The Role And Mechanism Of Stathmin In LPS-mediated Human Dermal Fibroblast Migration And Proliferation

Background:Wound healing is an interactive and dynamic biological process involving an inflammation phase,a proliferation phase,and a tissue remodeling phase.Dermal fibroblast is a critical executor during wound healing,and its proliferation and migration are essential steps to repair wounds due to its central role in the granulation tissue formation.If granulation tissue formation is dysfunction,wound healing may be delayed or wounds may not heal at all.Upon wounding,various cell types are activated by exogenous or endogenous stimuli to create conditions for wound healing.Although the inflammatory environment has been proven to be a key initial factor in wound healing and dermal fibroblasts play an essential role in this process,their correlation and potential regulatory mechanisms remain largely unclear.Therefore,a comprehensive understanding of the biological processes of dermal fibroblasts under inflammation is required.The microtubule(MT),a crucial component of the cytoskeleton,is composed of α-andβ-tubulin heterodimers.The transition between the depolymerization and polymerization of tubulin is recognized as MT dynamics,which are regulated by destabilizing proteins(e.g.,stathmin family)and stabilizing proteins(e.g.,microtubule-associated protein family).MT dynamics determine the biological behaviors of cells.Previous studies indicated that MT depolymerization led to increased epidermal or tumor cell migration and proliferation;whereas MT polymerization abolished cell migration and proliferation.However,some conflicting studies have also suggested that MT stabilization promotes carcinoma invasion.Thus,a deep exploration of MT dynamics is needed to understand its role in cell migration and proliferation.Stathmin destabilize MTs by both binding tubulin dimers as well as directly binding MTs and promoting depolymerization.Cell proliferation involves the progression from interphase to mitotic phase.Cytoplasmic MT regulatory proteins,such as the stathmin family,play a significant role in cell cycle regulation.Recent studies indicated that stathmin expression levels were closely correlated with cell proliferation and migration,with enhanced stathmin expression observed in highly proliferating neuroblastoma cells or gastric cancer cells.In addition,the involvement of signal transduction in regulating stathmin includes mitogen-activated protein kinases(MAPKs),microtubule affinity-regulating kinases,and protein kinase A.Previous studies indicated that p38/MAPK activation regulated stathmin in cardiomyocytes and that p38/MAPK activation affected gallbladder carcinoma cell and inflammatory cell proliferation and migration by regulating stathmin.However,the effect and mechanism of stathmin in inflammation-induced dermal fibroblast migration and proliferation during wound healing are still poorly understood.In this study,we aimed to explore the role of stathmin in human dermal fibroblast(HDF)migration and proliferation under inflammation.Our study aimed to provide a potential way for the progress of therapeutic interventions for wound healing.Materails and Methods1.Human dermal fibroblast(HDFs)and lipopolysaccharide(LPS)were used to simulate the cell inflammatory model.HDFs were stimulated with LPS at 7 concentrations(0ng/ml,5ng/ml,10ng/ml,50ng/ml,100ng/ml,500ng/ml,1000ng/ m L)for 24 h,and the cell migration was observed by scratch wound healing assay.Then,we selected CCK-8 kit to detect the effects of LPS(500ng/ m L)on HDFs proliferation at 4 time points(0h,12 h,24h,48h),and screened the most suitable LPS concentration and time for simulated cell inflammatory model.2.HDFs was stimulated according to the previously slected LPS conditions simulating the inflammatory microenvironment of cells,and then the changes of HDFs migration were observed by cell scratch experiment.Cell proliferation was detected by CCK-8 kit,Edu kit and western blot,and the changes of microtubule structure were observed by immunofluorescence staining.3.HDFs was stimulated according to the previously screened LPS conditions stimulating the inflammatory microenvironment of cells.The relationship between microtubules and migration and proliferation of human dermal fibroblasts was determined by using the microtubule stabilizing agent paclitaxel after intervention,cell migration was detected by the scratch assay,cell proliferation was detected by CCK-8 kit,Edu kit and western blot,and HDFs of microtubule structure was observed by immunofluorescence staining.4.The significance of stathmin inflammation in wound healing was verified by detecting the expression of stathmin in normal mouse skin