The Role Of Inflammatory Microenvironment In The Directional Migration Of Bone Marrow Mesenchymal Stem Cells

Background:Bone marrow-derived mesenchymal stem cells(BMSCs)are non-hematopoietic pluripotent stem cells that originate from the mesoderm of an embryo and are found in bone marrow with the potential of self-replication and multidirectional differentiation.Because it can migrate to the injured parts of the body and regulate the recovery of tissue and function in the injured areas through the way of differentiation and paracrine,making it an ideal cell for cell transplantation treatment.In the central nervous system,a variety of diseases can cause brain damage,cause the ineffective function of the nerve.At the cellular level,the central nervous system injury is mainly manifested as apoptosis of nerve cells and activation of microglia cells.At the same time,activated microglia can further aggravate the apoptosis of nerve cells,so regulating the activation of microglia has become a focus of current research on neurological diseases.The activated microglia mainly showed local inflammatory reaction.Characterized by increased secretion of inflammatory factors such as IL-6,TNF-?,IL-1?,and the formation of inflammatory microenvironment.Studies have shown that transplanted BMSCs can regulate the activation of microglia and promote the recovery of nerve function by migrating to the site of nerve injury.However,whether the inflammatory microenvironment is involved in the migration of BMSCs,and which components in the inflammatory microenvironment are involved in the migration of BMSCs and the specific mechanism are still unclear.As a result,only a small number of transplanted BMSCs could reach the target area in the end,and the transplantation efficiency was not high.Therefore,it is very important to elucidate the inflammatory microenvironmental factors and their regulatory mechanisms that affect the directional migration of BMSCs in order to improve the efficiency of transplantation and accelerate its clinical application.In this study,lipopolysaccharide(LPS)was used to establish an in vivo and in vitro inflammatory model of the nervous system.The changes of inflammatory cytokines in the inflammatory microenvironment of the nervous system were further detected.At the same time,the supernatant rich in inflammatory factors was collected to simulate the inflammatory microenvironment of the damaged central nervous system(CNS)in vivo to explore its influence on the migration of BMSCs and its specific regulatory mechanism.The experimental results were further verified by in vivo experiments.The significance of this study was to improve the transplantation efficiency of BMSCs and provide more experimental basis for clinical application of BMSCs.Methods1.To determine the effect of BVZ conditioned medium(CM)on BMSCs migration,and analyze the changes in cytokine levels secreted by BV,in the inflammatory microenvironment.Primary BMSCs were first obtained from bilateral femoral bone marrow of one-month old female rats,and the third to sixth generation was used for follow-up experiments.Meanwhile,BV2 cell lines were used to replace microglia cells in vitro.In order to obtain CM with different concentrations,the supernatant was collected by treating BV2 cell with LPS of 100ng/ml and 1000ng/ml,respectively.The effects of different concentrations of CM on BMSCs migration were evaluated by Transwell assay.Next,to determine whether BV2 cell affects BMSCs migration by changing the secretion profile of inflammatory cytokines in the inflammatory microenvironment,and to select specific inflammatory cytokines for the following in-depth exploration,we respectively used the method of enzyme-linked immunosorbent(ELISA)and RT-PCR experiment to measure the expression levels of tumor necrosis factor-a(TNF-?)interleukin-6(IL-6)and interleukin-1?(IL-1?)in different concentrations of CM and cells.2.To explore the effect of IL-6 on directed migration of BMSCs.11-6 specific neutralizing antibody and exogenous IL-6 cytokines were used to investigate the effect of IL-6 on BMSCs migration by Transwell.3.To explore the mechanism of IL-6 in directed migration of BMSCs.(1)Effects of p-STAT3,p-ERK1/2 and p-FAK on IL-6 promoting BMSCs directed migration.First,the expression of p-STAT3,p-ERK1/2 and p-FAK in BMSCs treated with different concentrations of CM was detected by Western blots.Then,BMSCs were pretreated with specific STAT3 inhibitor(AG490),ERK 1/2 inhibitor(PD98059)and FAK inhibitor(PF573228).Migration of BMSCs was detected by Transwell assay.Finally,adding IL-6 specific neutralizing antibody and exogenous IL-6 cytokines,the expression level of p-STAT3,p-ERK1/2 and p-FAK was further detected,to determine whether p-STAT3,p-ERK1/2 and p-FAK is involved in the process of IL-6 promoting the targeted migration of BMSCs.