茶多酚对脂代谢基因的调控作用及对人群血压影响的荟萃分析

发布时间：2018-05-07 23:08

本文选题：表没食子儿茶素没食子酸酯 + 胆固醇代谢　；参考：《北京协和医学院》2015年博士论文

【摘要】：研究背景及目的：冠状动脉粥样硬化性心脏病(冠心病)仍然是威胁人类健康的重要疾病。血中总胆固醇和低密度脂蛋白胆固醇(LDL-C)浓度的升高,已被确认为冠心病发生的最主要危险因素。健康的生活方式,如良好的饮食习惯可预防高脂血症的发生。绿茶是我国传统饮品,富含茶多酚,其中茶多酚单体表没食子儿茶素没食子酸酯(epigallocatechin gallate, EGCG)是绿茶中最具活性的分子。近年的流行病学研究提示饮用绿茶与心血管疾病死亡险呈负相关。一些基础研究表明茶多酚可通过抑制肠道内外源性胆固醇的吸收、增加肝脏LDL受体(LDLR)表达及活性、调节载脂蛋白的分泌等机制调节血LDL-C水平。但目前茶多酚对脂质代谢通路基因的直接作用以及可能的分子机制仍不清楚。长链非编码RNA (long nocoding RNA, IncRNA)是一类转录本长度超过200个核苷酸的RNA分子,能以RNA的形式在多种层面上(表观遗传、转录以及转录后水平)调控基因的表达,参与了剂量补偿效应、基因组印记、细胞发育分化等重要生物学过程。目前越来越多的研究揭示了IncRNA在心脏发育和心血管疾病(如冠心病)中发挥了重要作用,已成为心血管领域研究的新热点。因此,本研究通过生物芯片技术观察茶多酚EGCG对HepG2细胞脂质代谢通路基因和lncRNAs表达的影响,并初步探讨lncRNA对脂质代谢通路基因的调控作用。研究方法：人HepG2细胞经茶多酚EGCG (25μM)处理24小时后,抽提RNA,质检合格后杂交至Human Transcriptome Array 2.0(HTA2.0)芯片,进行mRNA和IncRNAs表达谱的分析并筛选出差异表达的基因。筛选标准为倍数变化(fold change)≥1.5 且 P0.05。通过生物信息学方法对差异表达的IncRNAs进行靶基因预测,建立IncRNAs和脂代谢相关基因的可能作用关系。为进一步研究IncRNA对靶基因的调控作用,我们利用RNA干扰技术降低相关IncRNA的表达,应用实时荧光定量多聚酶链反应和Western blot检测靶基因的mRNA和蛋白表达水平改变。研究结果：表达谱芯片结果显示与对照组相比,茶多酚EGCG处理后有27条脂质代谢相关基因表达差异具有统计学意义(P0.05),涉及脂质合成、转运与分解。其中EGCG可直接影响胆固醇平衡代谢调控的3个关键基因：羟甲基戊二酸单酰辅酶A还原酶(HMGCR)、LDLR及乙酰辅酶A乙酰转移酶2(ACAT2)的表达水平。同时EGCG处理后共有285条IncRNAs表达水平有统计学差异(P0.05)。生物信息学靶基因预测发现5个脂代谢相关基因包括HMGCR和ACAT2的表达可能受IncRNA的调控,其中IncRNA NONHSAT102202对HMGCR表达可能起着顺式调控作用。RNA干扰抑制IncRNA NONHSAT102202的表达后接受不同浓度EGCG (0,10,25 μM)处理,HMGCR mRNA和蛋白表达水平均显著性增高(P0.05)。结论：茶多酚EGCG可直接影响脂质代谢通路基因的表达,包括在胆固醇平衡调控中起关键作用的基因-LDLR, HMGCR和ACAT2。同时EGCG可上调或下调众多的IncRNAo。功能研究证实IncRNA NONHSAT102202可抑制HMGCR的表达。本研究结果提示IncRNAs可能是茶多酚降胆固醇作用的一个重要分子机制。研究背景及目的：高血压是心血管疾病的重要危险因素。在高血压防控中,尤其应关注正常高值血压(收缩压120-139 mmHg,舒张压80-89 mmHg).目前,我国约有3亿人为正常高值血压。正常高值血压者发展为高血压、发生心血管事件及死于心血管疾病的风险较120/80 mm Hg以下的血压正常者明显增高。正常高值血压在临床事件发生之前即可造成亚临床的心血管靶器官损害,因此需给予积极的干预。目前有关药物干预的循证依据不足,而采取生活方式的干预可能更具有公共健康意义。绿茶和红茶是世界上非常受欢迎的饮品,富含茶多酚,具有抗氧化应激和抗炎症的作用。本研究旨在系统评价茶多酚的摄入(绿茶或红茶)对正常高值血压者或高血压患者的降压作用。研究方法：计算机检索MEDLINE.EMBASE.Cochrane数据库(检索时间均从建库至2014年5月),语种限制为英文,全面收集有关茶多酚摄入对血压影响的随机对照试验,并追查所有纳入文献的参考文献。由两名评价者独立选择研究,进行文献的逐步筛查,并按照Cochrane系统评价方法,由2名评价者独立对纳入研究的质量进行评价和资料提取。运用加权均数差(weighted mean difference, WMD)和95%置信区间(condential interval.CI)作为血压前后变化的效应尺度。异质性采用I2和Cochrane Q检验,I250%或Q检验0.1认为存在异质性。若存在异质性,采用随机效应模型,否则运用固定效应模型进行统计。按照事先定义好的亚组分析探索临床特征(干预时程、种族、茶的类型、茶多酚的剂量、健康状况和咖啡因摄入)对茶多酚降压效应的影响。结果：共纳入25篇随机对照试验1,476例研究对象。其中6篇评价了茶多酚的即时降压效应(acute effect),平均观察时间为6.8小时；21篇评价了茶多酚的长期效应(long-term effect),中位观察时间为12周。荟萃分析显示茶多酚对收缩压和舒张压均无明显的即时降压效应(收缩压,95% CI-0.62,5.46；舒张压,95% CI-0.01,1.42)。而茶多酚的长期摄入可显著性地降低收缩压1.8 mmHg(95% CI-2.4.-1.1.I2=17.4%)和舒张压1.4 mmHg(95%CI-2.2,.0.6,I2=52.5%).根据茶的类型,荟萃分析显示绿茶可显著性地降低收缩压2.1 mmHg(95%CI-2.9,-1.2.I2=21.8%)和舒张压1.7 mmHg(95%CI-2.9,-0.5,I2=59.9%),红茶可降低收缩压1.4 mmHg(95%CI-2.4,-0.4,I2=9.7%)和舒张压1.1 mmHg(95%CI一1.9,-0.2.I2=22.9%).亚组分析显示显示摄入茶多酚≥12周后可更为显著地降低收缩压(WMD-2.6[95%CI-3.5,-1.7]mmHg,I2=0%)和舒张压(WMD-2.2[95%CI-3.0,-1.3]mmHg,I2=43.3%),而摄入的茶多酚的含量、种族、是否伴随心血管危险因素以及咖啡因的摄入对茶多酚的降压效应无明显作用。结论：长期的茶多酚摄入(≥12周)可显著降低正常高值血压者的血压水平。背景及目的冠心病是危害人类健康的头号杀手。我国目前有近4000万冠心病患者,每年死于冠心病及并发症者已超过100万。目前对冠心病尤其是急性冠脉综合征的患者(acute coronary syndromes, ACS)治疗中,经皮冠状动脉支架植入术(percutaneous coronary intervention, PCI)是最为行之有效的办法,极大地挽救了患者的生命。