CD26 Involves In The Promotion Of 640nm Red Light To The Skin Wound Repair

BackgroundSkin wound repair is a complex and orderly continuous process,which mainly includes three stages:inflammatory reaction stage,cell proliferation stage,and tissue remodeling stage.Multiple factors may affect the final wound healing outcome,such as age,neurovascular diseases,malnutrition,infection,and immune dysfunction.In recent years,low level laser/light therapy(LLLT)has been widely used in the clinical treatment of refractory wounds caused by burn,trauma,and diabetes.LLLT takes therapeutic effect by using laser or narrow spectrum light generated by low-power laser or light-emitting diode(LED),and regulating cell function through photobiomodulation(PBM).The most commonly used device for LLLT is LED which can generate 640-750 nm red light.Although a large amount of clinical evidence shows that 640-nm red light can promote wound healing and reduce inflammation and pain effectively,the specific mechanism of promoting wound repair by 640-nm red light remains unclear.As an essential and determining feature of wound repair,wound epithelialization,also called re-epithelialization,is conceptually viewed as the result of migration,proliferation,and differentiation of keratinocyte.It plays an important role in the restoration of integrity of the epidermal barrier after trauma.CD26 is a type II transmembrane glycoprotein and secretory protein,which is mainly expressed in epithelial cells,endothelial cells,and lymphocytes.CD26interacts with extracellular matrix to regulate the migration,adhesion and other functions of various cells.In this study,we used protein chip technology to exam the the differentially expressed proteins in keratinocytes irradiated with 640-nm red light compared to non-irradiated keratinocytes.After bioinformatics analysis,CD26 was selected as one of the differently expressed protein molecules induced by 640-nm red light for further verification.The possible mechanism of 640-nm red light involved in promoting skin wound repair was discussed.ObjectiveTo investigate the mechanism of 640-nm red light promoted skin wound repair preliminarily.To provide new clues for improving the understanding of the molecular mechanism of 640-nm red light promoted skin wound repair.MethodsPart one:Human skin keratinocytes were cultured in vitro and treated with 640 nm red light.They were divided into the LLLT group(640-nm red light irradiation)and the control group(NC group,non-irradiation).Cell scratch experiment and CCK8assay were used to compare the migration and proliferation of cells in the two groups.The protein chip technology was used to exam the differentially expressed proteins in the two groups of cells.Key signal molecules were selected by biostatistical analysis,which were further analyzed to confirm their roles in 640-nm red light promoted wound repair.Part two:Human skin keratinocytes were cultured in vitro and treated with CD26inhibitor Sitagliptin to determine the role of CD26 played in the induced migration by640-nm red light.Based on different treatments,the cells were divided into four groups:control group(NC group,non-irradiation and no Sitagliptin treatment),Sitagliptin group(non-irradiation and 2mM Sitagliptin treatment),LLLT group(640-nm red light irradiation,10mW/cm~2,8J/cm~2),and LLLT+Sitagliptin group(640-nm red light irradiation and 2mM Sitagliptin treatment).The expression levels of CD26 mRNA in the four groups of keratinocytes were detected by RT-PCR,and the expression levels of CD26 protein in these four group were detected by Western Blot.The migration ability of the cells in these four groups was evaluated by Transwell experiment and cell scratch experiment.Results.Part one:The 640-nm red light can accelerate the migration of keratinocytes,but has no effect on keratinocytes proliferation.It was found that there were 122 differentially expressed proteins detected by protein chip assay between non-irradiated cells and640 nm red light irradiated cells,Through GO analysis and Pathway analysis,these differentially expressed proteins were mainly enriched in biological processes of extracellular matrix degradation,proteolysis,and cell adhesion.The molecule CD26was inferred to play an important role in the process of 640-nm red light promoted cellular migration.Part two:According to RT-PCR results,CD26 mRNA expression level was increased in the 640 nm red light group compared with the control group(LLLT group 1.24±0.01398,NC group 0.95±0.02887,P<0.001).The CD26 mRNA expression level in Sitagliptin group was significantly lower than the control group(Sitagliptin group 0.34±0.03055,P<0.001).Western Blot results showed that the expression of CD26 protein in human keratinocytes treated with 640-nm red light was significantly increased compared to non-irradiated control group.The protein levels in Sitagliptin group and LLLT+Sitagliptin group were significant lower than those in control group and LLLT group respectively.Both Transwell and cell scratch assays showed that the keratinocytes in the 640-nm red light group showed enhanced migration ability compared to non-irradiated groups,while those in the Sitagliptin group and LLLT+Sitagliptin group showed decreased migration ability compared to control group and LLLT group without Sitagliptin treatment.The above results suggest that 640-nm red light can accelerate the migration of keratinocytes in vitro and might promote skin wound healing through promoting the expression of CD26.ConclusionThe 640-nm red light can accelerate the migration of keratinocytes by promoting the expression of CD26 in vitro,which might shorten the re-epithelialization time and thus promote the healing of skin wounds.