The Mechanism Of Regulation On DC2.4 Cells Treated With Oxygen-glucose Deprivation Based On CDK5-FOXO1 Crosstalk

Acute myocardial infarction?MI?is currently the world's most dangerous and fatal coronary artery disease.Heart protection and prognosis after myocardial infarction are still a question in clinical practice which need more attention.At present,for the treatment of myocardial infarction,more clinical treatment is focused on the development of myocardial cells after infarction and the area of infarction.Ischemic reperfusion injury is known as the cause of microvascular damage in myocardial infarction which will increase the area of infarction and promote inflammation,Therefore,more attention on the development of inflammation after ischemic reperfusion injury is a way of vital importance to help patients have better prognosis and fewer infarction area.The inflammatory reaction after the acute myocardial infarction includes the initial pro-inflammatory reaction and subsequently anti-inflammatory reaction,the continuous pro-inflammatory reaction and the transition period of the pro-inflammatory and anti-inflammatory have a bad effect on myocardial remodeling and improve the risk of heart failure.As a result,the study about the cells which play a role in the period of pro-inflammation and anti-inflammation after the ischemic reperfusion injury will become a point to help reduce worse cardiac remodeling.Dendritic Cells is an important kind of antigen-presenting cells that connects initial and adaptive immunity,and studies have shown that DCs migrate in large numbers to ischemic regions after myocardial infarction.The subsets tolerance DCs?tDCs?were involved in the cardiac remodeling process after infarction and help decrease the infarction area,so regulating the activity and function of DCs after myocardial infarction will become an effective treatment to improve prognosis.Studies have shown that FOXO1,a member of the Fork head Box transcription factor?FOX?family,regulates DCs after bacterial infection.FOXO1 can promote the ability of migration pinocytosis in DCs in order to activate initial T-cells while the absence of FOXO1 causes the inhibition of inflammation after infection.In addition,FOXO1 regulated DCs'ability to produce inflammatory cytokines,and overexpression of FOXO1 causes elevated expression of cytokines such as IL-12?TNF-?.Cyclin-dependent kinase?CDK5?has been found to be involved in regulation activity in many diseases,such as neurological diseases and tumor immunity?rheumatoid arthritis and other diseases.CDK5 can regulate several downstream targets,including phosphorylation FOXO1 as to inhibit its transcription activity.However,CDK5 is less been researched in some different diseases,and its regulatory function in more disease models needs to be further identified.In summary,we speculate that CDK5 can regulate FOXO1 transcription activity,inhibit downstream transcription activity by phosphorylation FOXO1,and thus affect DCs cell-related function,affect the maturation of DCs cells?phagocytosis?cytokine secretion etc.This project aims to clarify the mechanism of regulation in DCs cells after myocardial infarction by observing CDK5's control of oxygen glucose deprivation and the DC2.4 cell-related function given to the infarction heart lysate protein in mice,and provide new ideas for DCs cells as an important target to improve the adverse prognosis of myocardial infarction and reduce the area of infarction.Objectives:In the current experiment,mouse DC2.4 cells line were used as research subjects,and we use oxygen-glucose deprivation and infarction heart lysate to imitate the microenvironment after myocardial infarction.By the detection of the ability of maturation,pinocytosis,migration,and the secretion of cytokines,we try to find out the changes of DC2.4 cells function through CDK5-FOXO1 crosstalk in the microenvironment after myocardial infarction.The studies attempt to provide a new theoretical basis for the application of DCs in ischemic diseases.Method:The mouse DC2.4 cell line was used as the subject.The object was divided into Normoxia and Hypoxia,in each subsets we set Control?Roscovitine,?infarction lysate,?Roscovitine+infarction lysate 4 groups.MTT assay was used to detect the survival of DC2.4 cells as to assure the concentration of Roscovitine;Flow cytometry detects the maturation of DC2.4 cells in every group?CD80,CD86,MHC II?and pinocytosis was detected by Transwell experiment under given conditions;RNA-seq technology detects differential genes in DC2.4 cells in every group;RT-qPCR was used to detect the expression of different genes in each group;and Western blot was used to detect protein expression of CDK5,FOXO1 and p-Ser249 FOXO1;Immunofluorescent detects FOXO1 distribution in every group under given conditions;ELISA kit was used to detect the secretion of IL-1??IFN-??IL-10 in supernatant in different groups of DC2.4 cells.Results:1)The effects of Roscovitine on DC2.4 cells proliferation capacity:MTT results show that cell activity has a significant decrease when the concentration of Roscovitine is 30?M while IC₅₀ of 72h was 19.49?M.Therefore,we chose the concentration 10?M as the effective concentration of Roscovitine.2)The effects of the infarction heart lysate on DC2.4 cell:Compared with the Control group,the area of