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

Background and objectivesRecently, the trend of atherosclerosis(AS) in the younger age has been greatly concerned.It was found that vascular endothelial cells (VEC) injury and endothelium dysfunction(ED) are the initial events in the development of AS. Many studies have found that high-fat obese children have already existed vascular endothelial injury and arterial intima-media thickness change, while the injury of VEC and ED appear reversible pathological change. Therefore, it is crucial to early detect and prevent vascular endothelial dysfunction for the prevention of obesity vascular lesion and improvement of the live quality of young high-fat obese patients.Lipid metabolism disorder is an independent risk factor for the occurrence and development of AS. It is proved that oxidized low-density lipoprotein (Oxidized low-density lipoprotein, oxLDL) plays an important role in ED and AS. That endothelial cells uptake of LDL, especially oxLDL is the crucial progress of causing and development of AS. OxLDL damages the function of VEC by decreasing vascular diastolic function, inducing thrombosis and inflammatory reaction. Endothelial toxicity of oxLDL is executed by its specific endothelial cell surface receptor, lectin-like oxLDL receptor-1(LOX-1). The binding of oxLDL to LOX-1 can activate nuclear transcription factor kappa B (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. p38 mitogen-activated protein kinase (p38 mitogen activated protein kinase, p38MAPK) is one of mitogen-activated protein kinase (MAPK) family member. It regulates cellular response to external stimulation through the transmission of intracellular signals. The studies have shown p38MAPK pathway activated the nuclear transcription factor NF-κB, promoting endothelial cell secretion of VCAM-1, E-selectin, ICAM-1, increasing adhesion of neutrophils cells to endothelial cell, and then initiated AS. Therefore, reducing oxidative stress, blocking oxLDL cytotoxicity, reducing the expression of chemokines and intervention of macrophage accumulation, are the key to choose drugs for anti-atherosclerosis.During the development of AS, the early pathological changes including endothelium dysfunction before lipids streak in young patients are reversible, and early intervention can reverse the tendency of development of fibrous or atherosclerotic plaque. Statins as the first choice to anti-AS, has a large number of related research, and our group has applied the statin. Because of side effects of the statin such as liver and muscle toxicity,we choose niacin.Niacin has a unique and comprehensive lipid-lowering effect, so it has been applied to the clinical lipid-lowering treatment since 1960'. It was still the recommendation drug of Cardiovascular Disease Prevention Guidelines by the American Heart Association (AHA) in 2004, and it was recommend as the clinical lipid-lowering drug in recently pubilished "The prevention guide of adult dyslipidemia in China ". In recent years, clinical trials indicated that niacin owns not only the effection of regulating lipid, but also the effection of anti-inflammatory, antioxidant capacity. It was studied that niacin can improve endothelial function and hold back AS progress. But the exact mechanism of the improvement of endothelial dysfunction with niacin has not been clarified.The aim of the study is to explore effection of signaling pathway including oxLDL/LOX-1 and p38-MAPK/NF-KB and early intervention with niacin in endothelium dysfunction.This study consisted of two parts:obese rat model was induced by the high-fat diet in the first part, and human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVECs) model was cultivated with lysophosphatidylcholine (Lysophosphatidylcholine, LPC) in the other part. The aim of the study is to explore effection of signaling pathway including oxLDL/LOX-1 and p38-MAPK/NF-κB and early intervention with niacin in endothelium dysfunction.Methods1. In Vivo1.1 High-fat obese rats model: 21-day-old male Wistar rats (weighing approximately 65-75g) (total n=30) were divided randomly into three groups. The control group (CG; n=10) was fed with pellet chow and high-fat group (HF; n=10) with a high-fat atherogenic diet consisting of chow enriched with 10% lard oil and 2% cholesterol for 12 weeks. The drug control group (DG; n=10) was fed with the same high-fat diet and treated with niacin (100mg/kg·days) by gavage technique for 12 weeks. The rats were freely given drinking water during 12 weeks.8 rats in three groups were randomly picked and anesthetized. The