氯吡格雷与阿司匹林联合应用已经成为PCI术后的标准治疗方案,能明显减少心血管事件的发生率,降低支架内血栓形成的风险。但是,临床上仍然有2-3%的患者发生支架内血栓事件。这部分患者往往存在氯吡格雷治疗后的血小板高反应性(high on-treatment platelet reactivity, HTPR)。大量数据证实HTPR与PCI术后主要心血管不良事件(major adversecardiovascular events, MACE)发生有很强的相关性。其中残余血小板的活性可能是主要关键因素之一。替格瑞洛是一种新型的血小板ADP受体P2Y12拮抗剂,与同样作用于P2Y12受体的氯吡格雷不同,它无需经过肝脏代谢,本身已是活性状态,能够更加迅速、强效、持久的血小板抑制作用。PLATO(抑制血小板与患者转归)研究表明与氯吡格雷相比,替格瑞洛可显著降低ACS患者主要复合终点事件(心血管死亡、再发心梗和卒中)的发生风险,且不增加总体出血的风险。但是,PLATO研究主要基于国外人群,此外关于替格瑞洛在HTPR患者应用的研究也不多。因此,我们通过随机对照研究,运用快速血小板功能分析仪(VerifyNow-P2Y12)来评价替格瑞洛对PCI术后HTPR患者的抗血小板作用。并对替格瑞洛的安全性作一初步探讨,从而为PCI术后HTPR患者的合理治疗提供依据。研究方法连续入选2014年4月至2015年4月阜外心血管病医院12病区住院经冠脉造影确诊为冠心病并有介入指征行PCI术的患者。所有患者术前均服用氯吡格雷常规剂量75mg至少1周或少于一周但给予300mg负荷。根据血栓弹力图(Thrombelasography, TEG)进行初筛,当ADP抑制率50%且最大聚集幅度MA-ADP47mm,高度怀疑为HTPR,并于术后24h内采集空腹静脉血5m1运用VerifyNow-P2Y12系统检测P2Y12反应单位(P2Y12 reaction unit, PRU), PRU≥235定义为HTPR。将HTPR患者随机分为氯吡格雷组(150 mg qd)和替格瑞洛组(首次180 mg负荷,继以90mg bid维持),各组分别于服药后2、8、24 h再次采血运用VerifyNow-P2Y12分析比较两组对血小板活性的抑制作用。所有患者进行CYP2C19*2基因型鉴定。随访1个月,记录两组MACE、出血事件及药物不良反应情况。两组患者均常规接受阿司匹林100mg qd。研究结果1.本研究共入选465例经我院冠脉造影术确诊为冠心病(稳定型心绞痛或ACS)并行PCI术的患者其中有317例患者术后成功完成了TEG检测,共有53例怀疑为HTPR。经VerifyNow-P2Y12系统检测为HTPR患者45例,依据排除标准,最后纳入40例,随机分为氯吡格雷组(n=20)和替格瑞洛组(n=20)。两组在基线特征方面均无统计学差异,具有良好的可比性。2.两组服用抗血小板药物后,均可显著性地降低血小板残余活性(PRU)。与氯吡格雷组相比,替格瑞洛对血小板的抑制更为明显(P0.001)。在各个时间点上(2,8,24 h)替格瑞洛均比氯吡格雷更有效地降低PRU水平(P0.001)。3.服用替格瑞洛后2 h,所有患者PRU位于切点值(235)以下,在8,24 h检测PRU分别有1例(5%)及3例(15%)患者为HTPR。而氯吡格雷组,2,8,24 h检测HTPR分别为9例(45%),8例(40%),10例(50%),两组间HTPR发生率差异均有统计学差异(P0.05)。4.替格瑞洛组9例患者(45%)携带至少一个CYP2C19*2能缺失等位基因。在携带者和非携带者中,替格瑞洛均可显著降低PRU水平和减少HTPR的发生率(P0.001)。提示CYP2C19*2基因变异不影响替格瑞洛的抗血小板效应。5.随访1个月,两组均未发生MACE事件。替格瑞洛组有6例发生轻微出血,主要为皮肤瘀斑。1例发生小出血,为少量消化道出血。氯吡格雷组有5例轻微出血。两组在总体出血风险方面差异无统计学意义。此外,替格瑞洛组有5例患者(25%)出现呼吸困难,与氯吡格雷组相比,差异具有统计学意义(P0.05)。结论1.PCR术后HTPR患者应用替格瑞洛较高剂量氯吡格雷更能快速强效地抑制血小板活性,减少HTPR发生率。2.替格瑞洛的抗血小板效应不受CYP2C19*2基因变异的影响。3.替格瑞洛没有增加总体出血的风险,但呼吸困难发生率高于氯吡格雷组。
[Abstract]:Background and purpose: coronary atherosclerotic heart disease (coronary heart disease) remains an important disease that threatens human health. The increase in the concentration of total cholesterol and low density lipoprotein cholesterol (LDL-C) in blood has been identified as the most important risk factor for coronary heart disease. Healthy lifestyle, such as good eating habits, can be prevented. The occurrence of lipidemia. Green tea is a traditional Chinese drink rich in tea polyphenols and tea polyphenols (epigallocatechin gallate, EGCG) is the most active molecule in green tea. In recent years, epidemiological studies suggest that green tea drinking is negatively related to cardiovascular disease death risk. Some basic studies show tea. Polyphenols can inhibit the absorption of endogenous cholesterol and increase the expression and activity of LDL receptor (LDLR) in the liver, regulate the secretion of apolipoprotein and regulate the level of blood LDL-C. However, the direct effect of tea polyphenols on the lipid metabolism pathway and the possible molecular mechanism are still unclear. Long chain non coded RNA (long nocoding RNA, In) CRNA) is a class of transcriptional transcriptional