS mice.In Morris water maze,the escape latency of the Sirt3^-/-BCAS group began to significantly increase from day 5 compared to the Sirt3^+/-BCAS group,and the number of platform crossings and the percentage of time spent in the target quadrant significantly decreased,suggesting that Sirt3 knockout exacerbated cognitive impairment in VCI mice.Iba-1 IF staining results showed a significant increase in microglia in the Sirt3^-/-BCAS group compared to the Sirt3^+/-BCAS group.ELISA results showed that the expression of IL-1β,IL-6,and TNF-αin the Sirt3^-/-BCAS group was significantly higher than in the Sirt3^+/-BCAS group,while the expression level of IL-4 was significantly lower,suggesting that Sirt3 knockout exacerbated the neuroinflammatory response in the hippocampus of VCI mice;（3）Compared with the Sirt3^+/-BCAS group,the Sirt3^-/-BCAS group had a lower fluorescence intensity of MBP in the corpus callosum,a higher white matter damage score,and more myelin structure damage.In the hippocampal region,the fluorescence intensity of MBP was lower,and the number of GST-pi positive cells further decreased,suggesting that Sirt3 knockout exacerbated white matter damage in VCI mice.After knocking down the expression of Sirt3 in BV2 microglia,after OGD/R,Ed U staining suggested that cell proliferation in the knockdown group was more significant,and the expression levels of IL-1β,IL-6,TNF-αin cell supernatants increased,while the expression level of IL-4 decreased.Part Ⅳ:After enriching the differential genes in microglia from the hippocampus of Sirt3^+/-BCAS and Sirt3^-/-BCAS mice,the results showed that they were mainly enriched in mitochondria,glycolysis,NF-κB,and HIF-1 signaling pathways.WB results suggested that compared with Sirt3^+/-mice,the expression of PKM2,HK2,LDHa,HIF-1α,and p-p65in the hippocampal tissue of Sirt3^+/-BCAS mice significantly increased,and these glycolysis-related gene expressions further increased after Sirt3 knockout.In BV2 cell studies,PKM2,HK2,LDHa,and HIF-1αin BV2 cells significantly increased after OGD/R,and these glycolysis-related protein expressions further increased after Sirt3knockdown compared to the simple OGD/R group.Colorimetric assay kit detection suggested that compared with the Sirt3^+/-group,the expression of pyruvate and lactate in the hippocampal tissue of Sirt3^+/-BCAS mice significantly increased,and the expression of pyruvate and lactate further increased after Sirt3 knockout.After OGD/R,the expression levels of pyruvate and lactate in BV2 cells significantly increased,and Sirt3 knockout further increased the expression levels of pyruvate and lactate in BV2 cells after OGD/R modeling.ConclusionThe activation of microglia and the associated neuroinflammation play a significant role in the pathogenesis of VCI,with the NF-κB pathway,HIF-1 pathway,glycolysis,and mitochondria significantly enriched in VCI microglia.Sirt3 expression was found to be reduced in the brain tissue of VCI patients in the GEO dataset,hippocampal tissue of BCAS mice,primary microglia and BV2 cells after OGD/R.In vivo,Sirt3 knockout significantly exacerbated cognitive impairment and white matter damage in VCI mice,promoted microglial proliferation and neuroinflammatory response.In vitro,knocking down of Sirt3 promoted pro-inflammatory activation of BV2 cells after OGD/R.Furthermore,Sirt3 knockout exacerbated the level of glycolysis in the hippocampal tissue of VCI mice,and knocking down of Sirt3 increased the level of glycolysis in microglia after OGD/R.This study reveals that Sirt3 can regulate the activation of microglia to influence the pathological progression of VCI.