Hydrogen-Rich Saline Combined With Vacuum Sealing Drainage Promotes Wound Healing by Altering Biotin Metabolism

Abstract

Impaired wound healing affects the life quality of patients and causes a substantial financial burden. Hydrogen-rich medium is reported to have antioxidant and anti-inflammatory effects. However, the role of hydrogen-rich saline (HRS) in cutaneous wound healing remains largely unexplored, especially by metabolomics. Thus, untargeted metabolomics profiling was analysed to study the effects and mechanism of HRS combined with vacuum sealing drainage (VSD) in a rabbit full-thickness wound model. Our results indicated that the combination treatment of HRS and VSD could accelerate wound healing. In vitro experiments further confirmed its effects on HaCaT keratinocytes. We found that 45 metabolites were significantly changed between the VSD + HRS group and the VSD + saline-treated group. Pathway enrichment analysis indicated that biotin metabolism was the potential target pathway. The biochemical interpretation analysis demonstrated that combining HRS and VSD might enhance mitochondrial function, ATP synthesis, and GSH homeostasis by altering biotin metabolism. The detection of representative indicators of oxidative stress supported the critical metabolic pathway analysis as well. In summary, VSD combined with HRS might provide a new strategy to enhance wound healing.

Keywords: biotin metabolism; hydrogen‐rich saline; metabolomics; oxidative stress; vacuum sealing drainage; wound healing.

FIGURE 1

(A) The schedule of a rabbit full‐thickness wound model establishment, the treatment, and the sample harvest. (B) Representative wound pictures of rabbits' dorsal from each group were taken on post‐injury days 1, 4, 7, and 14. Scale bar, 1 cm. (C) Wound healing rate at different times from each group. Data are shown as means ± SD (n = 6–8). Variables in the five groups were compared by the one‐way ANOVA test, Welch's ANOVA, and the Kruskal–Wallis rank‐sum test. The Games‐Howell test (a post hoc test) and Dunn's test were used for multiple comparisons when appropriate. (D) The time to complete wound closure in different groups was measured and compared. The Kaplan–Meier survival curve was generated, and the Chi‐square test analysed statistical differences. (E) Expression of cytokines, TNF‐α, IL‐1β, and IL‐10. n = 3–4. Variables in the five groups were compared using the one‐way ANOVA test. The Tukey honest significant difference (Tukey HSD) test was used for multiplecomparisons. ***, **, and * represent significance at the significance level of 1%, 5%, and 10% respectively.

FIGURE 2

(A) HE staining of wound section on day 7. The scale bar of the upper panel is 100 μm; the lower, 50 μm. (B) Histopathological scores were calculated by HE staining on days 1, 4, and 7. (n = 3, data = mean ± SD.) Variables in the five groups were compared using the Kruskal–Wallis rank‐sum test. Dunn's test was used for multiple comparisons. (C) Cell viability on HaCaT cells with different interventions was evaluated by CCK‐8 assay. (n = 10, data = mean ± SEM.) Variables in the four groups were compared using the Welch one‐way ANOVA test. A Games‐Howell post hoc analysis was applied to determine multiple comparisons. ***, **, and * represent significance at the significance level of 1%, 5%, and 10% respectively.

FIGURE 3

The column graphs show the top 20 content of metabolites in positive (A) and negative ion mode (B). The unsupervised multivariate principal component analysis (PCA) of the studied metabolites in the 5 groups (C). The plots represent the first principal component (PC1) against the second principal component (PC2). The unsupervised data analysis between the Saline group and HRS group (D) and between the VSD + Saline group and the VSD + HRS group (E).

FIGURE 4

The Permutation test implicated the validity of the OPLS‐DA model among all groups (A) and between the VSD + Saline group and VSD + HRS group (B). OPLS‐DA score plots among all groups (C) and between the VSD + Saline and VSD + HRS groups (D). Variable importance in the projection (VIP) values of OPLS‐DA analysis among all groups (E) and between the VSD + Saline group and the VSD + HRS group (F). The points represent the metabolites, and the abscissa and ordinate represent the Value Importance in Projection (VIP) values and −log₁₀ (FDR‐adjusted p‐value) of the quantitative difference of metabolites.

FIGURE 5

(A) Heat maps of differential metabolites from the cutaneous specimens. Rows: Samples; Columns: Metabolites. Red means the metabolites is expressed at a higher level, and blue means the metabolites is expressed at a lower level. (B) The 15 most important metabolites in the SVM model between HRS and Saline groups. The importance of metabolites was assessed by the degree of average importance (bottom abscissa). The heat map of the 15 most important metabolites was displayed on the right side. Red: Higher expression levels; green: Lower expression levels. (C) KEGG enrichment analysis between the VSD + Saline group and the VSD + HRS group. The horizontal coordinates are fold changes. It refers to the ratio of the differential metabolite numbers in the corresponding pathway to the total identified metabolite numbers in this pathway. The higher the ratio value, the more differential metabolites are enriched in this pathway. The darker the colour, the smaller the p‐value.

FIGURE 6

Levels of SOD (A), GSH (B), CAT (C), and MDA (D) content in the wound tissues of different groups. (n = 3–4, data = mean ± SEM.) Variables in the five groups were compared using the one‐way ANOVA test and the Welch one‐way ANOVA test. The Games‐Howell test and Tukey HSD test were used for multiple comparisons when appropriate. ***, **, and * represent significance at the significance level of 1%, 5%, and 10% respectively.

FIGURE 7

Graphical abstract of the therapeutic effects and the metabolic mechanism of HRS combined with VSD in wound healing (created with BioRender.com; Agreement number: IH267FWL7V).