RNA molecules with more than 200 nucleotides and can regulate gene expression in a variety of layers (epigenetic, transcriptional, and post transcriptional levels) in the form of RNA, and participates in important biological processes such as dose compensation, genomic imprinting, and cell development differentiation. At present, more and more studies have revealed that IncRNA is in the heart. The important role of dirty development and cardiovascular disease (such as coronary heart disease) has become a new hot spot in the research of cardiovascular field. Therefore, the effect of tea polyphenols EGCG on the gene of lipid metabolism pathway and the expression of lncRNAs in HepG2 cells is observed by biochip technology, and the effect of lncRNA on the regulation of lipid metabolism pathway is preliminarily discussed. Methods: after 24 hours' treatment of human HepG2 cells through tea polyphenols (25 u M), RNA was extracted and RNA was extracted and hybridized to Human Transcriptome Array 2 (HTA2.0) chip after quality examination. The expression profiles of mRNA and IncRNAs were analyzed and the differentially expressed genes were screened. The screening criteria were multiple (fold change) above 1.5 and through bioinformatics. The target gene was predicted for differentially expressed IncRNAs, and the possible relationship between IncRNAs and lipid metabolism related genes was established. In order to further study the regulation of IncRNA on target gene, we use RNA interference technique to reduce the expression of related IncRNA, and use real-time fluorescent quantitative polymerase chain reaction and Western blot to detect mRNA of target gene. The results of the protein expression level change. The results of the expression spectrum chip showed that compared with the control group, there were 27 differences in the expression of lipid metabolism related genes after the treatment of the tea polyphenols EGCG (P0.05), involving lipid synthesis, transport and decomposition. Among them, EGCG could directly affect the 3 key genes of the regulation of cholesterol balance metabolism: hydroxymethyl. The expression level of glutaric acid monoyl coenzyme A reductase (HMGCR), LDLR and acetyl coenzyme A acetyltransferase 2 (ACAT2). At the same time, there was a total of 285 IncRNAs expressions after EGCG treatment (P0.05). The prediction of bioinformatics target gene found that the expression of 5 lipid metabolism related genes, including HMGCR and ACAT2, may be regulated by IncRNA, including IncRN A NONHSAT102202 may play a cis role in the expression of HMGCR in the expression of.RNA interference and the expression of IncRNA NONHSAT102202 with different concentrations EGCG (0,10,25 u M) treatment, HMGCR mRNA and protein expression levels are significantly increased (P0.05). Conclusion: tea polyphenols can directly affect the expression of lipid metabolism pathway genes, including cholesterol level. Genes -LDLR, HMGCR and ACAT2., which play a key role in the regulation and control, can up or down a large number of IncRNAo. functions, which