| Objectives1.Establishment of skin trauma model in rats with type 1 diabetes mellitus2.To observe the effect of AAT on skin wound healing in diabetic rats3.To explore the mechanism of AAT promoting skin wound healing in diabetic rats Methods:1.Establishment of rat model of type 1 diabetes mellitus(DM model): Sixty male SD rats were fasting for 12 hours after one week of adaptive feeding,they were randomly divided into diabetic model group(DM model group)(4xN=48)and normal control group(N=12).Intraperitoneal injection of sodium citrate buffer in normal control group(60mg/kg),streptozotocin solution administered with sodium citrate buffer in diabetic model group by one-time intraperitoneal injection(STZ solution,60mg/kg).After 72 hours,blood sugar in tail vein of rats was measured.2.Wound Surface Experiment and Grouping in Rats : Aftermonitoring the successful model of type I DM rats and confirming that their DM characteristics were stable,all rats including the normal control group were prepared before operation.Two full-thickness round skin defect wounds with a diameter of 1.0 cm were prepared at the same position on the back of each rat using surgical scissors.Successful establishment of normal rat wound model and diabetic wound model(DCU model).48 DCU model rats were randomly divided into four groups: blank control group,negative control group,high concentration AAT treatment group and low concentration AAT treatment group,12 rats in each group.Drug application method and dosage : No treatment was given to the wounds in blank group;The negative control group smeared with gel stroma;In the AAT low concentration treatment group,AAT was allocated to 25mg/ml solution and smeared with the ratio of AAT: gel =2:3;In the AAT high concentration treatment group,AAT was allocated to 100mg/ml solution and smeared with the ratio of AAT:gel =2:3.3.Observation index : Blood sugar,body weight,Wound dressing and photograph were recorded on the day of operation,3,7,11,14 and16 days after operation.Six rats in each group were sacrificed 7 and 16 days after operation to get wound tissue and prepare specimens for use.Indirect calculation of wound area and evaluation of wound healing with Image J software.Hematoxylin-eosin(HE)staining was used toobserve wound healing and the number of inflammatory cells and blood vessels in each group.The levels of inflammatory factors,tumor necrosis factor-alpha(TNF-?),interleukin-1alpha(IL-1?)and interleukin-6(IL-6)in the peri-wound tissues of each group were detected by Western blotting.All results were averagedąSD.Means were analyzed by variance analysis and t-test.SPSS19.0 software was used for data analysis.P < 0.05 is considered to have statistical significance Results:1.A single intraperitoneal injection of streptozotocin 60 mg/kg in rats can successfully induce type I diabetes mellitus model.Compared with the normal control group,the diabetic rats in the model group gradually showed weight loss,excessive drinking,polyuria,yellowish hair,mental depression,and blood sugar maintained above 16.7 mmol/L.On the basis of this model,a diabetic wound model can be successfully established by making full-thickness skin defect through the symmetrical part of the back skin,and the wound healing of diabetic rats is significantly delayed compared with the normal control group.2.Compared with blank control group and negative control group,wound healing in AAT treatment group was accelerated(p < 0.05).The healing rate of AAT group was dose dependent,that is,the healing rate of high concentration group was higher than that of low concentrationgroup(p < 0.05).3.Compared with the blank control groupand negative control group,the wound tissue of the AAT treatment group showed a decrease in inflammatory cell infiltration and the number of capillaries,an increase in the content of subcutaneous collagen fibers,and a local mass accumulation.4.Compared with the blank control group and the negative control group,the number of inflammatory cells and the content of blood vessels in AAT treatment group decreased,and the difference was statistically significant(p < 0.05).Compared with the low concentration AAT treatment group,the number of inflammatory cells in high concentration AAT treatment group was fewer,and the content of blood vessels was higher(p < 0.05).There was no significant difference between negative control group and blank control group.5.Compared with blank control group and negative control group,the contents of IL-1,IL-6 and TNF-? in AAT high and low concentration treatment group decreased significantly(p < 0.05).Compared with low concentration treatment group,the contents of three factors in AAT high concentration treatment group were lower(p <0.05).There was no significant difference between negative control group and blank control group.Conclusions:1.The diabetic model of type I diabetes mellitus can be successfully induced by intraperitoneal injection of streptozotocin solution according to the standard of 60mg/kg in rats.On this basis,a stable diabetic trauma model can be successfully established.2.AAT can promote the healing of diabetic skin wound in a dose-dependent manner..3.AAT can promote the healing of diabetic skin wound,which may be related to its inhibition of the production of inflammatory cytokines such as IL-1,IL-6 and TNF-?. |
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