| Objective: Establish a rat model of diabetic ulcer wounds,using cellular autophagy and PI3K-Akt-mTOR signaling pathway as the research entry point,to explore the mechanism of moist exposed burn therapy /moist exposed burn ointment(MEBT/MEBO)promoting the repair of diabetic foot ulcer wounds,to provide a theoretical basis for the clinical application of MEBT/MEBO in the treatment of diabetic ulcer wounds.Methods: Eighty healthy male wistar rats were randomly divided into 60 in the diabetic ulcer wound group,20 in the control group.The diabetic ulcer wound group was given adaptive feeding for 1 week,then fed with high fat diet for 4 weeks and fasted for 16 hours,intraperitoneal injection of streptozotocin(STZ)45 mg/kg,with fasting blood glucose ?16mmol/L as a successful model for diabetes,we established a full-thickness skin resection model with a diameter of about 2.5 cm × 2.5 cm on the back of the rat 1 week later,the wound depth required to reach the fascia,then smear Staphylococcus aureus suspension in wounds to cause a diabetic rat wound model,which was randomly divided into three groups: MEBO group,kangfuxin group,model group,each group had 20 rats,the three groups were treated with MEBO,kangfuxin and saline respectively.The rats in the control group underwent full-thickness skin resection on the back.The size and the depth was basically the same as that of the diabetic ulcer wound group.However,STZ was not injected,and the wound was not smear with S.aureus suspension.After successful modeling,each group dynamically observed the healing of the wound,and measured?calculated the wound healing rate of each group of rats,and on the first day,5th day,11 th day,18 th day,and 25 th day respectively.Four rats were randomly selected from each group at the time point,and the wound tissue was collected from the model area and sacrificed.The collected tissue samples were observed by routine pathological techniques to observe the morphology of the wound tissue,electron microscopy to observe autophagosome changes,Western blot to detect PI3 K,Akt,mTOR protein expression levels,and real-time quantitative PCR(RT-PCR)analysis of PI3 K,Akt,mTOR mRNA expression.Results: 1.Wound healing: The wound repair of the control group was the best and the healing time was the shortest;the repair of the wound in the MEBO group and the kangfuxin group was significantly better than that in the model group.Wound healing rate: The wound healing rate of each group increased gradually with the increase of each phase point.On the 5th day,the wound healing rate was in the control group> kangfuxin group>MEBO group>model group.The difference was statistically significant(P<0.05);On the 11 th day,the wound healing rate was control group >MEBO group> kangfuxin group>model group,the difference was statistically significant(P<0.05);On the 18 th day,the 25 th day,the wound healing rate was control group> MEBO group> kangfuxin group>model group,the difference was statistically significant(P<0.05),but there was no significant difference between MEBO group and kangfuxin group on the 18 th and 25 th day(P= 0.76,0.23).2.Pathological changes of wounds: On the 5th day,the morphology of fibroblasts and angiogenesis in the granulation tissue of ulcer wounds in MEBO group,kangfuxin group and control group were better than those in model group;On the day11 and 18,the fibroblasts and collagen fibers of the MEBO group and the rehabilitation group were densely formed,while the collagen fibers in the model group control group were arranged disorderly.3.In the process of wound healing,the autophagosomes of each group were first large,then became smaller,and the early morphology was more full and round,and the double capsule structure was obvious.The middle and late stages gradually became smaller or disappeared,and the double capsule structure was blurred.Among them,the changes in the MEBO group are more obvious.4.RT-PCR and Western blot analysis showed that the mRNA and protein expressions of PI3 K,Akt and mTOR in MEBO group,kangfuxin group and model group were highest on the 5th day.The expression of MEBO group> kangfuxin group > Model group,the difference between each two groups was statistically significant(P<0.05);on the 11 th day and the 18 th day,the expression of MEBO group and kangfuxin group showed a significant downward trend,but still higher than the model group,the difference was statistically significant(P<0.05),but there was no significant difference between MEBO group and kangfuxin group(P>0.05).Conclusion: MEBT/MEBO activates the PI3K-Akt-mTOR signaling pathway during the repair of diabetic ulcer wounds,which is more prominent in the middle and late stage of wound repair,thereby inhibiting autophagy,promoting ulcer wound healing,significantly improving healing rate and improving repair quality. |
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