| Backgroud and objectives:In recent years,many investigations have demonstrated that vascular endothelial cells(VEC) injury and endothelium dysfunction(ED) is the initiating and early event in the development of atherosclerosis(AS).So it's very important to study the early changes of endothelial function for the pathogenesy of AS.At the same time,as more is learned about the natural course of the development of AS,it is clear that AS is a pathological process that progressed slowly but steadily,in which the damage of endothelium cellular integrity and function begins in childhood,while becomes symptomatic during the adults.Diabetes mellitus(DM) in childhood mostly belongs to insulin-dependent diabetes mellitus(IDDM), which is easily complicated diabetes vasculopathy because of its early onset and long course.Of the total,diabetic macroangiopathy is the main reason of death and diasability for diabetes patients,which pathological change is vescular AS in essence.Equally,early vascular ED in children with diabetes also is the early key step for priming AS process. While,injury of VEC and ED appear reversible pathological change.Therefore,it is crucial to detect and prevent vascular endothelial dysfunction earlier for the prevention of diabetic vasculopathy and improve the live quality of young diabetes patients.In DM,hyperglycaemia and dyslipidemia increased oxidative stress and decreased antioxidative activity of body or partial tissue,which is viewed the main cause of vascular endothelium dysfunction in diabetic conditions.As one important product of oxidative stress,oxidized low-denstity lipoprotein(oxLDL) was a key factor to cause endothelium dysfunction.The endothelial phagocytosis of oxLDL damages the function of VEC by decreasing vascular diastolic function,inducing thrombogenesis and inflammatory reaction. On the other hand,more and more studies have confirmed that endothelial cells internalize and degrade the oxidized LDL mainly by its specificially endothelial cell surface receptor, lectin-like oxLDL receptor-1(LOX-1).The binding of oxLDL to LOX-1 can activate nuclear factor-KappaB(NF-κB),then further induce the expression of intercellular adhesion molecule-1(ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) as well as monocyte chemoattractant protein-1(MCP-1) in transcriptional levels,which facilitate the adhesion of neutrophil and monocytes to endothelium,and affect the product of endothelium-derived NO and diminish endothelium-dependent vasorelaxation,initiating atherosclerosis.The previous study have confired that oxLDL/LOX-1 was highly expressed in AS rabbits induced by high-cholesterol die.But it was very few about study in vivo on the expression and role as well as its related signal pathway of oxLDL/LOX-1 in early vascular endothelial dysfunction of diabetes.LOX-1 is a single transmembrane protein expressed on endothelial cells,and it belongs to the C-type lectin family in structure,which includes a NF-κBgene-like sequence and a AP-1 binding site in its 5'domain.In the physiological conditions,the basal expression of LOX-1 in endothelial cells is very low,while it can be induced in vitro by ox-LDL and other oxidant species.More importantly,LOX-1 expression is highly upregulated in cultured endothelial cells by its ligand,oxidized LDL,while this upregulation of LOX-1 was blocked by LOX-1 antibody and antioxidant.Recent studies in vivo or vitro showed that many drugs such as stains and ACEI(angiotensin converting enzyme inhibitor) may prevent the development of AS by regulating oxLDL/LOX-1 system,and this effect of regulation relys on their intracellular antioxidant abilities rather than the original hypolipidemic or hypotensive activities.All these results indicate that redox-sensitive regulation of oxLDL/LOX-1 actually occurs in vivo,which gives us a deep insight into the pathogenesis of some cardiovascular diseases and provides a potential selective therapeutic approach for clinical physicians. During the development of AS,the early pathological changes including endothelium dysfunction before lipids streak in young patients are reversible,and early intervention can reversed the tendency of development into fibrous or atherosclerotic plaque.While the drug choice of fit intervention focus on it's availability and safety.At present,chronic administration of insulin to control hyperglycaemia is still the basically clinical treatment for IDDM.At the same time,recent studies showed that anti-oxidative treatment is also an effective therapy to obtain a better control of diabetic complication.Taurine,also called 2-aminoethane sulfonic acid,acts as an important endogenous antioxidant,and its content is abundant in organs or tissues of high excitability,such as nerves,muscle,crystalline lens, glomerulus and cardiovascular system.Taurine exhibits a diversity of actions such as an anti-oxidative,anti-inflammatory,antiplatelet activity,osmoregulator and intracellular Ca2+ regulator et al.Recent studies confirmed that the taurine levels of plasma and tissues were dereased in type 1 diabetes conditions,and