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The Study on the Effection of Signaling Pathway Including oxLDL/LOX-1 and p38-MAPK/NF-κB and Early Intervention with Niacin in Endothelium Dysfunction

Author: NiuNa
Tutor: WangYi
School: Shandong University
Course: Pediatrics
Keywords: Oxidized low-density lipoprotein Lectin-like oxidized low-density lipoprotein receptor 1 p38 mitogen- activated protein kinase Nuclear factor-κB Niacin
CLC: R543
Type: PhD thesis
Year: 2010
Downloads: 453
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Abstract


Background and purpose in recent years, atherosclerosis (Atherosclerosis, AS) younger age is increasingly subject to the re-identification, the study found that the vascular endothelial cells (Vascular endothelial cell, VEC) damage and endothelial dysfunction (endothelial dysfunction, ED) is to start AS early event. The study confirmed that the fat obese children vascular endothelial injury and arterial intima - media thickness changes, vascular endothelial injury and dysfunction is still reversible pathological changes. Therefore, early detection and intervention endothelial dysfunction occurred For prevention of the fat obese vascular lesions, development and quality of life is essential for improving fat in obese adolescents. Lipid metabolism disorder is an independent risk factor for AS development, more and more evidence to suggest that the oxidation of low-density lipoprotein (Oxidized low-density lipoprotein, oxLDL) play an important role in the cause of ED and start AS. Endothelial cells to LDL, especially oxLDL uptake is the most critical step in the start and progress of the AS. OxLDL endothelial cell uptake of oxLDL lipids can reduce vasodilation, thrombosis and inflammatory response induced damage the VEC its function. The study confirmed the toxic effects of oxLDL endothelial - Lectin-like oxidized low-density lipoprotein receptor 1 (Lectin-like oxLDL receptor-1, LOX-1) mediated by specific endothelial cell surface receptor. oxLDL through LOX-1 binding, activation of nuclear transcription factor (Nuclear Factor, NF)-κB induces endothelial cells at the transcriptional level of intercellular adhesion molecule-1 (intercellular adhesion molecule-1, ICAM-1) and vascular cell adhesion Expression of the adhesion molecule 1 (Vascular cell adhesion molecule-1, VCAM-1), monocyte chemoattractant protein-1 (monocyte chemoattractant protein-1, the MCP-1), etc.. p38 mitogen-activated protein kinase (p38 mitogen activated p rotein kinase, p38MAPK) is one of the mitogen-activated protein kinase (MAPK) family members, involved in the response of cells to external stimuli regulation by intracellular signaling transfer studies show the p38MAPK pathway activated can activate NF-κB, promote endothelial cells secretion of VCAM-1, E-selectin, ICAM-1, exacerbate neutrophil and monocyte endothelial cell adhesion, start AS. So, reduce oxidative stress, blocking the cytotoxic effects of oxLDL down chemokine macrophage accumulation factor expression and intervention organization, is the protection of vascular endothelial function, the key to prevention and treatment of atherogenic progress. In the occurrence and development of the AS lesions occur in childhood and adolescence fatty streaks before the formation of the lesions, including endothelial dysfunction, is a reversible process, early intervention can reverse the progress or even to the fibrous plaque atherosclerotic plaque. Statins as the first choice of anti-AS, there are a lot of research, and the research group the statin intervention treatment has been applied, this topic chosen as the research object has a unique and comprehensive lipid-lowering effects of niacin. 