(2)Effect of p-STAT3 and p-ERK1/2 on p-FAK expression in the process of IL-6 promoting the targeted migration of BMSCs.BMSCs were pretreated with AG490 and PD98059,and p-FAK expression was detected by Western blots to determine whether p-STAT3 and p-ERK1/2 affect the targeted migration of BMSCs by regulating the expression of p-FAK.2.In vivo experiments on mice were conducted to verify the effect of IL-6 on BMSCs migration and its mechanism.(1)A model of brain inflammation in 57BL/6 mice was established and changes in inflammatory cytokines were analyzed.57BL/6 male mice with weight of 20-30g and a similar age were selected,each group of five.A model of brain inflammation was established by injecting 0.25mg/kg LPS into the lateral ventricle.Levels of the inflammatory cytokine IL-6,TNF-? and IL-1? in the brain after inflammation were measured by Western blots.(2)To verify the role and mechanism of IL-6 in directed migration of BMSCs.Twenty-five male 57BL/6 mice weighing 20-30g and of the same age were selected.They were divided into the LPS group,LPS+BMSCs group,LPS+BMSCs+IL-6 antibody group,LPS+BMSCs+ AG490 group,LPS+BMSCs+ PD98059 group,with 5 mouse in each group.The in vivo models of each group were constructed by injecting 106 GFP labeled BMSCs(GFP-BMSCs)or not?pretreating with inhibitors or not?giving 0.1mg/kg specific IL-6 neutralizing antibodies or not.The number of GFP-BMSCs in each group was counted by immunohistochemical staining(IHC)to further verify the effect of IL-6 on the directional migration of BMSCs and its mechanism in vivo.Results1.Through Transwell experiment,it was found that CM indeed promoted the migration of mesenchymal stem cells,and compared with 100-cm,1000-cm significantly enhanced the migration ability of mesenchymal stem cells,showing a concentration dependence.ELISA analysis showed that LPS stimulation significantly increased the secretion of IL-6,TNF-? and IL-1? in CM and the abundance of three inflammatory factors in 1000-CM was higher than that in 100-CM.The expression levels of the three inflammatory factors in BV2 cell were detected by RT-PCR,and it was found that 1000ng/ml,100ng/ml LPS significantly promoted the mRNA expression of the three inflammatory factors,but there was no concentration dependence.2.By Transwell assay,the number of BMSCs migrating cells was lower after specific anti-IL-6 antibody was added to 1000-CM to neutralize IL-6 compared with the control group.On the contrary,when the exogenous IL-6 cytokines were added to the simple DMEM,the number of BMSCs migrating cells increased significantly.3.(1)Western blots showed that CM significantly increased the expression of p-STAT3,p-ERK1/2 and p-FAK in BMSCs,and the expression of p-STAT3,p-ERK1/2 and p-FAK in BMSCs gradually increased with the increase of CM concentration.After the use of specific pathway inhibitors,Transwell assay showed that the inhibitors significantly inhibited the migration of BMSCs induced by 1000-CM,while the inhibitors themselves had no significant effect on the migration of BMSCs.BMSCs were treated with simple DMEM with specific exogenous IL-6 cytokines or 1000-CM with anti-IL-6 antibodies.Western blots revealed that exogenous IL-6 cytokines significantly up-regulated the expression of p-STAT3,p-ERK1/2 and p-FAK,and specific anti-IL-6 antibodies significantly down-regulated the expression of p-STAT3,p-ERK1/2 and p-FAK.(2)BMSCs were treated with AG490 or PD98059 in 1000-CM,and Western blots showed that AG490 or PD98059 could significantly inhibit the expression of p-FAK.4.(1)Western blots revealed that the expression of inflammatory cytokines IL-6,TNF-? and IL-1? was significantly up-regulated after intracerebral inflammation.(2)IHC staining showed that compared with the simple inflammatory group,the number of targeted migration cells in the brain was significantly reduced after the use of anti-IL-6 neutralizing antibody.With the use of specific pathway inhibitors,the number of BMSCs that migrate in the brain was significantly reduced.ConclusionIL-6 level was increased in CM of BV2 cells activated by LPS.When BMSCs were incubated in this CM,IL-6 further triggered phosphorylation of ERK1/2?STAT3 and downstream FAK,promoting BMSCs migration.Our study further explored the molecular mechanism of BMSCs migration,which is of great significance for clinical application of BMSCs in therapeutic transplantation.