A proline derivative-enriched methanol fraction from Sideroxylon obtusifolium leaves (MFSOL) stimulates human keratinocyte cells and exerts a healing effect in a burn wound model

Abstract

It was previously demonstrated that the methanol fraction of Sideroxylon obtusifolium (MFSOL) promoted anti-inflammatory and healing activity in excisional wounds. Thus, the present work investigated the healing effects of MFSOL on human keratinocyte cells (HaCaT) and experimental burn model injuries. HaCaT cells were used to study MFSOL's effect on cell migration and proliferation rates. Female Swiss mice were subjected to a second-degree superficial burn protocol and divided into four treatment groups: Vehicle, 1.0% silver sulfadiazine, and 0.5 or 1.0% MFSOL Cream (CrMFSOL). Samples were collected to quantify the inflammatory mediators, and histological analyses were performed after 3, 7, and 14 days. The results showed that MFSOL (50 μg/mL) stimulated HaCaT cells by increasing proliferation and migration rates. Moreover, 0.5% CrMFSOL attenuated myeloperoxidase (MPO) activity and also stimulated the release of interleukin (IL)-1β and IL-10 after 3 days of treatment. CrMFSOL (0.5%) also enhanced wound contraction, promoted improvement of tissue remodeling, and increased collagen production after 7 days and VEGF release after 14 days. Therefore, MFSOL stimulated human keratinocyte (HaCaT) cells and improved wound healing via modulation of inflammatory mediators of burn injuries.

Figure 1. Typical base peak intensity (BPI) chromatogram of methanol fraction from leaf decoction of Sideroxylon obtusifolium (MFSOL) in the negative ionization mode. ni: not identified; ud: up to 1000 DA.

Figure 2. Effect of methanol fraction of Sideroxylon obtusifolium (MFSOL) on cell viability of human keratinocytes (HaCaT). Increased viability of keratinocytes was produced by MFSOL (6.25-100 μg/mL) after 24 (A), 48 (B), and 72 (C) h of incubation by the MTT assay and SRB assay (D, E, and F), respectively. To illustrate the HaCaT density, photographs were taken at 200× magnification (scale bar 100 μm) 48 h after treatment with 25 and 50 μg/mL of MFSOL (G). Data are reported as means±SE of at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001, compared to the Control (Vehicle) group (one-way ANOVA followed by the Tukey post-test).

Figure 3. Methanol fraction of Sideroxylon obtusifolium (MFSOL) stimulated cell proliferation and migration rates of HaCaT in the scratch assay. A scratch was produced in a monolayer of HaCaT cells and photographs were taken, before (0 h) and 24 (A), 48 (B), and 72 h (C) after treatment with MFSOL (100× magnification; scale bar 200 μm) (D). Pretreatment with mitomycin C (10 μg/mL) was also performed one hour before the introduction of the scratch to inhibit cell proliferation, allowing only evaluation of cell migration after 24 h (E) and 48 h (F). The wells were photographed (100×, scale bar 200 μm) after 24 and 48 h (G). The data are reported as means±SE. *P<0.05, **P<0.01, ***P<0.001 compared to the Control group (water) (one-way ANOVA followed by the Tukey test).

Figure 4. Effect of methanol fraction of Sideroxylon obtusifolium (MFSOL) on the contraction of induced burns in mice. The percentage of contraction of the burns was measured after 3, 5, 7, 9, 12, and 14 days (A, B, C, D, E, and F, respectively). The animals were treated with a single daily application of dermatological creams (Cr) for 14 days. The CrMFSOL group received 0.5 and 1.0% MFSOL, the Sulfa group received 1% silver sulfadiazine cream, and the Vehicle group was exposed to the base vehicle cream without any addition of active substance. The superficial burns were photographed for macroscopic monitoring on days 3, 5, 7, 9, 12, and 14 after the burn was produced (scale bar 10 mm) (G). One animal per group was chosen to represent the group according to the lesion contraction results. The data are reported as mean±SE. *P<0.05, **P<0.01 compared to the Sham group; ^#P<0.05, ^##P<0.01 compared to the Vehicle group, (n=10 animals/group) (one-way ANOVA followed by the Tukey test).

Figure 5. Cream of methanol fraction of Sideroxylon obtusifolium (CrMFSOL) promoted improved tissue remodeling, reepithelialization, and stimulated collagen production on superficial burns 7 days after treatment. Photomicrographs of histological slides (hematoxylin-eosin staining) of burn lesions on the 7th day after injury for the groups Sham (A), Vehicle (B), silver sulfadiazine (C), 0.5% CrMFSOL (D), and 1.0% CrMFSOL (E) (n=6 animals/group). Black arrows indicate the presence of acute inflammatory infiltrate in groups A and B, while red arrows indicate the presence of atrophic epithelium, presence of a corneal layer, and absence of ulcer in groups C, D, and E. The lesions were stained with picrosirius red and photographed in six fields (200×, scale bar 200 μm). The percentage of total collagen present on the 7th day was determined by the ImageJ® software (F). The data are reported as mean±SE of the six areas of each wound per group. *P<0.05 compared to the Sham group; ^#P<0.05 compared to the Vehicle group (n=6 animals/group per day) (one-way ANOVA followed by the Tukey test).

Figure 6. The treatment with methanol fraction of Sideroxylon obtusifolium cream (CrMFSOL) reduced myeloperoxidase activity (MPO) and stimulated release of interleukin (IL)-1β and IL-10 after 3 days, and vascular endothelial growth factor (VEGF) after 14 days. The levels of tumor necrosis factor (TNF)-α were not reduced significantly. The data are reported as mean±SE. ^#P<0.05, ^##P<0.01, ^###P<0.001 compared to the Vehicle group; ^aP<0.05 and ^aaaP<0.001 compared to the Sulfa group n=6 animals/group per day) (one-way ANOVA followed by the Tukey test).