blood samples and abdominal arteries were collected, respectively for different detections.1.2 Measurement:1.2.1 Body weight and height:The body weight and height of every rat were measured three times every week during the study.1.2.2 Determination 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) and plasma endothelin (ET) were determined by nitrate reductase indirect assay and radioimmunity homogeneous phase competition assay respectively.1.2.3 Ultrastructure observation of vascular endothelium:Ultrastructure changes of aorta endothelium were observed by double staining transmission electron microscope.1.2.4 Determination of LOX-1 and ICAM-1 protein expression on aortic tissues:Expressions of LOX-1 and ICAM-1 protein on aortic endotheilium were qualitatively determined by immunofluorescent staining with frozen section and by SP immunostaining kit with paraffin sections respectively.1.2.5 The protein levels of LOX-1 and ICAM-1 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 ICAM-1 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 to B-actin protein.1.2.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR):Total RNA of aortic tissues was isolated using Trizol reagent and single stranded-cDNA was synthesized according to the manufactures instruction. Then Polymerase chain was reacted. Each specific mRNA band was normalized with a band of relative internal reference B-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 of B-actin band.2. In Vitro2.1 Model of human umbilical vein endothelial cells (HUVECs):Human umbilical vein endothelial cell line was cultured using Medium200 medium (containing low serum growth supplement LSGS) in incubator at 37℃and 5% CO2 condition. It was digested and passaged with 0.125% trypsin. Endothelial cells were polygonal, single paving stone-like tightly arranged. Cell viability was determined by 2% trypan blue staining, total cell number of viable cells accounted for 96%, and used in experiments. Experimental groups:(1) the negative control group:medium; (2) LPC different time groups:the medium adding 20μmol/L final concentration of LPC, were cultured for 0,10,30,60 min and 4,8 h; (3) different niacin dose and 10μmol/L p38-MAPK inhibitor (SB203580) group:After separately added with 0,0.25,0.5,lmmol/L niacin, cells were cultured forl8h and then cultured for 1h after added with 10μmol/L SB203580, then HUVECs added with the LPC were cultured for lOmin,8h and 24h. Cell concentration in each group is 5 x 105/ml, inoculated in 6-well plates, each well is 1ml.2.2 Measurement:2.2.1 Quantitative analysis with Western blot of pp38-MAPK, p38-MAPK, ICAM-1 protein content in endothelial cells:The same methods as 1.2.5.2.2.2 Reverse transcription-polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR) quantitative detection of ICAM-1 mRNA expression levels in endothelial cells:The same methods as 1.2.5.2.2.3 Real-time quantitative PCR (Real-time Quantitative Polymerase chain Reaction, Real-time PCR) to detect endothelial cells ICAM-1 gene expression and nicotinic acid, SB203580 its expression:Total RNA was prepared by Trizol according to the manufacturer's instruction. Reverse transcription (RT) was performed with 1μg RNA using Superscript II. For real-time PCR, a primer mastermix was prepared using 1μmol/L of forward and reverse primer in 2×SYBR(?) Green PCR Master Mix. Each reaction was performed in triplicate. Samples were amplified with a two step reaction 95℃for 15 s,60℃for lmin for 40 cycles using ABI PRISM 7700 Sequence Detector. Signal of a gene was normalized withβ-actin using the formula ACT=CT target-CT reference. The differential expression signal was calculated as AACt=ACt (gene of LPC treated group)-ACt (gene of untreated group) and expressed as relative fold of change using the formula:2-AACT.2.2.4 Immunofluorescence detection of LPC-induced ICAM-1, NF-κB protein expression:Cells were fixed in 4% paraformaldehyde at 4℃for 15 min and washed with PBS for 5min×3 times. After incubation in 0.2% Triton X100 in PBS for 5 min, and washed, cells were blocked with 1%BSA for 1h. Immunostaining was carried out with first antibodies diluted 1:200 inl%BSA incubating at 4℃overnight. After washing, FITC-second antibodies diluted 1:200 in 1%BSA, and incubated at room temperature for 1h away from light and washed with PBS for 5min×3 times. Specimens were embeded with glycerine and examined using an argon/krypton laser confocal microscope.3. Statistical analysisData are presented as means±SD. Statistical analysis was performed using SPSS software (SPSS 16.0). The differences were determined by one-way ANOVA