| Objective:Atrial fibrillation is the most common clinical arrhythmia, as to our statistics; the prevalence rate of atrial fibrillation is 0.77 % to the population whose ages are older then thirty. The morbidity rate of atrial fibrillation is increasing year by year. The morbidity rate of atrial fibrillation is closely related to gender and age, but the difference between men and women diminishes after female goes through their menopause. Atrial fibrillation also increases the mortality rate of heart failure. Usually, the abnormal atrial electrical conduction leads to atrial fibrillation. In clinical the symptom always shows like heart beat very fast, pulse rate disorder, and the main danger event of atrial fibrillation is stroke. Stroke usually occurs when thrombus falls down from atrial into the blood, and flows with the blood to the whole body, it can jam at some vessel of important organs, especially in the brain. Atrial fibrillation often occurs with some other existing diseases, such as hypertension, coronary heart disease, valvular heart disease, heart failure. The etiology of atrial fibrillation is still unclear because of the complicated pathogenesis. So we consider genetic changes maybe one of that. Some studies confirmed that 14% of atrial fibrillation happened together with hypertension, and high blood pressure is an independent predictor of new-onset atrial fibrillation. So we consider hypertension as a common complication in patients with atrial fibrillation. Our research will be aim to find if there is any relationship between uric acid and atrial fibrillation.Serum uric acid is a byproduct of purine catabolism, the terminal steps of which are catalyzed by xanthine oxidoreductase. It is an independent predictor of cardiovascular disease and mortality. Our hypothesis is in the atrial, endothelial cell dysfunction cause vasoconstriction, and reduces ability of vasodilation. Then vessels continued contraction will cause regional myocardial ischemic and fibrosis, which lead to the changing in the structure of the atrium, finally atrial fibrillation will happen. It is known that e NOS is one of the most important molecules to maintain cardiovascular functions. Normal endothelial cells constitutively express e NOS, which catalyzes the production of nitric oxide(NO) from L-arginine. NO directly mediates vasorelaxation. When e NOS downregulated, the function of vascular relaxation will be downregulated. We want to us uric acid to treat human endothelial cells in order to find if it will regulate e NOS gene expression.In addition, the etiology of atrial fibrillation is still unclear because of the complicated pathogenesis. So lots of studies were aim to find the cause of atrial fibrillation. Some studies referred to the association between the CYP11B2-344T>C polymorphism and atrial fibrillation, but different studies could not have the same conclusion. So the exact relationship between the CYP11B2-344T>C polymorphism and susceptibility to atrial fibrillation is not entirely established. Therefore, we performed a meta-analysis of all eligible studies to derive a more precise estimation of the association between the CYP11B2-344T>C polymorphism and atrial fibrillation.Methods:1.Clinical data retrospective study(1) In this retrospective study we recruited consecutive patients with essential hypertension in the Department of Cardiology of Southwest Hospital between January 2010 and December 2014. Hypertension was diagnosed as blood pressure level more than 140/90 mm Hg(mean of 3 measurements) in the supine position or the use of antihypertensive medications. The arrhythmia diagnosis required documentation from an official medical record, a 12-lead ECG, or a 24-hour Holter recording and its classification was based on authoritative international consensus statements.(2) Exclusion criteria were history of coronary artery disease, valvular heart disease, congenital heart disease, cardiomyopathy, left ventricular systolic dysfunction, previous cardiac surgery, diabetes, thyroid disease, serum creatinine >110 μmol/L, recent infection, autoimmune or inflammatory diseases, respiratory diseases, administration of drugs which affect UA metabolism(apart from diuretics).(3) Each clinical process note, which contained patient’s basic condition and the blood and urine test, results, especially cardiac ultrasound, renal function and cholesterol. Use different methods to get statistical results.2. The effect and mechanism of uric acid to the function of human endothelial cells(1) To find if uric acid reduces e NOS expression in human endothelial cells. HCAEC were treated with uric acid in the manner of concentration-dependent and time-dependent. ENOS m RNA and protein levels were detected through Real-time PCR and Western blot respectively. HCAECs and HUVECs were treated with uric acid(400 umol/L, 600 umol/L, 800 umol/L, 1000 umol/L) for 24 h or 48 h. According to the results we had chosen one proper concentration for the experiment after that.(2) To find how uric acid affects e NOS m RNA degradation in human endothelial cells. HCAECs and HUVECs were treated with 1 ug/ul Actinomycin D, a direct inhibitor of RNA polymerase II, and 1 ug/ul Actinomycin D together with uric acid(600 umol/L); e NOS m RNA level was detected through Real-time PCR.