The Mechanism Of ADAM-10 In Diabetic Wound Repair

Background and ObjectiveDiabetic wound is a common complication of diabetes.Its long duration,and easy recurrence have caused great distress to patients.It is important to explore its pathological mechanism and therapeutic drugs.Macrophages and fibroblasts play an important regulatory role in wound healing.ADAM-10 involves in the regulation of various cellular functions,such as cell adhesion,growth and migration.However,the specific mechanism of ADAM-10 in diabetic wounds is still unclear.This study explored the regulatory role of ADAM-10 in macrophages and fibroblasts;and ADAM-10 regulatory mechanism in the healing process of diabetic wounds.The ADAM-10 regulatory mechanism would be verified by drug treatment on diabetic wounds.Methods1.Clinically chronic refractory wounds were screened and grouped.The groups are MEBT/MEBO(MM)and rb-b FGF.On the 3rd and 10 th day of treatment,granulation tissue and a small amount of epithelial tissue were collected.The progressive tissue was IF stained with ADAM-10,MMP-12 to observe the characteristics of protein changes.2.Construct sh-ADAM-10 RAW264.7 cell line.The proteomics technology was used to screen differential proteins between groups.Differential proteins were analyzed by bioinformatics.3.Stimulate with LPS or MMP-12 inhibitor + LPS(MMP-12 inh + LPS)on the cell line RAW264.7 and J774 a.1.The changes were observed in cell viability and gene transcription Under the same stimulation conditions,the normal,sh-ADAM-10(sh),or EX ADAM-10(EX),cells was adopted.We tested cell viability,gene transcription,protein secretion,protein expression and other indicators4.EX intervention was performed on L929 fibroblasts to observe the cell viability,ADAM-10 secretion and protein expression levels after LPS stimulation.5.Normal rats were prepared for acute wounds(Acute group).Diabetic rats wounds were divide into groups: Model group,MM group and rb-b FGF group.After intervention,we observed the wound healing,histopathological structure,cell ultrastructure,ADAM-10 gene transcription level,protein expression at 3rd,7th,10 th and 14 th wounds periods by Western Blot or IF.Results1.On the 3rd day of clinical chronic refractory wound treatment,the expressions of ADAM-10 and MMP-12 in the MM group were lower than those in the rb-b FGF group.Compared to the 3rd day,ADAM-10 expression increased on 10 th day,MM increased ADAM-10 effect less than rb-b FGF.The rb-b FGF significantly reduces MMP-12.The amount of MMP-12 protein in the MM group was always low.2.The results of TMT relative quantitative proteomic analysis in sh RAW264.7cells showed that sh ADAM-10 affected multiple trauma-related biological processes,sh ADAM-10 reduced MMP-12 and AKT expression.3.In macrophages,(1)Combined to CON,LPS at 1000 ng/ml for 9 h～24 h increased cell viability(P<0.05);MMP-12 inh at 1.5 μmol/L for 12 h～36 h had no effect on viability(P>0.05).LPS action for 12 h inhibited ADAM-10 transcription and increased MMP-12 transcription most significantly(P<0.01).LPS increased MMP-12,i NOS,TNF-α transcription levels and ADAM-10 secretion(P<0.05).LPS promoted the protein expression of ADAM-10,MMP-12,i NOS,IL-10 and cyclin D1(P<0.05).MMP-12 inhibitor promoted the protein expression of pro-ADAM-10,IL-10 and cyclin D1(P<0.05).(2)Under LPS,MMP-12inh+LPS stimulation,the viability of sh cells was lower than the sh negative(P<0.01).The groups containing LPS in sh cells were all lower than the sh negative +LPS group(P<0.01).Similar changes in cell viability displays between J774 a.1 and RAW264.7.In contrast to the sh negative group,ADAM-10 transcription was reduced in the LPS group,the MMP-12inh+LPS group,and all groups of sh cells(P<0.01);MMP-12 transcript levels were increased in all LPS-containing groups and reduced in the sh group(all P<0.01).In contrast to the sh negative +LPS group,MMP-12 transcripts were decreased in the MMP-12inh+LPS,sh+LPS,and sh+MMP-12inh+LPS groups(P<0.01).(3)In contrast to the negative transfection group,cell viability was higher in all EX groups than in the EX negative group(P<0.01).The cell viability was further increased in EX+LPS,EX+MMP-12inh+LPS(vs EX negative +LPS,P<0.01).TNF-α secretion was decreased in the MMP-12inh+LPS and EX+MMP-12inh+LPS groups compared with the EX negative and EX negative +LPS groups(both P<0.05).The gene transcripts and protein expression of ADAM-10,MMP-12,and inflammatory factors were increased in the EX group(P<0.05).However,the opposite is true in the sh group.The transcription levels of genes MMP-12,i NOS,TNF-α,Arg1,TGF-β1,cyclin D1 and CD206 were all increased by LPS stimulation(vs EX negative,P<0.01);and this increased effect was further enhanced in EX ADAM-10cells(vs EX negative +LPS,P<0.01).The secretion of ADAM-10 was increased within the EX+LPS group and EX+MMP-12inh+LPS group(vs EX negative,P<0.05).The EX group increased the protein expression of ADAM-10,MMP-12,IL-6,and IL-10(vs EX negative,P<0.05).EX+LPS increased the protein expression of ADAM-10,MMP-12,i NOS,IL-6,TGF-β1,and IL-10(vs EX negative,P<0.05).IF showed experimental results similar to Western Blot.4.Compared with the EX negative group of L929 cells,the cell viability of EX group increased(P<0.01);LPS increased ADAM-10 secretion in EX cell(P<0.05);LPS and EX increased ADAM-10,p-AKT,AKT,TNF-α protein expression(P<0.05).The increased protein expression effect of LPS was further increased in the EX+LPS group(vs EX negative +LPS,P<0.05)5.In the diabetic wound experiment,the wounds rates were higher in the Model group than in the Acute group(P<0.05);the wounds rates were lower in the MM and rb-b FGF groups than in the Model group(P<0.05).The MM and rb-b FGF treatments can reduce the infiltration of inflammatory cells in diabetic wounds,promote collagen production,and improve cell ultrastructure.In MM and rb-b FGF groups,the amount of TNF-α protein in the wound tissue was decreased,while the protein expression of TGF-β1,ADAM-10,IL-10,Arg1 and p-AKT was increased,and the expression of MMP-12,i NOS and IL-6 was decreased(vs Model,P<0.05).IF showed that ADAM-10 on 10 th day was higher in the wounds of the administered group than in the Model group.Arg1 expressing in macrophages on 3rd day of the administered group was higher than in the Model group.The expression of i NOS was consistently higher in the Model group than in the other groups on10 th day of the wounds.ADAM-10 is mainly expressed in myofibroblasts.Conclusion1.In macrophages,ADAM-10 involves in LPS-induced macrophage activation by regulating MMP-12 and increasing inflammatory cytokines.2.In fibroblasts,ADAM-10 promotes cell proliferation by increasing AKT and its phosphorylation,increasing TNF-α.3.In vivo experiments,ADAM-10 can promote wound healing in diabetic rats.The specific mechanism is that the increase of ADAM-10 in fibroblasts promotes cell proliferation;the decrease of MMP-12 reduces the inflammatory response of wounds.And the ADAM-10 mechanism of action has been verified in the treatment of diabetic wounds with MM and rb-b FGF.