The Role And Mechanism Of Protein Modification In MAFLD Rats

Objective:This study is based on proteomics sequencing and proteomics sequencing techniques.The model of MAFLD rats was constructed with a high-fat and high-sugar diet.Using mass spectrometry to detect the presence of proteins and modified sites in normal and MAFLD rats’liver tissues.Define differential proteins and differential modification sites with a difference multiple greater than 1.5 times.The function of the differential protein and its post-translational modification site in the development of metabolic liver disease was discussed.Through a combination of proteomics and proteomics,Screening for different proteins with multiple posttranslational modifications.and verified the changes of these proteins in the liver tissue of MAFLD rats by western blot,Further search for potential target proteins in MAFLD progression.Methods:1.Model construction and validation of MAFLD ratsThe 16 SD male rats were randomly divided into two groups the normal control group（n=8,standard feed feeding）the and MAFLD group（n=8,high sugar high fat feed feeding）.After 12 weeks of group feeding,it was executed and tested for weight,weight of liver,liver index,serological index,HE staining,oil red "O" staining and Masson staining.2.Proteomics and proteomics sequencing,differential protein analysis,and functional annotationThree samples of fresh liver tissue were randomly selected in the normal control group and MAFLD group,protein peptide segment expression was obtained based on mass spectrometry,sample-specific protein database was constructed,and Spectronaut was used（v 16.0）Search Engine Through Database（Rattus_norvegicus₁0116_PR₂0230103.fasta）After searching,the results are corrected and the protein is quantified using the MSstats R language package.Quality control is carried out by peptide segment length,peptide segment quantity and protein molecular weight.Quantitative analysis of protein repeatability and differential analysis to identify proteins with significant differences,using the MoMo analysis tool to analyze differential modification sites with significant differences.GO annotation analysis of differential proteins using eggnog-mapper software（v2.1.6）,including biological processes,cell components and molecular functions;KEGG pathway annotation of differential proteins via KEGG pathway database.Using Fisher’s precise test to analyze pathway enrichment salience of differential expression proteins.The P values obtained from the enrichment analysis use the hierarchical clustering-related function.The-Log10-based logarithmic transformation is screened.The results are analyzed by one-sided clustering,and the clustering results are enriched with KEGG.3.Joint analysis of proteomics and protein modification histologyThe protein with significant differences was identified by proteomics and the modification sites of four protein modifiers were analyzed.KEGG pathway annotation of the abovementioned differential proteins is performed through the KEGG pathway database.By STRING（v.11.5）Protein interaction network database to extract four groups of differential proteins with differential modification sites,Using the R package "networkD3" tool to map the protein interaction network,With the four algorithms in CytoHubba,Degree,Closeness,EPC,and MNC,the top 10 proteins are selected.Overlap 4 algorithms to screen for proteins with more connections.And the four groups of proteins with more connections were analyzed again.get potential targets and validate the potential targets from the above methods in the liver tissue of the experimental animal.Use the western blot to verify that the results match the sequencing results.Results:1.Model construction and validation of MAFLD ratsSignificant increase in body weight,liver humidity,and liver index in the MAFLD group compared to the normal group（P<0.01）;Increased ALT,AST,TG,and LDL-C levels（P<0.05）;swelling of hepatic mass cells in the MAFLD group,Cytoplasm is replaced by lipid droplet,and voids of varying size are visible in the cytoplasm.A small number of lymphocytes in the lobe were infiltrated with small focal,and fat degeneration and inflammatory lesions were higher than those in the normal group.（P<0.01）,significant oil-red "O" dye deposition in the MAFLD group,positive area/mirror area significantly higher than normal group（P<0.01）,TC,HDL-C,and fibrosis scores are not statistically different.2.Proteomics sequencing,differential protein analysis,and function annotationProteomics detected 641 differential proteins（207 proteins upregulated,434 proteins downregulated）GO analyzes many biological processes that are enriched into organic matter metabolism processes,cellular metabolism processes,primary metabolism processes,regulation of biological processes,and nitrogen compound metabolism processes;KEGG analyses enriched many pathways such as steroid synthesis,oxidative phosphorylation,heat production,NAFLD and amino acid synthesis.3.Protein modification sequencing,differential protein analysis,and function annotation（1）Lactic acidification modification histologyLactic acidification modification histology detected 61 differential modification sites,of which 50 were upregulated and 11 were downregulated.GO analyzes many biological processes such as enrichment into organic matter metabolism,cellular metabolism,primary metabolism,nitrogen compound metabolism,and small molecule metabolism.KEGG analysis enriched pathways such as fatty acid breakdown,CoA synthesis,AMPK signaling pathways,tyrosine metabolism,and fructose/mannose metabolism.