Role And Mechanism Of Pin1/BRD4 Pathway In Diabetic Atherosclerosis

Objective:To observe the effect of Pin1 inhibitor（Juglone）on diabetic atherosclerosis and Pin1/BRD4 expression of aorta in streptozotoci（STZ）-induced diabetic ApoE^-/-mice,as well as the effect of Juglone on cell proliferation and migration and Pin1/BRD4expression in PDGF-BB cultured vascular smooth muscle cells（VSMC）.To explore the potential mechanism of Pin1/BRD4 in diabetic atherosclerosis.Methods:Animal experiments:Diabetic atherosclerosis model was established with intraperitoneal injection of STZ.Mice were divided into 5 groups（10 mice per group）:C57BL/6 group,ApoE^-/-group,DM group(STZ-ApoE^-/-),Juglone group(STZ-ApoE^-/-+Pin1 inhibitor Juglone)and JQ1 group(STZ-ApoE^-/-+BRD4 inhibitor JQ1).Fasting plasma glucose levels were measured with Glucose Oxidase Method.Blood lipid levels were measured with automatic biochemical analyzer.Enzyme Linked Immunosorbent Assay（ELISA）was performed to measure PDGF-BB levels in serum and tissue homogenate.Hematoxylin-eosin（HE）staining were used to observe thoracic aorta atherosclerotic plaque.Pin1,BRD4,NF-κB p65,cyclin D1 and MMP-9expression were measured by Western blotting.Cell experiments:VSMC was cultured with thoracic aortic tissue sticking method and identified by morphology and immunocytochemistry.VSMC cells were divided into different treatment groups:control group,PDGF-BB group,Juglone group（PDGF-BB+Juglone）and JQ1（PDGF-BB+JQ1）group.Cell proliferation was measured by MTT assay.Cell migration was examined by migration assay and wound healing assay.Pin1,BRD4,NF-κB p65,cyclin D1 and MMP-9 expression were measured by Western blotting.Overexpression of Pin1 with plasmid DNA transfection was used to observe the effect of Pin1 on BRD4 expression and VSMC cells proliferation and migration.Results:Animal experiments:1)Compared with ApoE^-/-group,the plasma glucose level of DM group was significantly higher.Compared with the DM group,the plasma glucose level was significantly lower in the Juglone group.There was no difference in plasma glucose levels between the DM group and JQ1 group.2)Compared with C57BL/6 group,the levels of LDL-c,TC and TG in ApoE^-/-group were significantly higher and the HDL-c level was significantly lower.The levels of LDL-c and TC in DM group were significantly higher than those of ApoE^-/-group,and there was no significant difference for HDL-c and TG levels.The levels of LDL-c and TC were significantly lower in Juglone group than in DM group,while the HDL-c and TG levels were significantly higher.Compared with DM group,the LDL-c and TC levels were significantly lower in JQ1 group,and there was no significant difference for HDL-c and TG levels.3)Compared with C57BL/6 group,PDGF-BB level in thoracic aorta tissue was significantly higher in ApoE^-/-group,and PDGF-BB level was further increased in DM group.Both Juglone and JQ1 decreased PDGF-BB levels in thoracic aorta of DM ApoE^-/-mice.Compared with DM group,PDGF-BB levels in Juglone group and JQ1group were significantly decreased.4)Vascular histomorphology determined by H-E staining showed that no plaques were found in the C57BL/6 group and the thickness of the media was uniform.Lipid deposition was observed in the blood vessels of the ApoE^-/-group and the thickness of the media was not uniform,and at the bottom of the lipid deposition,only a small amount of proliferation was seen.In the DM group,large plaques were seen,and the mesangial hyperplasia at the bottom of the plaques was irregular.In the Juglone group and the JQ1 group,only lipid deposition and a small amount of proliferation in the media were seen.The area of plaque in DM group was significantly higher than that in ApoE^-/-group.Both Juglone and JQ1 decreased the area of thoracic aortic plaque in STZ-induced diabetic ApoE^-/-mice.5)Compared with C57BL/6 group,Pin1,BRD4,NF-κB p65,cyclin D1 and MMP-9 expression in thoracic aorta in ApoE^-/-group increased,and the expression was further increased in DM group.Compared with DM group,Pin1 expression in Juglone group was significantly reduced,and BRD4 expression was also significantly reduced.However,BRD4 expression in JQ1 group was significantly decreased,but there was no significant difference in Pin1expression.Compared with DM group,NF-κB p65,cyclin D1 and MMP-9 expression in JQ1 group was significantly lower.Cell experiments:1)Compared with the control group,Pin1 expression was significantly increased in PDGF-BB group,and BRD4expression was also increased significantly.Compared with PDGF-BB group,Pin1expression was significantly decreased and BRD4 expression was also decreased significantly in Juglone group.Compared with PDGF-BB group,BRD4 expression in JQ1 group was significantly decreased,but there was no significant difference in the Pin1 expression.2)Compared with the control group,NF-κB p65,cyclin D1 and MMP-9 expression in PDGF-BB group was increased.JQ1 inhibited PDGF-BB-induced NF-κB p65,cyclin D1 and MMP-9 expression,as well as VSMC cells proliferation and migration.3)Up-regulation of Pin1 expression with the transfected DNA plasmid impaired the inhibitory effect of Juglone on of PDGF-BB-induced BRD4 expression and impaired the inhibitory effect of Juglone on PDGF-BB-induced VSMC cells proliferation and migration.Conclusions:1)Both inhibitions of Pin1 and BRD4 attenuate diabetic atherosclerosis.2)Both inhibitions of Pin1 and BRD4 reduce the level of PDGF-BB,NF-κB p65,cyclin D1 and MMP-9 in thoracic aorta of DM ApoE-/-mice.3)Inhibition of Pin1 reduces PDGF-BB-induced BRD4,NF-κB p65,cyclin D1 and MMP-9 expression.4)Inhibition of Pin1 reduces PDGF-BB-induced VSMC cells proliferation and migration.5)Inhibition of Pin1 reduces smooth muscle proliferation and migration thereby improving diabetic atherosclerosis,possibly through regulation of the BRD4 pathway.