DC2.4 cells increased significantly after giving50?g?ml^-11 infarction heart lysate protein solution individually,and the cell produced pseudo-foot,branched entrees,no longer growing in grape string,showing the characteristics of mature DCs.As a result,we chose 50?g?ml^-1as the concentration of the infarction heart lysate.3)The effects of Roscovitine on the maturation of DC2.4 cells induced by the infarction heart lysate protein:DC2.4 cells in the OGD group were expressed more in both CD80 and MHC II compared to the normoxia group.The expression of CD80,CD86 and MHC II was reduced in each group after giving Roscovitine,and the expression of CD80,CD86 and MHC II was significantly improved in the group which was added the infarction heart protein lysate?P<0.001?.4)The effects of Roscovitine on the pinocytosis of DC2.4 cells induced by infarction heart lysate protein:the ability of pinocytosis of DC2.4 cells decreased compared to the control group,while it decreased further after adding the infarction heart lysate.With the addition of Roscovitine,the pinocytosis was significantly increased?P<0.05,P<0.01?.5)The effects of Roscovitine on the ability of migration in DC2.4 cells induced by the infarction heart lysate:DC2.4 cells in the OGD group were significantly increased in the ability to migrate compared with normoxic group,while the migration of cells induced by the infarction heart lysate was also significantly enhanced.6)The effects of Roscovitine on the secretion of cytokines in DC2.4 cells:compared to the normoxic group,the secretion of IL-10 was slightly reduced and the secretion while IFN-?increased.After adding Roscovitine,the cells in the OGD group secrets more IL-10 than the control group.The expression of IL-1?and IFN-?increased significantly compared to the control group with the adding of infarction heart lysate.7)RNA-seq detects differential genes and mRNA validation results:ribosome protein?oxidation phosphorylation?lysosome-related processes?Tollage receptor pathways?FOXO signaling pathways?NK cell-mediated cytotoxicity and other related gene expression were found in a dynamic changes in the DC2.4 cells,and we choose several genes with significant differences,including CCL3?CCL4?CCL7?TNF?Vegfa?FOXO1?Cox6a2,a total of 7 genes for mRNA transcription levels detection.The results showed that the Vegfa?TNF?FOXO1 mRNA transcription was significantly decreased?P<0.01?compared with the normoxic group while the level of CCL3?CCL4?Cox6a2?CCL7 increased.Meanwhile,the expression of Vegfa?CCL3?CCL4?TNF?Cox6a2?FOXO1 was decreased with Roscovitine under oxygen-glucose deprivation.The expression of Vegfa?FOXO1 and Cox6a2was increased with infarction heart lysate under oxygen-glucose deprivation.8)The effects of Roscovitine on FOXO1 intracellular distribution and expression in infarcted heart lysate-induced DC2.4 cells:there was a slight decrease in FOXO1expression with Roscovitine.After Roscovitine was given,we were able to observe a significant decrease in the level of expression in p-FOXO1 and CDK5?P<0.05,P<0.01?,while the expression levels of p-FOXO and CDK5 increased in the group given the infarction heart lysate.In immunofluorescent results,compared with the control group,the expression of FOXO1 was strengthened in the nucleus.9)Changes in FOXO1 phosphorylation and intracellular distribution in DC2.4cells after interference with CDK5:Compared to DC2.4 that transfected with nt siRNA,the FOXO1 phosphorylation level of DC2.4 cells transfected with CDK5 siRNA was significantly reduced in CDK5 expression levels?P<0.01?.The above results are consistent with the trend of protein changes after giving Roscovitine.10)The effects of DC2.4 cell migration capacity after CDK5 interference:The migration capacity increased with infarction heart lysate compared with control group while the migration capacity decreased with CDK5 interference.11)The effects of DC2.4 cell pinocytosis with CDK5 interference:DC2.4 cells in the CDK5 siRNA group's ability of pinocytosis was increased compared with control group,while in groups with infarction heart lysate,the ability was significantly reduced,while interfering CDK5 were able to suppress the weakening of pinocytosis.12)The effects of DC2.4 cell maturation with CDK5 interference:DC 2.4 cell expression of CD80?CD86 and MHC II with CDK5 siRNA was weaker than control group?P<0.01?,while the expression of CD80?CD86?and MHC II was significantly higher?P<0.01?in groups that added infarction heart lysate.In addition,DC2.4 cells in the hypoxic group showed higher CD80 expression than the normoxic group.The trend of change in results in each group was consistent with the results of the addition of Roscovitine.Conclusions:1)Infarcted heart protein lysate can promote the maturation of DC2.4 cells,enhance their migration ability,and cytokine secretion.2)Roscovitine can reduce the ability of maturation and migration in DC2.4 cells induced by infarcted heart lysate protein with oxygen-glucose deprivation,and enhance the pinocytosis ability of DC2.4 cells.3)Roscovitine can increase IL-1?secretion and reduce IL-10 and IFN-?secretion in DC2.4 cells induced by infarcted heart lysate protein with oxygen-glucose deprivation.4)Roscovitine may reduce the level of FOXO1 phosphorylation by inhibiting CDK5,thereby regulating the function of DC2.4 cells.