Octenidine-based hydrogel shows anti-inflammatory and protease-inhibitory capacities in wounded human skin

Abstract

Octenidine dihydrochloride (OCT) is a widely used antiseptic molecule, promoting skin wound healing accompanied with improved scar quality after surgical procedures. However, the mechanisms by which OCT is contributing to tissue regeneration are not yet completely clear. In this study, we have used a superficial wound model by tape stripping of ex vivo human skin. Protein profiles of wounded skin biopsies treated with OCT-containing hydrogel and the released secretome were analyzed using liquid chromatography-mass spectrometry (LC-MS) and enzyme-linked immunosorbent assay (ELISA), respectively. Proteomics analysis of OCT-treated skin wounds revealed significant lower levels of key players in tissue remodeling as well as reepithelization after wounding such as pro-inflammatory cytokines (IL-8, IL-6) and matrix-metalloproteinases (MMP1, MMP2, MMP3, MMP9) when compared to controls. In addition, enzymatic activity of several released MMPs into culture supernatants was significantly lower in OCT-treated samples. Our data give insights on the mode of action based on which OCT positively influences wound healing and identified anti-inflammatory and protease-inhibitory activities of OCT.

Figure 1

Proteins are differentially regulated among wounded OCT-treated skin and controls. Abdominal skin biopsies of donors (age range: 38–45 years, n = 5) were TS, treated with OCT, control gel or left untreated, cultivated for 48 h and subsequently prepared for LC–MS analysis. Volcano plots show differences in LFQ values (fold change, logarithmic scale to the base of two) on the x-axis including their corresponding p-values (logarithmic scale) on the y-axis. Proteins marked in red are significantly differentially regulated (Log₂FC < -1 = downregulated; Log₂FC > 1 = upregulated) in OCT treated samples compared to (A) control gel-treated samples or (B) untreated samples. Larger sized dots indicate proteins that are differentially regulated in OCT treated samples compared to both, control gel-treated samples and untreated samples. Registered proteins were further analysed. Data presented in the plots was generated performing a two-sided t-test (p < 0.05) using Perseus and vulcano plots were generated using Graphpad Prism. Extended information on the proteins can be found in Tables S1 and S2.

Figure 2

OCT regulates proteins involved in wound healing and immune response. Full skin biopsies of donors (age range: 38–45 years, n = 5) were TS, treated with OCT, control gel or left untreated, cultivated for 48 h and subsequently prepared for LC–MS analysis. Classification by the GO term biological process shows pathways and larger processes made up of the activities of multiple proteins. Fold enrichment values for individual GO terms, count (proteins involved in the term), p-value and FDR (false discovery rate, calculated using the Benjamini–Hochberg procedure), listed next to the graph, were calculated using DAVID bioinformatics resources. Biological processes downregulated in OCT-treated TS samples are shown in dark grey, processes upregulated in OCT-treated TS samples are shown in light grey.

Figure 3

OCT significantly inhibits IL-6 secretion in wounded human skin. IL-6 levels of TS human skin biopsies treated with OCT were evaluated by LC–MS and were significantly lower compared with TS biopsies which were untreated or treated with the control gel (left panel). Label-free quantification (LFQ) intensities in a logarithmic scale to the basis 2 are indicated. LFQ intensities for proteins not detected in a replicate were replaced by 15. IL-6 concentration in supernatants was tested in duplicates with an ELISA (right panel). Data in both graphs is presented as a mean ± SD (n = 5). An unpaired t-test was performed with GraphPad Prism. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 4

OCT significantly reduces secretion and activity of proteases in wounded human skin. (A) Indicated MMP levels, analysed by LC–MS, were significantly lower in human TS skin biopsies treated with OCT compared to biopsies which were untreated or treated with a control gel, with exception of TIMP-1, which is not affected upon OCT treatment (left panel). Label-free quantification (LFQ) intensities in a logarithmic scale to the basis 2 are indicated. LFQ intensities for proteins not detected in a replicate were replaced by 15. Proteases and a typical protease inhibitor in culture supernatants were tested in duplicates with an ELISA (right panel). (B) Enzymatic activity of MMPs was significantly lower in culture supernatants of OCT-treated TS human skin biopsies compared to controls (untreated, control gel-treated). Relative fluorescence units (RFU) are indicated. All data is presented as mean ± SD (n = 5). An unpaired t-test was performed with GraphPad Prism. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns = not significant.

Figure 5

More proteins are significantly differentially regulated upon OCT treatment in the dermis than in the epidermis. Full skin biopsies of healthy female donors (age range: 36–44 years, n = 3) were TS, treated with OCT, control gel or left untreated and cultivated for 48 h. Subsequently, epidermis was detached from dermis and the two compartments were separately prepared for LC–MS analysis. Volcano plots show differences in LFQ values (fold change, logarithmic scale to the base of two) on the x-axis including their corresponding p-values (logarithmic scale) on the y-axis. Proteins marked in red are significantly differentially regulated (Log₂FC < -1 = downregulated; Log₂FC > 1 = upregulated) in OCT treated TS samples compared to control gel-treated TS samples or untreated TS samples. Data presented in the plots was generated performing a two-sided t-test (p < 0.05) using Perseus and Volcano plots were generated with Graphpad Prism using exclusively proteins with a p-value < 0.01.

Figure 6

OCT regulates proteins involved in immune response in the epidermal compartment and proteins involved in wound healing and immune response in the dermal compartment. Full skin biopsies of donors (age range: 36–44 years, n = 3) were TS, treated with OCT, control gel or left untreated and cultivated for 48 h. Subsequently, epidermis (A) was detached from dermis (B) and the two compartments were separately prepared for LC–MS analysis. Classification by the GO term biological process shows pathways and larger processes made up of the activities of multiple proteins. Fold enrichment values for individual GO terms, count (proteins involved in the term), p-value and FDR (false discovery rate, calculated using the Benjamini–Hochberg procedure), listed next to the graph, were calculated using DAVID bioinformatics resources. Biological processes downregulated in OCT-treated TS samples are shown in dark grey, processes upregulated in OCT-treated TS samples are shown in light grey.

Figure 7

OCT reduces cytokines as well as protease activities in wounded human skin (this study, black font), is not apoptotic for skin cells, does not change the skin architecture and prevents the upregulation of CD83 on Langerhans cells as well as their emigration (white font).