The Mechanism Research Of Shikonin On Regulation Of Proliferation And Apoptosis Of Human Dermal Fibroblasts And Inhibition Of Hypertrophic Scar Formation

Objective:To reveal the active status of Shh signal pathway in human fibroblast cell line and primary cell.The respective effect of Shh signal pathway,Shikonin and cyclopamine combined with Shikonin on the proliferation,migration,cell cycle,apoptosis of fibroblast cell and expression of dense scarring associated genes and apoptosis genes were demonstrated.Through research Shikonin,which the main component of traditional Chinese medicine ointment of lithospermum on fibroblast proliferation,migration,apoptosis and expression of some key genes and biological characteristics,and combined with Shh signal pathway inhibitor cyclopamine molecules on human HSF cell proliferation and influence of the key genes expression,to further explore the formation mechanism of the hypertrophic scar and the mechanism of Shikonin in promoting skin wound healing process,and provide basic evidence for further application development.Methods:Quantitative PCR and western-blot methods were used to assay the expression level of essential genes in Shh signal pathway including Ptch-1,Smo and Gli-1 in human fibroblast cell line and primary cell.Recombinant human rhShh of different concentraion,Shikonin of different concentraion and Shh signal pathway combined with Shikonin were treated on Fibroblast cell,separately.MTT and invasion assay were employed to test cell proliferation and cell migration.Cell cycle and apoptosis were determined by using flowcytometry.RT-qPCR and western-blot were used to detect the expression of dense scarring associated gene VEGF and collagenase I encoding gene and apoptosis genes:CyclinD1 and Bcl-2.Results:I.The expression of Siganl pathway gene Ptch-1?Smo and Gli-1 were both detected in two human-derived fibroblasts.The expression of HHSF group increased significantly.2.Recombinant human derived rhShh could promote the proliferation of fibroblasts in concentration-dependent manners.However,no significant enhanced promotion on proliferation of fibroblasts was observed after the concentration of rhShh exceed 1?g/ml.The inhibition of cyclopamine on the proliferation of fibroblasts was also in concentration depend-ent manners.No obvious enhanced inhibition on proliferation of fibroblasts was observed after the concentration of rhShh exceed 8?mol.Meanwhile,rhShh could promote the cell migration,inhibited apoptosis.It could improve the expression of the scarring related genes of VEGF and type I collagenase;apoptosis related genes of CyclinD1 and Bcl-2.The effect of cyclopamine is opposite.3.Shikonin could inhibited on proliferation of two kinds of fibroblasts.About 50%and 90%of cells were inhibited when the Shikonin concentration reached 5pM and lOFpM,respectively.Meanwhile,Shikonin could inhibit the cell migration and enhance apoptosis.It could down regulate the expression of the scarring related genes of VEGF and type I collagenase;apoptosis related genes of CyclinDl and Bcl-2.Shikonin can partially down regulate the level of shh pathway gene Ptch-1 and Gli-1;the inhibitory effect was more obvious especially in HHSF group.4.The treament of Shikonin combined shh signal pathway inhibitor cyclopamine could enhance proliferation of fibroblasts,improve cell apoptosis,significantly down regulate the expression level of scarring related genes of VEGF and type I collagenase,strongly down regulate apoptosis related genes of Bcl-2.Conclusions:The Shh signal pathway was activated in human fibroblast cell.Interdiction of Shh signal pathway could change the physiological characteristics of human fibroblast cell.Shikonin could repress proliferation of human.fibroblast cell,and inhibit the expression level of VEGF,type I collagenase,Bcl,2 and CyclinD1.Interdiction of Shh signal pathway could enhance repression of fibroblast cell for Shikonin.