Quantitative Proteomic Analysis Of Epidermal Stem Cells In Oxygen-glucose Deprivation Condition

Background:Chronic hard healing wounds refer to wounds that have not healed after 1 month of treatment and still have no tendency to heal.The rise of prevalence of diabetes,vascular diseases has led to the occurrence of a large number of chronic hard healing wounds,which are gradually becoming a major medical problem plagued the world.Epidermal stem cells(ESCs)play a key role in the process of wound healing.Therefore,studying the protein expression changes of ESCs in chronic wounds is of great significance for studying the formation mechanism of chronic refractory wounds and finding relevant therapeutic targets.Studies have shown that ischemia is the main cause of chronic hard-to-heal wounds,and mitophagy also plays an important role in it.Therefore,in this experiment,we used oxygen-glucose deprivation(OGD)condition to simulate the ischemic environment of chronic hard healing wounds.Quantitative proteomics research is used to find the key proteins and pathways leading to the dysfunction of ESCs in chronic hard healing wounds.Objective:Explore the effects of OGD conditions on the protein expression of ESCs,look for important differentially expressed proteins(DEPs)to obtain the important proteins and pathways,so as to further find related therapeutic targets.Methods:ESCs were extracted from neonatal rats and cultured for 0h,3h,6h,9h and 12h under oxygen and glucose deprivation conditions.Quantitative proteomics assays were performed on ESCs of different groups using Tandem mass tag(TMT)technology,and the DEPs in the four comparison groups OGD3_VS_OGD0,OGD6_VS_OGD0,OGD9_VS_OGD0 and OGD12_VS_OGD0 were screened.Then,these DEPs are analyzed for Gene Ontology(GO),KEGG pathway,protein-protein interaction(PPI),gene overlap,and specific mitophagy-related pathway and proteins to find the proteins and pathways that play a key role in the dysfunction of ESCs.Results:A total of 4,852 quantifiable proteins were detected in the experiment.In the four comparison groups OGD3_VS_OGD0,OGD6_VS_OGD0,OGD9_VS_OGD0,and OGD12_VS_OGD0,the number of up-regulated DEPs were 1、225、346 和 386,respectively,while the number of down-regulated DEPs were 26、229、330 和 462,respectively.In the analysis of the comparison group OGD6_V S_OGD0,key pathways included" chromatin binding"(GO:0003682)"cytoplasmic ribosomal large subunit(GO:0022625)" and "ribosome(ko03010)",and the 21 hub proteins screened through PPI analysis are all structural proteins of the ribosome,of which the most important seed protein is the mitochondrial ribosomal protein Mrpl24.In the comprehensive analysis of the four comparison groups,it was found that there was a large amount of overlap between the DEPs of each comparison group,and key pathways were all related to protein processing.In the analysis of mitophagy-related pathway and proteins,it was found that the expression of Tbcldl5,Rab7a related to lysosomal maturation and p53 continued to be up-regulated,while the expression of LC3 related to the level of autophagy flow did not show a definite trend.Conclusion:The key protein Mrp124 obtained through TMT quantitative proteomics analysis and the enrichment pathways related to protein processing are the key to the dysfunction of ESCs under OGD conditions.They will be the breakthrough point for further research on the formation mechanism of chronic hard healing wounds.At the same time,it also provides strong support for finding relevant therapeutic targets.Whether the level of mitophagy in chronic hard healing wounds really increases remains to be further verified.