The Role Of TFPI2-regulated PPARγ Axis In Diabetic Atherosclerosis

Purpose:Atherosclerosis(AS)is currently recognized as a kind of chronic inflammation.Atherosclerosis itself manifests as the damage of the intimal layer of the arterial wall and the accumulation of plaque.The long-term accumulation of plaque under the arterial endothelium leads to significant stenosis of the terminal blood vessels,accompanied by severe tissue hypoxia.As an independent risk factor,diabetes has been proved to accelerate the development of atherosclerosis,and diabetes is closely related to the aggravation of atherosclerosis.Macrophages have been found to exist at all stages of plaque formation,affecting plaque stability by regulating collagen production,matrix degrading enzyme release,and induction of smooth muscle cell apoptosis.Macrophage polarization is critical to the stability of atherosclerotic plaques in diabetes,and promoting reasonable changes in macrophage polarization phenotype can help reduce cardiovascular risk.In this study,we explored whether high glucose upregulated DNMT1 to inhibit the expression of TFPI2/PPARγ axis,thereby inducing the polarization of M1 macrophages,leading to AS plaque instability,and the effect of TFPI2 on the progression of diabetic atherosclerosis.Materials and methods:Peritoneal-derived macrophages were extracted from C57BL/6 mice and cultured in DMEM medium to construct an in vitro "cell model".Eight-week-old,male apo E-/-mice in the diabetic group were injected intraperitoneally with streptozotocin for 5 consecutive days,and mice in the non-diabetic group were injected intraperitoneally with the same volume of citrate buffer,and fed with high fat and high cholesterol for16 days.weeks to establish a model of diabetic atherosclerosis.Extract total protein and total RNA from cell models and animal models.The m RNA expression of DNMT1 and TFPI2 was detected by real-time quantitative PCR.The expressions of INOS,Arg-1,DNMT1,TFPI2 and PPARγ were detected by Western blot.The expression of macrophage polarized phenotype was detected by flow cytometry.Oil red O lipid staining,macrophage markers,smooth muscle cells,TFPI2,and Sirius red staining were used for immunohistochemical staining to quantify the collagen content and further calculate the plaque vulnerability.Result:1.High glucose environment promotes further polarization of macrophages Compared with the atherosclerosis control group(NC group)mice,the protein levels of INOS and DNMT1 in the aortic tissue of the diabetic atherosclerosis model group mice(DM group)were significantly increased,and the Arg-1 level was also slightly increased,while the levels of TFPI2 and PPARγ decreased significantly.Compared with the NC group,the m RNA level of DNMT1 in the DM group was significantly increased,while the m RNA level of TFPI2 was significantly decreased.2.High glucose stimulation inhibits the expression of TFPI2 by up-regulating DNMT1The peritoneal-derived macrophages were divided into normal glucose control group(NC),hyperosmotic control group(OC),high glucose group(HG)and high glucose+DNMT1 inhibitor group(HG+5-Aza).There was no significant difference in the expression levels of INOS,Arg-1,DNMT1,TFPI2 and PPARγ between the NC group and the OC group.Compared with the NC group,the levels of INOS and DNMT1 in the HG group were significantly increased,Arg-1 was slightly increased,while the expressions of TFPI2 and PPARγ were significantly down-regulated.Compared with the HG group,the levels of INOS and DNMT1 were significantly down-regulated,the levels of Arg-1 were significantly increased,and the expressions of TFPI2 and PPARγ were significantly up-regulated in the HG+5-Aza group.Compared with the NC group,there was no significant difference in the m RNA expression levels of DNMT1 and TFPI2 in the OC group.Compared with the NC group,the m RNA level of DNMT1 was significantly increased and the m RNA level of TFPI2 was significantly suppressed in the HG group.Compared with the HG group,the m RNA level of DNMT1 was significantly suppressed and the m RNA level of TFPI2 was significantly up-regulated in the HG-5Aza group.3.TFPI2 enhances aortic plaque stability in diabetic atherosclerotic mice Eight-week-old,male apo E-/-mice were randomly divided into diabetic group(DM),non-diabetic group(NC)and diabetic TFPI2 overexpression group(DM+TFPI2).After the model was successfully established,the aortic root was taken for calculation of plaque vulnerability index and immunohistochemical staining for TFPI2 expression,and the aortic tissue was retained to extract total protein and RNA. Compared with the NC group,the plaque vulnerability index in the DM group was significantly increased,and the expression level of TFPI2 was significantly decreased.Compared with the DM group,the plate vulnerability index of the DM+TFPI2 group decreased significantly.Compared with the NC group,the expression of INOS and Arg-1 in the DM group were significantly increased,while the expressions of TFPI2 and PPARγ were significantly inhibited.Compared with the DM group,the expression of INOS in the DM+TFPI2 group was significantly inhibited,the expression of Arg-1 was significantly increased,and the expressions of TFPI2 and PPARγ were also significantly increased.4.TFPI2 regulates the polarization of macrophages to M2RAW cells were divided into NC,OC,HG and HG+TFPI2 groups.Compared with the NC group,there was no significant difference in the m RNA expression levels of DNMT1 and TFPI2 in the OC group.Compared with the NC group,the m RNA level of TFPI2 was significantly suppressed in the HG group.Compared with the HG group,the m RNA level of TFPI2 was significantly up-regulated in the HG+TFPI2 group.Flow cytometry showed that the phenotypes of M1 and M2 cells in the OC group had no significant changes compared with those in the NC group.Compared with the NC group,the M1 macrophage phenotype was significantly elevated in the HG group.Compared with the HG group,the phenotype of M1 macrophages in the HG+TFPI2 group was significantly decreased,and the phenotype of M2 macrophages was significantly increased.There was no significant difference in the expression levels of INOS,Arg-1,TFPI2 and PPARγ between the NC group and the OC group. Compared with the NC group,the INOS level in the HG group was significantly increased,Arg-1 was slightly increased,while the expressions of TFPI2 and PPARγ were significantly down-regulated.Compared with the HG group,the level of INOS in the HG+TFPI2 group was significantly down-regulated,and the level of Arg-1 was significantly increased.In conclusion:(1)High-glucose environment stimulation inhibits the expression of TFPI2/PPARγ by up-regulating the expression of DNMT1,and promotes the polarization of macrophages to the M1 type.(2)The plaque stability of diabetic atherosclerotic mice is significantly decreased,and the overexpression of TFPI2 can effectively protect the plaque stability.(3)Overexpression of TFPI2 can promote the polarization of macrophages to M2 and inhibit the polarization of macrophages to M1.