[Proteomics analysis of the effect and mechanism of ADSCs on full-thickness skin defects in diabetic rats].

Objective: To explore the effect and mechanism of adipose-derived mesenchymal stem cells (ADSCs) on full-thickness skin defects in diabetic rats using proteomics analysis. Methods: This study was a self-control design experimental study. Four 8 to 10 weeks old male Sprague-Dawley rats were selected, and ADSCs were extracted from their epididymal adipose tissue and successfully identified. The third passage of ADSCs were used for the following experiments. Twenty-four 4 to 6 weeks old male Sprague-Dawley rats were selected and the type 2 diabetes model was successfully established. Among them, 16 diabetic rats weighing 350 to 400 g were chosen, and a full-thickness skin defect wound was created on each side of the spine on their backs at the same level. Using the random number table method, the two wounds of each rat were included in experimental group and control group (with 16 wounds in each group), and the cell suspensions containing ADSCs and phosphate buffered saline were injected at multiple points around and at the base of the wounds immediately after injury, respectively. The wound healing rates were calculated at day 7, 10, and 14 after injury in rats. At day 7 after injury in rats, wound tissue was collected and proteins were extracted. Four-dimensional data-independent acquisition label-free quantitative proteomics technology was used for quantitative proteomics analysis and bioinformatics analysis to screen differentially expressed proteins (DEPs) in the two groups of wound tissue. Then, key DEPs were screened through protein-protein interaction networks, gene ontology (GO) was used for functional annotation and enrichment analysis, and Kyoto encyclopedia of genes and genomes (KEGG) was used for pathway enrichment analysis to further screen target DEPs. The wound tissue of rats 7 days after injury was collected, and Western blotting was used to detect the protein expression of thymocyte differentiation antigen 1 (Thy-1) and G protein-coupled receptor 177/Wnt ligand secretion mediator (GPR177/Wls). Results: At day 7, 10, and 14 after injury in rats, the wound healing rates in experimental group were (66±16)%, (83±8)%, and (93±4)%, respectively, which were significantly higher than (30±8)%, (62±6)%, and (77±8)% in control group (with t values of -4.41, -7.46, and -6.65, respectively, P<0.05). At day 7 after injury in rats, compared with those in control group, a total of 474 DEPs were screened from the wound tissue in experimental group (P<0.05). A total of 224 key DEPs were further screened out, among which 78 DEPs were significantly upregulated and 146 DEPs were significantly downregulated. GO functional annotation analysis showed that the most significantly upregulated and downregulated DEPs mainly affected protein expression under cellular processes and biological regulation conditions, and were related to cell anatomical entities and protein-containing complexes, as well as specific binding and catalytic activity between biomolecules. GO functional enrichment analysis showed that the most significantly upregulated DEPs were significantly enriched in Wnt-protein binding. KEGG pathway enrichment analysis showed that the significantly upregulated and downregulated DEPs were enriched in pathways such as starch and sucrose metabolism, nucleotide metabolism, p53 signaling pathway, and extracellular matrix -receptor interaction, and so on. A total of 4 target proteins were screened out, including Thy-1, GPR177/Wls, Fer/CIP4 homology domain only 2, and mitochondrial ribosomal protein L21, with the first two being significantly upregulated DEPs while the latter two being significantly downregulated DEPs. At day 7 after injury in rats, the protein expressions of GPR177/Wls and Thy-1 in the wound tissue in experimental group were 0.93±0.07 and 0.96±0.05, respectively, which were significantly higher than 0.39±0.07 and 0.36±0.12 in control group (with t values of 11.61 and 9.41, respectively, P<0.05). Conclusions: Based on proteomics analysis, it was revealed that ADSCs from rats can promote the wound healing of full-thickness skin defects in diabetic rats by upregulating the protein expression of GPR177/Wls and Thy-1.