| Dendritic cells(DCs)are the most powerful antigen-presenting cells(APCs)in the immune system,and it can rapidly start the immune response and inflammatory response of the innate immune system.DCs are a class of heterogeneous cell populations that can be divided into two categories: the matured DCs and the immatured DCs.From the phenotypic point of view,matured DCs overexpress MHC class II molecules,CD80,CD86 and CD83;the immatured DCs,however,express these markers at relatively low levels.From the functional point of view,matured DCs have a powerful antigen presenting function,do not have phagocytosis;and the immatured DCs have a strong phagocytic function,can induce immune tolerance.It has been reported that in animals with myocardial ischemia and viral myocarditis,DCs are infiltrated in the lesion.DCs can regulate the inflammatory response and participate in the repair process of myocardial tissue after damage.It has been reported that the level of histone acetylation in DCs is closely related to its maturation,differentiation and function.Trichostatin A(TSA)is a broad spectrum histone deacetylase inhibitor(HDACIs).Our previous work proved that TSA can regulate the progress of tissue repair,reducing myocardial infarct size,reducing tissue damage in the infarcted myocardium.The number of DCs infiltrated into the myocardium was significantly increased during the repair of myocardial injury induced by TSA.How TSA affects DCs during these processes remains unclear.Studies have shown that locally infiltrated DCs existed mostly in their matured state.Therefore,we speculate that TSA may affect the maturation and function of DCs in the local microenvironment after myocardial infarction.Like most cells,DCs produce energy by oxidative phosphorylation(OXPHOS).Studies have shown that DCs under hypoxic conditions,will enhance its the glycolytic pathway through a process called metabolic reprogramming,which helps DCs to improve the ability to adapt to the external environment.Pyruvate kinase(PK)is one of the key enzymes in glycolysis.M1 type pyruvate kinas(PKM1)can promote the oxidation of pyruvate in mitochondria to produce acetyl-CoA where as the M2 pyruvate kinase(PKM2)can promote the expression hypoxia-inducible factor 1α(Hif1α),therefore facilitating the conversion of pyruvate to lactose.Studies have shown that TSA can enhance the expression and activity of PKM2 after MI,thereby improving tissue ischemic damage in rats.Based on the above,we hypothesized that TSA can alter the phenotype and function of DCs by affecting the expression and activity of PKM2 during the glycolysis of DCs in the local environment of myocardial ischemia,therefore protect the myocardium.Objectives:In the present study,we will use mouse DC2.4 cell line as the research subjects.By utilizing the use of oxygen glucose deprivation conditions in vitro,to simulate the oxygen glucose deprivation environment after myocardial infarction,and to uncover the mechanism by which TSA modulates the phenotype and function of DC2.4 cells,thus providing new strategies for ischemic heart diseases.Methods:The cell viability was determined by MTT assay at different durations and under different concentrations of TSA.The time of oxygen glucose deprivation,the gas compositions were also determined.Flow cytometry was used to study the effects of TSA on the phenotype of DC2.4 cells under given conditions.The effect of TSA on the phagocytosis of DC2.4 cells under OGD was detected by flow cytometry using FITC-dextran as substrate.The effects of TSA on the migration of DC2.4 cells under OGD was performed using scratch wound assay.The effects of TSA on the expression of IL-1β,IL-10,IL-12 and TGF-β in supernatant of DC2.4 cells were detected by ELISA.The effects of TSA on the production of ATP and lactose in DC2.4 cells were detected by respective detection kits.The effects of TSA on the expression of PKM1 / PKM2 and their upstream splicing factor SRSF3 under OGD were detected by Western-blot as well as RT-QPCR,meanwhile,the expression of HIF-1α,Tpi1,HK2 and LDHAAwere detected by RT-QPCR.Results:(1)The protective effect of TSA on DC2.4 cells under oxygen-glucose deprivation:MTT method was used to measure the survival rate of DC2.4 cells under models conditions with various concentrations and hypoxic durations.Control group,hypoxia group(OD)and oxygen-glucose deprivation(OGD)were set.Dosages of TSA include 200 nM,400 nM,800 nM,1000 nM.And the cells were treated for 4h,8h,12 h,and 24 h.According to MTT results,the cell viability was too low at 8h:(32.03 ±1.04)%,12h:(12.19 ± 3.35)%;24h:(8.56 ± 2.29)%;4h:(44.76±2.34)%.Under normoxic conditions,TSA didn’t affect the viability under 12 h.Therefore,TSA 200 nM with 4h OGD(Viability44.76%±2.34%)were used for next experiments.