| Objective: To observe the effects of vitamin D on the structure and function of thoracic aorta,as well as the expressions of prolyl isomerase-1(Pin1),pro-oxidant adaptor p66 Shc,nuclear factor-kappa B p65 subunit(NF-?B p65),and endothelial nitric oxide synthase(e NOS)in aortic tissue of streptozotocin-induced diabetic mice;to further test the effects of vitamin D on apoptosis,mitochondrial translocation of p66 Shc,mitochondrial oxidative stress,nuclear translocation of NF-?B p65,and nitric oxide(NO)generation in high glucose-cultured human umbilical vein endothelial cells(HUVECs),and explore the potential mechanisms implicated in the protection of vitamin D treatment against diabetes vasculopathy.Methods: Animal experiments: 10 male C57BL/6 mice were served as Control group(n = 10),30 male diabetic mice induced by intraperitoneal injection of STZ in C57BL/6 mice were randomly assigned to DM group(STZ-C57BL/6 + vehicle,n=10),Vit D group(STZ-C57BL/6 + calcitriol 10?g/kg/d,n=10)or Juglone group(STZ-C57BL/6 + Pin1 inhibitor Juglone 1mg/kg/d,n=10).Calcitriol was administrated by gavage 2 times per week and Juglone by intraperitoneal injection 3 times per week for eight weeks.Fasting plasma glucose levels were measured by glucose oxidase method;Thoracic aorta histomorphology was determined using Hematoxylin-Esosin(H-E)staining;Organ chamber experiments were performed in aortic rings to assess endothelium-dependent and endothelium-independent relaxations to acetylcholine(ACH 10-9 to 10-5 mol/L)and sodium nitroprusside(SNP 10-9 to 10-5 mol/L),respectively;Pin1 activity in supernatant of aortic homogenate was assessed by using a commercially available kit;Protein expression levels of Pin1,p66 Shc,p-p66 Shc,caspase3,NF-?B p65 and e NOS in thoracic aorta were examined by Western blotting;Superoxide dismutase(SOD)activity in serum was measured by chemiluminescence assay and malondialdehyde(MDA)by thiobarbituric acid(TBA)method;Serum levels of IL-1?,IL-6 and NO were measured by ELISA method.Cell experiments: Human umbilical vein endothelial cells(HUVECs)were isolated by type ? collagenase and identified by immunocytochemistry,immunofluorescence and flow cytometry.HUVECs were treated with high glucose(33 m M glucose)in the presence or absence of vitamin D or Juglone;Cell apoptosis rate were measured by flow cytometry and TUNEL staining method;Intracellular reactive oxygen species(ROS)levels were examined by flow cytometry and fluorescence microscopy;NF-?B p65 nuclear translocation was determined by immunocytochemistry;Protein expression levels of Pin1,p66 Shc,p-p66 Shc,mitochondria to cytoplasm ratio of p66 Shc,caspase3,nuclear to cytoplasm ratio of NF-?B p65 and e NOS in HUVECs were measured by Western blotting;Pin1 activity in HUVECs lysate was assessed by using a commercially available kit;Knockdown of VDR by si RNA was used to evaluated the role of VDR in the regulatory effects of vitamin D on Pin1 protein expression and activity in HUVECs under hyperglycemia conditions.Results: Animal experiments: 1)Compared with control mice,the fasting plasma glucose levels in vehicle-treated diabetic mice were significantly elevated(P < 0.01),and Pin1 inhibitor Juglone treatment significantly reduced the plasma glucose levels as compared with vehicle-treated diabetic mice(P < 0.05).However,no significant difference of plasma glucose levels were observed between vitamin D-treated and vehicle-treated diabetic mice;2)Vascular histomorphology determined by H-E staining showed that the tunica intima of aorta in control mice was smooth and continuous,and the thickness of tunica media was uniform in which smooth muscle cells were arranged regularly;However,the tunica intima of aorta in vehicle-treated diabetic mice was distinctly uneven with lining continuity interrupted,and the thickness of tunica media was nonuniform with fewer smooth muscle cells and irregular arrangement;Both Vitamin D and Juglone treatments