THE ROLE OF NITROGEN OXIDE AND NITROSATIVE STRESS IN BURN WOUND HEALING IN DIABETES MELLITUS
Authors: Myronenko O.I. , Natrus L.V. , Panova T.I. 
Published: December 2019

Relevance. The risk of developing of diabetic foot ulcers is 25% over the lifetime of patients with diabetes mellitus. Inhibition of wound healing is associated with impaired macrophage polarization, decreased bioavailability of growth factors, enhanced activation of matrix metalloproteinases, which in turn is probably associated with severity of not only oxidative stress, but also nitrosative one.

Objective. To investigate the indices of nitrosative stress in connective tissue regeneration in the healing of burn wound in rats with simulated diabetes, compared with rats without diabetes.

Materials and methods. Two groups of laboratory rats of the Wistar line were used: diabetes mellitus and thermal burns were simulated in the experimental group (n=24), and only burns were modelled in the control group (n=24).  Diabetes mellitus was reproduced by administering of streptozotocin at a dose of 50 mg/kg to rats of the experimental group.  Thermal burns with a total area of ​​18-20% of the body surface were reproduced by applying of copper plates heated in water to 100°C to the skin of rats.  Animals were removed from the experiment by decapitation under thiopental anesthesia, 6 rats of each group at a time on 3, 7, 14 and 21 day of observation.  Concentration of inducible NO-synthase (iNOS), 3-nitrotyrosine proteins and vascular endothelial growth factor (VEGF) were measured in tissue regenerate samples by Western-blot analysis.  The content of malondialdehyde (MDA) was determined in the blood serum of rats by reaction with thiobarbituric acid and subsequent spectrophotometric analysis.  The measured values ​​are expressed in arbitrary optical density units (arbitrary units, a.u.).

Results. The expression of inducible NO-synthase (iNOS) was shown to be significantly decreased during burn wound healing in diabetes mellitus compared with controls at 7 and 14 day of burn wound healing.  Thus, iNOS level was 1.24±0.05 a.u. in the control group and 0.72±0.03 a.u. in the experimental one at 7 day of observation; 0.93±0.08 a.u. and 0.32 ± 0.02 a.u., respectively, at 14 day.

 Statistically significant differences in the level of 3-nitrotyrosine compounds between the control and experimental groups were detected only at 3 day of observation: 1.0±0.05 and 0.69±0.04 a.u., respectively.

 The level of malondialdehyde (MDA) increased significantly in the experimental group from 3 to 14 day of observation, from 0.77±0.01 a.u.  to 8.17±0.07 a.u., which indicates the severity of lipid peroxidation in diabetes.  A statistically significant difference in MDA levels between the control and experimental groups was observed at all observation periods: from 4.13±0.09 and 0.77±0.01 a.u., respectively, at 3 day of observation to 4.18±0.09 and 3,46±0.25 a.u., respectively, at 21 day.

 A positive correlation between the level of malondialdehyde and 3-nitrotyrosine compounds (R=0.647; р<0.05) in diabetes mellitus is described, while in the non-diabetic group, it was negative (R=-0.528, p<0.05).

 A decrease in the level of VEGF in tissue regeneration samples in diabetes mellitus was found compared with the control group at 3, 7 and 21 days of observation, which correlated with impaired expression of iNOS (R=-0.703; p<0.05) in diabetes mellitus. Thus, at 3 day of observation, VEGF level was 1.0±0.06 a.u. in the control group and 0.4±0.03 a.u. in the experimental group; on 7 day - 2.76±0.05 and 1.46±0.08 a.u., respectively;  on 21day  - 2.61±0.09 and 1.26±0.06 a.u., respectively.

Conclusions. It is shown that in diabetes mellitus during healing of the burn wound, there is a local decrease in the level of iNOS expression, which leads to inhibition of NO formation and its physiological effects. Increased oxidative stress, while further reducing the bioavailability of NO through the formation of peroxynitrite. One of the important effects of NO during wound healing is the enhancement of VEGF formation. Shown reduction of NO (due to the reduced expression of iNOS) is correlated with the decrease of VEGF level in tissue regeneration in diabetes, which is one of the important factors of impaired wound healing.