taurine- supplemented diet had beneficial effect to ameliorate diabetic cardiomyopathy,diabetic polyneuropathy and retinopathy.Also,in vitro it had been manifested that taurine could protect against endothelial dysfunction induced by oxLDL.However,the exact molecule mechanisms of these actions are not very clear.As an endogenous antioxidant,the intervention effects of taurine in vivo on diabetes-induced early vascular dysfunction and the possible mechamism involved oxLDL/LOX-1 are worthwhile to study.In the present study,type 1 diabetes model was induced by STZ and given intervetion of insulin or taurine at once after the onset of diabetes.Then,the damages of endothelial ultramicrostructure and function,the expression of LOX-1,NF-κBp65 and ICAM-1 on aorta as well as the circulating levels of oxLDL were examed,aiming to find the effects of oxLDL/LOX-1 and NF-κB signal pathway on early vascular endothelium dysfunction as well as the endothelial protective effects of taurine and the possible mechanism in type 1 diabetes.Methods:1.The establishment of type 1 diabetes model:8-week-aged male Wistar rats (180-200g) were divided randomly into control group(NC,n=16) and diabetic models group.Diabetic models were induced by a single intraperitoneal injection of STZ 60 mg/kg and blood glucose level was measured after 72 hours of the injection.Rats with blood glucose(BG)≥16.7mmol/L and urine glucose≥3+ were indicated as diabetes. Then randomly picked 40 diabetic rats and subdivided into three groups:diabetes group (DM group,n=20),insulin-treated diabetes group(In group,n=10) and taurine-treated diabetes group(TAU group,n=10).Insulin-treated rats were injected subcutaneously with long acting insulin once daily at 17 o 'clock and then measured the non-fasting BG at about 9 o'clock of the second day,the dose of insulin(3～10unit/rat) was adjusted for individual animals to keep the non-fasting BG between 3.9～10mmol/L.While the rats in TAU group were given freely drinking water containing 1%taurine.The whole study lasted for 6 weeks.8 rats were randomly picked and killed in NC and DM rats at the end of week 2 and week 6,while 8 rats of other groups were killed at the end of week 6 respectively,then blood and tissues were collected.2.Measurement Methods:①Body weight and BG:The body weight and fasting BG of every rat were measured every week during the study.BG was determined by blood glucose meter OneTouch Ultra.②Determination of circulating indexes:the levels of total cholesterol(TC), low-density lipoprotein(LDL),high-density lipoprotein(HDL) and triglyceride(TG) in serum were determined by Olympus AU-1000 biochemical instrument.The levels of serum oxLDL and sICAM-1 were measured by enzyme-labeled immunosorbent assay(ELISA) kit, and the level of serum nitric oxide(NO) as well as plasma endothelin(ET) were determined by nitrate reductase indirect assay and radioimmunity homogeneous phase competition assay respectively.③Functional assessment of isolated thoracic aortas:After the rats were killed and blood samples were collected,the thoracic aortas were rapidly isolated at once.Segments of the suprema aortas were rapidly cut into rings of 3～5mm length,and the endothelium-dependent vasodilator responses induced by acetylcholine(Ach) were determined.Then the percentage of accumulative concentration-vasodilatator response to aeetylcholine(1nM～100μM) were calculated.Rmax was the Ach-induced maximum relaxation of arteries,and EC50 represents the Ach concentration needed to induce 50%maximum relaxation calculated by linear regression. ④Ultrastructure observation of vascular endothelium:Ultrastructure changes of aorta endothelium were observed by double staining transmission electron microscope.⑤Determination of LOX-1,NF-κBp65 and ICAM-1 protein expression on aortic tissues:Expression of LOX-1 and NF-κBp65 as well as ICAM-1 protein on aortic endotheilium were qualitatively determined by immunofluorescent staining with frozen section and by SP immunostaining kit with paraffin sections respectively.⑥The protein content of LOX-1 and NF-κBp65 on aortic tissues were determined by Western blotting assay:freshly frozen samples of partial aortic tissues were subsequently homogenized and lysed in lysis buffer,then protein samples were resolved and extracted. Immunoblotting was performed using diluted LOX-1 or NF-κBp65 antibody.The membranes were subsequently probed with horseradish peroxidase-conjugated secondary antibody and developed by chemiluminescence.The membranes were then exposed to X-ray film and subsequently developed.Densitometry was performed with gel imaging system and levels of protein expression were measured as the ratio toβ-actin protein.