| BackgroundCoronary artery disease is one of the major causes of death all over the world. Endothelial dysfunction is an early pathophysiological feature and an independent predictor of poor prognosis in atherosclerosis and coronary artery disease. Previous studies have demonstrated that estrogen can stimulate the activation of endothelial nitric oxide synthase (eNOS) and result in the production and release of nitric oxide (NO). Postmenopausal women, with the decline of estrogen production, have been proved to suffer three-fold higher risk of cardiovascular diseases than same age men. Isoflavone, mainly produced by soybeans, has been suggested to have estrogenic and potentially cardioprotective effects and improved endothelial function in many animal experimental studies. Therefore, isoflavone, as a replacement of estrogen, may protect the endothelial function and reduce the risk of coronary artery disease in postmenopausal women.However, the results of previous clinical studies were inconsistent, van der Schouw et al have shown that dietary isoflavone is not associated with aortic stiffness in postmenopausal women, but another study reports that the infusion of isoflavone can cause the dilation of the targeted artery and increase the blood flow. Double-blind, randomized and placebo-controlled trials also cannot give us a consistent answer, resulting in the precise effect of isoflavone supplementation on endothelial function has not yet been established. ObjectiveThe objective of the present study was to evaluate the effect of oral isoflavone supplementation on endothelial function measured by using flow-mediated dilation (FMD) in postmenopausal women.MethodsA meta-analysis of randomized, placebo-controlled trials was conducted to evaluate the effect of oral isoflavone supplementation on endothelial function in postmenopausal women. Trials were searched in PubMed, Embase, Cochrane Library database, reviews and reference lists of relevant articles. Summary estimates of weighted mean differences (WMDs) and 95% CIs were obtained by using random-effect models. Meta-regression and subgroup analysis were performed to identify the source of heterogeneity.ResultsA total of 9 trials were involved in the present meta-analysis. The results of overall nine trials showed that isoflavone significantly increased FMD (WMD:1.75%; 95% CI:0.83,2.67;P=0.0002). Significant heterogeneity for this outcome was found (heterogeneity X2=101.83, I2=92%, P<0.00001). To detect the sources of heterogeneity, Meta-regression and subgroup analyses were performed in our present study. Meta-regression analysis indicated that the age adjusted baseline FMD was inversely related to effect size. Subgroup analysis showed the oral supplementation of isoflavone had no influence on FMD if the age adjusted baseline FMD≥5.2%(4 trials, WMD:0.24%; 95%CI:-0.94,1.42; P=0.69). This improvement seems to be significant when the age adjusted baseline FMD levels< 5.2%(5 trials, WMD:2.22%; 95%CI:1.15,3.30; P<0.0001). The subgroup meta-analysis showed that there was no heterogeneity (heterogeneity X2=0.73,I2=0%, P=0.87) in the 4 trials with the high age-adjusted baseline FMD (>≥5.2%)ConclusionOral isoflavone supplementation can not improve endothelial function in postmenopausal women with high baseline FMD level, but the improvement seems to be significant in those with low baseline FMD. BackgroundAtherosclerosis has become one of the leading causes of death all over the world. Based on the inflammatory theory, atherosclerosis has been considered as a chronic inflammatory process with multiple immune cells involvement, such as monocytes, macrophage cells and lymphocytes. Accumulating data from molecular studies and animal studies have shown that circulating monocytes differentiating into macrophages in the subendothelial space is one of the pivotal steps in the development of atherosclerosis. Varieties of proteins and cytokines are involved in the differentiation of monocytes into macrophages. Nuclear factor I-A (NFI-A) is one of the important proteins. Rosa et al have demonstrated that NFI-A can inhibit the differentiation of monocytes to macrophages, and the down-regulation of NFI-A can increase the mRNA levels of the monocyte terminal differentiation gene M-CSFr and promote the differentiation of monocytes into macrophages.MicroRN As are one of the recently discovered, highly conserved, non-coding small RNAs. They can suppress protein synthesis by inhibiting the translation of protein from mRNA or by promoting the