confirm that IncRNA NONHSAT102202 can inhibit the expression of HMGCR. The results of this study suggest that IncRNAs may be an important molecular mechanism of the effect of tea polyphenols on cholesterol lowering. Research background and purpose: hypertension is the heart and blood. In the prevention and control of hypertension, in the prevention and control of hypertension, we should pay special attention to normal high blood pressure (systolic pressure 120-139 mmHg, diastolic pressure 80-89 mmHg). At present, about 300 million people in our country are normal high blood pressure. Normal high blood pressure people develop hypertension, the risk of cardiovascular events and death of cardiovascular disease is less than 120/80 mm Hg Normal high blood pressure can cause subclinical cardiovascular target damage before the occurrence of clinical events. Therefore, a positive intervention is needed. The evidence of drug intervention is not evidence-based, and the intervention of lifestyle may be more public health. Green tea and black tea are very happy in the world. The purpose of this study is to systematically evaluate the antihypertensive effects of tea polyphenols (green tea or black tea) on normal high blood pressure or hypertensive patients. Research methods: the computer retrieval MEDLINE.EMBASE.Cochrane database (retrieval time from construction to May 2014) A randomized controlled trial of tea polyphenols intake on the influence of tea polyphenols intake on blood pressure was collected and all the references were reviewed. Two evaluators selected the study independently, screened the literature, and evaluated and funded the quality of the study independently by 2 evaluators according to the Cochrane system evaluation method. Material extraction. Using weighted mean difference (weighted mean difference, WMD) and 95% confidence interval (condential interval.CI) as the effect scale of the changes before and after blood pressure. Heterogeneity uses I2 and Cochrane Q test, I250% or Q test 0.1 considers the existence of heterogeneity. If heterogeneity exists, the random effect model is used, otherwise the fixed effect model is used. The effect of clinical features (intervention duration, race, tea type, tea polyphenols dose, health status and caffeine intake) on the antihypertensive effect of tea polyphenols was investigated in accordance with a pre defined subgroup analysis. Results: a total of 1476 subjects were included in 25 randomized controlled trials. 6 of them evaluated the immediate hypotension effect of tea polyphenols (acute Effect), the average observation time was 6.8 hours; 21 articles evaluated the long-term effect of tea polyphenols (long-term effect), and the median observation time was 12 weeks. The meta-analysis showed that there was no obvious immediate hypotensive effect on systolic and diastolic pressure (systolic pressure, 95% CI-0.62,5.46; diastolic pressure, 95% CI-0.01,1.42). The systolic pressure was significantly reduced by 1.8 mmHg (95% CI-2.4.