A Study On The Mechanism Of Biglycan In Keloid

Background:Keloid is a pathological wound healing by fibroblastic proliferation and accumulation of extracellular matrix.Biglycan(BGN)is a proteoglycan composed of a core protein and two chondroitin sulfate(CS)/dermatan sulfate(DS)glycosaminoglycan(GAG)chains,and known to be involved in structural and many physiological regulations in the skin.Although it is abundant in many tissues,its biological functions remain poorly understood.BGN is considered to have an organizing role in the assembly of the ECM.Targeted disruption of the BGN gene results in abnormal collagen fibril morphology and an osteoporosis-like phenotype.BGN interacts via its core protein or GAG chains with numerous components of the extracellular matrix.Keloids behave like tumors as they grow beyond the boundaries of the original wound margin,invade the surrounding normal tissue,do not regress spontaneously.Recently,a great number of studies have found that BGN increases in a few of tumors and BGN can promote the proliferation and metastasis of tumor cells.Therefore,BGN is regarded as a therapeutic target for tumor.So we hypothesize that increased activity of BGN may be associated with keloid fibroblast proliferation and accumulation of extracellular matrix.We think BGN is also a therapeutic target for keloid.PART ONEObjectives:To confirm the expression of BGN in keloid tissues and fibroblasts.To provide new pathogenetic mechanisms for keloid formation.Methods:The histology of keloid and adjacent normal skin was observed using HE staining.The expression of BGN and extracellular matrix related proteins were determined by immunohistochemistry and Western blot assay.Results:Histologically,there were excessive extracellular matrix and infiltrated cells in keloid.In keloid tissue,the expression of BGN,collagen Ⅰ and Ⅲ were up-regulated.The expressions of BGN,Pro-collagen Ⅰ and collagen Ⅲ protein were markedly increased in keloid fibroblasts including internal and external fluid compared to normal fibroblasts.Conclusions:BGN protein was overexpressed in keloid tissues and fibroblasts included internal and external fluid.PART TWOObjectives:To investigate the influence of BGN knockdown on the collagen synthesis and phosphorylation of related signaling pathway proteins in keloid fibroblasts.To observe the effect of BGN knockdown on cell migration in keloid fibroblasts,and further confirm its anti-keloid migration.Methods:SiRNA was used to knockdown the BGN in keloid fibroblasts including internal and external fluid.After successful knocking down BGN,the expression of extracellular matrix protein and the phosphorylation of related signaling pathway proteins were determined by Western blot assay.The cell migration ability of keloid fibroblasts was observed by the method of in vitro scratch assay.Results:BGN knockdown inhibits the expression of Pro-collagen Ⅰ and collagen Ⅲ in keloid fibroblasts including internal and external fluid.BGN knockdown also significantly inhibits the phosphorylation level of Akt and ERK in keloid fibroblasts.Furthermore,BGN knockdown significantly inhibits the cell migration of keloid fibroblasts.Conclusions:In keloid fibroblasts,BGN knockdown significantly inhibits collagen synthesis including internal and external fluid and the phosphorylation levels of Akt and ERK.Furthermore,BGN knockdown significantly inhibits the cell migration of keloid fibroblasts.Taken together,these results indicate that BGN might be an important regulatory factor for anti-keloid migration.PART THREEObjectives:To investigate the influence of BGN knockdown on the TGF-β1 induced secretion of extracellular matrix and phosphorylation of signaling pathway related proteins in keloid fibroblasts.Methods:To simulate keloid microenvironment,fibroblasts were cultured in medium containing 10 ng/ml concentration of TGF-β1.After0,0.5,1,3,6,12,24,48,72 h following TGF-β1 treatment,the expression of BGN,Pro-collagen Ⅰ,collagen Ⅲ and signal pathway related protein were detemined.In TGF-β1 culture condition,BGN was also knockdown and the secretion of extracellular matrix and the effect of phosphorylation of signal pathway related proteins were detected following BGN knockdown by Western blot.Results:Western blot:The expression of BGN was up-regulated quickly after treatment of TGF-β1 for 24 h in keloid fibroblasts including internal and external fluid.The expression of Pro-collagen Ⅰ was up-regulated after cultured for 24 h,and achieved the peak at 72 h in the internal of keloid fibroblasts,but the expression of Pro-collagen Ⅰ was up-regulated after cultured for 48 h in the external fluid of keloid fibroblasts.The expression of collagen Ⅲ was up-regulated quickly after cultured for 3 h and 24 h in the internal of keloid fibroblasts,but the expression of collagen Ⅲ was up-regulated after cultured for 24 h,and achieved the peak at 72 h in the external fluid of keloid fibroblasts.The expression of p-Akt(Thr308)was up-regulated quickly after cultured for 0.5 h and 1 h,then gradually decreased,24 h began to gradually increased,72 h reached another peak,while p-ERK up-regulated significantly for 1h,then gradually decreased,12 h began to appear gradually increased,48 h reached another peak.In TGF-β1 culture condition,BGN knockdown can inhibit the expression of Pro-collagen Ⅰ and collagen Ⅲprotein in keloid fibroblasts including internal and external fluid,and also inhibit the phosphorylation level of Akt and ERK in keloid fibroblasts.Conclusions:In TGF-β1 culture condition,BGN knockdown can inhibit the expression of Pro-collagen Ⅰ and collagen Ⅲ protein in keloid fibroblasts included internal and external fluid,and effectively inhibit the phosphorylation level of Akt and ERK.These results suggest BGN may be involved in keloid development,it may be through the TGF-β1-mediated Akt and ERK signaling pathways,and been a potential therapeutic target for keloids.