Dehydrozingerone promotes healing of diabetic foot ulcers: a molecular insight

Abstract

Introduction: One of the most common problems of diabetes are diabetic foot ulcers (DFUs). According to National Institute for Health, initial management of DFUs can decrease the complication of limb amputations and can improve the patient's quality of life. DFU treatment can be optimized with the help of multidisciplinary approach. Based on many studies, control of glucose levels in blood, antioxidant activity, reduction in cytokine levels, re-epithelialization, collagen formation, migration of fibroblasts are major phases involved in managing DFU. Dehydrozingerone (DHZ), has been known for its anti-inflammatory, antioxidant and wound healing properties.

Methodology: Three months high-fat diet and low dose of streptozotocin-induced type-II diabetic foot ulcer model was used to evaluate the effectiveness of dehydrozingerone. DHZ was given orally to rats for 15 days post wounding. TNF-α, IL-1β and antioxidant parameters like lipid peroxidation, glutathione reductase were estimated. Immunoblotting was done to investigate the effect of DHZ on the expression of ERK, JNK, HSP-27, P38, SIRT-1, NFκB, SMA, VEGF and MMP-9 in skin tissue. Histopathology was performed for analyzing DHZ effect on migration of fibroblasts, formation of epithelium, granulation tissue formation, angiogenesis and collagen formation.

Results: DHZ decreased the levels of malondialdehyde, TNF-α, IL-1β and increased glutathione levels in wound tissue. Western blotting results suggested that DHZ activated ERK1/2/JNK/p38 signaling, increased expression of HSP-27, SIRT-1, VEGF, SMA thus facilitating the migration and proliferation of fibroblasts, angiogenesis and decreased inflammation. Masson Trichrome & histopathology showed an increase in collagen, epithelial and granulation tissue formation.

Conclusion: DHZ significantly accelerates the healing of diabetic foot ulcers in high fat diet fed plus low dose streptozotocin induced type-II diabetic Wistar rats.

Keywords: Cellular mechanism; Dehydrozingerone; Diabetic foot ulcers; High fat diet; Inflammation.

Fig. 1

HFD effect on body weight (in gms), represented as Mean ± SEM. ****p < 0.0001 when compared to normal pellet fed diet group. Analysis done by using two-way ANOVA with Sidak’s post-hoc test

Fig. 2

Effect of HFD on OGTT (mg/dl). Data represented as Mean ± SEM. *p < 0.01, ***p < 0.0001 when compared to normal pellet fed diet group. Analysis done by using two-way ANOVA with Sidak’s post-hoc test

Fig. 3

Effect of HFD on Lipid profile (mg/dl). Data represented as Mean ± SEM. **p < 0.001, *p < 0.01 when compared to HFD group and ^##p < 0.001, ^###p < 0.0001 compared to normal pellet fed diet. Analysis done by using two-way ANOVA with Tukey’s post-hoc test

Fig. 4

A Representative images of effect of DHZ treatment on different days of DFU B Effect of DHZ on re-epithelization of wound. Data represented as Mean ± SEM. **p < 0.001, ****p < 0.0001 when compared to HFD group and ^#p < 0.001 compared to normal pellet fed diet. Analysis done by using two-way ANOVA with Tukey’s post-hoc test.

Fig. 5

A Effect of DHZ on Malondialdehyde (MDA) levels on diabetic foot ulcer tissue B Effect of DHZ on GSH levels in diabetic foot ulcer tissue in type-II diabetic Wistar rats. Data represented as Mean ± SEM. **p < 0.001, *p < 0.01 when compared to HFD group and ^###p < 0.0001, ^##p < 0.001 compared to normal pellet fed diet. Analysis done by using one-way ANOVA with Tukey’s post-hoc test

Fig. 6

A Effect of DHZ on TNF-α levels on day 5 (post wounding) diabetic foot ulcer tissue B Effect of DHZ on IL-1β levels on day 5 (post wounding) in diabetic foot ulcer tissue. Data represented as Mean ± SEM. *p < 0.01, **p < 0.001 when compared to DFU group and ^##p < 0.001 compared to normal control. Analysis done by using one-way ANOVA with Tukey’s post-hoc test

Fig. 7

Effect of DHZ on MAPK, HSP-27, SIRT-1, NFkB, SMA-α, MMP-9, VEGF-B, COL-1 A Representative images of blots B p-ERK/ERK ratio, C p-JNK/JNK ratio, D p-P³⁸/P³⁸ ratio, E HSP-27/α-tubulin ratio F SIRT-1/α-tubulin ratio G p-NFkB/NFkB ratio H SMA- α /α-tubulin ratio I MMP-9/ α-tubulin ratio J VEGF-B/ α-tubulin ratio K COL-1. Data represented as Mean ± SEM. ***p < 0.0001, **p < 0.001, *p < 0.01 when compared to DFU group. Analysis done by using one-way ANOVA with Tukey’s post-hoc test.

Fig. 7

Effect of DHZ on MAPK, HSP-27, SIRT-1, NFkB, SMA-α, MMP-9, VEGF-B, COL-1 A Representative images of blots B p-ERK/ERK ratio, C p-JNK/JNK ratio, D p-P³⁸/P³⁸ ratio, E HSP-27/α-tubulin ratio F SIRT-1/α-tubulin ratio G p-NFkB/NFkB ratio H SMA- α /α-tubulin ratio I MMP-9/ α-tubulin ratio J VEGF-B/ α-tubulin ratio K COL-1. Data represented as Mean ± SEM. ***p < 0.0001, **p < 0.001, *p < 0.01 when compared to DFU group. Analysis done by using one-way ANOVA with Tukey’s post-hoc test.

Fig. 8

A Effect of DHZ on histopathology (H&E staining). Images captured at 100 × optical zoom B Effect of DHZ on histopathology (H&E staining). Images captured at 400 × optical zoom

Fig. 9

Effect of DHZ on Collagen deposition (Masson trichrome staining). Images captured at 400 × optical zoom