Preliminary Study On The Role Of Autophagy In Promoting The Healing Of Diabetic Wounds Treated With MEBT/MEMO

Objective: To observe the effect of Moist Exposed Burn Therapy/Moist Exposed Burn Ointment(MEBT/MEBO)on the expression of ULK1,p70S6 K and 4EBP1 in ulcer wound tissues of diabetic rats,and to explore its mechanism,so as to provide theoretical basis for clinical application.Methods: Two hundred and fifty SPF Wistar male rats were randomly divided into blank group,control group,model group,Kangfuxin group and MEBO group,with 50 rats in each group.The rats in the blank group were only treated with skin preparation,while conventional wound models were established in the control group,diabetic wound models were respectively established in the model group,Kangfuxin group and MEBO group.After the establishment of the model,the rats in the control group and the model group were treated with external application of saline,while the Kangfuxin group and MEBO group were treated with external application of Kangfuxin and MEBO respectively.The following changes were observed in each group on day 1,day 5,day 11,day 18 and day 25 after treatment:(1)The wound healing status,size of wound area was recorded and wound healing rate was calculated;(2)Pathological changes of wound tissues stained with HE were abserved.(3)The expression of ULK1,p70S6 K and 4EBP1 in wound tissues was detected by immunofluorescence.(4)Changes in the number,morphology and structure of autophagosomes in the wound tissues were observed by electron microscopy.(5)ULK1,p70S6 K,4EBP1 and their phosphorylation status were detected by Western blotting.(6)QRT-PCR was used to analyze the expression ofULK1 m RNA,p70S6 K m RNA and 4EBP1 m RNA.Results:(1)During the treatment,the wound healing status and wound healing rate of the control group,the Kangfuxin group and the MEBO group were all better than that of the model group.(2)During the treatment,the histopathological morphology of the wound in the control group,the Kangfuxin group and the MEBO group improved significantly compared with the model group.(3)During the treatment process,the number of positive ULK1,p70S6 K and 4EBP1 fluorescent staining cells and their OD value in the wound tissues of each group showed a trend of increasing and then decreasing.The number of positive cells and OD value were the highest in the MEBO group at the early stage of treatment,and the number of positive cells and OD value were the lowest in the model group.(4)The number of autophagosomes in the control group,the model group,the Kangfuxin group and the MEBO group increased and then decreased,the number of autophagosomes in the MEBO group at the early stage of treatment was the largest.(5)During the treatment process,the expression levels of ULK1,p70S6 K,p-p70S6 K,4EBP1 and p-4EBP1 in the wound tissues of the control group,the model group,the Kangfuxin group and the MEBO group all increased and then decreased,in the early stage of treatment,the expression level of the model group was the lowest,while the expression of p-ULK1 decreased and then increased,the expression level of the model group was the highest in the early stage of treatment.(6)During the treatment process,the expressions of ULK1、p70S6K、4EBP1 m RNA in the control group,the model group,the Kangfuxin group and the MEBO group all increased and then decreased,in the early stage of treatment,the expression level of the model group was the lowest.Conclusion: MEBT/MEBO can promote wound healing of diabetic ulcer wound,improve the expression level of ULK1,p70S6 K and 4EBP1 in wound tissue,and promote gene transcription and protein synthesis may be one of its mechanisms.