白藜芦醇通过重塑肠道菌群改善动脉粥样硬化的作用及机制研究
发布时间：2018-08-06 17:23
【摘要】：动脉粥样硬化(atherosclerosis,AS)引起的心血管疾病(cardiovascular disease,CVD)已经成为威胁人类健康的首要死因。寻找有效抗AS的策略对于降低CVD的发病率和死亡率具有十分重要的科学意义。白藜芦醇(resveratrol,RSV)是膳食中天然存在的多酚类植物化合物,主要富含于葡萄等浆果类水果、花生及红酒中,具有多种生物学活性。大量研究表明,RSV对AS的发生发展具有明显的防治作用,但机制尚未明确。同时许多证据表明,RSV的血浆浓度低、代谢速率快,其较低的生物利用率与其显著的生物学活性之间形成了一个明显的“悖论”。体内较低水平的RSV如何发挥显著的抗AS作用,其抗AS作用是否还存在其它机制,如何为这一“悖论”提供合理解释,是亟待解决的重要科学问题。近年来研究发现,肠道菌群组分改变和功能失调在AS的发生发展中起重要作用,已成为防治AS的新靶点。肠道菌群由上万亿个共生微生物组成,以人体内的营养成分维持生存和代谢,并与人体共同对外界的环境因素做出反应,进行代谢和免疫活动维持人体健康。研究证实,肠道菌群一方面通过代谢膳食卵磷脂产生三甲胺(trimethylamine,TMA),而后TMA经肝脏黄素单加氧酶(flavin monooxygenases,FMO)活化生成一种新的独立的AS危险因子氧化三甲胺(trimethylamine-N-oxide,TMAO),而诱发AS发生;另一方面肠道菌群可通过减少胆汁酸在肠道的水解,从而抑制肝脏胆汁酸的合成,引起胆固醇的代谢紊乱进而促进AS的进程。此外,越来越多的证据表明包括RSV在内的多种植物化学物能够通过影响和重塑肠道菌群进而发挥健康功效。因此,基于国内外研究进展,我们提出如下假说:RSV可能通过影响肠道菌群从而分别减少TMAO生成和调节胆汁酸代谢,进而降低AS发生风险。为了验证该假说,本课题以肠道菌群为靶点,从肠道菌群依赖的TMAO生成途径及胆汁酸代谢途径两个关键环节入手,以C57BL/6J和ApoE-/-小鼠为研究对象,利用肠道菌群研究的主要技术方法(16Sr RNA高通量测序、q PCR、细菌培养)和分子营养学技术方法(免疫印迹、qPCR、LC/MS)等,深入揭示肠道菌群在RSV抗AS中的作用及潜在的分子机制。本研究的主要实验结果和结论如下:1、rsv能显著抑制模型动物as斑块形成,肠道菌群在其中有重要作用。高胆碱膳食可显著促进apoe-/-小鼠as斑块的形成(p0.01),而加入rsv或抗生素后,可明显减轻高胆碱膳食诱导的as斑块形成(p0.01);同时,采用抗生素抑制肠道菌群后,可显著削弱rsv对高胆碱膳食诱导的as的改善作用(p0.05)。此外,rsv单独处理亦可显著抑制as的形成(p0.01)。2、rsv能够通过调控肠道菌群抑制tmao生成。(1)rsv可显著抑制tmao的生成。rsv可显著抑制单次或长期高胆碱膳食引起的血浆tma和tmao的生成(p0.01);同时,与普食组比较,rsv单独处理亦可显著降低小鼠血浆tma和tmao的含量(p0.01)。(2)rsv可显著改变肠道菌群结构,抑制肠tma的生成。rsv可明显增加拟杆菌(bacteroides)丰度而降低普雷沃氏菌(prevotella)丰度(p0.05);经相关性分析和多重比较发现,普雷沃氏菌(prevotella)和拟杆菌(bacteroides)的丰度与血浆tma和tmao水平具有显著的相关性(p0.05);同时,rsv可显著抑制肠道细菌代谢胆碱产生tma(p0.01)。此外,同一批小鼠在rsv干预一个月后,再利用普通膳食洗脱一个月,rsv对单次胆碱灌胃引起的血浆tma和tmao生成的抑制作用被显著的减弱(p0.01)。结果表明,肠道菌群在rsv抑制tmao生成中发挥关键作用。3、rsv能够通过调控肠道菌群影响胆汁酸代谢。(1)rsv能显著影响胆汁酸代谢,促进肝脏胆汁酸合成,维持胆固醇的代谢平衡。rsv可降低小鼠肝脏胆固醇的含量、增加胆汁酸池大小、促进粪便胆汁酸的外排、减少小肠肠壁组织中胆汁酸的含量、增加胆汁和肠道内胆汁酸的含量(p0.01),而对血浆和肝脏组织中的胆汁酸水平没有显著影响(p0.05);同时,rsv还能增加胆汁tca/tβmca的比值、促进肝脏cyp7a1的表达(p0.01)。(2)rsv可显著改变肠道菌群结构,促进肝脏胆汁酸合成。rsv可显著增加乳酸杆菌(lactobacillus)和双歧杆菌(bifidobacterium)的丰度,从而增加肠道胆汁酸水解酶活性,以促进胆汁酸在肠道的分解代谢,进而促进粪便胆汁酸的外排,最终促进胆汁酸在肝脏的合成。同时,使用抗生素抑制肠道菌群后,rsv对肝脏胆汁酸合成的促进作用被显著削弱(p0.05)。结果表明,肠道菌群在rsv调节胆汁酸代谢中发挥关键作用。4、RSV通过抑制肠肝FXR/FGF15轴促进肝脏胆汁酸合成。RSV能显著抑制肠道FGF15的表达(p0.01),而对肠FXR和肝FXR/小异二聚体伴侣(SHP)的表达没有显著作用(p0.05)。FXR抑制剂作用后,RSV不能进一步的抑制FGF15表达和促进肝CYP7A1表达;而FXR激动剂可逆转RSV引起的FGF15表达减少和CYP7A1表达增加。这些结果提示,肠肝FXR/FGF15轴在RSV调节肝脏胆汁酸合成中发挥重要作用。综上所述,RSV能够通过改变肠道菌群结构减少TMAO的生成;同时,通过影响肠道菌群结构,抑制肠肝FXR/FGF15轴,促进肝脏胆汁酸的合成,进而实现其抗AS的作用。该结果一方面为RSV的临床应用提供了新的科学试验依据,另一方面也将为CVD膳食营养防治提供新的策略和指导。
[Abstract]:The cardiovascular disease (cardiovascular disease, CVD) caused by atherosclerosis (AS) has become the primary cause of threat to human health. Finding an effective anti AS strategy is of great scientific significance for reducing the incidence and mortality of CVD. Resveratrol (resveratrol, RSV) is a natural polyphenol in the diet. Plant compounds, mainly rich in berries and other berries, peanuts and red wine, have a variety of biological activities. A large number of studies have shown that RSV has a clear prevention and control effect on the development of AS, but the mechanism is not clear. At the same time, many evidence shows that the plasma concentration of RSV is low and the metabolic rate is fast, and its lower bioavailability is more obvious. There is a clear "paradox" between the biological activity and how the lower level of RSV in the body plays a significant anti AS effect, and whether there are other mechanisms for its anti AS action. How to provide a reasonable explanation for this "paradox" is an important scientific problem to be solved. In