The Effects And Mechanisms Of Melatonin On Fibroblast Differentiation Of Human Hypertrophic Scar

BackgroundFibroblast differentiation into myofibroblast is important pathological change in the processof hypertrophic scar formation.A great number of researches showed that transforming growthfactor-β1(TGF-β1) plays a central role in the fibroblast differentiation.TGF-β1exerts its effectson the cell by binding TGF-breceptor type II and subsequent phosphorylation of a type1receptor subunit forming a heterodimeric complex,Smad2and Smad3are receptor-activatedSmad pro-teins (R-Smad) phosphorylated by TGF-β receptor type I,subsequently form aheteromeric complex with Smad4,translocates to the nucleus,where the dimer binds to thepromoter region of the a-SMA gene,expression of α-SMA and differentiation intomyofibroblasts.Recent research information identified that the production and release of ROSfollowing TGF-β1binding the cell surface is an important signaling pathway of fibroblastdifferentiation.Melatonin is neuroendocrine hormones secreted by the pineal gland，which modulation ofcell cycle,differentiation and apoptosis,regulating circadian rhythms,anti-tumor,anti-oxidationand radical scavenger.The skin is an important target organs of melatonin,while the skin and itscomponents can produce melatonin and express melatonin receptor.Therefore,melatonin plays animportant role in the maintenance of normal structure and function of the skin.Previous studiesof our research group showed that concentration of melatonin was decreased and expression ofmelatonin receptors was abnormal in hypertrophic scar,experimental researches have shown thatmelatonin inhibit fibroblast proliferation,while promote the apoptosis of fibroblasts from humanhyertrophic scar,the result indicated that formation of hypertrophic scar has a relationship todysfuction of the melatonin system.Fibroblast differentiation into myofibroblasts is importantpathological change in the process of formation of hypertrophic scar,therefore,the study furtherexplores the role and mechanism of melatonin on fibroblast differentiation of humanhypertrophic scar. PARTⅠ The effect of melatonin on α-SMA and TGF-β type I receptor expressio inhuman hypertrophic scar fibroblastsObjective To investigate the effect of melatonin on fibroblast differentiation of hypertrophicscar.Methods Fibroblasts from hypertrophic scar (n=6) were isolated with tissue culturingmethod.The cells used in the study were randomly divided into two groups: control group(simple medium) and melatonin treatment group (melatonin10-3mM),and cultured for48h.Western-Blot was used to qualify the expression of α-SMA,TGF-βⅠreceptor protein andthe expression of α-SMA,TGF-β Ⅰ receptor mRNA in the fibroblast were qualified withRT-PCR.The statistical significance was examined using the Paired-Sample T test.Results Administered with melatonin,content of α-SMA protein（0.40±0.08）and TGF-βⅠreceptor protein（0.46±0.25）in fibroblast from hypertrophic scar was significantly decreased thancontrol group(0.80±0.13and0.55±0.30,P<0.05）and exprsession of α-SMA mRNA（0.16E-2±0.22E-2）and TGF-βⅠreceptor mRNA（11.85±1.17）also lower than control group (0.50E-2±0.57E-2and17.15±0.58,P<0.05）.Conclusion The data indicated that melatonin can inhibit the fibroblast differentiation ofhypertrophic scar.PARTⅡ The effect of melatonin on TGF-β1stimulation of normal skin fibroblastdifferentiationObjective To investigate the mechanism of melatonin on fibroblast differentiation.Methods Fibroblasts from normal skin (n=6) were isolated with tissue culturing method,Thecells used in the study were randomly divided into four groups:(1) control group(simplemedium),(2)TGF-β1group(10ng/ml),(3) melatonin treatment group(10-3mM),(4)TGF-β1+melatonin group,and cultured for72h.Western-Blot was used to qualify the expressionof α-SMA,TGF-βⅠreceptor protein and the expression of α-SMA,TGF-βⅠreceptor mRNA inthe fibroblast were qualified with RT-PCR.The statistical significance was examined using Student-Newman-Keul＇s test.Results Content of α-SMA protein(0.86±0.02) and the expression of α-SMA mRNA（1.18±0.79）of fibroblast treated TGF-β1were both increased than control group(0.41±0.03,0.50±0.38,P <0.05);melatonin could revers TGF-β1stimulation,content of α-SMA protein(0.16±0.07) and the expression of α-SMA mRNA（1.18±0.79）in fibroblast of TGF-β1+melatonin group were both lower than TGF-β1group(P<0.05).Content of TGF-βⅠreceptor protein (0.72±0.09) and the expression of TGF-β ⅠreceptormRNA （5.86±3.41） of fibroblast treated TGF-β1were both increased than controlgroup(0.13±0.05,4.15±2.74,P <0.05）;melatonin reversed TGF-β1stimulation, content of TGF-βⅠreceptor (0.22±0.16) and the expression of TGF-βⅠreceptor mRNA（0.16±0.07）in fibroblastof TGF-β1+melatonin group were both lower than TGF-β1group(P<0.05).Conclusion The results suggested that melatonin suppress fibroblasts differentiation byinhibiting the role of TGF-β1.PARTⅢ The melatonin role on ROS level and activity of antioxidant enzymes inhypertrophic scar fibroblasts and human normal skin fibroblast administered withTGF-β1Objective To investigate mechanism underlying which melatonin supresses the differentiationstimulated by TGF-β1.Methods Fibroblasts from hypertrophic scar and normal skin (n=6) were isolated and withtissue culturing method.The cells in hypertrophic scar used in the study were randomly dividedinto two groups: control group(simple medium) and melatonin treatment group(10-3mM);Thecells in normal skin used in the study were randomly divided into4groups:(1)control group(simple medium),(2)TGF-β1group (10ng/ml),(3)melatonin treatment group(10-3mM),(4)TGF-β1+melatonin group.Immunofluorescence was used to mark ROS in fibroblas on thebasis of cells seeded,ELISA were used to assay the content of MDA and activity of SOD.Thestatistical significance was examined using the paired-sample t test or Student-Newman-Keul＇s test.Results Administered with melatonin,green fluorescence of fibroblast from hypertrophic scarwas weaker,content of MDA decreased (1.38±0.31) nmol/mgprot (P＜0.05) and the activity ofSOD increased (2.63E1±0.713) U/mgprot (P＜0.05) in fibroblasts from hypertrophic scar wassignificantly decreased than control group.Green fluorescence intensity was strengthened in fibroblasts treated TGF-β1,content ofMDA increased (0.86±0.06) nmol/mgprot (P＜0.05) and the activity of SOD decreased(192.47±11.91) U/mgprot (P＜0.05) in fibroblasts treated TGF-β1than control group(P<0.05);melatonin reversed TGF-β1stimulation,content of MDA was decreased (0.72±0.10)nmol/mgprot (P＜0.05)and the the activity of SOD increased (241.20±25.73) U/mgprot (P<0.05)in fibroblast of TGF-β1+melatonin group were than TGF-β1group.Conclusion Melatonin can inhibit ROS release,and increase activity of SOD of fibroblasttreated TGF-β1, indicating that melatonin can influence the fibroblast differentiation byinhibiting downstream of signal pathway of TGF-β1.