(3) To find if uric acid decreases human endothelial cells proliferation. Uric acid at 600uoml/L significantly reduced both HCAECs and HUVECs cell proliferation(Figure 6A, 6B). To further confirm if this effect depends on e NOS, HCAEC and HUVEC were treated with 600uoml/L uric acid for 1, 2, 3 days. Then both cells were treated with uric acid(600 umol/L), 100 u M L-NAME, 100 u M L-NAME(pretreated) together with uric acid(600 umol/L) separately for 1day, 2 days and 3 days. Cell proliferation was detected by MTT.(4) In order to understand the signaling pathway involved in uric acid-induced e NOS decreasing in human endothelial cells, we investigated the possible involvement of major MAPKs(P38 MAPK, JNK MAPK, ERK MAPK) Western blot was used to make these determinations. The activation of MAPKs was studied by measuring the increase in MAPKs phosphorylation.(5) To further confirm the relationship between MAPKs pathway activation and uric acid-induced e NOS decreasing. HCAECs were incubated with chemical inhibitors p38(SB203580) and ERK1/2(PD98059), and then treated with uric acid(600 umol/L) for 24 hours. ENOS m RNA expression levels was determined by Real-time PCR. Then HCAECs were infected with P38-DN-Adv 48 h, after that treated with uric acid(600 umol/L) for 24 h. The expression of e NOS protein levels was determined by Western blot.(6) To find if uric acid inhibits e NOS promoter activities in human endothelial cells. HCAECs were transfected with plasmid, which hold PGL2-331 to +109 region of e NOS promoter for 24 h. Then treated them with uric acid(600 umol/L) for 24 h. Luciferase activity was measured using a Dual-Luciferase®Reporter Assay System and normalized by the ratio of firefly and Renilla luciferase activities.(7) To identification if GATA-4 binding to e NOS promotor in human endothelium cells. HCAECs were treated with uric acid(600 umol/L) for 24 h. We performed Ch IP assay following the protocol of Magna Ch IPTM A/G, using 1ul GATA-4 antibody, 1ul anti-acetyl-histone H3 antibody as positive control, 1ul Normal mouse Ig G as negative control. Purify DNA and PCR of all the samples with primer. We use 2% agarose gel electrophoresis to analysis reaction product by using 100 bp DNA marker.(8) Rat celiac artery rings were treated with uric acid(600 umol/L) for 24 h, the vasomotor function was analyzed by vessel tension change in response to different concentration of bradykinin. Got results through statistical analysis.3. Relationship between CYP11B2-344T>C polymorphism and atrial fibrillation(1) We therefore carried out a meta-analysis of published case-control studies to investigate the association between CYP11B2-344T>C polymorphism and atrial fibrillation susceptibility. Electronic searches were conducted on links between this variant and atrial fibrillation in several databases. We excluded studies with the same data or overlapping data by the same authors. Only published studies with full text articles were included. When more than one of the same patient population was included in several publications, only the most recent or complete study was used in this meta-analysis.(2) Odds ratios(ORs) and 95% confidence intervals(CIs) for homozygous, dominant model, recessive model and allele were calculated to estimate the strength of associations in fixed and random effect models.(3) We set subgroups according to the source of heterogeneity. Begg’s funnel plot and Egger’s test were performed to assess the publication bias in the literature.Results:1.We found that uric acid level and atrial fibrillation in patients with hypertension was closely related(1) Nine hundred and forty patients were finally included in the analysis. We classify the study population according to the presence or absence of atrial fibrillation. There were no significant differences between the two groups regarding sex,WBC Count, potassium level, serum Cr levels, systolic and diastolic blood pressure, LVPWT and IVST. Compared with the patients without AF, patients with AF were older(67.13±10.59 years old vs. 56.78±14.92 years old), had a longer duration of hypertension(9.27±9.50 years vs. 3.85±3.57 years,p<0.01), higher uric acid level(388.11±112.51 umol/L vs. 311.54±122.51 umol/L,p<0.01), and larger LAD(45.79±7.56 mm vs. 34.08±4.36 mm,p<0.01).(2) The association between uric acid and atrial fibrillation was further assessed by logistic regression analysis. This analysis showed independent associations between atrial fibrillation and uric acid(OR = 1.014; 95% CI: 1.005-1.023, p = 0.002), and LAD(OR = 1.390; 95% CI: 1.221-1.582; p< 0.001)2.We found that uric acid had an influence on the function of human endothelial cells(1) When treated with uric acid(600 umol/L and 1000 umol/L) for 24 h, HCAECs e NOS m RNA levels were decreased by 39.22 % and 69.22 % respectively, compared with controls(p < 0.01). ENOS protein levels were decreased by 62.10 % and 65.67 % respectively, compared with controls(p < 0.05). Then use another endothelial cells(HUVECs) to find if uric acid had the same effect. HUVECs were treated with uric acid(600 umol/L and 1000 umol/L) for 24h; e NOS m RNA levels were decreased by 27.44 % and 51.78 % respectively, compared with controls(p < 0.01). When HUVECs were treated with 600umol/L uric acid for 24 hours e NOS protein levels just showed a tiny decreasing 7.21 % but if the time came to 48 hours e NOS protein levels were decreased by 40.31 % compared with controls(p < 0.05). When HUVECs were treated with 1000umol/L uric acid for 24 hours e NOS protein levels were significantly decreased as 52.88 %(p<0.01).