followed by LSD test of all groups. Some data were performed with Pearson correlation analysis and P<0.05 was considered as significant.Results1. Results of In Vivo1.1 General health state and changes of Lee Index: During the whole experimental period, one rat in HF group died. The body weight of high-fat group and niacin rats was significantly heavier than that of the normal control group (P<0.01).Compared with the control group, Lee index of high-fat group increased, the difference was statistically significant (P<0.01), while Lee index of the niacin group is between that of the high-fat group and that of the control group, but difference was not significant compared with high-fat group.1.2 Changes in serum lipid levels:TG, TC, LDL of high-fat group were significantly higher than these of control group, but HDL of high-fat group was lower than that of the control group. In niacin intervention group, TG, TC, LDL and oxLDL levels were significantly lower than those of high-fat group (P<0.01), and HDL was higher than that of high-fat group (P<0.01), the difference was significant.1.3 Circulating levels of NO, ET and sICAM-1:Peripheral blood ET and sICAM-1 of high-fat group were higher than these control group (P<0.01), NO was lower than that of the control group (P<0.01). ET and sICAM-1 of niacin group were lower than these of high-fat group (P<0.01); NO was higher than that of high-fat group (P<0.01); the difference was significantly. The level of NO, sICAM-1 of niacin group was similar to these of the control group. 1.4 Ultrastructure changes of aortic endothelial cell:Aortic tunica intima was very smooth. The arrangement and the number of endothelial cells were all normal. After 12 weeks, ultrastructure changes of aortic endothelial cell of HF obviously changing, presenting swelling of cell and partially showing amotic of endothelial cells or breaked of base plate and vanishing of microvilli.1.5 Aortic vessel wall protein expression of LOX-1:1.5.1 Immunofluorescent staining:Normal control group, rat aortic vascular wall basically little or no LOX-1 protein expression (bright green fluorescent staining), while the high-fat obese rats at 12 weeks of intra-aortic cortex shows a clear bright green fluorescent staining signal; niacin intervention group within the cortex of rat aortic bright green fluorescence staining signals were reduced significantly decreased. 1.5.2 Western Blot quantitative analysis of aortic wall of LOX-1 protein expression:The protein content of LOX-1 in aortas tissues were significantly enhanced in high-fat obese rats (P<0.01 vs control group), while niacin treatment largely suppressed this enhancement of LOX-1 protein expression (P<0.01 vs HF group).1.6 Expression of ICAM-1 protein on aorta tissues:1.6.1 Immunohistochemistry SP method detected ICAM-1 protein expression in the aortic wall:ICAM-1 was mainly expressed in rat aortic vascular cells within the cortex. Normal expression of ICAM-1 protein rarely, and only a small number of endothelial cells with pale staining cytoplasm of the particles; high-fat group can be seen within the cortex, continuous, dense granules stained brown; niacin intervention group, endothelial cell expression of ICAM-1 protein significantly decreased.1.6.2 Western Blot quantitative analysis of aortic wall of ICAM-1 protein expression:The protein content of ICAM-1 in aortas tissues were significantly enhanced in high-fat obese rats (P<0.01 vs control group), while niacin treatment largely suppressed this enhancement of ICAM-1 protein expression (P<0.01 vs HF group).1.7 RT-PCR quantitative assay:Expression of LOX-1 and ICMA-1mRNA on aortas were very low in normal control rats, but all of them dramatically upregulated in high-fat obese rats (P<0.01 vs control group) while niacin treatment markedly inhibited this upregulation (P<0.01 vs HF group).1.8 Correlation analysis:In HF group, oxLDL levels were significantly correlated positively with the levels of LOX-1 mRNA. OxLDL levels clearly correlated positively with the parameters of serum and characteristics of endothelium secretion function, and negatively with HDL and NO (P<0.001).2. Results of In Vitro2.1 The effects of niacin/SB203580 on LPC-induced ICAM protein expression:2.1.1 Western blot quantitative analysis:Compared with control group, LPC 24h group can significantly increase ICAM-1 protein expression (P<0.01). The level of ICAM-1 decreased notably in niacin/SB203580 intervention group when compared with that in LPC group (P<0.01).2.1.2 Immunofluorescence staining:In control group, there is no positive staining cells; In LPC group, cells