1960s, niacin that began to be used lipid-lowering therapy clinical Half a century later, the American Heart Association (AHA) 2004 still listed as one of the cardiovascular disease prevention and treatment guidelines recommended drugs our newly introduced \In recent years, clinical trials showed that niacin except Lipid Metabolism, also has anti-inflammatory, and enhance the role of the LDL antioxidant capacity has been shown that niacin can improve endothelial function to prevent the progress of AS, but improve endothelial dysfunction The exact mechanism is not yet clear. The purpose of this study is to investigate the effect of niacin intervention the oxLDL/LOX-1 system and p38MAPK/NF-KB of signal pathway mediated endothelial dysfunction and its possible mechanism. This study is divided into two parts: the first part through the establishment of high-fat diet-induced fat obese rat model, and given niacin intervention in endothelial dysfunction in body research oxLDL/LOX-1 system, observe niacin interfere with the protective effect of vascular endothelial; second part by in vitro application lysophosphatidylcholine (Lysophosphatidylcholine, LPC) to cultivate human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVECs) model and give niacin intervention vitro observations p38-MAPK/NF-KB signaling pathway in endothelial dysfunction observed protective effect and the possible mechanism of niacin intervention on vascular endothelial cells. Research methods in vivo animal experiments 1.1 fat obese rat model and grouping: 21 days of age, weaning 30 male Wistar rats, weighing 65 to 75g were randomly divided into a control group, the high-fat group and the niacin group , 10 in each group. Which the control group (CG), to be conventional commercial rat feed; fat group (HF), be a high fat diet (10% lard, 2% cholesterol); niacin group (DG), to be the high fat diet and smoke Acid Sustained Release Tablets 100mg / (kg-d), gavage fed for 12 weeks. Throughout the experimental period all rats free access to water, weighed three times a week in each group, were killed 8 the rat derived and detection experiments of the 12th week, respectively. The weight and length of the 1.2 detection indicators and methods 1.2.1: groups three times body weight and length were measured weekly. 1.2.2 For the detection of peripheral blood indicators: peripheral blood triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL) and high density lipoprotein (HDL) levels using automatic biochemical analyzer; Serum levels of soluble ICAM-1 (sICAM-1), oxLDL level using the ELISA method; colorimetric detection of serum levels of NO by nitrate reductase; plasma endothelin (endothelin, ET) content were measured by radioimmunoassay. 1.2.3 vascular endothelial ultrastructure observation: thoracic aorta in turn glutaraldehyde and osmium tetroxide fixation, dehydration, embedding, toluidine blue ultrathin sections underwent transmission electron microscopy uranium-lead double staining, observed aortic endothelial changes of cell ultrastructure. 1.2.4 aortic wall of LOX-1, ICAM-1 protein expression: frozen slice immunofluorescence staining statutory testing thoracic aortic wall LOX-1 protein expression; paraffin sections immunohistochemical staining SP method to detect aortic wall ICAM 1 protein expression. 1.2.5 Western blot method for quantitative analysis of the aortic wall LOX-1, ICAM-1 protein content: protein extracted aortic tissue samples, denatured, electrophoresis, transfer film and closed, has joined the diluted LOX-1, ICAM -1 anti-horseradish peroxidase-labeled secondary antibodies were incubated at room temperature by the ECL developing gel imaging analysis system scans protein strip optical density analysis. Β-actin as an internal reference, with the absorbance value as the intensity of protein to the target protein / B-actin protein bars. 1.2.6 reverse transcription - polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR) method for detection of aortic wall LOX-1, ICAM-1 mRNA expression levels: extract groups aorta total RNA, the mRNA reverse transcribed into cDNA underwent PCR amplification. The PCR products were analyzed by agarose gel electrophoresis, the imaging. LOX-1, ICAM-1 and Β-actin amplification stripe absorbance ratio as: mRNA expression intensity. Vitro test 2.1 in human umbilical vein endothelial cell model (HUVECs) Establishment and grouping: human umbilical vein endothelial cell line, with Medium200 medium (containing low serum growth supplement LSGS) at 37 ° C, 5% CO2 in culture tank culture, digested with 0.125% trypsin, passaged. Endothelial cells were polygonal monolayer cobblestone closely spaced. 