The Empirical Study About The Effect And Generating Mechanisms Of Oxidative Stress In Wound Healing Of Diabetes Mellitus

Oxidative stress was a condition which was the imbalance of prooxidant and antioxidants,abnormally high levels of free radicals and/or the decline of antioxidant defense mechanisms.Excessive oxidative stress could lead to damage of tissue,which played an important role in the development of many kinds of diseases.Many cells in microenvironment of wound could product reactive oxygen species(ROS) in different extent.A series of changes of pathology and physiology happen in diabetes mellitus(DM),such as anomal-glycometabolism,the products of nonenzymatic glycoxidation of proteins/lipids and so on.Recently,during glycometabolism and its intermediate metabolite could lead to oxidative stress by many ways.Hard healing happen in DM wound.Oxidative stress may participate in the forming of DM hard wound healing.[objective]To study the effect and generating mechanisms of oxidative stress in wound healing of diabetes mellitus.[Methods]Male Sprague-Dawley were enrolled in the experiment and randomized into control and experimental group.The experimental groups were injected with Streptozotocin(STZ,65mg/kg)to induce diabetic animal.The shaved rat skin specimens were harvested on post STZ-induced weeks.We also used the skin of diabetes patient as specimens.One-Touch Ultra was used for monitoring the amount of blood sugar;the skin glucose contents were measured by Beckman Autoanalyzer; skin AGEs concentrations were assessed by detecting total fluorescence in tissue collagen and immunohistochemistry assay.Glycosylated plasma protein contents,H₂O₂ contents,MDA contents and GSH contents in blood were deteceted by biochemistry technique.The amount of MDA were detected by using assay kit.In vitro study,H₂O₂ was as the model of oxidative stress to investigate the mechanism and effect of the biological behavior of dermal fibroblasts of normal rats;ROS was deteceted when neutrophils exposing to the different concentrations (0.315,0.625,1.25mg/ml)of AGEs modified human serum albumin(AGE-HSA) and high glucose.Different glucose and/or human serum albumin(HAS) incubated together,observated H₂O₂ contents from glucose-oxidation.[Result]The histological examinations showed that the diabetic skin has a series of pathology-physiology changes,including the thickness of dermis layer,the degenerated collagen fibers with a focal chronic inflammatory cells infiltration,the high content of glucose and AGEs in the diabetic skin.The levels of MDA in diabetic skin were increased significantly;The levels of GSP,H₂O₂ and MDA in diabetic blood were increased significantly(p＜0.01,p＜0.01,P＜0.05);while the GSH contents were lower than that of controlled group(P＜0.05).The proliferations of neutrophils were obviously stimulated when exposed to AGE-HSA at the concentrations of 0.315,0.625 and 1.25mg/ml for 30 minutes.AGEs and high glucose can prime neutrophils to release higher ROS than that of control groups(P＜0.05,P＜0.05)。Different concentrations of AGEs and/or HAS incubated together could generate H₂O₂ to different extent(p＜0.01);With the increasing of concentreation of H₂O₂,cell survival of fibroblasts was lower and lower(0.385±0.120,0.359±0.007,0.301±0.005,0.282±0.013,0.251±0.009,P＜0.01);compared with that in control group,apoptosis was not changed in 20μmol/L H₂O₂ group,while abundant,apoptotic dermal fibroblasts were observed in 100μmol/L H₂O₂ group(,P＜0.01);ROS was produced more than that in control group(P＜0.01,p＜0.01);the Content of MDA in 100μmol/L H₂O₂ group increased(P＜0.01),while in 20μmol/L H₂O₂ group the the Content of MDA was not changed.[Conclusions]1.A pathophysiologic disorder has already been occurred in diabetic skin before injury.That's why we call it "Underlying disorder" This phenomenon may be result from the local biochemical factors such as high concentrations of glucose and AGEs,which should be an important mechanism in the pathogenesis of impaired wound healing in diabetes.2.High glucose and AGEs acting with neutrophils and glucose-oxidation led to ROS,and made oxidative stress in the diabetic skin increasing。3.The increasing of oxidative stress resulted in the abnormal changes of cellular biological behaviors of dermal fibroblasts of rats, and led to hard wound repairing。Oxidative stress may participated in the forming and development of the diabetic hard wound repairing。4.It may supply theory base that Anti-oxidation promote diabetic wound healing.