The Role And Mechanism Of IL-1β In Promoting The Migration Of Human Dermal Fibroblasts By Regulating Stathmin/FAK Pathway

Background:The primary goal of wound healing is to repair the damaged barrier,restore the structural and functional integrity of tissues,and thus maintain homeostasis of the internal environment.Its process can be roughly divided into three stages: inflammation stage,proliferation stage and tissue remodeling stage.The early inflammatory response of skin wound can activate neutrophils and macrophages,and secrete various inflammatory factors to accelerate fibroblast migration,thus promoting wound healing.fibroblast is a class of myofibroblast cells with contractile function.Its proliferation and migration function play a key role in skin wound healing,but the molecular interactions and potential regulatory mechanisms remain unclear.Therefore,it is necessary to further explore how fibroblasts regulate the process of wound healing in the inflammatory microenvironment.Microtubule is a kind of dynamic polymer,which is an important part of cytoskeleton.The dynamic transition of depolymerization and polymerization is called microtubule dynamics.Previous studies have shown that microtubule depolymerization can promote the proliferation and migration of tumor cells;on the contrary,the proliferation and migration of tumor cells will be inhibited in the process of microtubule polymerization.Stathmin is a microtubule depolymerization protein,widely expressed in a variety of tissue cells,involved in the regulation of microtubule dynamics and many cell biological processes,such as cell cycle,cell proliferation,intracellular signal transduction,substance transport and cell migration.It has been found that stathmin/FAK pathway may be involved in the migration and invasion of tumor cells.Focal adhesion kinase(FAK),a non-receptor tyrosine kinase,plays a key role in the regulation of cell adhesion,migration,proliferation and survival.Therefore,it plays an important role in tissue regeneration and repair.Similarly,FAK protein can regulate microtubules in cells to affect cell polarization,thus carrying out a series of cell activities such as proliferation and migration.Our previous studies have found that the expression level of stathmin is closely related to the proliferation and migration of skin fibroblasts,suggesting that stathmin plays an important role in regulating skin wound healing,but the underlying mechanism is far from clarified.Previous studies have shown that FAK plays an important role in promoting cell migration,but whether FAK is involved in regulating the migration of skin fibroblasts in inflammatory states remains unclear,and whether stathmin affects cell migration by mediating FAK expression remains unclear.Therefore,it is very important for clinical intervention of early wound healing to explore and elucidate the role of early inflammatory stimulation on human dermal fibroblast migration and its molecular mechanism.This study aims to clarify the role and preliminary mechanism of IL-1β mediating human dermal fibroblast migration by regulating stathmin/FAK pathway,and provide new targets and ideas for clinical intervention in the treatment of early skin wounds.Materials and Methods:1.Human dermal fibroblasts(HDF)and interleukin-1β(IL-1β)were used to establish a cell inflammation model.First,the cells were treated with blank group,low concentration(10ng/ml)and high concentration(500ng/ml)of IL-1β for 24 hours,and the cell migration was observed by cell scratch test to screen out the appropriate concentration of IL-1β for subsequent experiments.2.HDF cells were treated with IL-1β at the concentration that mimics the inflammatory microenvironment selected above,and the expressions of stathmin and FAK proteins were detected by western blot and immunofluorescence experiments to explore the possible molecular mechanism of IL-1β promoting cell migration.3.HDF cells were treated with the concentration of IL-1β selected previously to mimic the inflammatory microenvironment of HDF cells,and HDF cells were interfered with small interfering stathmin(si STMN)and small interfering FAK(si FAK),respectively.The effects of stathmin and FAK proteins on HDF cell migration were observed by scratch test.The effect of stathmin and FAK on cell migration was further verified.4.After HDF cells were treated with small interfering stathmin(si STMN),the expression of stathmin was detected by western blot to test the interference efficiency,and the expression of FAK protein was detected to explore the possible pathway of IL-1β in promoting HDF cell migrationResult:1.Compared with the control group,the migration of HDF cells treated with low concentration of IL-1β(10ng/ml)for 24 hours was promoted,whereas the migration of HDF cells treated with high concentration of IL-1β(500ng/ml)for 24 hours was inhibited.Therefore,IL-1β(10ng/ml)was used to mimick the inflammatory microenvironment in vitro for subsequent experiments.2.After treatment of HDF cells with IL-1β(10ng/ml),stathmin and FAK protein were significantly increased compared with the control group,suggesting that stathmin and FAK play an important role in the migration of HDF cells promoted by IL-1β.3.When si STMN was used to interfere with stathmin expression,cell migration was significantly decreased in the si STMN group and IL-1β+si STMN group.Similarly,when si FAK was used to interfere the expression of FAK,the migration of HDF cells in the si FAK group and IL-1β+si FAK group was also significantly reduced,further suggesting the key role of stathmin and FAK protein in the process of IL-1β-promoted HDF cell migration.4.When si STMN was used to interfere with the expression of stathmin,the expression of stathmin was significantly reduced,indicating that the interference was effective.At the same time,the expression of FAK protein was also significantly reduced after si STMN was used to interfere with the expression of stathmin,suggesting that FAK may be a downstream protein molecule of stathmin.Together with stathmin,they are involved in the regulation of IL-1β-mediated migration of HDF cells.Conclusion:1.IL-1β(10ng/ml)promoted the migration of human dermal fibroblasts,while IL-1β(500ng/ml)inhibited the migration of human dermal fibroblasts.2.IL-1β(10ng/ml)promoted the expression of stathmin and FAK protein in human dermal fibroblasts.3.IL-1β(10ng/ml)promoted the migration of human dermal fibroblasts through stathmin/FAK pathway.