2% trypan blue staining to determine the cell activity, and the number of live cells accounted for more than 96% of the total number of cells used in the experiment. Experimental groups: (1) Negative control group: medium; (2) LPC different time group: medium was added to a final concentration of 20μmol / L the LPC were cultured 0,10,30,60 min and 4,8 h; ( 3) different doses of nicotinic acid (niacin) and 10μmol / L of p38-MAPK inhibitor (SB203580) Group: medium were added to a final concentration of 0,0.25,0.5,1 mmol / L niacin culture 18h. SB203580, an inhibitor of p38 MAPK, 10μmol / L culture 1h, and then were joined LPC culture 8h and 24h. The cells in each group concentration of 5 × 105/ml, seeded in 6-well plates, each hole 1ml. The 2.2 detection indicators and methods 2.2.1 Western blot method for quantitative analysis of the endothelial cells pp38-MAPK, p38-MAPK, ICAM-1 protein content: the same as 1.2.5. 2.2.2 reverse transcription - polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR) was used to detect endothelial cell ICAM-1 mRNA expression: the same way. 2.2.3 1.2.6 Real-time quantitative PCR (Real-time Quantitative Polymerase chain Reaction, Real-time PCR) assay endothelial cell ICAM-1 gene expression: extraction of endothelial cell total RNA, mRNA reverse transcribed into cDNA, amplified PRISM 7700 automatic real-time PCR instrument. The reaction conditions were: 95 ℃ 15 s, 60 ℃ 1min, 40 cycles. Quantitative analysis of gene expression relative amount is calculated according to the ABI user manual the comparative method, the gene expression of the different signals of the β-actin standardized formula: the ΔCT = CT target-CT refernce, Gene Expression: ΔΔCt = ΔCt (Gene of LPS - treated Group)-ΔCT (gene of untreated group), the gene expression relative multiples: 2 - △ △ CT. 2.2.4 immunofluorescence method detects the LPC induced ICAM-1, NF-κB protein: in the use of the slides before the sterilization, a sterilized and stored under sterile conditions. Cells (400 × g centrifugation 5min) were washed with PBS, and resuspended in PBS. Adjust the concentration to 1 × 106/ml; slides were fixed the thrown piece machine turned on, and then added 0.1ml cell suspension; quickly the centrifugal force of 1200 × g, centrifugal ~~ 10min; single layer of cells on the slide and dried in air for 15-20min. 4% formaldehyde were fixed 15min, washed 3 times with 1 × PBS, every 10min, 0.2% Triton X-100 permeabilization 5min, washed. 1% BSA at room temperature closed 1h. Plus 1:200 primary antibody (diluted with 1% BSA) on the wet box, spend the night. Washing, plus 1:200 FITC-second antibody (diluted with 1% BSA) 1h, dark. Washing, 95% glycerol were mounted, observed under a fluorescence microscope. Statistical analysis nean ± D: collected measurement data statistically analyzed using SPSS16.0 software. Differences between the groups using single-factor analysis of variance (one-way ANOVA) was used to compare between any two LSD test, correlation analysis using Pearson test, P lt; 0.05 Differences were considered statistically significant. Results. Changes in the body results 1.1 General and Lee index: during the entire experiment, obese rats death of a cause of death was severe high blood lipids coinfection. High-fat group was significantly higher body weight of rats with niacin group (P lt; 0.01), high-fat group compared with control group increased index Lee, the difference was statistically significant (P lt; 0.01), while niacin Group Lee index ranged between a high-fat group and the control group, but no significant difference between the high-fat group. 1.2 Change in lipid levels: high-fat group peripheral blood TG, TC, LDL were significantly higher than that of the control group, HDL lower than the control group. Compared with the high-fat group, niacin group TG, TC, LDL, oxLDL water on average significantly lower (P lt; 0.01), and HDL higher than the high-fat group (P lt; 0.01), the difference was statistically significant. 1.3 peripheral blood endothelial secretion of functional indicators of change: the fat in peripheral blood ET, sICAM-1 levels were higher than that of the control group (P lt; 0.01) NO lower than the control group (P lt; 0.01). ET in the niacin group, sICAM-1 water lower than the average high-fat group (P lt; 0.01) NO higher than the high-fat group (P lt; 0.01), the difference was statistically significant, NO, sICAM-1 has reached the control group level. 