Study On The Mechanism Of Hippocampal Dysfunction In Mice Induced By Gut Microbial Absence Based On Lysine Acetylation

Background Major depressive disorder(MDD)is a leading cause of disability around the world and contributes greatly to the global burden of disease,with over 350 million people of all ages affected globally.MDD is caused by gene-environment interactions.There is growing evidence to suggest that gut microbiota is an important environmental factor that can shape brain function and behavior through the gut–microbiota–brain axis.Thus,gut microbiota alterations may be a key trigger for the development of many neuropsychiatric diseases.Recent research suggests that epigenetic modifications might relate to depression.In addition,many studies have reported that the microbiome and the epigenome interact,and gut microbiota activity can modify the host’s epigenome,thereby affecting gene expression.Here,we aimed to investigate whether there are epigenetic changes in acetylation under conditions of absence of gut microbiota.Objective By analyzing the changes of acetylation modification levels in the hippocampal tissues of germ-free mice and specific pathogen-free mice to explored the potential mechanism of gut microbiota regulating hippocampal functional.Methods 1.Male GF Kunming mice and SPF Kunming mice.The GF mice were kept in flexible film gnotobiotic isolators.The SPF mice were housed in standard animal facilities.The experiment were conducted when mice were 8 weeks old.2.Using tandem mass tag(TMT)labeling and Kac affinity enrichment followed by high-resolution liquid chromatography-tandem mass spectrometry(LC-MS/MS),quantitative analysis the levels of protein acetylation in hippocampus between GF and SPF mice.3.To assess the gut microbiota absence whether have a structural preference in regulating protein modification,Motif-X analysis software was used to analyze the motif of Kac-modified quantitative peptide sequences.4.The significantly changed modified sites were screened according to the threshold of fold change≥1.2 or ≤0.83 and p-value <0.05.To reveal the effect of differential acetylation modification induced by gut microbiota absence on protein function,we used Net Sur FP to predict thesecondary structure of significant differential Kac proteins.5.In order to reveal the function of Kac proteins in gut microbiota absence,we performed subcellular localization analysis using Wolf PSort software.In addition,based on the PFAM domain database,the functional analysis of the domains of the differential Kac proteins was performed.6.To further identify the biological functions of Kac proteins,we performed Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis,Ingenuity Pathway Analysis(IPA)and protein-protein interaction(PPI)network analysis.Results 1.A total of 986 lysine acetylation sites in 543 protein groups were identified,among which 747 sites in 427 proteins were quantified.Based on the threshold of fold-change ≥ 1.2 or ≤ 0.83 and p-value <0.05,a total of 138 Kac sites in 101 proteins were up-regulated and 40 Kac sites in 30 proteins were down-regulated in the GF mice.2.The results of motif analysis showed that D*Kac,Dkac,Kacy,Kacd,and D**Kac conserved sequences had significant differences.Most of the acetylated sites were located on the Coil(62.1%),followed by the α-helix(31.63%)and the β-strand(6.26%),and 34.17% of the acetylated lysine sites were exposed to the protein table.Lys acetylation influenced by gut microbiota absence can affect the surface property of proteins.Differentially acetylated modified proteins were mainly localized inmitochondria(35%)and cytoplasm(32%).3.The biological function enrichment analysis showed that the differential Kac protein involved a variety of biological functions,mainly located in the mitochondria.The changes in acetylation modified proteins induced by the absence of gut microbes are mainly related to mitochondrial dysfunction,oxidative phosphorylation,tricarboxylic acid cycle and Sirtuin signaling pathway.These results suggest that gut microbiota may affect mitochondrial function by regulating the modification level of acetylation proteins.Conclusion A large number of Kac sites were identified in mice with an absence of gut microbiota.A total of 986 Lysine Acetylation sites in 543 protein groups were identified,among which 747 sites in 427 proteins were quantified.Based on the threshold of fold-change ≥ 1.2 or ≤ 0.83 and pvalue <0.05,a total of 138 Kac sites in 101 proteins were up-regulated and 40 Kac sites in 30 proteins were down-regulated in the GF mice.Differential Kac protein are involved a variety of biological processes,including mitochondrial dysfunction,oxidative phosphorylation,tricarboxylic acid cycle and Sirtuin signaling pathway,which are mainly located in the mitochondria.This study reveal that absence of gut microbiota may affect the function of mitochondria by regulating the level of acetylation modification.This study lays the foundation for exploring the regulation of mouse hippocampal tissue function based on gut microbiota.