The Experimental Study About The Regulation Of The TIR/BB-loop Mimetic AS-1 On Wound Healing Quality Of Deep Partial-thickness Scald And Its Enlightenment For Clinical Application Strategies

BackgroundIn terms of the burn treatment,China was among the leading nations of the world.Although the lethal area 50 had already been reached over 98%,most patients had unfavourable prognosis in hypertrophic scar that greatly affected the quality of life.There were still many key issues needed to be researched and resolved about the mechanism of rehabilitation and formation of wounds and the prevention.The cure without scar healing was a long-term goal still far from being achieved.Professor Lu SL.from Shanghai Ruijin hospital had pointed out that a moderate inflammatory reaction was the essential condition of favorable healing in 2007.Due to the complexity of the mechanism of wound rehabilitation and formation,the relevant domestic and foreign researches were still lack of complete and systematic awareness.The interpretation on moderate inflammatory reaction was not the same.Therefore,to further explore the mechanism of tissue damage repair and scar formation,to explore the effective prevention and control of the risk of scar formation in the early stage,to improve the quality of wound healing,was of great significance for clinical.It was the objective requirement of the development of humanistic medicine and regenerative medicine under the modern medical mode,and it was also the embodiment of the clinical application value of transformed medicine in burn practice.A previous study from our team,Professor Li YH.had found that TIR/BB-loop mimetic AS-1 could protect pressure overload-induced cardiac hypertrophy by inhibiting IL-1R mediated MyD88 singnaling pathway.Although there were some similarities between pressure overload-induced cardiac hypertrophy and deep scald injury,diseases with different causes,different stages,different organizations,the same intervention may not be able to cause the same outcome,and the mechanism of biological regulation also may vary.Could AS-1 control excessive inflammatory reaction in depth of burned tissue and prevent fibrosis and scar formation in order to improve wound healing quality?Through Which mechanism AS-1 could achieve such biological effect?Was there any significance inspiration and guiding to solve these clinical problems?They all needed further experimental demonstration.ObjectiveTo observe the effects of TIR/BB-loop mimetic AS-1 on wound healing and scar formation of deep scald wound in mice,and to verify the molecular signal transduction pathway and to explain its mechanism in some extent.Cases of different outcomes from deep scald wounds healing were compared and analyzed,in order to explore some potential control points which reflect moderate inflammatory reaction and the prevention of hypertrophic scars.MethodsPart One:6-8 weeks,42 C57BL/6 mice were randomly divided into six groups:Sham Group,Burn Group,AS-1 treatment in acute inflammatory stage group(TA Group),AS-1 solvent dimethyl sulfoxide(DMSO)treatment in acute inflammatory stage(DA Group),AS-1 treatment in chronic inflammatory stage group(TC Group),AS-1 solvent DMSO treatment in chronic inflammatory stage(DC Group),7 in each group,respectively.Experimental groups of mice dorsal first to unhair with barium sulfide(100g/L)s then anesthetized mice with the intraperitoneal injection of 20g/L sodium pentobarbital 0.5ml,and made the second degree deep scald model by using a metal sheet of the electric scald apparatus close to the dorsal skin of mice at 80℃ for 8 seconds.Each dorsal wound was treated with saline and vaseline gauze dressing,daily wound cleaning and dressing changing.Form 8 hours postscald of TA Group and from 2 weeks postscald of TC Group were injected AS-1 50mg/Kg each day while Form 8 hours postscald of DA Group and from 2 weeks postscald of DC Group were injected DMSO at the same dose.Burn Group was only cleaned wound and changed dressing each day.The general situation of wound healing was observed at 3 weeks after injury,and the wound healing rate was calculated.Tissue samples were collected,and the H.E staining and Masson staining were performed to observe the changes of the wound tissue.Image analysis software was used to analyze the fibrosis area of each group under Masson staining.Conventional extraction wound tissue mRNA,by qRT-PCR method to detect the IL-1β,TNF-α,TGF-β1,MMP-1,TIMP-1 and CTGF mRNA expression level and understood the cytokines which affect wound healing and scar formation.qRT-PCR and Western Blot were used to detect the expression of collagen type I and type III collagen mRNA and protein in wound tissues of 3 weeks after injury.Part Two:28 mice were randomly divided into four groups,named sham group,Burn group,AS-1 treatment group(TA Group),AS-1 solvent DMSO control group(DA Group),with 7 mice in each group,respectively.After the intraperitoneal anesthesia,scald model made was the same to Part One.Each dorsal wound was treated with saline and vaseline gauze dressing,daily wound cleaning and dressing changing.TA Group was injected AS-1 at the 8 hours postscald,the 24 hours postscald,the 48 hours postscald and the 72 hours postscald,respectively.While DA Group was injected DMSO at the same point of time.The dose was 50mg/Kg each day.After 96 hours of injury,the mice were anesthetized and fixed,weighed,and the whole skin tissues involving of the wounds were taken.Conventional extraction wound tissue mRNA,by qPCR method to detect the IL-1β,TNF-α,TGF-β1,MMP-1,TIMP-1 and CTGF mRNA expression level,Western Blot were used to detect the expression of P38 MAPK、ERK1/2、JNK、TAK1 phosphorylation and protein expression levels of TGFβ1 TRAF6、CTGF.Then made a summary of the possible molecular mechanism for these regulation of AS-1.Part Three:From September 2014 to March 2015,46 cases of second degree deep scald patients were included in sequence.All patients were collected peripheral blood to detect the IL-1β、TNF-α levels at 8 hours,24 hours,48 hours,72 hours,7 days,14 days,21 days postscald.Healthy volunteers set up the control group for serum collection at the same point.All