Figure 1.

Metabolome profile identifies accumulation of phenylpyruvate in DFUs, which is found to impair wound healing and prolong inflammation

(A) Score plot presenting the metabolic separation for pair splitting between the NDW group and the DFU group through OPLS-DA (n = 12).

(B) A volcano plot shows the differential metabolites in the comparison of DFUs to NDWs.

(C) Overview of the top 25 terms in the metabolic enrichment analysis of the differential metabolites.

(D) Pathway enrichment analysis of differential metabolites between DFUs and NDWs. The ordinate represents the p value of pathway enrichment analysis (negative logarithm). The abscissa represents the influencing factor of pathway topological analysis. The larger the circle is, the higher the influence factor. A deeper red color indicates a larger −log(p) value, indicating more enrichment.

(E) Schematic representation of phenylalanine, tyrosine, and tryptophan biosynthesis.

(F) Histogram and heatmap generated by relative fold change of differential metabolites in the phenylalanine, tyrosine, and tryptophan biosynthesis pathways.

(G) Representative IHC staining images of NLRP3 in the DFU group and NDW group.

(H) Correlation analysis between phenylpyruvate levels and NLRP3 expression in DFUs (n = 12).

(I) Representative images of cutaneous wounds of age-matched C57BL/6JGpt mice on days 0, 3, 6, 9, and 12 after wound model generation by surgical excision. n = 12 for each group.

(J) Ratio of wound sizes were quantified by using ImageJ software and were calculated by the percentages of wound closure compared to day 0 wound size.

(K) Cutaneous wound sections were subjected to H&E and Masson’s trichrome staining, and IHC staining using Ki-67, α-SMA, CD31, and NLRP3 antibodies was performed. Samples were collected 6 days after wound model generation. n = 3 mice for sampling at the indicated time points. Scale bar, 100 μm.

(L) Histological staining analysis showing relative epidermal thickness (n = 3).

(M) Statistical analysis of NLRP3 staining in cutaneous wounds (n = 3).

(N and O) Representative immunofluorescence images and analysis showing macrophage infiltration in cutaneous wounds on day 6. n = 3 mice in each group. Scale bar, 20 μm.

(P and Q) Flow cytometry analysis of macrophage infiltration in cutaneous wounds on days 6 and 9. The representative histogram shows the levels of F4/80+CD86+ cells in cutaneous wounds from these groups. Statistical analysis indicates the mean fluorescence intensity (MFI) of CD86 (n = 3).

(R) Western blot image and analysis showing NLRP3 protein expression levels in cutaneous wounds from these groups (n = 3). Data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01; n.s., not significant.