-1.1.I2=17.4%) and diastolic pressure 1.4 mmHg (95%CI-2.2,.0.6, I2=52.5%). According to the type of tea, the meta-analysis showed that green tea could significantly reduce the systolic pressure 2.1 mmHg (95%CI-2.9, -1.2.I2=21.8%) and diastolic pressure 1.7 mmHg (95%CI-2.9, -0.5, and 1.4), and black tea could reduce the systolic pressure 1.4 .4, -0.4, I2=9.7%) and diastolic pressure of 1.1 mmHg (95%CI 1.9, -0.2.I2=22.9%). Subgroup analysis showed that after intake of tea polyphenols more than 12 weeks, the systolic pressure (WMD-2.6[95%CI-3.5, -1.7]mmHg, I2=0%) and diastolic pressure (WMD-2.2[95%CI-3.0, -1.3]mmHg, I2= 43.3%) were significantly reduced, and the content of tea polyphenols intake, race, and cardiovascular risk Factors and caffeine intake have no obvious effect on the antihypertensive effect of tea polyphenols. Conclusion: long-term tea polyphenols intake (more than 12 weeks) can significantly reduce the level of normal high blood pressure. Background and objective coronary heart disease is the leading killer of human health. There are nearly 40 million coronary heart disease patients in China, died of coronary heart disease and the same year. More than 1 million of the patients have been diagnosed with coronary artery disease (acute coronary syndromes, ACS). Percutaneous coronary stent implantation (percutaneous coronary intervention, PCI) is the most effective way to save the patient's life. The combination of clopidogrel and aspirin should be combined. The use of a standard treatment program after PCI can significantly reduce the incidence of cardiovascular events and reduce the risk of thrombosis in the stent. However, stent thrombosis in patients with 2-3% is still in the clinic. These patients often have high on-treatment platelet reactiv after clopidogrel treatment. Ity, HTPR). A large number of data confirmed that HTPR has a strong correlation with the major cardiovascular adverse events (major adversecardiovascular events, MACE) after PCI. The activity of residual platelets may be one of the major key factors. The vililo is a new type of platelet ADP receptor P2Y12 antagonist, which is also used in P2Y12 receptor. Unlike clopidogrel, which does not need to be metabolized by the liver and itself is active, a more rapid, powerful and persistent platelet inhibition.PLATO (inhibition of platelet and patient outcomes) studies show that, compared with clopidogrel, velogelo significantly reduces the main complex endpoint events in ACS patients (cardiovascular death, recurrent myocardial infarction and death). However, PLATO studies are mainly based on foreign populations, and there are few studies on the use of tigreloo in HTPR patients. Therefore, we used a randomized controlled study to evaluate the resistance of tigreloe to HTPR patients after PCI with a rapid platelet function analyzer (VerifyNow-P2Y12). Thrombocytopenia, and a preliminary study of the safety of T - greelo, provides a basis for the rational treatment of HTPR patients after PCI. The methods of study were continuously selected from April 2014 to April 2015 in 12 patients in Fuwai Hospital of