Correlation Between Adipocytokines Proneurotensin, Clusterin And Obesity And Premature Coronary Heart Disease

Part 1:the relationship between serum proneutensin,clusterin and premature coronary artery diseaseObjective:Coronary artery disease(CAD)remains the commonest cause of mortality in the world.There is a general agreement on a multifactorial etiology of CAD and that the incidence of disease increases with age.However,with the development of the economy,we changed eating habits and lifestyle.It has been documented that younger age group people are developing the diseases frequently than before in the last couple of years.The premature coronary artery disease(males<55 years and females<65 years)may have the poor prognosis and few study.So we focused on the risk factors of premature coronary artery disease(PCAD).Previous studies have found that obesity is closely related to coronary artery disease,and adipocytokines play an important role in it.Proneurotensin(PNT)and clusterin(CLU)are both adipocytokines closely related to obesity.But their relationship with PCAD has not been reported,so our study focusd on the relationship between serum clusterin,proneutensin and premature coronary artery disease.Methods:We selected 359 patients with PCAD(male<55 or female<65,and any coronary artery stenosis>50%)from 3364 patients underwent coronary angiography in PUMCH from November 2011 to April 2016.According to age,gender,and BMI,we selected 139 non-premature CAD(who did coronary angiography in PUMCH from 2011 to 2016)and 193 physical examing controls.We collected all the subjects' routine blood test,liver and kidney function,lipid(HDL-C,LDL-C,TG and TC)and fasting blood glucose.We also detected serum proneutensin and clusterin with ELISA.The associations between these data and premature CAD were analyzed.Result:(1)Within the 359 PCAD patients,22.0%(79/359)were obesity,42.1%(151/359)were overweight,34.5%(124/359)were normal,1.4%(5/359)were lean.The proportion of overweight and obesity in PCAD was 64.1%(230/359),which was significantly higher than the obesity/overweight guideline of China.(2)Comparison of serum proneurotensin and clusterin in obesity,overweight and normal BMI group:The level of proneurotensin in obesity group was significantly higher than overweight and normal group[obesity 35.27(8.42,108.12)vs.overweight 11.52(7.85,85.34)vs.normal 9.14(7.79.74.51),p<0.05].The level of clusterin in obese group was higher than that in overweight and normal group,but there was no significant difference[obesity 84.15(34.05,141.96)vs.overweight 44.87(30.74,147.77)vs.normal 47.86(31.77,156.22),p>0.05].(3)Serum proneurotensin and clusterin in physical examination(PE),nonPCAD(NPCAD)and PCAD group:The serum proneurotensin was significantly higher in the patients of PCAD than the other two groups[PE 9.05(8.53-54.52)vs.NPCAD 12.90(7.49-98.89)vs.PCAD 14.60(7.83-105.89),p<0.05].Serum clusterin increased combined with PE/NPCAD/PCAD group,however there was no sigigicant difference.[PE 40.64(28.00-135.91)Vs.NPCAD 54.56(31.13-147.92),p<0.05;NPCAD 54.56(31.13-147.92)vs.PCAD70.25(33.40-162.07),p=0.07].(4)Risk factors of proneurotensin and multiple linear regression analysis:proneurotensin was positive correlated with female(r=0.12),PE/NPCAD/PCAD(r=0.12),BMI(r=0.10),total cholesterol(r=0.16),and HDL-C(r = 0.09)(p<0.05).And clusterin was negatively correlated with LDL-C(r=-0.15,p<0.05).Multivariate linear stepwise regression analysis showed that the PE/NPCAD/PCAD(?= 0.30,p<0.05)and BMI group(? = 0.14,p<0.05)entered the equation.Indicating that proneurotensin independently correlated with PCAD and BMI.(5)Risk factors of clusterin and multiple linear regression analysis:clusterin was positive correlated with female(r=0.13,P<0.05)and PE/NPCAD/PCAD(r=0.13,P<0.05).And clusterin was negative correlated with total cholesterol(r =-0.12),LDL(r=-0.19)(p<0.05).We found that PE/NPCAD/PCAD(?= 0.30,p<0.05)and total cholesterol(? =-0.13,p<0.05)were entered into the equation by multiple linear stepwise regression analysis.Indicating that serum clusterin was positively correlated with PCAD and negatively correlated with total cholesterol.