1.4 aortic endothelial cells: ultrastructural changes in the normal rat aortic intimal surface is flat, flat endothelial cells, arranged normal rules, the number of organelle morphology nucleus of rules; endothelial ultrastructural changes in the high-fat diet for 12 weeks Obviously, swelling of endothelial cells, lipid deposition, endothelial cell necrosis, dissolution, detachment or shedding of the part or a large area with the basement membrane. LOX-1 protein expression 1.5 aortic vascular wall: 1.5.1 Immunofluorescence staining the statutory testing aorta wall tissue expression of LOX-1 protein: control group rat aortic wall basically no or very little LOX-1 protein expression (bright green staining), 12 weeks of high-fat obese rat aorta cortex visible obvious bright green staining signal; cortex in the niacin group rat aortic bright green staining signal was significantly weakened . 1.5.2 Western Blot quantitative analysis of the aortic wall of the LOX-1 protein expression: ECL developing results and B-actin expression as a reference, results of quantitative analysis of the image system, display, each group abdominal aortic wall in LOX-1 protein levels on the expression of very low, LOX-1 protein expression levels of the control group, 12 weeks of high-fat obese rat LOX-1 protein expression levels were significantly higher than the normal control group (P lt; 0.01); niacin group LOX-1 protein expression levels were significantly lower in comparison with the high-fat group differences are significant (P lt; 0.01). 1.6 aortic vascular wall ICAM-1 protein expression levels: 1.6.1 Immunohistochemical SP method detecting aortic wall ICAM-1 protein expression: ICAM-1 is mainly expressed in the rat aorta endodermis. Control group, ICAM-1 protein expression rarely, only a small number of endothelial cells intracytoplasmic particles lightly stained; continuous brown stain particles within the cortex visible in the high-fat group; niacin group, endothelial cell ICAM-1 protein expression weakened. 1.6.2 Western Blot quantitative analysis of the aortic wall ICAM-1 protein expression: ECL development results, and B-actin protein expression as a reference image system quantitative analysis showed, the aortic wall ICAM-1 protein levels expression, the extremely low level of ICAM-1 protein expression in the control group, 12 weeks of high-fat obese rats ICAM-1 protein expression levels were significantly higher than the normal control group (P lt; 0.01); niacin group, ICAM-1 protein expression levels were significantly reduced in comparison with the high-fat group differences are significant (P lt; 0.01). 1.7 RT-PCR quantitative detection of aortic wall LOX-1 expression of ICAM-1 mRNA expression levels: the control group rat aortic vascular wall LOX-1, ICAM-1 mRNA expression is extremely weak; 12 weeks, the high-fat group LOX- 1, ICAM-1 mRNA expression was significantly enhanced expression levels were significantly higher (P lt; 0.01); compared with the high-fat group, niacin group LOX-1, ICAM-1 mRNA expression in the aortic wall was significantly affected by inhibition (P lt; 0.01). 1.8 Correlation Analysis: fat obese rat serum oxLDL NO, HDL levels were negatively correlated positively correlated with plasma ET, serum sICAM-1 levels; Pearson correlation analysis showed that the above correlation is still statistically significant. Aortic wall LOX-1 mRNA expression levels of serum oxLDL levels and ICAM-1 mRNA expression level is also a significant positive correlation was negatively correlated with NO. 2.1 niacin and p38MAPK blocker (SB203580) in vitro findings LPC-induced ICAM-1 protein expression: 2.1.1 Western blot the quantitative Analysis: compared with the control group, LPC24h group a significant increase in force (?) ICAM-1 protein expression (P lt; 0.01), while the the niacin, SB203580 intervention group of ICAM-1 protein expression levels were significantly lower compared with the LPC group was statistically significant (P lt; 0.01). 2.1.2 immunofluorescence staining: control group cells basically no positive staining signal; LPC cells visible bright green fluorescent signal; niacin, SB203580 intervention group, cell only see weak green fluorescence signal. 