Antibacterial Activity of Juglone Revealed in a Wound Model of Staphylococcus aureus Infection

Abstract

A serious problem currently facing the field of wound healing is bacterial infection, especially Staphylococcus aureus (S. aureus) infection. Although the application of antibiotics has achieved good effects, their irregular use has resulted in the emergence of drug-resistant strains. It is thus the purpose of this study to analyze whether the naturally extracted phenolic compound, juglone, can inhibit S. aureus in wound infection. The results show that the minimum inhibitory concentration (MIC) of juglone against S. aureus was 1000 μg/mL. Juglone inhibited the growth of S. aureus by inhibiting membrane integrity and causing protein leakage. At sub-inhibitory concentrations, juglone inhibited biofilm formation, the expression of α-hemolysin, the hemolytic activity, and the production of proteases and lipases of S. aureus. When applied to infected wounds in Kunming mice, juglone (50 μL juglone with a concentration of 1000 μg/mL) significantly inhibited the number of S. aureus and had a significant inhibitory effect on the expression of inflammatory mediators (TNF-α, IL-6 and IL-1β). Moreover, the juglone-treated group promoted wound healing. At the same time, in animal toxicity experiments, juglone had no obvious toxic effects on the main tissues and organs of mice, indicating that juglone has good biocompatibility and has the potential to be used in the treatment of wounds infected with S. aureus.

Keywords: Staphylococcus aureus; biofilm; cell membrane integrity; juglone; wound healing.

Figure 1

(A) Molecular structural formula of juglone. (B) The effect of juglone on the growth curve of S. aureus.

Figure 2

Effect of juglone on intracellular substances of S. aureus. (A) The effect on the membrane potential of S. aureus in the presence of juglone. (B) The effect of juglone treatment on the intracellular ATP concentration of S. aureus. (C,D) Detection of S. aureus intracellular whole protein after juglone treatment. (E) Effects of juglone treatment on S. aureus membrane integrity. MIC is 1000 μg/mL. Results are shown as the mean ± standard deviation of three independent experiments. When p < 0.01, it is represented by **.

Figure 3

Laser confocal observation of the effect of juglone on the membrane integrity of S. aureus. The PI dye label shows red fluorescence and the SYTO 9 dye label shows green fluorescence. The magnification was 200×.

Figure 4

Effect of juglone on the cell morphology of S. aureus observed by field emission scanning electron microscopy. The scale bar is 3 μm.

Figure 5

The effect of juglone on S. aureus biofilm formation. (A) After treatment of S. aureus with sub-inhibitory concentration of juglone for 24 h, the formation of biofilm was observed by crystal violet staining. (B) Statistical analysis of the effects of sub-inhibitory concentrations of juglone on S. aureus biofilm formation. MIC is 1000 μg/mL. When p < 0.01, it is represented by **, when p < 0.05, it is represented by *.

Figure 6

The effect of juglone on the hemolytic activity of S. aureus. (A,B) Six percent fresh sheep erythrocytes were used to analyze the hemolytic activity of S. aureus when sub-inhibitory concentrations of juglone were treated with S. aureus for 24 h; (C,D) Western blot analysis of the effect of sub-inhibitory concentration of juglone on protein Hla expression of S. aureus. MIC is 1000 μg/mL. When p < 0.01, it is represented by **.

Figure 7

Juglone inhibited the expression of protease and lipase in S. aureus. (A) The effect of juglone on the protease secretion of S. aureus; (B) the effect of juglone on the lipase secretion of S. aureus. MIC is 1000 μg/mL. When p < 0.01, it is represented by **, when p < 0.05, it is represented by *.

Figure 8

Effect of juglone on wound healing in S. aureus infection. (A) Representative pictures of S. aureus infected wounds on days 3, 7 and 14 of topical treatment with juglone; (B) Image J calculation and analysis of the relative wound healing percentage. When p < 0.01, it is represented by **, when p < 0.05, it is represented by *.

Figure 9

Effect of juglone topical treatment on wound bacterial load and wound histological analysis. (A) Bacterial load of wounds after 2 d of treatment in different treatment groups. (B) Histological evaluation of different treatment groups after 14 d of treatment. Wound tissue uninoculated with S. aureus (Control group); untreated wound tissue infected with S. aureus (S. aureus group); jugone-treated wound tissue with S. aureus contamination (S. aureu + juglone group); vancomycin-treated S. aureus infected wound tissue (S. aureu + vancomycin group). Scale bar was 100 μm. When p < 0.01, it is represented by **.

Figure 10

The effect of juglone on the levels of inflammatory factors in mice with S. aureus-infected wounds. The expression level of TNF-α (A), IL-6 (B) and IL-1β (C) in the serum of mice after three days of local treatment with juglone. The expression level of TNF-α (D), IL-1β (E) and IL-6 (F) in the wound tissue of mice after three days of local treatment with juglone. When p < 0.01, it is represented by **, when p < 0.05, it is represented by *.

Figure 11

Safety assessment of juglone. The effect of juglone on the main organs (heart, liver, spleen, lung and kidney) of mice after 14 d of topical treatment. Scale bar was 100 μm.