Juglone Regulates The Mechanism Of Atherosclerosis In STZ-induced Diabetes ApoE^-/- Mice

Objective:In vivo（animal）experiments:To observe the effect of the Pin1 receptor inhibitor Juglone on thoracic aortic intima plaque and Pin1/FOXD3 expression in Streptozotocin（STZ）-induced diabetes apolipoprotein E knockout mice(Apo E^-/-).In vitro（cell）experiments:To investigate the effects of Pin1 receptor inhibitor Juglone on Pin1/FOXD3signaling pathway,cells proliferation and migration in PDGF-induced VSMCs（vascular smooth muscle cells,VSMCs）under high glucose（33 m M）condition.To explore the possible regulation mechanism of Juglone in improving diabetic Apo^-/-mice arteriosclerosis by the Pin1/FOXD3 signaling pathway.Methods:Animal grouping:40 male Apo E^-/-mice were randomly divided into four groups（10 mice per group）:Apo E^-/-group,STZ-Apo E^-/-group,STZ-Apo E^-/-+Juglone（0.5mg/kg/3d）group,STZ-Apo E^-/-+Juglone（1mg/kg/3d）group.Juglone is administered intraperitoneally once every 3days for 3 weeks.A model of arteriosclerosis in diabetic Apo E^-/-mice was established by intraperitoneal injection of streptozotocin（STZ:60 mg/kg）.Blood glucose level was measured by blood glucose test strip.Blood lipid level was determined by our laboratory.WB（Western blotting,WB）was used to measure the level of Pin1,FOXD3.HE（Hematoxylin eosin,HE）staining were used to measure the area of atherosclerotic plaque.Cell experiments:Primary VSMCs were isolated by tissue adherence method from thoracic aorta of rats.Immunofluorescence method was used to determineα-SMA.Proliferation of VSMCs was measured by BRDU assay.Transwell method was used to determine level of VSMCs migration.The levels of Pin1,FOXD3 of VSMCs were measured by WB（Western blotting,WB）.The effect of Juglone mediated Pin1/FOXD3 signaling pathway on proliferation and migration of VSMCs was observed by Transfection of specific FOXD3Si RNA（mall interfering RNA,Si RNA）.Results:Animal experiments:1)Compared with Apo E^-/-group,the level of bloodglucose,TC,HDL-C,LDL-C in STZ-Apo E^-/-group were significantly increased（P<0.05）,and there was no significant difference between two groups in TG（P>0.05）.Pin1 inhibitor Juglone had no significant effect on blood glucose,LDL-c,HDL-c,TC and TG levels in diabetic Apo E^-/-mice（P>0.05）;Oil red O staining showed:there were a small amount of red-like lipids in thoracic aortic intima of Apo E^-/-group;in STZ-Apo E^-/-group,thoracic aortic intima there were a large number of flaky red-stained lipids;in STZ-Apo E^-/-+Juglone（0.5mg/kg/3d）,a small amount of flaky red-stained lipid was found in thoracic aorta intima;STZ-Apo E^-/-+Juglone（1mg/kg/3d）only a small amount of red stains were found in the thoracic aorta intima.HE staining showed:plaque formation in the intima of the thoracic aorta in the Apo E^-/-group,thick hyperplasia around the plaque,formation of cavities（lipids）inside the plaque and uneven thickness in the arteries;STZ-Apo E^-/-group of thoracic aortic intima plaques increased,hyperplasia around the plaque,the bottom of the plaque irregular venous membrane,enlarged voids（lipids）inside the plaque;in STZ-Apo E^-/-+Juglone（0.5mg/kg/3d）and STZ-Apo E^-/-+Juglone（1mg/kg/3d）group chest aortic intima plaque is small,proliferative tissue around the plaque is reduced,there are small holes（lipids）inside the plaque and the medial membrane of the plaque is more regular.Juglone could reduce the area of plaque and inhibit the deposition of plaque internal lipid in the thoracic aorta of diabetic Apo E^-/-mice.In STZ-Apo E^-/-+Juglone（0.5mg/kg/3d）group thoracic aortic intima plaque area significantly decreased compared with that in STZ-Apo E^-/-group（P<0.05）.In STZ-Apo E^-/-+Juglone（1mg/kg/3d）group plaque area was further reduced（P<0.01）compared with Apo E^-/-group,and STZ-Apo E^-/-group.The expression of Pin1 and FOXD3 protein in the thoracic aorta were significantly increased（P<0.05）.Juglone decreased the expression of Pin1 and FOXD3 proteins in the aorta of diabetic Apo E^-/-mice in a dose-dependent manner.The expression of Pin1 and FOXD3 protein in STZ-Apo E^-/-+Juglone（0.5mg/kg/3d）group were significantly lower than that in STZ-Apo E^-/-group（P<0.05）.There were significant differences in the expression levels of Pin1 and FOXD3 between Juglone（1mg/kg/3d）group and STZ-Apo E^-/-+Juglone（0.5mg/kg/3d）group（P<0.05）.Cell experiments:PDGF（10 ng/ml）could promote the proliferation and migration of VSMCs in high glucose environment.Compared with Ctr group,the proliferation and migration of VSMCs in PDGF（10ng/ml）group were significantly increased（p<0.01）;Juglone（10-8M）decreased PDGF（10ng/ml）induced proliferation and migration of VSMCs cells.Compared with PDGF（10ng/ml）group,the proliferation and migration of VSMCs in PDGF（10ng/ml）+Juglone（10-8M）group were significantly decreased（p<0.01）.PDGF（10 ng/ml）promoted Pin1 andFOXD3 expression in VSMCs in high glucose（glucose 33 m M）.Compared with the Ctr group,the level of Pin1 and FOXD3 protein was significantly increased（p<0.05）.Pin1inhibitor Juglone could decrease the level of Pin1 and FOXD3 in VSMCs induced by PDGF（10ng/ml）in high glucose（33m M）.Compared with the PDGF（10 ng/ml）group,the level of Pin1 and FOXD3 in Juglone（10-8M）group was significantly decreased（p<0.05）,FOXD3-specific si RNA reduced FOXD3 gene expression,attenuate proliferation and migration of VSMCs induced by PDGF（10ng/ml）in high glucose condition.Conclusions:1.Juglone could reduce the lipid deposition and lipid plaque formation in the thoracic aorta of diabetic Apo E^-/-mice;2.Juglone could improve the imbalance of the Pin1/FOXD3signaling pathway in the thoracic aorta of diabetic Apo E^-/-mice;3.Juglone could inhibit the proliferation and migration of VSMCs induced by PDGF in high glucose condition,and reduce the activity of Pin1/FOXD3 signaling pathway;4.Juglone may improve diabetic arteriosclerosis by regulating Pin1/FOXD3 signaling pathway.