2.2 nicotinic acid, and p38MAPK blockers (SB203580) ICAM-1 mRNA expression induced by LPC: RT-PCR and Real-time PCR analysis: compared with the control group, LPC4h group, and LPC8h of group can be a significant increase of ICAM-1 mRNA expression (P lt; 0.01), while the the niacin intervention group ICAM-1 expression levels were significantly lower, compared with LPC8h group was statistically significant (P lt; 0.01), and a dose-dependent manner; SB203580 group failed to reduce the expression of ICAM-1 mRNA (P gt; 0.01). 2.3 Western Blot detection LPC-induced p38-MAPK activity: The results showed that peaked in cultured 10min, 60min when diminished; treated with different concentrations of niacin and 10μmol / L SB203580 intervention, niacin and SB203580 could reduce p38 -MAPK activity, while no effect on p38-MAPK itself. 2.4 different interventions impact of the HUVECs NF-KBp65 protein: 2.4.1 Western Blot quantitative test results: Compared with the control group, LPC can significantly increase of NF-KBp65 protein expression (P lt; 0.01), while niacin intervention group the of NF-KBp65 protein expression levels were significantly reduced compared with the LPC group was statistically significant (P lt; 0.01). 2.4.2 immunofluorescence staining: control group, fewer positively stained cells showed no nuclear staining cells LPC24h group within the endothelial cell cytoplasm seen obviously bright green deeply stained particles, we can see more nuclear staining; niacin intervention group the number of green cells and staining intensity was significantly reduced, weakened, and only a very small cell nuclei staining; the SB203580 group endothelial cells intracytoplasmic obvious bright green deep dye particles still visible. Research conclusions. Hyperlipidemia obesity early can appear endothelial function and ultrastructural damage and excessive oxidation of LDL; peripheral blood of NO, ET, sICAM-1 and oxLDL levels as endothelial dysfunction early screening indicators . 2 fat obesity early endothelial dysfunction, synchronization occurs aortic wall LOX-1 protein and gene upregulation, and is closely related with the level of serum oxLDL, prompt oxLDL/LOX-1 system is induced by high-fat obesity early vascular endothelial dysfunction important molecular factors. Niacin intervention not only improved endothelial function, but also reduce the level of serum oxLDL significantly lowered aortic endothelial LOX-1 expression; suggesting that niacin may by downregulating oxLDL/LOX-1 system to play to improve endothelial function role. 4. LPC induced HUVECs, ICAM-1 protein: mRNA expression were significantly enhanced the activity of p38-MAPK and NF-KBp65, were also significantly enhanced. The the niacin intervention can reduce the above expression, p38-MAPK inhibitor SB203580 can only reduce the expression of ICAM-1 protein, and not affect the expression of mRNA and NF-KBp65, prompted Niacin may on the one hand through the intervention p38 -MAPK activity regulation of ICAM-1 protein expression, on the other hand, through the inhibition of other pathways intervention the NF-KBp65, regulation of ICAM-1 mRNA expression. Niacin This endothelial protective effect, at least in part, with inhibition (?) P38MAPK/NF-KB system-mediated endothelial injury. Innovation and significance of in vitro studies found that the main ingredient of oxLDL induced by LPC HUVECs, ICAM-1 protein and p38-MAPK activity synchronization enhance p38-MAPK inhibitors reduce the ICAM-1 protein expression of ICAM- no effect; 1 mRNA expression of NF-κB and ICAM-1 mRNA and NF-κB expression always synchronized. Therefore, this study is the first in vivo Foreign Minister combination confirmed of oxLDL can be combined with LOX-1 to, activate p38MAPK/NF-KB pathways mediated fat obese vascular endothelial injury. The first time, in vitro and in vivo comparative study found that niacin can reduce the damage of the the fat obese vascular endothelial cell function and synchronization to reduce oxLDL and LOX-1, p-of p38MAPK, NF-κB, ICAM-1 protein and gene expression, suggesting that vascular endothelial protective effects of nicotinic acid and its antioxidant, inhibiting p38MAPK activity and reduce the expression of NF-κB. This is early prevention of the fat obese vascular lesions develop new ideas.

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CLC: > Medicine, health > Internal Medicine > Heart, blood vessels ( circulatory ) disease > Vascular disease
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