⑦Reverse Transcription-Polymerase Chain Reaction(RT-PCR):Total RNA of aortic tissues were isolated using Trizol reagent and single stranded-cDNA were synthesized according to the manufactures instruction.Then Polymerase chain was reacted.Each specific mRNA band was normalized with a band of relative internal referenceβ-actin mRNA.Relative intensity of band of interest was analyzed by gel imaging system and the levels of mRNA were expressed as the ratio to the optical density ofβ-actin band.3.Statistical analysisAll data are expressed as mean±S.D and analyzed using statistical package SPSS 11.0 for Windows.One-way analysis of variance(ANOVA) was used to determine statistically significant differences among different groups.Some data were performed with Pearson correlation analysis and Partial correlations analysis.A level of P<0.05 was considered statistically significant.Results:1.General health state or changes of body weight and blood glucose(BG):During the whole experimental period,one rat in DM group died.The BG of all diabetic rats were kept on high levels until the end of experiment.The body weight failed off and were largely lower than normal control rats(P<0.01).But the blood glucose and body weight levels of diabetic rats were normalized by insulin treated,while there were no obvious improvement by taurine treated.2.Serum lipid levels:At 6 weekend of experiment,the levels of serum TC,TG and LDL in DM group were markedly higher than NC group(P<0.01),while insulin-treatment restored all these changes almost to normal levels(P>0.05) and taurine intervention also markedly decreased the serum levels of TC and TG(P<0.01).3.Circulating levels of oxLDL,NO,ET and sICAM-1:at 2 weekend of experiment, the levels of serum oxLDL,NO and plasma ET were all higher in DM group than those in NC group(P<0.05).Also at 6 weekend,the serum levels of oxLDL,sICAM-1 and plasma ET were markedly increased,and the serum NO levels were largely decreased(all P<0.01). Insulin and taurine intervention markedly reversed these changes,but there still had differences with NC group.4.Ultrastructural changes of aortic endothelial cell:in NC group,aortic tunica intima was very smooth,and the arrangement or shape of endothelial cells or organella were all normal.At 2 weekend of diabetic course,the changes were not very obvious.But at 6 weekend,the changes became obvious,presenting swelling of cell and organella,partially showing amotic of endothelial cells or breaked of base plate and vanishing of microvilli, bluring of crista mitochondriales as well as dilation of perinuclear cisterna.5.Endothelium-dependent relaxation of isolated aortic rings:At 2 weekend of diabetic course,the Ach-induced endothelium-dependent relaxations became blunted in DM groups,but the difference of Rmax between DM and NC groups wasn't obvious. While at 6 weekend,compared with normal control,the Ach induced concentration-dependent vasorelaxation of diabetic rats were markedly blunted,Rmax was markedly decreased and EC50 was largely increased(P<0.01).The function of endothelium-dependent relaxation were all markedly improved by insulin and taurine intervention(P<0.01),and the effect of insulin was extremely obvious,showing the completely restoration of EC50 to normal level(P>0.05).6.Expression of LOX-1 protein on aorta tissues: Immunofluorescent staining:almost no LOX-1 immunostaining were found on the whole layers of aortas in NC group.On the other hand,significantly positive LOX-1 immunostaining(bright green fluorescent staining) were observed in DM group at 6 weekend and mainly localized on the endothelial layers.While LOX-1 immunoreactivities were very weak in In and TAU groups,and the positive staining area and strength of LOX-1 were extremely weak in In group and also markedly weakened in TAU group.Western blot quantitative assay:the protein content of LOX-1 in aortas tissues were significantly enhanced in STZ-induced diabetic rats(P<0.01 vs control group),while insulin and taurine treatment largely suppressed this enhancement of LOX-1 protein expression(P<0.01 vs DM group),also the inhibition of LOX-1 expression was most significant in In group.7.Expression of NF-κBp65 protein on aortic tissues:Immunohistochemistry staining:In every group,NF-κBp65 protein were all expressed on aortic endothelium and muscular layers.But there were very weak staining in NC group and only presenting in partial intracytoplasm.But on diabetic aortas at 6 weekend,thickly brown positive staining could be seen in most endothelial and muscular cells,part cellular nucleus of endothelium showed positive staining.While the positive staining in cells or cellular nucleus were markedly weakened in In and TAU groups.Western blot quantitative assay:the protein content of NF-κBp65 in aortic tissues were significantly enhanced in STZ-induced diabetic rats(P<0.01 vs control group),while insulin and taurine treatment largely suppressed this enhancement(P<0.01 vs DM group), in which the NF-κBp65 protein in In group was reduced nearly to that of NC group(P>0.05).8.Expression of ICAM-1 protein on aorta tissues:Immunohistochemical staining for ICAM-1 on aortas showed that seldom ICAM-1 immunostaining were found on the whole layers of aortas in NC group,but significantly positive ICAM-1 immunostaining(brown,continuous staining) were observed in DM group and mainly localized on the endothelial layer.While ICAM-1 immunoreactivities were very weak in In and TAU groups,and the optical density integration calculated by positive area and the