can be seen a clear bright green fluorescent signal, while in niacin group and SB203580 intervention group, the cells only can be seen weak green fluorescent signal.2.2 The effects of niacin/SB203580 on LPC-induced ICAM mRNA expression:RT-PCR and Real-time PCR analysis:Compared with control group, LPC4h group and LPC8h group can significantly increase ICAM-1mRNA expression (P<0.01), In niacin intervention group, the expression of ICAM-1 levels were significantly decreased (P <0.01vs LPC8h group), and it is concentration-dependent; while SB203580 group failed to reduce ICAM-1mRNA expression (P> 0.01).2.3 Western Blot detection of LPC-induced p38-MAPK activity:The results showed that when cultured 10min, p38-MAPK activity peaked, but when cultured 60min, it decreased; Giving different concentrations of niacin and 10μmol/L of SB203580 intervention, niacin and SB203580 are able to reduce the p38-MAPK activity, while p38-MAPK in itself had no effect.2.4 Different intervention methods on the aortic wall NF-κBp65 protein expression:2.4.1 Western Blot of quantitative test results:The protein content of NF-κBp65 were significantly enhanced in LPC group (P<0.01 vs control group), while niacin treatment largely suppressed this enhancement (P<0.01 vs LPC group).2.4.2 Immunofluorescence staining:Positive staining cells are less in control group, there are no significant nuclear staining cells, while the LPC24h endothelial cells can be seen clearly in the cytoplasm deeply stained bright green particles, we can see more nuclear staining; In niacin intervention group, the number and staining intensity of transfected cells were significantly reduced, weakened, and only very few cell nuclear staining; In SB203580 intervention group, it also can be seen clearly in the cytoplasm deeply stained bright green particle.Conclusions1. Vascular endothelium dysfunction and ultramicrostructure injury have been present in early high-fat induced rats. Circulating levels of NO, ET and sICAM-1 may be used as early indirect markers to screen endothelium dysfunction. Excessive oxidative modification of LDL occurred in early high-fat obesity. OxLDL was the key risk factor to induce early vascular endothelium dysfunction in high-fat obesity condition. Circulating level of oxLDL can be used as an early predictor of high-fat obesity vascular dysfunction.2. When vascular endothelium was injuried in high-fat obesity, expression of LOX-1 protein and gene of artery was up-regulated and also closely correlated with circulating oxLDL level that indicated oxLDL/LOX-1 system was the important agent inducing early vascular dysfunction of high-fat obesity.3. Early intervention by niacin relieved the vascular endothelium dysfunction. Niacin can lower serum levels of oxLDL, also significantly reduced aortic endothelial expression of LOX-1. Correlative analysis showed that niacin improved endothelial function by reducing oxLDL/LOX-1 system.4. In LPC-induced HUVECs, the expression of ICAM-1 protein and mRNA was significantly increased. The activity of p38-MAPK and the expression of NF-KBp65 were also enhanced. The above mentioned expression can be reduced by niacin intervention. The inhibitor SB203580 of p38-MAPK can reduce ICAM-1 protein expression, but can not affect its mRNA and NF-κB expression. Niacin may regulate ICAM-1 protein expression by interfering the activity of p38-MAPK. It regulate the mRNA expression of ICAM-1 by interfering NF-KBp65 through another pathway. Endothelial protective function of niacin may be partly related to inhibition p38MAPK/NF-KB system.Innovations and meanings1. When LPC which is the main component of oxidized low density lipoprotein induced HUVECs,ICAM-1 protein and the activity of p38-MAPK increased simultaneously, and the inhibitor of p38-MAPK can reduce the ICAM-1 protein expression, but can not affect ICAM-1 mRNA and NF-κB expression, ICAM-1 mRNA expression and NF-κB changed simultaneously. The study in vivo and in vitro firstly indicated that oxLDL binding LOX-1 activates p38MAPK/NF-KB pathway, and then mediates endothelial injury in high-fat obesity.2. The comparative study in vivo and in vitro firstly found that niacin can improve the damage of vascular endothelial cell function in high-fat obesity, and simultaneously lower levels of oxLDL, LOX-1, p-p38MAPK, NF-κB, ICAM-1 protein and gene expression, suggesting that protective effect of niacin on vascular endothelial is related with its antioxidant effect and blocking p38MAPK/NF-KB pathway. The study provides a new idea for early prevention and treatment of vascular lesions in high-fat obesity.