recent years, the study found that the intestinal flora changes and works. Dysregulation plays an important role in the development of AS and has become a new target for the prevention and control of AS. Intestinal microflora consists of trillions of symbiotic microbes that maintain survival and metabolism in the human body, react with the human body to the external environmental factors, carry out metabolic and immune activities to maintain human health. On the one hand, the group produces trimethylamine (trimethylamine, TMA) by the metabolic dietary lecithin, and then TMA is activated by the liver flavin monooxygenase (flavin monooxygenases, FMO) to generate a new independent AS risk factor (trimethylamine-N-oxide, TMAO), which induces AS; on the other hand, intestinal microflora can be reduced by reducing bile acids. The hydrolysis of the intestinal tract, which inhibits the synthesis of bile acids in the liver, causes the metabolic disorder of cholesterol and thus promotes the process of AS. In addition, more and more evidence suggests that many phytochemicals, including RSV, can affect and reshape the intestinal microflora and then play a healthy role. Hypothesis: RSV may reduce the TMAO generation and regulation of bile acid metabolism by affecting the intestinal microflora and thus reducing the risk of AS. In order to verify this hypothesis, the subject takes the intestinal flora as the target, from the two key links of the intestinal microflora dependent TMAO generation and bile acid metabolism, and C57BL/6J and ApoE-/- mice. The main technical methods of intestinal microflora (16Sr RNA high throughput sequencing, Q PCR, bacterial culture) and molecular nutrition technology (Western blot, qPCR, LC/MS) were used to reveal the role of intestinal microflora in RSV anti AS and its potential molecular mechanism. The main experimental results and conclusions of this study were as follows: 1, RSV can significantly inhibit the model. The intestinal flora plays an important role in the formation of as plaque in type animals. High choline diet can significantly promote the formation of as plaque in apoe-/- mice (P0.01), while RSV or antibiotics can significantly reduce the formation of as plaque induced by high choline diet (P0.01). At the same time, the use of antibiotics to inhibit intestinal microflora can significantly weaken the RSV to the high choline diet. The improved effect of induced as (P0.05). In addition, RSV alone can significantly inhibit the formation of as (P0.01).2, RSV can inhibit TMAO generation by regulating intestinal flora. (1) RSV significantly inhibits TMAO production.Rsv can significantly inhibit the production of plasma TMA and production caused by a single or long-term high cholinergic diet; meanwhile, compared with the general food group, The content of TMA and TMAO in the plasma of mice was significantly reduced (P0.01). (2) RSV could significantly change the structure of intestinal flora, and the inhibition of the formation of.Rsv in intestinal TMA could significantly increase the abundance of Bacillus (Bacteroides) and decrease the abundance of Poulet Was (Prevotella), and the correlation analysis and multiple comparison found that the bacteria (Prevotella) and Poulet Was (Prevotella) The abundance of Bacteroides has a significant correlation with plasma TMA and TMAO levels (P0.05); at the same time, RSV can significantly inhibit the production of TMA (P0.01) by choline metabolism in intestinal bacteria. In addition, a