Fig latex inhibits the growth of pathogenic bacteria invading human diabetic wounds and accelerates wound closure in diabetic mice

Abstract

Impaired wound healing is one of the most critical complications associated with diabetes mellitus. Infections and foot ulcers are major causes of morbidity for diabetic patients. The current treatment of diabetic foot ulcers, commonly used antibiotics, is associated with the development of bacterial resistance. Hence, novel and more effective natural therapeutic antibacterial agents are urgently needed and should be developed against the pathogenic bacteria inhabiting diabetic wounds. Therefore, the current study aimed to investigate the impact of fig latex on pathogenic bacteria and its ability to promote the healing process of diabetic wounds. The pathogenic bacteria were isolated from patients with diabetic foot ulcers admitted to Assiut University Hospital. Fig latex was collected from trees in the Assiut region, and its chemical composition was analyzed using GC‒MS. The antibacterial efficacy of fig latex was assessed on the isolated bacteria. An in vivo study to investigate the effect of fig latex on diabetic wound healing was performed using three mouse groups: nondiabetic control mice, diabetic mice and diabetic mice treated with fig latex. The influence of fig latex on the expression levels of β-defensin-1, PECAM-1, CCL2 and ZO-1 and collagen formation was investigated. The GC‒MS analysis demonstrated the presence of triterpenoids, comprising more than 90% of the total latex content. Furthermore, using a streptozotocin-induced diabetic mouse model, topical treatment of diabetic wound tissues with fig latex was shown to accelerate and improve wound closure by increasing the expression levels of β-defensin-1, collagen, and PECAM-1 compared to untreated diabetic wounds. Additionally, fig latex decreased the expression levels of ZO-1 and CCL2.

Figure 1

Gas chromatography and mass spectrometry chromatogram of fig latex analysis. The analysis demonstrated several peaks with different retention times to 47 min of twenty-four active constituents. A, B, C and D are zoomed regions of the chromatogram, RT, retention time.

Figure 2

Inhibition zone showing the antibacterial activity of fresh fig latex on bacteria isolated from diabetic wounds. Causing a clear zone with diameter against the pathogenic bacteria (A) Paenibacillus sp., (B) Bacillus sp., (C) Pseudomonas sp. MS2, (D) Pseudomonas sp. MS4 and (E) Staphylococcus haemolyticus AUMC b-331.

Figure 3

Minimum inhibition concentration (MIC) of fig latex for gram-positive and gram-negative bacteria. MIC reduction is presented as the decrease in the OD600 of the bacterial suspension treated with latex at various concentrations (0.97, 1.95, 3.9, 7.81, 15.62, 31.25, 62.5, 125, 250 and 500 μg/ml) against St. haemolyticus, Ps. MS2, Ps. MS4, Bacillus sp. and Paenibacillus sp. showing the MIC at 250 mg/ml with a red circle.

Figure 4

The impact of fig latex on bacterial biofilm using a microtiter plate biofilm assay against five pathogenic bacterial isolates from diabetic foot ulcers. (A) Microtiter plate showing the differences in the color intensity; (B) Quantitative determination of the optical density of the biofilm from the isolated bacteria as control (open bars) and after treatment with latex (closed bars); (C) Percentage of the antibiofilm activity of fig latex against the bacterial isolates (St. haemolyticus, Ps. MS2, Ps. MS4, Bacillus sp. and Paenibacillus sp.).

Figure 5

Survival curve of DFU pathogenic bacteria in the presence of fresh fig crude latex. The curves represent the bacterial isolates supplemented with the MIC of fresh fig latex in TSB media (dashed line) and show a significant decrease in OD600 for (A) Paenibacillus sp., (B) Bacillus sp., (C) Pseudomonas sp. MS2, and (E) Staphylococcus haemolyticus AUMC B-331 but not for (D) Pseudomonas sp. MS4. The curves represent the bacterial isolates in TSB media without latex (solid line) and show an increase in OD at 600 nm. The results are the mean from triplicate independent measurements.

Figure 6

Effect of fig latex on wound healing and wound closure in diabetic mice. (A) Photographs showing the effect of fig latex on accelerating the healing process in diabetic wounds at each time point from Day 0 (the day of wound creation) until Day 15 (final point). (B) The accumulated data from three individual mice in each group at each time point post-wounding represent the change in diameter (mm). (C) The accumulated results from three animals in each group for each time point post-wounding were calculated by the previously mentioned equation in the Methods section, and the results show the percentage of wound closure. The data are expressed as the mean ± SEM. *P < 0.05, Diab. versus cont., ⁺P < 0.05, diabetic mice treated with Latex versus cont., ^#P < 0.05, diabetic treated with Latex versus diabetic group.

Figure 7

Collagen deposition in the wounded skin tissues stained with Sirius red. Collagen formation (red color) was detected in the wounded skin tissues of the three animal groups using Sirius red staining. One representative experiment is shown at 3 (A), 6 (B), 9 (C), 12 (D) and 15 days (E) post-wounding. (F) The percentages of collagen formation were quantified in the three animal groups, and the accumulated data from three animals from each group are expressed as the mean percentage of collagen formation ± SEM in control mice (open bars), diabetic mice (closed black bars) and diabetic mice treated with fig latex (hatched bars). *P < 0.05, diabetic versus control; and ⁺P < 0.05, diabetic-treated with fig latex versus diabetic animals.

Figure 8

Effects of fig latex on the expression levels of PECAM-1 in wounded diabetic skin tissues. The expression levels of PECAM-1 were detected in the wounded skin tissues of the three animal groups using anti-PECAM-1 and IHC assays. One representative experiment is shown at 3 (A), 6 (B), 9 (C), 12 (D) and 15 days (E) post-wounding. (F) The PECAM-1⁺ cells were quantified in the three animal groups, and the accumulated results from three animals from each group are expressed as the mean percentage of PECAM-1⁺ cells ± SEM in control mice (open bars), diabetic mice (closed black bars) and diabetic mice treated with fig latex (hatched bars). *P < 0.05, diabetic versus control; and ⁺P < 0.05, diabetic-treated with fig latex versus diabetic animals.

Figure 9

Topical application of fig latex restored the expression levels of CCL2 in wounded diabetic skin tissues. (A, B, C, D and E) show one representative experiment of CCL2 expression in the control mice, diabetic mice and diabetic mice treated with latex at Days 3, 6, 9, 12 and 15 post-wounding. (F) The CCL2⁺ cells were quantified in the three animal groups, and the accumulated results from three animals from each group are expressed as the mean percentage of CCL2⁺ cells ± SEM in control mice (open bars), diabetic mice (closed black bars) and diabetic mice treated with fig latex (hatched bars). *P < 0.05, diabetic versus control; and ⁺P < 0.05, diabetic-treated with fig latex versus diabetic animals.

Figure 10

Topical application of fig latex altered the expression of β-defensin-1 and ZO-1. The levels of β-defensin-1 (A) and ZO-1 (B) in the wounded skin tissues of control mice (open bars), untreated diabetic mice (closed black bars), and diabetic mice treated with fig latex (hatched bars) were measured using ELISA. The collected data from three mice in each group are expressed as the mean ± SEM (n = 3). *P < 0.05 for diab. versus cont.; ⁺P < 0.05 for diab. treated with fig latex versus cont.; and ^#P < 0.05 diab. treated with fig latex versus diab. (ANOVA with Tukey’s post-test).