Cardiovascular Disease, which were diagnosed as coronary heart disease with coronary artery angiography and had interventional indications for PCI. All patients were pretreated before operation. The routine dose of clopidogrel was given for at least 1 weeks or less than one week but given 300mg load. The initial screening was carried out according to the thrombus elasto map (Thrombelasography, TEG), when the ADP inhibition rate was 50% and the maximum aggregation amplitude was MA-ADP47mm, and the HTPR was highly suspected, and the 5m1 using VerifyNow-P2Y12 system in 24h in 24h was detected in 24h, and the P2Y12 reaction was detected in 24h. P2Y12 reaction unit (PRU) and PRU > 235 were defined as HTPR. to randomly divide HTPR patients into clopidogrel group (150 mg QD) and tenogelo group (first 180 mg load, followed by 90mg bid). Each group was divided into two groups to compare the inhibitory effect on platelet activity. 1 months followed up for 1 months, two groups of MACE, bleeding events and adverse drug reactions were recorded. The two groups were routinely received aspirin 100mg qd. results in 1. study, and 465 patients with coronary heart disease (stable angina or ACS) and 317 patients with coronary heart disease (stable angina or ACS) were enrolled in this study. After successful completion of the TEG test, 53 cases were suspected to be HTPR. by VerifyNow-P2Y12 system for 45 cases of HTPR patients, according to the exclusion criteria, and finally included in 40 cases, randomly divided into the clopidogrel group (n=20) and tenogelo group (n=20). The two groups were not statistically different in the baseline characteristics, with a good comparability of the.2. two groups taking antiplatelet. Compared with the clopidogrel group, the inhibitory effect of Grillo on platelets was significantly lower than that of the clopidogrel group (P0.001). At all time points (2,8,24 h), tgrel was more effective than clopidogrel in reducing PRU level (P0.001).3. after taking the 2 h of togelo, and all patients were at the cut point value (235). In 8,24 h, 1 (5%) and 3 (15%) patients were HTPR. and clopidogrel, 2,8,24 h was used to detect HTPR in 9 cases (45%), 8 (40%), 10 (50%), and there were statistically significant differences in the incidence of HTPR in group two (P0.05).4. (45%) carrying at least one CYP2C19*2 allele. In non carriers, Grillo could significantly reduce the level of PRU and reduce the incidence of HTPR (P0.001). It was suggested that the CYP2C19*2 gene mutation did not affect the platelet effect of the antiplatelet effect of 1 months, and there were no MACE events in the two groups. There were 6 cases of slight bleeding in the group of the greelo group, which was mainly caused by small bleeding in the.1 cases of skin ecchymosis, and a small amount of elimination. There were 5 cases of mild bleeding in clopidogrel group. There was no significant difference in the overall bleeding risk between the two groups.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R544.1

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