(6)The correlation between serum clusterin and proneurotensin in PCAD group:Serum proneurotensin was positive correlated with hyperlipidemia(r = 0.13)and diabetes mellitus(r = 0.18)(p<0.05).There was a positive correlation between serum clusterin and hyperlipidemia(r = 0.11).Taking statins might reduce both levels.Conclusion:1.Serum proneurotensin was positively correlated with PCAD and BMI.2.Serum clusterin increased in obesity and was positively correlated with PCAD,independent negative correlation with total cholesterol,suggesting that clusterin may affect the development of coronary heart disease through downtrend cholesterol.Part 2:Expression of neurotensin and clusterin in inflammatory 3T3-L1 differentiation cells,human adipocytes and their effects on HUVECsObjective:Recently,it is becoming apparent that adipose tissue is an active endocrine and paracrine organ that releases several bioactive mediators,which influence body weight homeostasis,inflammation,coagulation,fibrinolysis,insulin resistance,diabetes and atherosclerosis.The cellular mechanisms linking obesity and atherosclerosis are complex and have not been fully elucidated.In our study,different concentrations of lipopolysaccharide(LPS)were used to induce inflammatory status of 3T3-L1 and human adipocytes.Then we analyzed inflammatory cytokines(neurotensin,clusterin,IL6,TNF alpha)expression levels.What's more,we used neurotensin,clusterin and LPS affect endothelial cell adhesion,chemotaxis and inflammatory factor expression.Methods:(1)Adding insulin(100?g/ml),dexamethasone(10uM),IBMX(0.5mM)into 3T3-L1 preadipocytes for 3 days,and then adding insulin(100?g/ml)for 4 days.Adding 1×10-6mol/1 dexamethasone,50?g/ml ascorbic acid and 100?g/ml IBMX induced subcutaneous adipose mesenchymal stem cells into mature adipocytes for 12 days.Then using 1,2,5ug/ml LPS for 24 hours respectively.The expressions of neurotensin,clusterin,IL-6 and TNF-a were detected by RT-qPCR.(2)The expression of ICAM-1,VCAM-1,IL-6,TNFa,MCP-1 and IL-10 mRNA was detected by RT-qPCR after 200 ?g/ml neurotensin or 5ug/ml clusterin for 24 hours.Statistical analysis was performed by statistical software,with p<0.05 as statistically significant.Results:(1)Different concentrations of LPS on the expression of neurotensin,clusterin,IL-6 and TNFa of 3T3-L1 adipocytes:? The expression of neurotensin mRNA was dose-dependent with LPS in 3T3-L1 adipocytes.The expression of neurotensin mRNA was 1.75 ±0.16 times(p<0.01,1?g/ml),2.41±0.28times(p<0.01,2?g/ml),5.32±0.33times(p<0.01,5?g/ml).? The expression level of clusterin mRNA was higher than the control group[2.16±0.06times(p<0.01,1?g/ml),2.14±0.04 times(p<0.01,2?g/ml),1.77±0.09times(p<0.01,5?g/ml)].? The expression of IL-6 and TNFa mRNA in the LPS group was significantly higher than that in the control group.(2)Different concentrations of LPS on the expression of neurotensin,clusterin,IL-6 and TNFa of human adipocytes:? The expression level of nurotensin mRNA was increased in different concentrations of LPS group[6.17±0.58 times(p<0.01,1?g/ml);5.02±0.60 times(p<0.01,2?g/ml);5.34±0.84 Times(p<0.01,5?g/ml)].? The expression level of clusterin mRNA in different concentrations of LPS group was similar to the control group[1.10±0.15(p>0.05,?g/ml);0.91±0.13 times(p>0.05,2?g/ml);0.91±0.06 times(p>0,05,5?g/ml)].? The expression of IL-6 mRNA in the LPS group was significantly higher than that in the control group[5.52±0.45 times(p<0.01,?g/ml);5.58±0.57(p<0.01,2?g/ml;3.64±0.53(p<0.01,5?g/ml).? TNFa mRNA expression level in different concentrations of LPS group was no significant difference with control group.(3)Effects of LPS,neurotensin and clusterin on adhesion,chemotaxis and expression of inflammatory cytokines in human umbilical vein endothelial cells:? The expression of HUVEC adhesion,chemotaxis and inflammatory factor was significantly increased in 1?g/ml LPS group[ICAM-1(18.01 ± 1.60 times,p<0.01),VCAM-1(10.11 ± 1.17times,p<0.01),IL-6(24.61 ±2.22 times,p<0.01),TNF?(20.83±2.47,p<0.01),MCP-1(12.72 ± 2.024 times,p<0.01)].? The expression level of HUVEC adhesion,chemotaxis and inflammatory factors was decreased by 200?g/ml neurotensin intervention[VCAM-1(0.77±0.05 times,p<0.01),IL-6(0.62±0.05 times,p<0.01),TNFa(0.55±0.12 times,p<0.01),MCP-1(0.45±0.04 times,p<0.01)].? The expression of HUVEC chemotaxis and inflammatory factors were significantly decreased by 5?g/ml clusterin intervention.[IL-6(0.77±0.05times,p<0.05),TNFa(0.55±0.12 times,p<0.01),MCP-1(0.52±0.03 times,p<0.01).Conclusion:(1)The expression of neurotensin and IL-6 mRNA was up-regulated in 3T3-L1-induced differentiation cells and human adipocytes.Neurotensin may have the relationship with obesity.(2)The expression of clusterin in 3T3-L1 cells was up-regulated after LPS,but no expression was up-regulated in human adipocytes.Clusterin expression in different adipocytes maybe different.(3)Application of neurotensin and clusterin in HUVEC cells,inflammatory,chemokines,and adhesion factors showed a downward trend,suggesting that they may protect the endothelial cell.