month after the intervention of RSV in the same group of mice, the inhibition of TMA and TMAO production of plasma by RSV to the single choline perfusion of a single month. The production was significantly weakened (P0.01). The results showed that intestinal flora played a key role in RSV inhibition of TMAO production.3, RSV could affect bile acid metabolism by regulating intestinal microflora. (1) RSV could significantly affect bile acid metabolism, promote liver bile acid synthesis, and maintain the metabolic balance of bile solid alcohols,.Rsv can reduce the content of liver cholesterol in mice. Increase the size of bile acid pool, promote the excretion of bile acid in the feces, reduce the content of bile acid in the intestinal wall tissue, increase the bile acid content in the bile and intestinal tract (P0.01), but have no significant effect on the bile acid level in the plasma and liver tissues (P0.05); at the same time, RSV can increase the ratio of bile tca/t beta MCA to the liver CYP7A1. (P0.01) (2) (2) RSV can significantly change the intestinal microflora structure, promote the liver bile acid synthesis of.Rsv to significantly increase the abundance of Lactobacillus (Lactobacillus) and Bifidobacterium (Bifidobacterium), thereby increasing the activity of bile acid hydrolase in the intestinal tract, promoting the catabolism of bile acids in the intestinal tract, and thus promoting the excretion of fecal bile acids, and eventually promoting the excretion of bile acids. The effect of RSV on liver bile acid synthesis was significantly weakened after the use of antibiotics to inhibit intestinal microflora (P0.05). The results showed that the intestinal flora played a key role in RSV regulating bile acid metabolism,.4, RSV could significantly inhibit the intestinal bile acid synthesis by inhibiting the FXR/ FGF15 axis of the intestinal liver, which could significantly inhibit the intestinal bile acid synthesis of the intestine. The expression of FGF15 (P0.01) had no significant effect on the expression of FXR and FXR/ small two polymer chaperone (SHP) in the intestine (P0.05). After the action of P0.05.FXR inhibitor, RSV could not further inhibit the expression of FGF15 and promote the expression of the liver CYP7A1. F15 axis plays an important role in the RSV regulation of liver bile acid synthesis. To sum up, RSV can reduce the formation of TMAO by changing the intestinal microflora structure; at the same time, by affecting the intestinal flora structure, inhibiting the intestinal liver FXR/FGF15 axis, promoting the synthesis of liver bile acid, and then realizing its anti AS effect. This result is the clinical application of RSV on the one hand. It provides a basis for new scientific experiments, and on the other hand, it will provide new strategies and guidance for the prevention and control of dietary nutrition in CVD.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.5
[Abstract]:The cardiovascular disease (cardiovascular disease, CVD) caused by atherosclerosis (AS) has become the primary cause of threat to human health. Finding an effective anti AS strategy is of great scientific significance for reducing the incidence and mortality of CVD. Resveratrol (resveratrol, RSV) is a natural polyphenol in the diet. Plant compounds, mainly rich in berries and other berries, peanuts and red wine, have a variety of biological activities. A large number of studies have shown that RSV has a clear prevention and control effect on the development