strength of ICAM-1 positive stainings were markedly lower(P<0.01 vs DM group).The reduction of ICAM-1 protein expression by insulin treated was mostly obvious, but there still had difference with NC group(P<0.05).9.RT-PCR quantitative assay:Expression of LOX-1,NF-κBp65 and ICMA-1mRNA on aortas were very low in normal control rats,but all of them dramatically upregulated in diabetic rats(P<0.01 vs NC group).While insulin and taurine treatment markedly inhibited this upregulation(P <0.01 vs DM group).Also,there was no difference of NF-κBp65 and ICAM-1mRNA expression between In group and NC group(P>0.05).10.Correlation analysis:In DM group,serum oxLDL level was negatively correlated with NO,NO/ET and Rmax,but positively correlated with serum sICAM-1 and plasma ET.Rmax was negatively correlated with ET,oxLDL and sICAM-1,but positively correlated with NO and NO/ET.Controlling the interference of body weight and blood lipid,partial correlation analysis showed these correlations still had statistical significance.Also,the level of LOX-1mRNA expression was positively correlated with the levels of serum oxLDL, NF-κBp65 and ICAM-1mRNA expression,but negatively correlated with Rmax.Conclusions:1.Vascular endothelium dysfunction and ultramicrostructure injury have been present in early diabetes.Endothelium-dependent vasodilation was the most direct and sensitive measurement,and circulating levels of NO,ET,NO/ET and sICAM-1 may be used as early indirect markers to screen endothelium dysfunction in diabetes.2.Excessive oxidative modification of LDL was occurred in early diabetes,oxLDL was the key risk factor to induce early vascular endothelium dysfunction in diabetes condition.Circulating levels of oxLDL can be used as a early predictor of diabetic vascular dysfunction.3.Aortic expression of LOX-1 protein and gene were all up-regulated and also closely correlated with circulating oxLDL level,indicating that the interaction of oxLDL and LOX-1 mediated the damage of vascular endothelium.OxLDL/LOX-1 system was the important agent that induced early vascular dysfunction of diabetes. 4.Aortic expression of NF-κBp65 were markedly enhanced and existing nuclear translocation,at the meantime,its mRNA expression were possitively correlated with the expression of LOX-1 and ICAM-1mRAN,indicating that aortic NF-κB was activated in early diabetes.ICAM-1 expression induced by activation of NF-κB signal pathway may be involved in the endothelial toxic effect of oxLDL/LOX-1.5.Strictly control of blood glucose by insulin treatment could restore the dyslipidemia and vascular endothelium dysfunction to some extent,at the same time could greatly inhibit LDL oxidative modification and LOX-1 expression in early diabetes.However insulin treatment couldn't completely restore the vascular dysfunction to the normal level.6.Although early intervention by taurine couldn't control hyperglycemia in diabetic rats,it may relieve the vascular endothelium dysfunction induced by oxLDL/LOX-1.This endothelium protection were possibly involved in the blockage of oxidative modification of LDL,the inhibition of oxLDL/LOX-1 and NF-κB activation as well as downregulation of ICAM-1 expression on aortic endothelium,relying on its antioxidation and anti-inflammatory action.Innovations and meanings:1.This study dynamically and directedly investigated in vivo the early occurrence of vascular endothelium dysfunction in type 1 diabetes as well as the levels of circulating oxLDL and other endothelium-derived markers,which provided referenced evidence for early discovery and intervention of diabetic vascular endothelium dysfunction in clinical practice.2.This study firstly in vivo comfirmed that the level of circulating oxLDL was increased,at the same time the aortic LOX-1 and NF-κBp65 were accordingly overexpressed.Correlation analysis also comfirmed that the expression of LOX-1mRNA was closely correlated with the level of circulating oxLDL,endothelium-dependent vasodilation as well as expression of NF-κBp65 and ICAM-1mRNA.All these indicated that during the process of diabetic vasculopathy,the activation of NF-κB signal pathway was involved in the damage of endothelium induced by oxLDL/LOX-1 in diabete,which may also provide a new clue for prevention of diabetic vasculopathy by mediating the expression of NF-κB and blocking the interaction of oxLDL/LOX-1. 3.This study firstly study the protection of insulin and taurine treatment against the early injury of vascular endothelium,discovering that strict control of hyperglycaemia by insulin treatment couldn't completely avoid the endothelium dysfunction in diabetes,and early intervention by taurine could significantly relieve the diabetic endothelium-dysfuntion,simultaneously decrease circulating oxLDL level and downregulate LOX-1,NF-κB,ICAM-1 protein as well as gene expression on aortas.All these indicated that endothelial-protection of taurine was related to its antioxidant and anti-inflammatory action,and taurine may be a good choice for the early prevention of diabetic vasculopathy. |
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