AMELIORATIVE ROLE OF SELECTED MICRONUTRIENT SUPPLEMENTS ON OXIDATIVE STRESS AND WOUND HEALING IN DIABETIC RATS AND TYPE 2 DIABETICS

Abstract

Oxidative Stress (OS) has been implicated in aetiology of Diabetes Mellitus (DM) and its complications including Diabetic Foot Ulcer (DFU). The role of antioxidant micronutrient supplementation on enzymatic and non-enzymatic markers of OS was investigated in streptozotocin (STZ)-induced DM in rats and Type-2 diabetes with and without DFU. One hundred and sixty rats were divided into: control (Group A); diabetic rats without-ulcer (Group B); diabetic rats with-ulcer (Group C); glibenclamide-treated diabetic rats without-ulcer (Group D) and glibenclamide-treated diabetic rats with-ulcer (Group E) which were either supplemented with selenium (50μg/kg) + vitamin C (500mg/kg) + vitamin E (200mg/kg) for 12weeks or not. Diabetes was induced with STZ (30mg/kg) in 0.1M sodium citrate buffer i.p. To create ulcers, after confirming diabetes, Groups C and E were anaesthetized with sodium pentobarbital (5mg/100g), depilatory cream was applied to rats' back, washed off, and swabbed with 70% alcohol. Thereafter, four full thickness skin deep wounds were inflicted using acupunch (8mm). Human study comprised consenting 50 non-diabetic controls (Group one), 50 DM without-DFU (Group two) and 50 DM with-DFU (Group three). Twenty-five subjects each in Groups two and three were given selenium (100μg) + vitamin C (1000mg) + vitamin E (400mg) for 16weeks while the rest received no supplements. Wound healing in rats was evaluated using histological parameters while ‘ABDEFS’ tools were used for evaluating chronic ulcers in humans. In rats and humans, non-enzymatic OS markers: plasma 4-hydoxy-2′-nonenal (4-HNE) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) were determined by ELISA while lipid peroxides (LPO), Total Antioxidant Status (TAS) were determined spectrophotometrically. Data were analysed using Friedman and Wilcoxon tests at p=0.05. Before supplementation, 4-HNE, LPO and 8-OHdG were higher while TAS was lower in Group B by 1.1, 20.7, 44.4 and 8.1% respectively compared with Group A. Similar differences were respectively observed in Groups C (6.8, 58.4, 57.7 and 24.2%), D (2.2, 34.5, 55.7 and 14.6%) and E (12.8, 81.5, 88.2 and 28.9%). However, supplementation resulted in significant decreases in 4-HNE, LPO and 8-OHdG with increase in TAS of supplemented Groups: A (2.9, 7.5, 13.0 and 2.2% respectively), B (4.9, 11.2, 21.4 and 6.0% respectively), C (8.8, 4.7, 14.8 and 6.2%), D (10.5, 21.7, 28.6 and 14.0% respectively) and E (20.3, 23.4, 32.6 and 13.4% respectively) compared with non-supplemented groups. Wounds in supplemented Groups C and E displayed better re-epithelisation and granulation tissue formation than non-supplemented groups. In the studies for humans before supplementation, significant increases in LPO and 8-OHdG were observed in Groups two (42.6 and 46.7%) and three (76.6 and 56.8%) with decreased TAS of 53.5 and 52.8% respectively in Groups two and three relative to Group one. Following supplementation, LPO decreased by 10.5 and 22.5% and 8-OHdG by 10.2 and 22.5% while TAS increased by 12.5 and 9.1% in supplemented Groups two and three respectively, when compared with non-supplemented groups. A lower ABDEFS-score (7.0±1.3) was observed in supplemented Group three which resulted in better wound healing than non-supplemented group (10.7±1.2). Antioxidant micronutrient supplementation demonstrated ameliorative effect on oxidative stress and wound healing in diabetic rats and humans.