（2）β hydroxymethylation modification histologyβ hydroxymethylation modification histolothe gy detected 426 differential odification sites,ohistology2 differential modification sitmodification regulated and 174 differential modification sites downregulated.GO analyses biological processes such as enrichment to organic matter metabolism,cellular metabolism,primary metabolism,regulation of biological processes,and nitrogen compound metabolism;KEGG is enriched in purine/pyrimidine metabolism,primary bile acid biosynthesis,lipid,and atherosclerosis,pentose phosphate pathways and protein processing in the endoplasmic reticulum.（3）Acetylation modification histologyAcetylation modification histology detected 1167 differential modification sites,of which 715 differential modification sites were raised and 452 differential modification sites were downregulated.GO analyses biological processes such as enrichment to organic matter metabolism,cellular metabolism,primary metabolism,nitrogen compounds metabolism,and regulation of biological processes;KEGG enriches the tricarboxylic acid cycle,fatty acid prolongation,arginine biosynthesis,multiple amino acid metabolism,and unsaturated fatty acid biosynthesis.（4）Phosphorylation modification histologyPhosphorylation modification histology detected 408 differential modification sites,of which 206 differential modification sites were upregulated and 202 differential modification sites downregulated.GO analyses biological processes such as regulation of enrichment-tobiological processes,cellular metabolic processes,organic substance metabolic processes,primary metabolic processes,and nitrogen compound metabolic processes.KEGG is enriched in a variety of amino acid metabolism,nitrogen metabolism,primary bile acid biosynthesis,starch/sucrose metabolism,and pentose phosphate pathways.4.Joint analysis of proteomics and protein modification histology（1）Combined analysis of proteomics and lactic acidification modificationThe significant difference in protein and the significant lactic acidification difference in modification site were analyzed,and the proteins such as Ndufs3,Eci2,Gludl,At,p5pd,and Dbt were found.KEGG enriinto reactive oxygen and heat generation pathways.（2）A combined analysis of proteomics and β hydroxybutyl modified histologyThe significant difference in protein was analyzed in conjunction with the significant βhydroxymethylation difference modification site.It was found that Acat2,Aco2,Eci2,Fdft,and Ecil were proteins with hydroxymethylation tion modification sites.KEGG enriches the carbon metabolic pathway,fatty acid breakdown,pyruvate metabolism,PPAR signaling,pathway and tryptophan metabolism.（3）Combined analysis of proteomics and acetylation modification histologyThe acetylation modification sites of Aco2,Aldh7a1,Glud,1,Hmgcl and Cth were analyzed.KEGG enriches propionate metabolism,amino acid metabolism,carbon metabolism,cofactor biosynthesis,and fatty acid degradation.（4）Combined analysis of proteomics and phosphorylation modificationThe phosphorylation modification sites of Gludl,Cps1,Aldh7a1,Dmgdh and Cth were analyzed.KEGG enriches the carbon metabolism pathway,amino acid metabolism,pyruvate metabolism,acetate and dicarboxylate metabolism and amino acid breakdown pathways.（5）4 combined analysis of protein modification histology and proteomicsThe PPI connections of 4 groups of proteins with modified differential sites were analyzed again.The results showed that Eci2 was involved in both lactic acidification and acetylation modification.Aco2 to participate in β hydroxybutylation modification and acetylation modification.Aldh7al and Cth are involved in both acetylation and phosphorylation.Gludl also participate in four post-translation modifications.（6）Verify the presence of several post-translational modification sites of differential proteins by western blotRelative expression of Eci2,Aco2,Aldh7a1,Cth and Glud1 proteins in liver tissues of rats with MAFLD group was lower than normal group（P<0.05）,consistent with proteomics sequencing results.（7）Correlation analysis of various post-translation modified proteins with rat general dataIn the liver tissue of MAFLD group Eci2 it was negatively correlated with weight,humidity weight,liver index and LDL-C.Aco2 and weight,liver wet weight,liver index,TG and LDL-C showed negative correlation;Aldh7al showed negative correlation with liver index.Cth and Gludl were negatively correlated with weight,humidity,liver index,AST,TG and LDL-C.Conclusion（s）:1.The model of MAFLD rats was successfully established by feeding SD rats with a high-fat and sugar diet for 12 weeks.2.Proteomics combined with proteomics showed that After protein translation,modification is involved in amino acid metabolism,fatty acid metabolism,and sugar metabolism.involved in energy metabolism and substance metabolism,Plays an important role in the development of MAFLD.3.Eci2,Aco2,Aldh7a1,Cth,and Glud1 are involved in a variety of post-translational modifications.It is suggested that differences in the expression of these proteins may result from changes in protein translation levels or post-translation modification levels.Potential targets for the diagnosis and treatment of MAFLD can be provided at the protein and protein modification levels.