Light-emitting Diode Irradiation(640nm) Regulates Keratinocytes Migration And Cytoskeletal Reorganization Via HIF-1?

Wound healing is a complicated process,the epithelial integrity is disrupted by injury or other causes.Which may be divided into four phases: hemostasis,inflammation,proliferation,and tissue remodeling,during the differents phases,several cell types were taken part in the actions.The extracellular matrix were secreted by fibroblasts which were stimulated to produce collagen and fibronectin,immediately after that,the keratinocytes initiated to migrate through the wound,then proliferated to form a skin barrier.The actin polymerization of the keratinocytes,such as lamellipodia,pseudopodia,and stress fibers,which drived the the cell forward,at the same time,the desmosomes and half bridge connection between the cell and the matrix were dissociation,the cycle of the adhesion and dissociation drive the cells migration forword.These process were influenced by growth factors,cytokines,chemokines,antibacterial peptides,oxygen partial pressure and many other factors.Wound healing is completed by a variety of cells.The re-epithelialization as the important process of the formation of skin layer plays an key role in the integrity of the recovery.The migration of keratinocytes is essential to discuss the molecular mechanism.HIF-1? is a hypoxia-inducible factor 1 which is encoded by the HIF1 A gene.HIF-1? is a very important transcriptional regulator of the cellular metabolism under hypoxia.HIF-1? takes part in the transcription of almost 60 genes and these genes are closely related with the regulation of celluar activity such as proliferation and migration,both of which are vital process in wound healing.The up-regulation of HIF-1? could accelerate the diabetic murine wound closure and overexpression of HIF-1? was found to accelerate the migration of keratinocytes.Furthermore,the overexpression of HIF-1? enhanced cell migration in human dermal fibroblasts,inversely the down-regulated of HIF-1? decreased fibroblasts migration.Thus,it is essential to demonstrate the molecular mechanism about the migration regulated by HIF-1?.Lots of therapeutic methods have been applicated to improve wound healing,such as growth factors,negative pressure,and dressings.Beyond that,some other methods have been used,including electricity,magnetism,and light therapy.Recently,photobiomodulation has been proved to improve wound healing both in vivo and in vitro.The low level laser therapy was effective to improve the wound healing in varity of reports,while the LEDs could reach the same treatment effect as the laser therapy.The photomodulation effect is due to the wavelength and energy of the light soures.So far,lots of reports have proved that light therapy can enhance the migration and proliferation of fibroblasts,but few have investigated the specific molecular mechanism.In this study,the LED irradiation(640nm)can significantly accelerate the primary human keratinocyte migration,meanwhile increase the expression level of the HIF-1?.The irradiation of LED could significantly regulate the F-actin reorganization in the endothelial cells and HeLa cells,we speculate that the irradiation could improve the expression of F-actin in human keratinocytes via HIF-1?.In this study,the up-regulation and down-regulation of the HIF-1? has been investigated to observe the the alteration of cytoskeleton.After these,We further survey that the influence of LED irradiation upon the keratinocytes cytoskeleton with the supression of HIF-1?.Methods1.keratinocytes were isolated from foreskins which were obtained from the circumcision operation,many of the single cell were cultured in the dishes and observed by the application of the imaging system,the migration tack were analyzed by using Image J software.The keratinocytes cell layers were made scratch wound healing model.the effects of red light irradiation on the migration of epidermal cells were observed.The effect of red light on cell proliferation was detected by CCK-8 kit.2.human primary keratinocytes were subjected to a single dose of irradiation at either 12J/cm2 or 24 J/cm2.the expression of HIF-1? were detected by western blot.Immunoblotting was done performed using an antibody against HIF-1?.At the same time Staining of the actin cytoskeleton were performed on human keratinocytes by using phalloidin fluorescence.the change of skeleton was analyzed by Using Image J software.3.the chemiclas(DMOG and COCL2)were used to improve the expression of HIF-1,and to observe the changes of cytoskeleton,the cells were cultured with the chemicals and then the expression of protein was confirmed by the method of WB.Staining of the actin cytoskeleton were performed on human epidermal cells by using phalloidin fluorescence.4.with the above methods,the YC-1 was used to block the expression of HIF-1 after the light irradiation to observe the changes of the human primary skin cytoskeleton.Result:1.The motility and migration of keratinocytes is promoted by LED irradiation.In the study,we chosen two different energy of the red LED irradiation(12 J/cm2 and 24J/cm2)to treat the keratinocytes,observed the motility of keratinocytes.Analyzed by image system,it is found that the keratinocyte motility was increased significantly by the LED with two different energy,espeacially,the 12 J/cm2 group has a more apparent effect.Since the 12J/cm2 energy had a more obvious effect,the 12 J/cm2 were choosen in the subsequent research.the cell migration on the scratch wound model was also accelerated by LED irradiation,but the proliferation of keratinocytes were no significant differences compared with the control group.2.LED radiation increases HIF-1? protein concentration and cytoskeletal actin reorganization HKCs0,3,6,12 h after the treatment of LED irradiation,we deteceted the expression level of HIF-1? protein by Western blot.The results showed that HIF-1? protein was significantly upregulated under the LED irradiation.Alexa Fluor 488 phalloidin could specifically dye the polymeric F-actin,the results of fluorescence expression indicated the LED light significantly enhanced cytoskeletal reorganization in HKCs.3.CoCl2 and DMOG regulated the HIF1? protein expression and induced cytoskeletal actin reorganization in HKCsBoth the treatment with CoCl2(100 ?M or 200 ?M)and DMOG(0.5 m M or 1 mM)could enhance the expression of HIF-1? protein in the keratinocytes,and increse the percentage of cells exhibiting F-actin.4.YC-1 suppressed upregulation of HIF-1? in LED-irradiated HKCs and the LED-dependent actin cytoskeleton reorganization via reducing HIF-1?YC-1(3-(5-hydroxymethyl-2-furyl)-1-benzylinda-zole)is a specific HIF-1 inhibitor.HKCs were cultured with YC-1(0,20 or 50 ?M).YC-1 was found to suppress HIF-1?expression by Western blot,meanwhile,the formation of actin-rich lamellipodia and pseudopods was apparently suppressed after YC-1 treatment even after the red light irradiation.ConclusionFirstly,the motility and migration of keratinocytes were apparently increased by the LED irradiation,but the proliferation has no differences.Secondly,We found that the red light irradiation could upregulate the expression of HIF-1? protein and increase the filamentous actin(F-actin)expresion which were closely related with the cell motility.Furthermore,Inhibition of HIF-1? by the chemical special inhibitor YC-1 could supress F-actin formation even upon the light irradiation,Conversely,activation of HIF-1? via by DMOG orand CoCcl2 increased F-actin expression.The light-emitting diode LED irradiation may regulate enhance keratinocyte migration via HIF-1? dependent cytoskeletal reorganization.