effectively alleviated the impaired structure observed in the vehicle-treated diabetic mice;3)Compared with control mice,the endothelium-dependent relaxation to ACH(10-9~10-5 M)and endothelium-independent relaxation to SNP(10-9~10-5 M)of isolated thoracic aorta from vehicle-treated diabetic mice were markedly blunted;When the concentration of ACH and SNP reached 10-7 M and 10-8 M,respectively,the relaxation potential showed significant difference between control mice and vehicle-treated diabetic mice(P < 0.05);Both the endothelium-dependent and endothelium-dependent relaxations were significantly and equally improved by Vit D and Juglone treatment compared with vehicle-treated diabetic mice(P < 0.05);4)Protein expression and activity of Pin1,as well as protein expression of p-p66 Shc,caspase3,and NF-?B p65 in thoracic aorta of vehicle-treated diabetic mice were significantly higher than those of control mice(all P < 0.01),while e NOS protein expression was significantly lower than those of control mice(P < 0.01);Vit D and Juglone treatment markedly reduced the protein expression and activity of Pin1,as well as the protein expression of p-p66 Shc,caspase3,and NF-?B p65 in thoracic aorta compared with those of vehicle-treated diabetic mice(P < 0.01),while significantly increased the e NOS protein expression(P < 0.01);Protein expression and activity of Pin1,as well as protein expression of e NOS were significantly higher in Vitamin D-treated diabetic mice than those of Juglone-treated diabetic mice(P < 0.01);5)Serum levels of Pin1,MDA,IL-1?,and IL-6 were significantly higher(P < 0.01),while SOD and NO were significantly lower in vehicle-treated diabetic mice compared with control mice(P < 0.01);Compared with vehicle-treated diabetic mice,both Vitamin D and Juglone treatment reduced the serum levels of Pin1,MDA,IL-1?,and IL-6(P < 0.01),while increased SOD and NO(P < 0.01);Serum levels of Pin1 and SOD were significantly higher,while MDA were significantly lower in Vitamin D-treated diabetic mice than Juglone-treated diabetic mice(P < 0.01).Cell experiments: 1)Vitamin D suppressed HUVECs apoptosis and intracellular ROS generation induced by high glucose in a dose-dependent(10-8~10-6 M)manner(P < 0.01);2)High glucose increased Pin1 protein expression and activity in HUVECs in a time-dependent(24 h to 72 h)manner,which were corrected by vitamin D in a dose-dependent(10-8~10-6 M)manner(P < 0.01);3)Vitamin D inhibited phosphorylation and mitochondrial translocation of p66 Shc and caspase3 expression induced by high glucose,reduced nuclear translocation of NF-?B p65,and increased expression of e NOS and NO generation(P < 0.01);4)Knockdown of VDR by si RNA abolished the inhibitory effects of vitamin D on high glucose-induced upregulation of Pin1 protein expression and activity.Conclusions: 1)Vitamin D alleviated thoracic aorta structure damage and improved vasodilatation function in STZ-induced diabetic mice,which were comparable to effects of Pin1 inhibitor Juglone;2)Vitamin D inhibited high glucose-induced upregulation of Pin1 protein expression and activity,as well as p66Shc-mediated mitochondrial oxidative stress and NF-?B-mediated inflammation,and consequently attenuated HUVECs apoptosis;3)The inhibitory effects of Vitamin D on Pin1 protein expression and activity depends on the activation of VDR receptor;4)Although the inhibitory effects of vitamin D on Pin1 protein expression and activity were significantly weaker than those of Pin1 inhibitor Juglone,its overall antioxidant effects were still more powerful than those of Juglone,suggesting possible multifaceted antioxidant effects of vitamin D;5)Juglone reduced blood glucose in diabetes mice,and produced equal amount of NO,although its enhancing effect on e NOS protein expression was weaker compared with vitamin D,which suggested a complex regulatory role of Pin1 inhibitors on e NOS activity. |
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