degradation of mRNA on the post-transcriptional level. NFI-A has been demonstrated to be one of the targets of miR-424. Rosa et al have shown that miR-424 can inhibit the expression of NFI-A in monocytes and promote the differentiation of monocytes to macrophages. Therefore, the level of miR-424 expression may indicate the level of monocytes differentiation. Since monocytes differentiating into macrophages is one of the pivotal steps in atherosclerosis, we hypothesized that miR-424 may have a potential pathophysiological role in the atherosclerosis.NF-kB pathway has been demonstrated to promote the differentiation of monocytes to macrophages and plays an important role in atherosclerosis. Isoflavone, extracted mainly from soybeans, is known as one of the agonist of PPARγ, and PPARγhas been proved as an inhibitor of NF-kB pathway. Many clinical trials have demonstrated that oral supplementation of isoflavone can help people prevent the development of coronary heart diseases. Therefore, in previous studies, the cardioprotective mechanism of isoflavone has been ascribed to the agonist of PPARγ, which can inhibit the NF-kB pathway. MiR-424 has been proved to attend the differentiation of monocytes to macrophages by inhibiting the expression of NFI-A, so we hypothesized that isoflavne might reduce the level of miR-424 in monocytes by inhibiting the NF-kB pathway to prevent the differentiation of monocytes to macrophages.In the present study, we determined the expression level of miR-424 in peripheral blood monocytes in patients with coronary artery disease (patients with unstable angina or acute myocardial infarction) and evaluated the expression of NFI-A and related cytokines in these subjects, in order to explore the possible mechanisms of miR-424 modulating the differentiation of monocytes/macrophages and inflammatory response in patients with coronary artery disease. Furthermore, we also tried to reveal whether isoflavone could reduce the level of miR-424 in ox-LDL stimulated monocytes in vitro to prevent the differentiation of monocytes to macrophages.Methods(1) In vivo study: We enrolled totally 50 subjects who visited our hospital for chest pain syndrome from July 2009 to August 2010 in the present study. Coronary arteriongraphy was performed on all enrolled subjects..Peripheral blood samples were drawn from all the patients with 21-gauge needle for clean venipuncture of an antecubital vein within 30 min rest on the following morning after arrival. Mononuclear cells were isolated by using centrifugation through a Ficoll-isopaque density gradient. To obtain the monocytes, the mononuclear cells were allowed to adhere to six-well plates with 5%autologous serum for 2 h at 37℃, in a 5%CO2 incubator. Non-adherent cells were removed and the monocytes adherenting to the culture dish were used for microRNA isolation and analysis.Total< 200bp RNA was extracted with the miRVana Isolation Kit (Ambion, CA, USA) according to the manufacturer’s instructions. The micoRNA was then reversed to cDNA by using miScript Reverse Transcription Kit (Ambion, CA, USA) and the level of miR-424 was assayed with the Taqman probes and primer sets in an Applied Biosystems PRISM 7900HT Fast Real-Time PCR System (Applied Biosystems, CA, USA). NFI-A protein was analyzed by Western blot. Serum IL-6 and TNF-a were determined by ELISA.(2) In vitro study:Isoflavone (Sigma, St Louis, MO, USA) was dissolved in DMSO and frozen in aliquots until use. Mononuclear cells were obtained from healthy donors by using Ficoll-isopaque density gradient as the above method. Then the obtained mononuclear cells were seeded at a density of 2x106/mL in RPMI 1640 medium supplemented with 10%foetal bovine serum,1%pen/strep solution, and incubated in a 5%CO2 incubator at 37℃for 2 h. After 2 h, non-adherent cells were removed and the adherent monocytes in 24-well tray were treated as follows:(1) Control group:0.5 ul DMSO was added in each well. (2) ox-LDL group:10 ul assay medium with ox-LDL at final concentration of 30 ug/mL was added in each well. (3) Isoflavone and ox-LDL group:0.5 ul assay medium with isoflavone at final concentration of 100 umol/L and 10 ul with ox-LDL at final concentration of 30 ug/mL in each well. (4) Isoflavone group:0.5 ul assay medium with isoflavone at final concentration of 100 umol/L in each well. The monocytes in 24-well tray were cultured in a 5%CO2 incubator at 37℃for 24 h and then ready for microRNA and cytokines analysis as mentioned above.ResultsClinical characteristics of the subjectsThere were