Facilitatory Effect Of Interleukin-1 Alpha On Matrix Metalloproteinases-1 Regulation In The Treatment Of Hypertrophic Scar With Fractional Carbon Dioxide Laser

Objective:Fractional carbon dioxide laser is widely used in treatment of hypertrophic scar with promising results.However,the specific mechanism remains unclear.To optimize the treatment protocol on hypertrophic scar,this study explored the exact mechanism of the application of fractional Carbon Dioxide（CO₂）laser on the treatment of hypertrophic scar.Methods:This study was divided into 3 parts.PartⅠ:Evaluation of the clinical effects of fractional CO₂laser on the treatment of hypertrophic scar.Patients with hypertrophic scar were recruited to participate in this trial to complete 3 sessions of fractional CO₂laser.Vancouver Scar Scale（VSS）and Patient and Observer Scar Assessment Scale（POSAS）were recorded in detail and the scar tissue were collected before the first treatment and after the last treatment to carry histological examination to evaluate the clinical effects of fractional CO₂laser on the treatment of hypertrophic scar.PartⅡ:Transcriptome sequencing of scar tissue from before and after fractional CO₂laser treatment and the following bioinformatics analysis.Five pairs of scar tissues were collected 24 hours after treatment for the expression of MMP-1 was higher in 24 hours than 48 hours after treatment.Differently expressed genes were characterized from the data of transcriptome sequencing in R software.Gene oncology（GO）analysis and Reactome pathway enrichment analysis were performed by Geneset Enrichment Analysis（GSEA）.The enrichment of these genes in genesets Degradation of extracellular matrix and Collagen degradation were also performed.Genes correlatedly express with matrix metalloproteinases-1（MMP-1）were figured out by batch correlation analysis.PartⅢ:Roles of interleukin-1α（IL-1α）on the regulated expression of MMP-1 via IL-1R/p38/JNK/AP-1 signal pathway.（1）m RNA expression of IL-24,IL-1αand MMP-1in the treated scar tissue was examined by Quantitative Real-time PCR（q PCR）.（2）Human hypertrophic scar derived fibroblast（HSFS）was cultured from scar tissue,obtained from patient during scar plastic surgery,via the method of typeⅠcollagenase digestion.HSFS were characterized by immunofluorescence methods.（3）HSFS were treated by different IL-24 concentration gradients（0,1,10,50,100 and 1000ng/ml）or different IL-1αconcentration gradients（0,1,10,50,100 and 1000pg/ml）for 24 hours.Expression of MMP-1 were examined by q PCR and Western Blotting（WB）.（4）HSFS were treated by different IL-1RA concentration gradients（0,10,100,500 and1000ng/ml）two hours before the application of IL-1αin 100pg/ml.Expression of MMP-1 were examined by q PCR and Western Blotting.（5）After HSFS were treated in different time（0、10min,20min,30min,1 hour and 2 hour）by IL-1αin 100pg/ml,expression of total and phosphorylated P38,ERK1/2 and JNK were detected by Western Blotting.（6）HSFS were treated by different p38 inhibitor concentration gradients（SB203580,10,20,50 and 100μM）,and different JNK inhibitor concentration gradients（SP600125,10,20,50 and 100μM）two hours before the application of IL-1αin100pg/ml.Expression of MMP-1 were examined by q PCR and Western Blotting.（7）HSFS were treated separately by IL-1RA（100ng/ml）,SB203580（50μM）and SP600125（100μM）two hours before the application of IL-1αin 100pg/ml.The transcriptional activity of transcription factor AP-1 was detected by Electrophoretic Mobility Shift Assay（EMSA）and dual-luciferase reporter assay.Results:PartⅠ:Compared to the time before treatment,VSS score was significantly reduced after three serious treatment and pigment,vascularization,itchy,pliability and thickness in VSS were significantly improved.Observer evaluated vascularization,pigmentation,thickness,relief,pliability and area size were and patient evaluated itchy,color,stiff,thickness and irregular were significantly improved.Both of the total score of observer scar scale,patient scar scale and total POSAS score were significantly reduced.The typeⅠcollagen predominance before treatment were significantly reduced and replaced by typeⅢcollagen.PartⅡScar tissues were collected for transcriptome sequencing 24 hours after treatment,the expression of MMP-1 was significantly higher in 24 hours than 48 hours.There total528 differently expressed genes after treatment,containing 160 down-regulated genes and 368 up-regulated genes.The result of GO indicated that all genes enriched in extracellular region part,extracellular space,extracellular region,cellular protein metabolic process,cell differentiation,cellular developmental process,cellular response to chemical stimulus and regulation of response to stimulus.The result of Reactome pathway enrichment analysis indicated that all genes enriched in keratinization,cytokine signaling in immune system,immune system,innate immune system,GPCR ligand binding,neutrophil degranulation,developmental biology and signaling by GPCR.Similarly,these genes were also enriched in Degradation of extracellular matrix and Collagen degradation.Batch correlation analysis indicated that there were 47 genes correlatedly express with matrix metalloproteinases-1（MMP-1）.PartⅢ（1）Expression of IL-24,IL-1αand MMP-1 was significantly increased in 24hours after treatment.（2）Human hypertrophic scar derived fibroblast,spindle-shaped and have a larger cell volume under microscope,was characterized by double-positive of Vimentin and S100A4 by immunofluorescence methods.（3）IL-24 do not regulate the expression of MMP-1 in different concentration.IL-1αpromoted the expression of MMP-1 in a concentration-dependent pattern that it was elevated in the concentration of10pg/ml and peaked at 1000pg/ml.（4）IL-1RA regulated the expression of MMP-1 in a concentration-dependent pattern that it was reduced in the concentration of 10ng/ml and totally inhibited at 100ng/ml.（5）p38 and JNK protein phosphorylation increased in 10minutes after application of IL-1α,peaked at 30 minutes,reduced in 1 hour and returned to prestimulus level in 2 hours after application of IL-1α.（6）SB203580 and SP600125regulated the expression of MMP-1 in a concentration-dependent pattern that SB203580totally inhibited at 50μM,and SP600125 totally inhibited at 100μM.（7）EMSA and dual-luciferase reporter assay indicated that IL-1αinduced the AP-1 transcriptional activity,and IL-1RA,SB203580 and SP600125 inhibited the activity.Conclusions（1）Fractional CO₂laser can significantly improve pigment,vascularization,itchy,pliability and thickness of hypertrophic scar and shift typeⅠcollagen to typeⅢcollagen in scar tissue.（2）Fractional CO₂laser alters the expression of many genes,especially the genes code proteins associated the degradation of extracellular matrix.The pathophysiology of fractional CO₂laser treated scar is associated with immune response and matrix degradation.（3）IL-1αbinds to IL-1R to induce the AP-1 transcriptional activity by phosphorylation of p38 and JNK,and finally regulate the expression of MMP-1.