Effect Of TFPIct32 On Migration Of Macrophage-derived Foam Celland Its Mechanism

Experimental background:Atherosclerosis?As?,a chronic inflammatory process of arteries,which is characterized by the deposition of modified low-density lipoproteins on the walls of blood vessels and leading to the formation of atherosclerotic plaques.It is the main cause of acute cardiovascular events such as angina pectoris and myocardial infarction.Despite great progress in the clinical diagnosis and treatment of atherosclerosis have been achieved,the complex process of plaque formation poses great challenges for effective diagnosis and treatment.Specific diagnostic methods and efficient treatment methods have become the research hotspot of atherosclerosis.A large number of studies have shown that macrophages,an important part of plaque,are important regulatory target that affecting plaque development.After phagocytosing a large amount of modified low-density lipoprotein,macrophages are transformed into macrophage-derived foam cells.The lower migration capacity of foam cells made it retented under the endothelial cells,and resulting in the formation of atherosclerotic plaques and necrotic core.Therefore,promoting foam cell migration helps to inhibit plaque progression.Tissue factor pathway inhibitor?TFPI?is a physiological inhibitor of tissue factor-mediated exogenous coagulation pathway.Studies have confirmed that TFPI inhibits the development of plaques by inhibiting the activation of vascular endothelial cells,the proliferation and migration of vascular smooth muscle cells,and the expression of proinflammatory factors.Studies have shown that TFPIct32 is a peptide of C-terminus of TFPI,has an anti-atherosclerotic effect similar to TFPI,but its specific mechanism of action is unknown.The preliminary experimental results of our group showed that after treatment with TFPIct32,the area of plaque at the aortic sinus of ApoE^-/-mice was reduced,and the number of macrophages in the plaque decreased.The mechanism may be related to the migration of macrophages.The excessive accumulation of foam macrophages will drive the development of atherosclerosis.The increased migration of foam cells in the plaque helps the plaque to subside.Combining previous experimental results and literature reports,this study intends to further observe the effect of TFPIct32 on the migration ability of macrophage-derived foam cells in vitro and explore its mechanism of action.Aims:By establishing a foam cell model,the effects of TFPIct32 on the behavior of foam cells were observed,and the mechanism of TFPIct32 on foam cells was initially discussed.Methods:Raw264.7 murine macrophages were used in this experiment.CCK8 test was used to observe the effect of TFPIct32 on the survival rate of macrophage cells.The experiment is divided into three groups:the control group?Control?,the negative control peptide group?TFPIs?and the experimental group?TFPIct32?.First,a foam cell model was constructed,and cells were treated with 50?g/ml oxidized low-density lipoprotein?oxLDL?for 24 hours.Different treatment groups were added with pure medium,TFPIct32?12.5nM?and the same molar negative control peptide--TFPI C terminal random arrangement of amino acid sequence peptides?TFPIs?.After 24 hours,the Transwell test and phalloidin staining were used to detect the effect of TFPIct32 on foam cell migration.Western blot was used to detect the protein expression level of CCR7,ICAM-1,VCAM-1.qPCR was used to detect the mRNA levels of CCR7,ICAM-1,VCAM-1,IL-1?,IL-6,MCP-1.MIF and TNF?.In order to determine whether the effect of TFPIct32 on foam cell migration is related to CCR7,siRNA transfection technology was used to establish a CCR7 knockdown model.The experimental grouping is as follows:the control group,the TFPIct32 group,the TFPIct32+siCCR7group.Transwell test and phalloidin staining were conducted to observe whether the effect of TFPIct32 on foam cell migration is affected after CCR7 knockdown.Experimental results:1.The results of CCK8 experiment showed that TFPIct32 had no effect on the survival rate of macrophages at a concentration from 0 to12.5nM for 24 hours,but at a concentration of 25nM,it caused the survival rate of cells to decrease by 19.45%?P<0.01?;After treatment of macrophages with 12.5nM of TFPIct32,the survival rate of macrophages was not significantly inhibited from 0 to 24 hours,but after 48 hours,the cell survival rate of macrophages decreased by 3.64%?P<0.01?.It shows that TFPIct32 has no effect on cell survival rate within a certain concentration and time range.2.The foam cell model is successfully constructed.Macrophages were treated with 50?g/ml oxLDL for 24h,and oil red O staining was performed on the blank group and oxLDL treated group.Under the light microscope,there was no red-stained particles in the macrophages of the blank treatment group,and there were more red-stained particles in the cytoplasm of the oxLDL treatment group,which was consistent with the characteristics of foam cells,which proved that the foam cell model was successfully constructed.3.The results of Transwell experiment showed that compared with the control group,there was no significant difference in the number of cells passing through the chamber in the TFPIs group;the number of cells passing through the chamber in the TFPIct32 group increased by146.94%?P<0.01?.The results of phalloidin staining experiment showed that the fluorescence intensity of cells treated with TFPIs was not significantly different from that of the control group;the fluorescence intensity of cells in the TFPIct32 group increased by 49.05%?P<0.05?.It shows that TFPIct32 can promote the migration of foam cells.4.Western blot?WB?and quantitative real-time PCR?Quantitative Real-time-PCR,qPCR?experiments showed that the expression of CCR7mRNA and protein in the TFPIs group was not significantly different from the control group.Compared with the control group,the mRNA and protein expressions of CCR7 in TFPIct32 group were up-regulated by397.21%and 72.28%?P<0.01?.In order to clarify the role of CCR7 in the process of TFPIct32 promoting foam cell migration,the transient transfection technology of siRNA was used to establish the CCR7knockdown model.The number of cells passing through the chamber in TFPIct32+siCCR7 treatment group was relatively reduced by26.4%?P<0.01?than the TFPIct32 group.The results of the phalloidin staining experiment showed that the fluorescence intensity staining of the TFPIct32+siCCR7 treatment group was reduced by 28.19%?P<0.01?when compared with the TFPIct32 group.This indicates that TFPIct32promotes the migration of foam cells by up-regulating CCR7.5.The expression of adhesion molecules,MIF and MCP-1 in the TFPIs group was not significantly different from that in the control group.Compared with the control group,the protein expression of ICAM-1 and VCAM-1 in the TFPIct32 treatment group decreased by 19.97%and46.42%?both P<0.01?,and the mRNA expression of ICAM-1 and VCAM-1 decreased by 34.86%?P<0.01?and 88.85%.?P<0.01?.Compared with the control group,the mRNA expression levels of MIF and MCP-1 in the TFPIct32 treatment group decreased by 14.20%and29.02%?both P<0.01?.It is suggested that TFPIct32 down-regulates the expression of adhesion molecules,MIF and MCP-1.6.The expression of inflammatory factors TNF?,IL-1?,IL-6 in the TFPIs group was not significantly different from that in the control group.Compared with the control group,the levels of TNF?,IL-1?,and IL-6 in the TFPIct32 treatment group decreased by 53.99%?P<0.01?,21.84%?P<0.05?,and 47.79%?P<0.01?.It is suggested that TFPIct32down-regulates the expression of inflammatory factors,such as TNF?,IL-1?,IL-6.Conclusion:1.TFPIct32 promotes foam cell migration by up-regulating the expression of chemokine receptor CCR7 on the surface of foam cells.2.TFPIct32 inhibits the expression of VCAM-1,ICAM-1,MIF,MCP-1 and inflammatory factors in foam cells.