(2) When HCAECs were treated with Actinomycin D alone and Actinomycin D together with uric acid separately for 24 hours, e NOS m RNA stability decreased but there were no different effects between these two treatment groups(p < 0.01). While HUVECs were treated with Actinomycin D alone and Actinomycin D together with uric acid separately for 48 hours e NOS m RNA stability decreased but there were no different effects between these two treatment groups(p < 0.01).(3) It is shown that the combination of specific e NOS inhibitor 100 u M L-NAME(pretreated) with 600uoml/L uric acid did not cause further reduction of cell proliferation compared with levels compared with 100 u M L-NAME treatment alone in both cells. These data indicated that uric acid decreases human endothelial cell proliferation by decreasing e NOS expression level.(4) Treating HCAEC with uric acid(600 umol/L) for 0, 10, 20, 30, 40 min, resulted in a significant increase in the phosphorylation levels of ERK MAPK(peaked at 10-20 min of treatment) and p38 MAPK(peaked at 20-30 min of treatment). No substantial phosphorylation was observed for JNK MAPK. When Treating HUVEC with uric acid(600 umol/L) for 0, 10, 20, 30, 40 min, resulted in a significant increase in the phosphorylation levels of JNK MAPK in and ERK MAPK in these cells(peaked at 10 min of treatment). No substantial phosphorylation was observed for P38 MAPK.(5) To further confirm the relationship between MAPKs pathway activation and uric acid-induced e NOS decreasing. First HCAECs were incubated with chemical inhibitors p38 MAPK(SB203580). After that treated HCAECs with uric acid(600umol/L) for 24 hours. ENOS m RNA expression level was determined by Real-time PCR. It was shown that uric acid decreased e NOS m RNA expression level in HCAECs compared to the controls and P38 MAPK chemical inhibitor reversed the downregulation effect.(6) HCAECs were infected with P38-DN-Adv for 48 h, after that treated with uric acid(600umol/L) for 24 h. The expression of e NOS protein level was determined by Western blot. It is shown that e NOS protein level decreased after treated with uric acid and the effect was reversed by P38-DN-Adv.(7) HCAECs were transfected with plasmids holding PGL2-331 to +109 region of e NOS promoter for 24 h. Then treated them with uric acid(0, 600 umol/L) for 24 h. Luciferase activity was measured by using a Dual-Luciferase® Reporter Assay System and normalized by the ratio of firefly and Renilla luciferase activities. It has shown that 600 umol/L uric acids downregulated e NOS promoter activities.(8) HCAECs were treated with uric acid(0, 600 umol/L) for 24 h. We performed Ch IP assay following the protocol of Magna Ch IPTM A/G. Analysis reaction products by 2% agarose gel electrophoresis with a 100 bp DNA marker. Uric acid increases transcription factor GATA-4 directly binding to the e NOS promoter-binding site in HCAECs when assayed by Ch IP with regular PCR.(9) Uric acid has a negative effect on relaxation of vessel. The effect was obvious when the concentration of bradykinin was 10-7.The rate of vessel rings relaxation was 48.53% compared to control group.3. A total of nine case-control studies were identified. The C allele was associated with an increased susceptibility risk of AF compared with the T allele among hypertension populations(OR=1.26; 95% CI=1.09–1.45). The contrast of homozygotes and the recessive model produced the same pattern of results as the allele contrast. In the hypertension population, a significant association was found for the genetic models that were examined.Conclusion:1. Uric acid could make P38 MAPK singnal pathway phosphorylation, increase transcription factors GATA-4 binding to e NOS promoter, downregulate the activity of e NOS promoter, which made HCAECs e NOS m RNA and protein level went down. Our results shown that in vitro, uric acid could decrease e NOS gene expression and NO releasing, which made the function of endothelial cells disorder, further more, cause artery tighten.2. We found uric acid levels are related with atrial fibrillation in hypertensive patients.no matter through clinical research or via experiment in vitro.3. In order to know more about the causes of atrial fibrillation we made a Meta-analysis to view this problem in a different angle. Our pooled data suggest a significant association existed between CYP11B2-344T>C polymorphism and atrial fibrillation among hypertension populations.4. Finally, the role of uric acid-lowering therapy in patients with hypertension will be a new insighs into preventing atrial fibrillation improvement in those patients. |
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