Topical combined Phyllanthus emblica Linn. and simvastatin improves wound healing in diabetic mice by enhancing angiogenesis and reducing neutrophil infiltration

Abstract

The present study aimed to investigate the effects of combined Phyllanthus emblica Linn. (PE) and simvastatin (SIM) on diabetic wounds in male BALB/C mice. Bilateral full thickness wound excisions were performed in the control and diabetic groups (45 mg/kg streptozotocin, intraperitoneally injected daily for 5 days). The diabetic mice received daily treatment with four different types of cream: Vehicle [diabetes mellitus (DM) + Vehicle group], 100% PE (DM + PE group), 5% SIM (DM + SIM group) and combined 100% PE + 5% SIM (DM + Combination group) for 4, 7 and 14 days. The tissue malondialdehyde (MDA) and IL-6 protein levels, the number of infiltrated neutrophils, and the percentages of wound closure (%WC), capillary vascularity (%CV) and re-epithelialization (%RE) were subsequently measured. The results indicated that in the DM + Combination group, %CV and %WC were significantly increased when compared with the DM + Vehicle group on days 7 and 14. The tissue MDA content on day 14, and the number of infiltrated neutrophils on days 4 and 7 were significantly reduced in the DM + Combination group compared with those in the DM + Vehicle group. Furthermore, a strong positive correlation was revealed between %CV and %WC in the five groups on day 7 (r=0.736; P=0.0003). These findings indicated that topical application of combined PE and SIM could enhance wound healing by upregulating angiogenesis and reducing neutrophil infiltration in mice with diabetic wounds.

Keywords: Phyllanthus emblica Linn; angiogenesis; capillary vascularity; diabetic wound; neutrophils; simvastatin; wound closure.

Figure 1

MDA content and correlation analysis between MDA and %WC. (A) Tissue MDA levels in the various groups at day 4, 7 and 14. Data are presented as the mean ± SEM. Pearson's correlation analyses between MDA contents (nmol/mg total protein) and %WC on day (B) 4, (C) 7 and (D) 14. Number of animals in each group, 4-5; number of wounds in each group, 3-5. ^*P<0.05. %WC, percentage wound closure; DM, diabetes mellitus; MD, malondialdehyde; ns, not significant; PE, Phyllanthus emblica Linn.; SIM, simvastatin.

Figure 2

Effects of topical application of combined PE and SIM cream on the number of infiltrated neutrophils in diabetic wounds. (A) Positive infiltrated neutrophils were indicated by yellow arrows (hematoxylin and eosin staining; x400 magnification; scale bar, 100 µm). (B) Number of infiltrated neutrophils (per 1,000 cells) in each group on days 4, 7 and 14. Data are presented as the mean ± SEM. Number of animals in each group, 4-5; number of wounds in each group, 3-5. ^*P<0.05. DM, diabetes mellitus; ns, not significant; PE, Phyllanthus emblica Linn.; SIM, simvastatin.

Figure 3

Effects of topical application of combined PE and SIM cream on IL-6 protein levels (pg/mg total protein) in diabetic wounds. (A) IL-6 protein levels in diabetic groups on days 4 and 7. Data are presented as the mean ± SEM. Number of animals in each group, 4-5; number of wounds in each group, 3-5. (B) Pearson's correlation analysis between number of infiltrated neutrophils (per 1,000 cells) and IL-6 protein levels on days 4 and 7. DM, diabetes mellitus; ns, not significant; PE, Phyllanthus emblica Linn.; SIM, simvastatin.

Figure 4

Effects of topical application of combined PE and SIM cream on tissue VEGF protein levels (pg/mg total protein). (A) VEGF protein levels in the groups on day 7 and 14 (n=5). Data are presented as the mean ± SEM. (B) Confocal image of capillary microvasculature. Scale bar, 100 µm. (C) %CV on days 7 and 14. Number of animals in each group, 4-5; number of wounds in each group, 4. Data are presented as the mean ± SEM. ^*P<0.05. (D) Pearson's correlation analysis between VEGF levels and %CV on day 7. %CV, percentage of capillary vascularity; CON, control; DM, diabetes mellitus; ns, not significant; PE, Phyllanthus emblica Linn.; SIM, simvastatin.

Figure 5

Effects of topical application of combined PE and SIM cream on %RE. (A) RE on days 4, 7 and 14. Black arrows indicate the wound edge with features of increased wound thickness and proliferated epidermis; yellow arrows indicate the tip of the endothelial cell in the wound area (hematoxylin and eosin staining; x400 magnification; scale bar, 1 mm). (B) %RE in diabetic groups on days 4, 7 and 14. Data are presented as the mean ± SEM. Number of animals in each group, 4-5; number of wounds in each group, 4-5. ^*P<0.05. %RE, percentage of re-epithelialization; CON, control; DM, diabetes mellitus; ns, not significant; PE, Phyllanthus emblica Linn.; SIM, simvastatin.

Figure 6

Effects of topical application of combined PE and SIM cream on %WC. (A) Images of wound area (scale bar, 1 mm). (B) %WC in the groups on days 7 and 14 post-wounding. Data are presented as the mean ± SEM. ^*P<0.05. (C) Pearson's correlation analysis between %CV and %WC in the five groups on days 7 and 14. Number of animals in each group, 4-5; number of wounds in each group, 4-9. %CV, percentage of capillary vascularity; %WC, percentage of wound closure; CON, control; DM, diabetes mellitus; ns, not significant; PE, Phyllanthus emblica Linn.; SIM, simvastatin.

Figure 7

Proposed mechanism of the effect of combined PE and simvastatin on promoting diabetic wound healing. MDA, malondialdehyde; PE, Phyllanthus emblica Linn; ROS, reactive oxygen species.