Study On The Mechanism Of MEBT/MEBO Regulating Myofibroblasts To Promote The Repair Of Diabetic Refractory Wound

Objective: To investigate the effect of Moist exposed burn therapy/moist exposed burn ointment(MEBT/MEBO)on diabetic refractory wounds,and to reflect the degree of myofibroblast transformation of diabetic refractory wounds with the expression level of α α-smooth muscle actin(α-SMA).The specific mechanism of MEBT/MEBO in promoting diabetic refractory wounds was explored through animal and cell experiments,which provided a theoretical basis for the clinical application and promotion of MEBT/MEBO.Method:(1)90 adult male Wistar rats with SPF were divided into five groups by random number table method,blank group,control group,model group,MEBO group,rb-b FGF group,18 rats in each group,and STZ intraperitoneal injection was used to induce diabetic rat models after 7days of adaptive feeding,and then wounds were uniformly prepared by full-thickness skin defect method after molding.The wound healing was observed,the rat wounds on days 0,3,7and 14 after wound formation were photographed,and the granulation tissue of the wounds of6 rats in each group was randomly cut,and the histopathological morphological changes of each group were observed by HE and Masson staining,the distribution and expression of α-SMA and COL-1 were observed by immunofluorescence,and the protein expression levels of α-SMA and COL-1 were detected by Western blot.(2)To explore the effect of MEBT/MEBO on rat dermal fibroblasts(RDFs).Firstly,Wistar rat skin dermal fibroblasts were isolated and extracted by double enzyme method(neutral protease + pancreatic enzyme),cell morphology was observed by HE staining,and the expression of waveform protein(Vimentin)was identified by immunofluorescence.The appropriate dose of MEBO to RDFs was explored by CCK8 method,and the cell experiments were divided into low glucose group,high sugar group,solvent group,low-dose MEBO group,high-dose MEBO group,DCFH-DA fluorescent probe method to detect the level of reactive oxygen species(ROS)in each group,real-time quantitative PCR to detect the gene transcription level of α-SMA and COL-1,Western blot method to detect the α-SMA of RDFs,COL-1 protein expression levels.Results:(1)After the STZ method induced the diabetes model,the rats developed typical "three more and one less" symptoms,and the blood sugar was significantly elevated to reach the diabetes standard.Compared with other groups,rats in the model group had delayed wound healing(P<0.05),continuous inflammatory infiltration,insufficient angiogenesis,low number of fibroblasts and reduced extracellular matrix deposition.Wound healing rate in the middle and late stages of treatment: control group,MEBO group = rb-b FGF group,model group.(2)Compared with the control group,the expression of α-SMA and COL-1 proteins in the wound tissue of the model group decreased significantly on the 3rd,7th and 14 th days of wound occurrence(P<0.05);Compared with the model group,on the 7th day of treatment,the expression of α-SMA in wound tissue in the MEBO group was significantly increased(P<0.05).On the 3rd,7th,and 14 th days of treatment,the expression of COL-1 in wound tissue in the MEBO group was significantly increased(P<0.05).(3)The isolation and culture of RDFs were good,HE staining showed typical RDFs morphological characteristics,and immunofluorescence identification showed that cells expressing rich Vimentin protein accounted for more than 99%;The results of cell proliferationtoxicity detection(CCK8 method)showed that MEBO could increase the activity of RDFs in the concentration range of 0.5～2.0mg/m L,and the maximum activity of cells at MEBO concentration of 1.0mg/m L reached the maximum.The ROS test results showed that the ROS level in the low-dose MEBO group(1.0mg/m L)and the high-dose MEBO group(1.5mg/m L)was significantly lower than that in the high-glucose group(P<0.05).The results of real-time quantitative PCR and WB showed that the transcription level and protein expression of dermal fibroblasts under high glucose culture α-SMA and COL-1 decreased significantly compared with those in the low sugar group(P<0.05),and the transcription level and protein expression of the two increased after MEBO intervention(P<0.05).Conclusion:(1)MEBT/MEBO can effectively promote the proliferation and maturation of collagen fibers,promote the growth of granulation tissue of wounds,thereby improving the wound healing rate and accelerating the repair process of diabetic refractory wounds.(2)MEBT/MEBO may accelerate the repair of diabetic refractory wounds by increasing the protein expression levels of α-SMA and COL-1,promoting myofibroblast transformation and extracellular matrix deposition.(3)MEBO may increase the transcription level and protein expression of α-SMA and COL-1 in RDFs by reducing the ROS level of RDFs in high glucose environment,promote myofibroblast transformation and extracellular matrix deposition,thereby accelerating the repair of diabetic refractory wounds.