| Diabetes is a major health problem afflicting millions of people in the world. Individuals with Type 2 diabetes have markedly increased risks of both macrovascular diseases (including coronary heart disease and stroke) and microvascular diseases (including nephropathy and retinopathy). Retrospective and prospective studies have demonstrated that hyperhomocysteinemia, a risk factor for cardiovascular disease, plays an important role in the development of macro- and microvasculopathy of diabetes. It has been suggested that homocysteine derivative of homocysteine thiolactone (HcyT), formed in various cells in human, may provide a plausible chemical mechanism in explaining Hcy toxicity to the human vascular endothelium. It has been reported recently that homocysteine thiolactonase(HTase), that hydrolyzes HcyT to Hcy, could be in fact paraoxonase(PON). The human serum PON is an antioxidative enzyme in high density lipoprotein (HDL), which eliminates radicals in the circulation. The extent of HcyT formation in human umbilical vein endothelial cells (HUVECs) depends on levels of Hcy, Met, folate and HDL.Clinical researchs on the correlation of human plasma Hcy,HcyT concentrations and HTase/PON activity have been reported rarely, especially the influence of Hcy, HcyT concentration and HTase/PON activity on type 2 diabetes mellitus(DM) and diabetic vasculopathy. The aim of this study was to investigate the role of Hcy, HcyT and HTase/PON in the development of diabetic vascular diseases.The Study of the Relationship between Plasma Homocysteine,Homocysteine Thiolactone Concentrations or HTase/PON Activity and Type 2 Diabetes Mellitus and Its VasculopathyObjective: To investigate the changes of plasma Hcy, HcyT concentrations and HTase/PON activity in subjects with various glucose tolerance and to study the role of Hcy, HcyT and HTase/PON in the development of vascular complications including microanginopathy and macroanginopathy in patients with type 2 DM.Methods: A total 225 subjects were recruited in this study and among them were 40 healthy controls with normal glucose tolerance(NGT), 35 cases of impaired glucose tolerance(IGT), 30 newly diagnosed patients with type 2 DM and other patients with diabetic duration more than one year. Plasma Hey levels were measured by Polarization Immunoassay(FPIA), HcyT concentrations were monitored using high-performance liquid chromatography(HPLC) on a reversephase C18 column with ultraviolet detection, serum HTase/PON activity was assayed using spectrophotometer, serum C Reactive Proteine(CRP) concentrations were determined with Enzymatic Linked Immunity Sorb Assay(ELISA), and plasma folic acid and Vitamin B12 levels were measured with radioimmunoassay method. Results: The serum CRP levels were increased, while HTase/PON activity and folic acid and Vitamin B12 levels were decreased gradually with the worsening of glucose tolerance(P<0.05). Plasma Hey concentrations in IGT and DM groups were higher significantly compared with NGT group(P<0.05). Plasma HcyT concentrations were not elevated in subjects with different stages of glucose tolerance(P>0.05). Plasma Hey, HcyT and CRP concentrations in type 2 DM patients were significantly higher than that in healthy controls, while serum HTase/PON activity was significantly lower(P<0.01). The HTase/PON activity in patients with macroanginopathy tended to be lower, while Hey, HcyT, CRP levels to be higher(P<0.05), compared with patients without macrovasculopathy. With the progression of diabetic retinopathy(DR), the Hey, HcyT and CRP concentrations were increased gradually, while HTase/PON activity decreased significantly(P<0.05). With the increase of urinary albumin/ creatinine(Alb/Cr), HTase/PON activity was decreased significantly(P<0.05), and plasma Hey and HcyT concentrations were positively correlated with urinary excretion of Alb/Cr. HTase/PON activity was negatively correlated with CRP and Hey level (r=-0.202, P=0.041;r=-0.196, P=0.043 respectively). Plasma HcyT concentration was positively correlated with CRP and Hey levels(r= 0.220,P=0.053;r=0.211,P=0.057respectively), negatively correlated with HDL-ch (r=-0.223,P=0.037). Conclusions: Hey, HcyT and HTase/PON were involved in the development and progression of diabetic maro/micro vasculopathy. HcyT might provide a plausible chemical mechanism in explaining Hey toxicity to the human vascular endothelium. HTase/PON, also a component of HDLs, exhibitedantioxidant activity and decreased HTase/PON activity was associated with an increased risk of vasculopathy in patients with type 2 DM.Effect of Intervention Therapy of Methylcobalamin and Folic acid on Plasma Homocysteine, Homocysteine Thiolactone Concentration and HTase/PON Activity in Patients with Type 2 Diabetes MellitusObjective: To study the effect of methylcobalamin and folic acid treatment on plasma Hey, HcyT levels and HTase/PON activity in patients with Type 2 DM. Methods: 120 patients with type 2 DM were recruited and divided into four groups randomly: Patients in group 1 received no intervention therapy, group 2 were given folic acid orally(5mg/d), group3 were supplemented with methylcobalamin(500ug,im,qd), group 4 were treated with methylcobalamin (500ug,im,qd) in addition to folic acid(5mg/d). In all groups, plasma total Hey, HcyT, Vitamin B12, folic acid and HTase/PON activity were assayed at baseline and after 2- week treatment. Results: After providing folic acid and Vitamin B12, the Hey levels were decreased and serum HTase/PON activity was increased significantly in the three groups receiving intervention treatment(P<0.05). Plasma HcyT levels were decreased significantly by 13.3% only in patients treated with methylcobalamin and folic acid. After two weeks, Hey levels were decreased by 2.75%, 37.3%, 21.73% and 14% in group 1, group 4, group3 and group 2 respectively. A 2.72% increase in HTase/PON activity was seen in group 1, 8.03% and 3.41% were detected, when folic acid(5mg/d) or methylcobalamin(500ug im qd) were given respectively. HTase/PON activity was increased by 17.59% in the combination treatment group. The CRP concentration was decreased by 56.11%, 28.59% and 16.8% in group 4, group 2 and group 3. Routine treatment could also reduce serum CRP concentration by 12.81%. Conclusions: In patients with type 2 DM, methylcobalamin(500ug im qd) or folic acid(5mg/d) two-week treatment alone could decrease Hey and CRP levels significantly, concurrently increase HTase/PON activity obviously. It seemed that methylcobalamin was more effective than folic acid in reducing Hey levels, while folic acid intervention affected HTase/PON activity and CRP concentration more greatly compared with methylcobalamin. Methylcobalamin and folic acid combination therapy was likely to be much more effective. Folic acid might affect HTase/PON activity and CRPconcentration through its antioxidant ability.Homocysteine Thiolactone Induced the Apoptosis of Human Umbilical Vein Endothelial CellsObjectives: Hey is an independent risk factor for cardiovascular disease in humans. HcyT, one of the Hey metabolites in vivo, is toxic both in vivo and in vitro. The aim of this study was to investigate the effects of HcyT on apoptotic damage in HUVEC and to study the role of some antioxidants (N-acetylcysteine, vitamin E, and pyrrolidine dithiocarbamate) in the reduction of HcyT -induced apoptosis. Methods: HUVECs were cultured in DMEM medium supplemented with 20% heat inactivated fetal bovine serum and antibiotics (100 U/ml penicillin and 100 ug/ml streptomycin). Cell cultures were maintained in a humidified 5% CO2 atmosphere at 37°C. Cytotoxicity was determined by MTT assay. Flow cytometer and agarose electrophoresis of DNA fragmentation were applied to observe cell apotosis, and mRNA and protein expression of apoptotic genes were determined by RT-PCR and Western blot and intracellular reactive oxygen species levels(ROS) using DCFH-DA as the probe by flow cytometry. Results: HcyT induced endothelial cell apoptosis in a time- and concentration-dependent manner(range: 250-2000umol/L). ROS levels rose in response to increasing HcyT concentrations at 24-h incubation. The mRNA and protein expression of Caspase3 and Bax were up-regulated in concordance with the increased HcyT concentration. N-acetylcysteine, vitamin E, or pyrrolidine dithiocarbamat reduced HcyT-indued apoptosis, concurrently down-regulated Caspase3 and Bax expression, increased Bcl-2 expression(P<0.05). A significant decrease in intracellular ROS levels occurred simultaneously with the reduction of cell apoptosis by antioxidants.Conclusions: HcyT induced apoptoic damage mediated by increased cellular ROS and Caspase3 activation in HUVEC. The capacity of N-acetylcysteine, vitamin E and pyrrolidine dithiocarbamate to scavenge HcyT-induced cellular ROS was correlated well with their efficiency to protect against HcyT-promoted apoptotic damage. The protective effect of pyrrolidine dithiocarbamate on cell apoptosis indicated that HcyT -generated hydrogen peroxide might provoke cell apoptosis via activating nuclear factor -kappa binding protein.Conclusions1. Hey, HcyT and HTase/PON were involved in the development and progression of diabetic maro/micro vasculopathy. HcyT might provide a plausible chemical mechanism in explaining Hey toxicity to the human vascular endothelium. HTase/PON, also a component of HDLs, exhibited antioxidant activity and decreased HTase/PON activity was associated with an increased risk of vasculopathy in patients with type 2 DM.2. Methylcobalamin seems more effective than folic acid in reducing Hey levels, while folic acid intervention affected HTase/PON activity and CRP concentration more greatly than methylcobalamin. Methylcobalamin and folic acid combination therapy was likely to be much more effective. Folic acid might affect HTase/PON activity and CRP concentration through its antioxidant ability.3. HcyT induced apoptoic damage mediated by increased cellular ROS and Caspase 3 activation in HUVEC. The capacity of N-acetylcysteine, vitamin E and pyrrolidine dithiocarbamate to scavenge HcyT-induced cellular ROS was correlated well with their efficiency to protect against HcyT-promoted apoptotic damage. The protective effect of pyrrolidine dithiocarbamate on cell apoptosis indicated that HcyT-generated hydrogen peroxide might provoke cell apoptosis via activating nuclear factor -kappa binding protein.
|
PDF Full Text Request