no significant difference in age, gender, height, weight, body mass index (BMI), systolic and diastolic blood pressure, fasting blood glucose, Cr, BUN, UA, TG, TC, HDL-C and LDL-C among the three groups. Although the amount of white blood cells showed no significant difference, monocytes in peripheral blood were significantly increased in the UA and AMI groups. High sensitivity C-reactive protein, as one of the classic inflammation marker, was obviously increased in UA and AMI groups, even higher in AMI group.Due to the standard therapy of coronary artery disease, the applications of cardiovascular medicine including beta-blokers, angiotension-converting enzyme inhibitors, calcium-channel blockers, nitrates and statins showed no significant differences between UA and AMI groups.Relative mRNA expression of miR-424 in peripheral blood monocytes The<200 bp RNA were isolated from each subjects and the expression of miR-424 in peripheral blood monocytes were determined by using real-time PCR.The relative levels of miR-424 expression were significantly increased in UA (1.38±0.33, P<0.05) and AMI (3.69±0.92, P<0.001) groups as compared to the control group (1.00±0.21). Compared with UA group, the relative miR-424 expression was further elevated in AMI group (3.69±0.92 vs 1.38±0.33, P<0.01).Correlation between the relative miR-424 expression and clinical characteristics Pearson correlation was performed between the relative miR-424 expression and multiple clinical characteristics including body mass index, systolic blood pressure, diastolic blood pressure, the amount of monocytes in blood sample, fasting blood glucose, TC, LDL-C and hs-CRP, in order to explore the mechanism of miR-424 increasing in UA and AMI groups. The results showed that the relative miR-146a expression was significantly associated with the level of plasma hs-CRP and the amount of peripheral blood monocytes, indicating that the mechanism of miR-424 increasing was related to inflammation.The protein expression of NFI-A in peripheral monocytes The protein expression of NFI-A was significantly reduced in UA and AMI groups, especially in patients with acute myocardial infarction (AMI group:0.07±0.03;UA group:1.08±0.03; Control group:1.76±0.09).The levels of inflammatory cytokines in plasma We determined the levels of IL-6 and TNF-a in plasma from each subjects and, which were the two main cytokines secreted by macrophages. The results showed that the levels of IL-6 and TNF-a were significantly high in UA and AMI groups. The levels of IL-6 and TNF-a were further elevated in AMI group as compared to UA group (IL-6:5.92±0.86 pg/ml vs 3.68±0.64 pg/ml, P<0.05; TNF-a:263.93±15.71 pg/ml vs 52.69±9.47 pg/ml, P<0.01).Correlation between the relative miR-424 expression and inflammatory cytokines The increased miR-424 expression could inhibit the translation and expression of NFI-A protein, and then promote the differentiation of monocytes into macrophages. The increased macrophages secreted more inflammatory cytokines to peripheral blood. Therefore, the miR-424 expression should correlate with the levels of IL-6 and TNF-a in subjects of three groups. In the present study, we performed Pearson linear correlation and found that there were significant linear correlation between the relative miR-424 expression and the levels of IL-6 and TNF-a (miR-424 with IL-6:γ=0.461, P< 0.01; miR-424 with TNF-α:γ=0.453, P<0.01).Isoflavone inhibited the level of miR-424 in vitro Monocytes from healthy donors were cultured with different assay media in different groups. The results showed that the level of miR-424 was significantly increased in ox-LDL group as compared to controls. However, the miR-424 in ox-LDL and isoflavone group was significantly reduced as compared to ox-LDL group but similar to the controls. The level of miR-424 in isoflavone treated group showed no significant difference from controls.Meanwhile, the levels of IL-6 and TNF-αwere significantly higher in ox-LDL group than in the controls, but were significantly lower in ox-LDL and isoflavone group. The levels of IL-6 and TNF-αin isoflavone treated group showed no significant difference from controls.ConclusionsIn the development of atherosclerosis, miR-424 and its target protein—NFI-A have been involved and play important roles in the differentiation of monocytes to macrophages. Isoflavone can reverse ox-LDL induced miR-424 in monocytes in vitro, but detailed mechanisms are still needed to explore in the future. |
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