specimens were sent to the laboratory for centrifugal treatment and suck out the upper serum and preserved under 80 degrees refrigeratoration,centralized detection data records in the database of patient.Followed all patients up for 1 year and scoring their hypertrophic scars according to the Vancouver Scar Scale(VSS).Then put them into different group as mild hypertrophic scars sub-group(score:1-5),moderate hypertrophic scars sub-group(score:6-10),severe hypertrophic scars sub-group(score:11-15)and healthy volunteer as sham group(score:0).Retrieved the information from the database of these patients,to analyze the difference between the groups and inner-group,to observe the relationship between the expression level of serum IL-1β,TNF-α and the hypertrophic scar in patients and to comprehensive evaluate the quality of wound healing.ResultsPart One:Compared with the Sham group,epithelium completely performed at three weeks postscald in TA group,part of the hair re-growth,and no obvious wound contraction happened.Burn group,DA group,TC group,DC group were not completely healed.There were still necrotic tissues existed,with varying degrees of scar formation.The Burn group,DA group,DC group also had obvious wound contraction.TC group had several hair re-growth.There was no significant difference between TA group and Sham group(P>0.05)in the wound healing rate while it was higher than the B group,DA group,TC group and DC group(P<0.05).H.E staining showed:epidermis homogeneity and granulation tissue hyperplasia were more obvious,disorder of collagen fibers with fascicular arrangement,containing many newborn capillaries and spindle formation in Burn group,DA group,TC group and DC group.Meanwhile,in the Burn Group,spine cell layer thickening,and there was still a more inflammatory cell infiltration.In TA group,The morphology of the Sham group was close to that of the group,the epidermis was less homogeneous,the new collagen fibers were arranged in a relatively orderly manner,and can be woven into a network.Masson staining showed:compared with Sham group,scar fibrosis in Burn group,DA group,TC group and DC group increased significantly than that of the TA group,the part of fiber connective tissue was replaced by substitution of granulation tissue,the formation of the blue staining areas were increased and collagen arranged in disorder were significantly,compared with Sham group and TA group.Quantitative analysis of fibrosis area in Burn group,DA group,TC group,DC group,fibrosis were significant difference with Sham group(P<0.05),and there was no significant difference between Sham group and TA group(P>0.05).In the tissue of 3 weeks postscald,the IL-1β mRNA expression in TA group,TC group were significantly decreased than that in Burn group(P<0.05);In the tissue of 3 weeks postscald,the IL-1β mRNA expression in TA group,TC group was significantly decreased than that in Burn group(P<0.05);TNF-α mRNA expression in TA group was significantly decreased than those in Burn group(P<0.05);TGFβ1 mRNA expression in TA group was significantly decreased than that in Burn group,DA group and DC group(P<0.05);MMP-1 mRNA expression in TA group was decreased significantly than in Burn group and TC group(P<0.05);TIMP-1 mRNA expression was significantly higher in TA group than that in Burn group(P<0.05);CTGF mRNA expression in TA Group,TC group was decreased significantly than that in Burn group(P<0.05).Part Two:In the tissue of 96 hours postscald,compared with Burn group,the IL-1βmRNA and TNF-a mRNA expression levels were significantly lower in Sham group and TA group.In the AS-1 intervention group,AS-1 intervention group IL-1β mRNA,TNFα mRNA and TGF-β1 mRNA and MMP-1 mRNA levels were significantly decreased than that in the Burn group and DMSO group(P<0.05);TIMP-1 mRNA expression level of AS-1 intervention group increased than the DMSO group(P<0.05);the CTGF mRNA was significantly decreased than the Burn group(P<0.01),while it was decreased than the DMSO group(P<0.05).Western Blot test showed that the degree of phosphorylation of P38 MAPK,ERK1/2,JNK,TAK1 in Burn group was higher than that of AS-1 group;the protein expression of TGFβ1,TRAF6 and CTGF was higher than that of AS-1 group.Part Three:Compared with the normal control group,the serum levels of IL-1β and TNF-a were significantly different(P<0.01)at the different time points postscald.Compared with each group inner hypertrophic scar sub-group,the serum levels of IL-1β at the 8 hours time point postscald in the mild hypertrophic scar sub-group was of no statistical significance with moderate and severe hypertrophic scar sub-group(P>0.05).The remaining indicates at each time point,there was significant difference(P<0.01).Compared with each group inner hypertrophic scar sub-group,the serum levels of TNF-α at the 8 hours time point and the 24 hours postscald in the mild hypertrophic scar sub-group was of no statistical significance with moderate and severe hypertrophic scar sub-group(P>0.05).The remaining indicates at each time point,there was significant difference(P<0.01).ConclusionTIR/BB-loop mimetic AS-1 can effectively promote the wound healing rate and reduce scar fibrosis in 3 weeks after the acute inflammation reaction in mice,which can improve the quality of wound healing.AS-1 can significantly down regulate theexpression of IL-1β,TNF-α,TGF-β1,MMP-1,CTGF mRNA,while up regulate the expression of TIMP-1 mRNA in the wounds of mice after deep partial thickness burn for three weeks.The type I collagen mRNA and protein levels were significantly reduced,and no obvious influence was found in the type Ⅲ collagen mRNA and protein.AS-1 regulation of the quality of wound healing of second degree deep scald in mice may be mediated by IL-1R and MyD8 8-dependent NF-kappaB signaling pathway and MAPKs/TGF beta 1/TRAF6 signaling pathway.We could regard the serum IL-1β level 8 hours postscald and the serum TNF-a level 8 hours and 24 hours postscald as the prognosis indicates of patients who suffered the second degree deep scald correlating with hypertrophic scars formation.Further implementation of clinical trials,AS-1 intervention may put into effect within 24 hours.