and 3-day wound.The expression of stathmin in HDF was detected by simulating the inflammatory microenvironment with LPS conditions selected in the early stage.Subsequently,the expression of stathmin was interfered by si STMN(si RNA targeted interference with stathmin),cell migration was detected by scratch assay,cell proliferation was detected by CCK-8 kit,Edu kit and western blotting assay,and the changes of microtubule structure were observed by immunofluorescence staining.To clarify the role of stathmin in promoting HDFs migration and proliferation in LPS-induced inflammatory microenvironment.5.The significance of P38/MAPK in wound healing inflammation was verified by detecting the expression of P38/MAPK in normal skin and 3-day wound skin of mice.P38 activation of HDF was detected by western blot.In addition,p38 inhibitor SB203580 was used to determine whether P38/MAPK is an upstream kinase of stathmin.Then,the cells were pretreated with CMV(null),MKK6,si NC,si STMN,and the cell migration was detected by scratch assay.Cell proliferation was detected by CCK-8 kit,Edu kit and western blot.Immunofluorescence staining was used to observe the changes of microtubule structure to determine whether the P38/MAPK pathway participates in and regulates LPS-induced HDFs migration and proliferation and microtubule depolymerization by regulating stathmin expression.Results1.LPS concentration of 500ng/m L was screened out in pre-experiment and treated for24 hours to construct inflammatory cell model.The migration of HDFs increased in a concentration dependent manner after different concentrations of LPS(0ng/ml,5ng/ml,10ng/ml,50ng/ml,100ng/ml,500ng/ml,1000ng/ml),and the migration was the fastest at500ng/ml.After LPS(500ng/m L)was treated with HDFs at 4 time points(0h,12 h,24h,48h),cell proliferation was the most significant at 24 h compared with the control group.Therefore,LPS(500ng/ml)was used to stimulate HDFs for 24 h for subsequent experiments to build cell inflammatory model.After the construction,the migration and proliferation of detected cells compared with the control group were significantly increased.The HDFs microtubule depolymerization in LPS treatment group was more obvious than that in control group.These results suggest that appropriate inflammatory stimulation promotes the migration and proliferation of human dermal fibroblasts and promotes microtubule depolymerization.2.HDFs were stimulated according to the previously screened LPS conditions stimulating the inflammatory microenvironment of cells.After pretreatment with paclitaxel,the migration and proliferation of normal cells did not change significantly,but microtubule depolymerization decreased compared with the matched group.However,stimulation with LPS(500ng/m L)at 24 h inhibited migration and proliferation and reduced microtubule depolymerization.3.The stathmin in the wounds of 3 days expressed significantly higher than the control group;LPS stimulated HDFs to increase the expression of stathmin,and si STMN inhibited this effect.HDFs were transfected with si STMN could inhibit its migration and proliferation,and attenuated the promoting effect of LPS induced inflammatory microenvironment on HDFs migration and proliferation.After HDFs transfection,si STMN not only increased microtubule density and weakened the depolymerization of microtubules,but also weakened the promoting effect of LPS induced inflammatory microenvironment on HDFs microtubule depolymerization.4.The expression of P38/MAPK in wounds of 3 days was higher than the control group;After stimulation of HDFs by LPS,p-P38 expression was increased,but after inhibition p38 pathway,p-P38 expression and stathmin expression were decreased.Inhibition of this pathway by SB203580,a P38/MAPK inhibitor,significantly inhibited HDFs migration and proliferation and microtubule depolymerization in LPS-induced inflammatory microenvironment.The cells were treated with CMV(null),MKK6,si NC and si STMN.MKK6 treatment group had the same effect as LPS to promote HDFs migration,proliferation and microtubule depolymerization.Whether,this effect was inhibited by si STMN,suggesting that p38/MAPK kinase pathway is involved in and regulates LPS induced HDFs migration and proliferation and microtubule depolymerization by regulating stathmin expression.Conclusions:The findings demonstrated in our present study reveal a novel role of stathmin in inflammation-mediated HDF migration and proliferation during wound healing,while p38/MAPK serves as the upstream kinase of stathmin-mediated MT depolymerization,followed by HDF migration and proliferation.Furthermore,our data provide a potential target for the development of therapeutic interventions for wound healing.