of AS, but the mechanism is not clear. At the same time, many evidence shows that the plasma concentration of RSV is low and the metabolic rate is fast, and its lower bioavailability is more obvious. There is a clear "paradox" between the biological activity and how the lower level of RSV in the body plays a significant anti AS effect, and whether there are other mechanisms for its anti AS action. How to provide a reasonable explanation for this "paradox" is an important scientific problem to be solved. In recent years, the study found that the intestinal flora changes and works. Dysregulation plays an important role in the development of AS and has become a new target for the prevention and control of AS. Intestinal microflora consists of trillions of symbiotic microbes that maintain survival and metabolism in the human body, react with the human body to the external environmental factors, carry out metabolic and immune activities to maintain human health. On the one hand, the group produces trimethylamine (trimethylamine, TMA) by the metabolic dietary lecithin, and then TMA is activated by the liver flavin monooxygenase (flavin monooxygenases, FMO) to generate a new independent AS risk factor (trimethylamine-N-oxide, TMAO), which induces AS; on the other hand, intestinal microflora can be reduced by reducing bile acids. The hydrolysis of the intestinal tract, which inhibits the synthesis of bile acids in the liver, causes the metabolic disorder of cholesterol and thus promotes the process of AS. In addition, more and more evidence suggests that many phytochemicals, including RSV, can affect and reshape the intestinal microflora and then play a healthy role. Hypothesis: RSV may reduce the TMAO generation and regulation of bile acid metabolism by affecting the intestinal microflora and thus reducing the risk of AS. In order to verify this hypothesis, the subject takes the intestinal flora as the target, from the two key links of the intestinal microflora dependent TMAO generation and bile acid metabolism, and C57BL/6J and ApoE-/- mice. The main technical methods of intestinal microflora (16Sr RNA high throughput sequencing, Q PCR, bacterial culture) and molecular nutrition technology (Western blot, qPCR, LC/MS) were used to reveal the role of intestinal microflora in RSV anti AS and its potential molecular mechanism. The main experimental results and conclusions of this study were as follows: 1, RSV can significantly inhibit the model. The intestinal flora plays an important role in the formation of as plaque in type animals. High choline diet can significantly promote the formation of as plaque in apoe-/- mice (P0.01), while RSV or antibiotics can significantly reduce the formation of as plaque induced by high choline diet (P0.01). At the same time, the use of antibiotics to inhibit intestinal microflora can significantly weaken the RSV to the high choline diet. The improved effect of induced as (P0.05). In addition, RSV alone can significantly inhibit the formation of as (P0.01).2, RSV can inhibit TMAO generation by regulating intestinal flora. (1) RSV significantly inhibits TMAO production.Rsv can significantly inhibit the production of plasma TMA and production caused by a single or long-term high cholinergic diet; meanwhile, compared with the general food group, The content of TMA and TMAO in the plasma of mice was significantly reduced (P0.01). (2) RSV could significantly change the structure of intestinal flora, and the inhibition of