Proteomics changes after negative pressure wound therapy in diabetic foot ulcers

Abstract

Label‑free quantitative mass spectrometry was used to analyze the differences in the granulation tissue protein expression profiles of patients with diabetic foot ulcers (DFUs) before and after negative‑pressure wound therapy (NPWT) to understand how NPWT promotes the healing of diabetic foot wounds. A total of three patients with DFUs hospitalized for Wagner grade 3 were enrolled. The patients received NPWT for one week. The granulation tissue samples of the patients prior to and following NPWT for one week were collected. The protein expression profiles were analyzed with label‑free quantitative mass spectrometry and the differentially expressed proteins (DEPs) in the DFU patients prior to and following NPWT for one week were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were conducted to annotate the DEPs and DEP‑associated signaling pathways. Western blotting and ELISA were performed to validate the results. By comparing the differences in the protein profiles of granulation tissue samples prior to and following NPWT for one week, 36 proteins with significant differences were identified (P<0.05); 33 of these proteins were upregulated and three proteins were downregulated. NPWT altered proteins mainly associated with antioxidation and detoxification, the cytoskeleton, regulation of the inflammatory response, complement and coagulation cascades and lipid metabolism. The functional validation of the DEPs demonstrated that the levels of cathepsin S in peripheral blood and granulation tissue were significantly lower than those prior to NPWT (P<0.05), while the levels of protein S isoform 1, inter α‑trypsin inhibitor heavy chain H4 and peroxiredoxin‑2 in peripheral blood and granulation tissue were significantly higher than those prior to NPWT (P<0.05). The present study identified multiple novel proteins altered by NPWT and laid a foundation for further studies investigating the mechanism of action of NPWT.

Keywords: cathepsin S; diabetic foot ulcer; differentially expressed proteins; inter α‑trypsin inhibitor heavy chain H4; label‑free quantitative mass spectrometry; negative‑pressure wound therapy; peroxiredoxin‑2; protein S isoform 1.

Figure 1.

Screening of DEPs. Based on selection criteria of an absolute log2-fold change (fold-change ≥1.2 difference) and P<0.05. DEPs before and after NPWT via (A) volcano plot. DEPs are indicated with green dots (downregulated proteins) or red dots (upregulated proteins), while proteins with insignificant changes are shown by black dots. (B) Cluster gram was used to show DEPs before and after NPWT. Hierarchical clustering analysis was performed to demonstrate the distributions of different protein expression levels in each sample. Color keys represent expression levels based on the log2 of the quantity values. DEPs, differentially expressed proteins; NPWT, negative-pressure wound therapy.

Figure 2.

Analysis of GO functional terms and enrichment for DEPs. (A) GO analysis of the distribution of differential proteins in three aspects: Biological processes, cellular localization and molecular functions. The ordinate is the number of proteins. The abscissa represents the classification description under each GO classification, i.e., the number of DEPs identified under a GO classification. (B) Significantly enriched GO terms of DEPs before and after negative-pressure wound therapy are listed and arranged by P-value. Enrichment factors were calculated and shown on horizontal ordinates. The size of bubbles indicates protein numbers, and different colors of bubbles indicate the value of -log10 (P-value). GO, Gene Ontology; DEPs, differentially expressed proteins.

Figure 3.

KEGG enrichment analysis for DEPs. Significantly enriched KEGG pathways of DEPs before and after negative-pressure wound therapy are listed and arranged by P-value. Enrichment factors were calculated and are shown on horizontal ordinates. The size of the dots indicates protein numbers and different colors of dots indicate the value of -log10 (P-value). KEGG, Kyoto Protocol Encyclopedia of Genes and Genomes; DEPs, differentially expressed proteins.

Figure 4.

Validation of the protein expression levels of CTSS, PROS1, ITIH4 and PRDX2. Wound granulation tissues from eight patients with diabetic foot ulcers before and after NPWT were collected and subjected to western blotting. Primary antibodies against (A) CTSS, (B) PROS1, (C) ITIH4 and (D) PRDX2 were used to detect differences in protein levels. (A1-D1) Protein bands from four representative patients prior to NPWT (designated as minus symbol ‘−’) and following NPWT (designated as plus symbol ‘+’) are presented in the left panel. Protein samples from the same patients were loaded next to each other. (A2-D2) Then, eight pairs of protein samples from eight patients were analyzed by ImageJ and are depicted as paired dots in the right panel. (A3-D3) The gray level of the target protein was compared with the internal reference (β-actin) gray level to obtain the relative expression value. Paired t-tests were performed and significant changes are indicated by ***P<0.001 vs. before NPWT. CTSS, cathepsin; PROS1, protein S isoform 1; ITIH4, inter α-trypsin inhibitor heavy chain H4; PRDX2, peroxiredoxin-2; NPWT, negative-pressure wound therapy.

Figure 5.

ELISA analysis of serum CTSS, ITIH4, PROS1 and PRDX2 levels in patients before and after NPWT. Blood samples from 17 patients were collected and serum CTSS, ITIH4, PROS1 and PRDX2 levels were measured with ELISA. Serum (A1) CTSS, (B1) ITIH4, (C1) PROS1 and (D1) PRDX2 prior to and following NPWT are depicted as paired dots. The changes in serum (A2) CTSS, (B2) ITIH4, (C2) PROS1 and (D2) PRDX2 levels prior to and following NPWT are shown by histograms. Paired t-tests were performed and significant changes are indicated by *P<0.05, **P<0.01, ***P<0.001 vs. before NPWT. CTSS, cathepsin; PROS1, protein S isoform 1; ITIH4, inter α-trypsin inhibitor heavy chain H4; PRDX2, peroxiredoxin-2; NPWT, negative-pressure wound therapy.