(2)The effect of TSA on the maturation of DC2.4 cellsCompared with the control group,the expression of CD80 and CD86 in GD group,OD group and OGD group was increased.In addition,compared with the model group,the expression of CD80 and CD86 was significantly increased by TSA(P<0.05).(3)The effect of TSA on the uptaking ability of DC2.4 cells:Compared with control group,the positive rate of FITC in GD group,OD group and OGD group were decreased.Compared with the model group,the positive rate of FITC in DC2.4 cells was significantly decreased(P<0.05).(4)The effect of TSA on the migration of DC2.4 cells:Compared with the control group,the migration distance of GD group,OD group and OGD group were significantly decreased.Compared with the model group,after treatment with TSA,the migration distance of the cells was significantly increased(P <0.05).(5)The effect of TSA on the secretion of cytokines in DC2.4 cells:Compared with their respective model group,cells in Control、GD、andOD group the secretion of IL-1β、IL-10、IL-12 and TGF-β were decreased Meanwhile,cells in OGD group,the secretion of IL-1β was decreased in OGD group whereass,the secretions of IL-10、IL-12 and TGF-β were increased after treatment with TSA(P<0.05).(6)The effect of TSA on ATP production in DC2.4 cells:Compared with their respective model group,the secretion of IL-1β decresed in control group,GD group,and OD group after TSA treatment;but in OGD group,the secretion of IL-1β was reduced after TSA treatment,and the secretion of IL-10,IL-12 and TGF-β were increased(P < 0.05).(7)The effect of TSA on lactose production in DC2.4 cells:Compared with the control group,cells in GD、OD and OGD group,the contents of lactose decreased,and the OGD group was more significantly(P<0.05);In addition to the Control group,compared with their respective model group,after treatment with TSA,the lactose content in the supernatant were increased,and GD group and OGD group were more significantly(P<0.01)(8)The effect of TSA on the glycolysis-related genes of DC2.4 cells:After administration of TSA,the mRNA levels of HIF-1α,Tpi1,HK2 and LDHAA were significantly higher than those of the corresponding model group(P <0.05).(9)The effect of TSA on the expression of PKM2,PKM1 and SRSF3 in DC2.4 cells:The expression of PKM2 and SRSF3 protein was significantly higher than that of the corresponding model group after TSA treatment.While the expression of PKM1 was significantly down-regulated(P <0.05).(10)The effect of TSA on the mRNA expression of PKM2,PKM1 and SRSF3 inDC2.4 cells:The mRNA expression of PKM2 and SRSF3 was significantly higher than that of model group after TSA treatment.While the expression of PKM1 was significantly down-regulated(P <0.05,P <0.01).(11)The effect of TSA on content of ATP and lactate in DC2.4 cells.The contents of ATP and lactate were decreased(P<0.05)after interference SRSF3 and PKM2.Compared with the Vetcor group,the contents of ATP and lactate were increased significantly after treatment with TSA(P<0.05).Conclusions:(1)TSA can improve the survival rate of DC2.4 cells under OGD condition;(2)TSA can reduce the uptake capacity of DC2.4 cells and promote the maturation of DC2.4 cells;(3)TSA can promote the migration of DC2.4 cells,reduce the secretion of IL-1β under OGD and increasing the secretion of IL-10、IL-12 and TGF-β.(4)TSA may promote the expression of PKM2 by upregulating the expression of splicing factor SRSF3 in DC2.4 cells,promote glycolysis,increased the production of ATP,and promote DCs cells survive,and then affect the maturation and function of DC2.4 cells. |
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