the formation of.Rsv in intestinal TMA could significantly increase the abundance of Bacillus (Bacteroides) and decrease the abundance of Poulet Was (Prevotella), and the correlation analysis and multiple comparison found that the bacteria (Prevotella) and Poulet Was (Prevotella) The abundance of Bacteroides has a significant correlation with plasma TMA and TMAO levels (P0.05); at the same time, RSV can significantly inhibit the production of TMA (P0.01) by choline metabolism in intestinal bacteria. In addition, a month after the intervention of RSV in the same group of mice, the inhibition of TMA and TMAO production of plasma by RSV to the single choline perfusion of a single month. The production was significantly weakened (P0.01). The results showed that intestinal flora played a key role in RSV inhibition of TMAO production.3, RSV could affect bile acid metabolism by regulating intestinal microflora. (1) RSV could significantly affect bile acid metabolism, promote liver bile acid synthesis, and maintain the metabolic balance of bile solid alcohols,.Rsv can reduce the content of liver cholesterol in mice. Increase the size of bile acid pool, promote the excretion of bile acid in the feces, reduce the content of bile acid in the intestinal wall tissue, increase the bile acid content in the bile and intestinal tract (P0.01), but have no significant effect on the bile acid level in the plasma and liver tissues (P0.05); at the same time, RSV can increase the ratio of bile tca/t beta MCA to the liver CYP7A1. (P0.01) (2) (2) RSV can significantly change the intestinal microflora structure, promote the liver bile acid synthesis of.Rsv to significantly increase the abundance of Lactobacillus (Lactobacillus) and Bifidobacterium (Bifidobacterium), thereby increasing the activity of bile acid hydrolase in the intestinal tract, promoting the catabolism of bile acids in the intestinal tract, and thus promoting the excretion of fecal bile acids, and eventually promoting the excretion of bile acids. The effect of RSV on liver bile acid synthesis was significantly weakened after the use of antibiotics to inhibit intestinal microflora (P0.05). The results showed that the intestinal flora played a key role in RSV regulating bile acid metabolism,.4, RSV could significantly inhibit the intestinal bile acid synthesis by inhibiting the FXR/ FGF15 axis of the intestinal liver, which could significantly inhibit the intestinal bile acid synthesis of the intestine. The expression of FGF15 (P0.01) had no significant effect on the expression of FXR and FXR/ small two polymer chaperone (SHP) in the intestine (P0.05). After the action of P0.05.FXR inhibitor, RSV could not further inhibit the expression of FGF15 and promote the expression of the liver CYP7A1. F15 axis plays an important role in the RSV regulation of liver bile acid synthesis. To sum up, RSV can reduce the formation of TMAO by changing the intestinal microflora structure; at the same time, by affecting the intestinal flora structure, inhibiting the intestinal liver FXR/FGF15 axis, promoting the synthesis of liver bile acid, and then realizing its anti AS effect. This result is the clinical application of RSV on the one hand. It provides a basis for new scientific experiments, and on the other hand, it will provide new strategies and guidance for the prevention and control of dietary nutrition in CVD.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.5
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