Effect Of MEBT/MEBO On The Expression Of ZEB1/LAMA3/ITGA3 Signaling Pathway In Refractory Wounds Of Diabetic Rats

Objective:To explore the expression mechanism of ZEB1,LAMA3 and ITGA3 in the re-epithelialization of diabetic refractory wounds and whether MEBT/MEBO influences this mechanism.Methods:12-week-old male Wistar rats were randomly divided into acute wound group(normal blood glucose)and diabetic wound group.After the preparation of type 1 diabetic rats,they were divided into MEBT/MEBO group,rb-bFGF group,and model group without drug intervention.Two full-thickness skin defect wounds with a diameter of 30mm were prepared on the back of rats in each group,and then different interventions were used to treat the wounds.The wound healing effect of each group was preliminarily observed by counting the wound healing area of rats on 3,7,and 14 days.HE staining and Masson staining were used to observe the granulation tissue regeneration,collagen fiber formation and keratinocyte re-epithelialization in the process of wound healing.The distribution and expression of ZEBland LAMA3 protein in the basement membrane of each group at different time periods(on the 3rd,7th and 14th day)were observed by immunofluorescence chemistry.The expression levels of LAMA3,ITGA3 and ZEB1 genes in wounds were detected by real-time PCR.Results:(1)Wound healing rate:On the third day of the experiment,there was no difference between the model group and the other three groups(P>0.05);On the 7th and 14th days of the experiment,the delayed wound healing of the diabetic rats in the model group was significantly different from that in the acute wound group(P<0.001),while the healing rate of diabetic rats after treatment with MEBT/MEBO and rb-bFGF was better than that in the model group(P<0.05 or P<0.001),while there was no difference in the healing rate between MEBT/MEBO group and rb-bFGF group(P>0.05).(2)Observation of pathological staining and epidermal migration:On the third day of experimental intervention,all the wounds in each group showed inflammatory infiltration,and the skin structure was defective,and there was no significant difference in the migration of the wound edge epidermis(P>0.05).On day 7,the acute wound group had a large number of granulation tissue,collagen fibers,small angiogenesis,slight inflammation,and epidermal keratinocytes at the edge of the wound proliferated and migrated to the wound center.The model group had a small amount of granulation tissue and blood vessels,a large number of necrotic cell fragments,inflammatory infiltration,and its reepithelialization migration was slower than that of the acute wound group(P<0.001).The MEBT/MEBO group and the rb-bFGF group had less inflammatory infiltration,cell necrosis,granulation tissue,collagen fibers and angiogenesis,and reepithelialization migration was better than that of the model group(P<0.05 or P<0.01).On day 14,most of the wounds in the acute wound group were covered with migrating epidermis,with rich granulation tissue,orderly distribution of collagen fibers,and the presence of new sebaceous glands,hair follicles,blood vessels,nerve fiber bundles,etc.The rb-bFGF group and MEBT/MEBO group were close to completing epithelialization,with rich granulation tissue,neat and uniform collagen fibers,and skin appendages generated.However,there was still a small amount of inflammatory infiltration in the model group,and the epithelialization was not completed,and its reepithelialization was significantly slower than that of the other groups(P<0.01 or P<0.05).(3)Immunofluorescence:ZEB1 protein was expressed in dermal nuclei,and compared with the acute wound group,the expression of ZEB1 protein was higher in the model group on day 3(P<0.05),the expression of ZEB1 protein in the model group on the 7th and 14th days of the experiment was high,and there was a significant difference(P<0.001).Compared with the model group,the expression of ZEB1 protein in the MEBT/MEBO group on days 3,7 and 14 of the experiment was comparable to that in the rb-bFGF group,and all were lower than those in the model group,which was statistically significant(P<0.001).The expression of LAMA3 protein was positive in the epidermal basement membrane where the wound edge migrated,and the expression of LAMA3 protein in the model group on the 3rd,7th and 14th day of the experiment was lower(P<0.05 or P<0.01 or P<0.001).(4)Real-Time PCR:On the 3rd day of experiment,LAMA3 gene expression in wounds of model group was lower than that of acute wound group(P<0.05),and there was no significant difference between model group and MEBT/MEBO group and rb-bFGF group(P>0.05),ITGA3 gene expression was lower than that of acute wound group and rb-bFGF group(P<0.05).There was no significant difference between MEBT/MEBO group and model group(P>0.05).On the 7th and 14th day of the experiment,the expressions of LAMA3 and ITGA3 genes in the model group were lower than those in the acute wound group,MEBT/MEBO group and rb-bFGF group(P<0.05 or P<0.01).The expression of ZEB1 gene in the model group was slightly higher than that in the other groups(P<0.05 or P<0.01 or P<0.001).Conclusions:The delay of reepithelialization of refractory wounds in diabetic rats may be related to overexpression of ZEB1 level and insufficient expression of LAMA3 and ITGA3.MEBT/MEBO may up-regulation of LAMA3,ITGA3 and down-regulation of ZEB1 in the reepithelialization zone to promote re-epithelialization of refractory wounds in diabetic rats.