| BackgroundWound healing is a fundamental problem in burning wounds,and promoting rapid wound healing is the key to solving the problem.After skin damage,various cells will act immediately to protect the wound tissue and promote healing.The completion of this process requires multi-tissue and multi-cell coordination,and re-epithelialization plays an important role in the whole process of wound healing.The re-epithelialization of skin wounds depends on the combined effects of many factors such as migration,proliferation and differentiation of keratinocytes.Studies have shown that wound tissue tissue is depleted of large amounts of oxygen after wound injury due to activation of a large number of cells,resulting in a hypoxic microenvironment.A large number of studies have shown that the hypoxic environment of the wound is beneficial to the migration of epidermal cells and promote wound healing.However,the underlying regulatory mechanism remains unclear.ADAM17 is a multidomain transmembrane metalloproteinase that regulates cell migration,adhesion,and cellular signaling.During skin development and homeostasis,ADAM17 is the basis for the regulation of many cellular signaling processes.However,its mechanism of action in the epidermis of the skin has not been revealed.Numerous studies have shown that p38/MAPK promotes keratinocyte migration during wound healing,and p38/MAPK is activated by hypoxia.Studies have shown that p38 can regulate the activity and expression of ADAM17 by regulating the phosphorylation of ADAM17 to mediate the cellular effects regulated by ADAM17.Whether ADAM17activity in epidermal cells is regulated by p38 and promote cell migration remains unclear.As a member of the transmembrane metalloproteinase family,ADAM17 regulates the physiological processes of cells in the cell primarily by detachment of the extracellular domain of the lytic membrane molecule.Epidermal growth factor receptor EGFR plays an important role in wound healing in skin.Studies have shown that the phenotype of ADAM17 knockout mice is similar to that of EGFR deletion,indicating that EGFR is the basic base of ADAM17,and the two are common.Maintaining the integrity of the skin plays an important role.The role of EGFR epidermal growth factor receptor is mainly activated and regulated by its ligand EGF family.The EGF family includes a plurality of molecules in which TGF-?,HB-EGF,and AREG are mainly regulated by ADAM17.However,under hypoxic conditions,whether the migration of epidermal cells is affected by the cleavage of ADA ligand by ADAM17,and whether this process is regulated by p38/MAPK,this important regulatory mechanism is still not clear enough.Therefore,this study will explore the mechanism of action of ADAM17 in epidermal cell migration based on this idea.This study proposes that hypoxia up-regulates the p38/MAPK signaling pathway and activates ADAM17 activity to promote the release of EGF family molecules,thereby regulating epithelial cell migration.By detecting the effect of ADAM17 on epidermal cell migration under hypoxic conditions,and exploring the effect of p38/MAPK on ADAM17 activity and cell migration in epidermal cells,the mechanism of ADAM17 promoting epidermal cell migration under hypoxic conditions was clarified.Method1.Make a model of in vitro epidermal cells to promote wound healing,simulate the hypoxic microenvironment of tissue healing in wound injury in vivo,observe the treatment of normoxia?21%O2?and hypoxia?1%O2:0h,6h,12h,24h?The motility and migration of epidermal cells before and after,as well as the expression and activity of ADAM17 protein and the changes of basal EGFR ligands;2.Inhibit the activity of ADAM17 in epidermal cells by TAPI-2 and silence the expression of ADAM17 protein by si RNA transfection technique,then scratch the treated epidermal cells to make a wound healing model,and further put the cells into a hypoxic chamber.The simulated experimental time?12h?of the cultured cells in a hypoxic environment?1%O2?was simulated,and then the wound healing rate and cell movement of the cells were counted.In this experiment,cells were first treated with drugs and cell supernatants were collected and extracted at specific experimental time points.Then,the molecular activity and protein expression of ADAM17 inhibitor TAPI-2 and si RNA were detected by kit and WB,respectively.Inhibition and silencing effect;the cell healing state simulated by the monolayer cell scratch test was recorded by microscopy at the experimental time point after the treatment,and the effect of inhibiting the molecular activity of ADAM17 on the migration of wound epidermal cells was observed;in addition,the treated epidermal cells were treated.The effect of downregulation of ADAM17 expression on cell motility in individual epidermal cells was observed under a living cell workstation.In summary,the activity level and protein level of ADAM17 were shown to regulate the motility of epidermal cells under hypoxic conditions.3.Epidermal cells were treated with p38/MAPK inhibitor SB203580 and kinase MKK6,respectively.The expression of p-p38/MAPK and the substrate of p-p38/MAPK in epidermal cells before and after hypoxia?1%O2:12h?treatment were detected by WB.The phosphorylation level of MK-2 was changed,and the effect of down-regulation and up-regulation of p38/MAPK on the expression and activity of ADAM17 protein in epidermal cells was detected under hypoxic conditions.Finally,p38/MAPK under hypoxic conditions was detected by scratch test and single cell exercise test.The inhibitory effects of SB203580 and kinase MKK6 on epidermal cell motility and migration indicate the regulation of ADAM17 on epidermal cell migration under hypoxic conditions from both cellular and molecular levels.Results1.Hypoxia treatment at different times within 24h significantly promoted the motility of epidermal cells,and hypoxia significantly up-regulated the expression of hypoxia transcription factor HIF-1?and the expression and activity of ADAM17.Related2.Using ADAM17 inhibitors to inhibit the activity of ADAM17 and its specific small interfering siRNA to silence its protein expression,through the wound healing model scratch test and single cell motility experiment found that the down-regulation of ADAM17 can significantly inhibit the migration of epidermal cells,Moreover,it can effectively reverse the hypoxia-promoted epidermal cell migration,indicating that hypoxia-promoted epidermal cell migration is regulated by the disintegrin metalloproteinase molecule ADAM17;3.The activity of p38/MAPK is activated by hypoxia.Treatment of epidermal cells with p38/MAPK inhibitor SB203580 and kinase MKK6 under hypoxia can significantly regulate the phosphorylation of p38/MAPK substrate MK-2.And also positively correlated with the protein expression and activity of ADAM17.In addition,inhibition of p38/MAPK activity can significantly inhibit epidermal cell migration under hypoxic conditions,and activation can enhance hypoxia promotion.This indicates that p38/MAPK promotes the migration of epidermal cells by promoting the ADAM17signaling pathway under hypoxic conditions.ConclusionHypoxia promotes the activation of p38/MAPK signaling pathway in epidermal cells and up-regulates the expression and activity of ADAM17,thereby promoting epidermal cell migration;while hypoxia up-regulates the protein activity of ADAM17translation through hypoxia transcription factor and p38/MAPK signaling pathway However,whether the expression of transcriptional level genes is still unknown is not known,and there is literature that p38/MAPK can phosphorylate the cytoplasmic domain of ADAM17,so whether p38/MAPK under hypoxic conditions also passes this signaling pathway.Need to explore in depth.In conclusion,this study demonstrates for the first time the role of ADAM17 in the re-epithelialization of skin wounds,elucidating its promoting mechanism in epidermal cell migration,and providing new research ideas for further research on the mechanism of regulating wound healing.Clinically,it can provide new potential therapeutic targets for the treatment of wound diseases,accelerate wound healing,and solve the complications of wound injury. |
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