| BACKGROUNDUric acid is the main metabolite of guanine and adenine metabolism,with antioxidant,regulate endothelial function,when the pathological conditions,uric acid levels continue to rise,induced inflammatory response,induced leukocyte activation leading to endothelial dysfunction.Epidemiological studies have found that uric acid increased in varying degrees is one of the risk factors of diabetes and coronary heart disease,but uric acid can be used as an independent risk factor,involved in the development and prognosis of the disease is still controversial.Recent studies at home and abroad suggest that elevated levels of uric acid in patients with stable angina are not significantly associated with the prognosis of the disease.In patients with acute coronary syndromes,hyperuricemia and disease recurrence and death.There is not a clear related relationship with the severity of coronary artery.The clinical study of this subject also shows that acute coronary syndrome in patients with elevated urinary acid levels and coronary heart disease prognosis,there are significant statistical differences,survival analysis model shows that uric acid levels in the 500-600umol/L,the patients with coronary heart disease poor prognosis,the relationship between uric acid and coronary heart disease and other traditional risk factors such as hypertension,diabetes,hyperlipidemia,smoking,its impact on cardiovascular disease is a separate role or the role of other factors is still unclear.At present,the study of uric acid at home and abroad is mostly in clinical analysis and a small amount of in vitro cell experiments.The effect of hyperuric acid on the inflammatory response of endothelial cells and its related mechanism is not clear and needs further study.OBJECTIVETo study the effects of different concentrations of uric acid on TNF-a-induced endothelial inflammatory changes and possible mechanism.OBJECTS&METHODS1.Objects Human umbilical vein endothelial cells(HUVEC).2.Experiment methods2.1 Cultured human umbilical vein endothelial cell line Human umbilical vein endothelial cells(HUVEC)were obtained from ordered from American Sciencell.HUVEC between passage 4 and 6 were used for all experiments.2.2 The effects of different concentrations of uric acid on the toxicity and proliferation activity of HUVEC cells were determined by Cell counting Kit-8 assay(CCK-8)and MTT assay and we accordingly set the concentration gradient of uric acid intervention.2.3 Pretreatment with different concentrations of uric acid and TNF-? for treatment of human umbilical vein endothelial cells:According to our previous collection of patients with coronary heart disease with high uric acid,the levels of high uric acid in the patients with coronary heart disease were higher than those in the patients with coronary heart disease.The slightly elevated mean level of hyperuricemia was 450 ?mmol/L,the moderate mean level of uric acid Value of 590?mmol/L,the higher mean level of high uric acid in the coronary heart disease group was 700?mmol/L,and the cells were treated with 80?g/mL,100?g/mL,120?g/mL uric acid,and 10ng/mL tumor necrosis factor-(TNF-?)in endothelial cell culture medium.The human tumor necrosis factor-a(hTNF-?,10ng/ml)was used as a positive control.H1:0 ng/ml,control groupH2:hTNF-a:10ng/ml,positive control groupH3:80?g/mL,the slightly elevated average level of serum uric acid in coronary heart diseaseH4:100?g/mL,the moderate mean level of high uric acid in patients with coronary heart diseaseH5:120pg/mL,higher mean serum uric acid levels in patients with coronary heart disease2.4 Determination of intracellular extracellular hydrogen peroxide using the relevant kit and intracellular ROS with a fluorescent microplate reader.2.5 The expression of ET-1(endothelin-1),TF(tissue factor)and other related factors were measured by ELIS A.2.6 The expression of ET-1(endothelin-1)and TF(tissue factor)mRNA was measured by q-PCR.2.7 The protein of ICAM-1?VCAM-1?IL-18 and t/p-IKKa,t/p-I?B? and?-actin was detected by Western Blotting.3.Statistical and analysisSPSS software,version 19.0,was applied for the results and statistical analyses.Data are reported as Mean±SD.Two groups was compared with one-way ANOVA.The significant level is 0.05.P<0.05 mean statistical signification.P<0.01 mean obviously statistical signification.4.RESULTS4.1 The viability of HUVEC did not show a significant decrease in uric acid concentration at 80?g/mL,100?g/mL and 120?g/mL(P>0.05),indicating that these three concentrations had no effect on the viability of human umbilical vein endothelial cells The viability of human umbilical vein endothelial cells was significantly decreased at 200?g/mL,400?g/mL and 800?g/mL(P<0.05).4.2 The expression of H2O2 in H1,H2,H3,H4 and H5 groups was 2.46 ±0.25?mmol/L,3.70 ± 0.07?mmol/L,4.75 ± 0.02?mmol/L,5.20 ± 0.23?mmol/L,3.72 ± 0.26 Mmmol/L.Compared with H2 group,the secretion of H2O2 in H3 and H4 group was significantly higher than that in H2 group(P<0.05).The OD value of intracellular reactive oxygen species(ROS)in H1,H2,H3,H4 and H5 were 36.5 ±6.1,638.2 ± 26.8,1196.5 ± 74,1400.8 ± 111,888.2 ± 87.2.Compared with H2 group,the secretion of ROS in H3 and H4 group was significantly higher than that in H2 group(P<0.01),indicating that uric acid could promote the production of reactive oxygen species and extracellular hydrogen peroxide induced by TNF-a.4.3 The expression of ET-1(endothelin-1)in H1,H2,H3,H4 and H5 was 199.2±13.0pg/mL,802.6 ± 72.7pg/mL,1134.4 ± 135.0 pg/mL,1255.6 ± 275.4 pg/mL,463.1 ± 147.0 pg/mL.Compared with H2 group,the secretion of ET-1 in H3 and H4 group was significantly higher than that in H2 group(P<0.05).The expression of TF(tissue factor)in H1,H2,H3,H4 and H5 groups was 74.5 ± 7.6 pg/mL,142.0 ± 15.0 pg/mL,211 ± 26.0 pg/mL,250 ± 37.3 pg/mL and 254.4 ± 30.2 pg/mL.Compared with H2 group,the secretion of TF in H3,H4 and H5 group was significantly higher than that in H2 group(P<0.01).ET-1 and TF(tissue factor)increased at the mRNA level,Consistent with that uric acid can cause TNF-a-induced endothelial cell inflammatory response levels increased.Compared with H2 group,the secretion of VCAM-1 in H3,H4,H5 group was significantly higher than that in H2 group(P<0.01).Compared with H2 group,the secretion of ICAM-1 in H4 and H5 group was significantly higher than that in H2 group(P<0.01).Compared with H2 group,the secretion of IL-18 in H3 and H4 group was significantly higher than that in H2 group(P<0.01).4.4 That uric acid induced TNF-a-induced NF-?B signal pathway protein t/p-IKKa,t/p-l?B? expression was not significantly different.5.CONCLUSIONSUric acid can increase TNF-?-induced oxidative stress in endothelial cells and the production of its products and inflammatory factors.Uric acid may had no effect on TNF-a-induced endothelial cell IKKa-I?Ba signaling pathway(NF-?B). |
PDF Full Text Request