白藜蘆醇通過(guò)重塑腸道菌群改善動(dòng)脈粥樣硬化的作用及機(jī)制研究
發(fā)布時(shí)間：2018-08-06 17:23
【摘要】：動(dòng)脈粥樣硬化(atherosclerosis,AS)引起的心血管疾病(cardiovascular disease,CVD)已經(jīng)成為威脅人類健康的首要死因。尋找有效抗AS的策略對(duì)于降低CVD的發(fā)病率和死亡率具有十分重要的科學(xué)意義。白藜蘆醇(resveratrol,RSV)是膳食中天然存在的多酚類植物化合物,主要富含于葡萄等漿果類水果、花生及紅酒中,具有多種生物學(xué)活性。大量研究表明,RSV對(duì)AS的發(fā)生發(fā)展具有明顯的防治作用,但機(jī)制尚未明確。同時(shí)許多證據(jù)表明,RSV的血漿濃度低、代謝速率快,其較低的生物利用率與其顯著的生物學(xué)活性之間形成了一個(gè)明顯的“悖論”。體內(nèi)較低水平的RSV如何發(fā)揮顯著的抗AS作用,其抗AS作用是否還存在其它機(jī)制,如何為這一“悖論”提供合理解釋,是亟待解決的重要科學(xué)問(wèn)題。近年來(lái)研究發(fā)現(xiàn),腸道菌群組分改變和功能失調(diào)在AS的發(fā)生發(fā)展中起重要作用,已成為防治AS的新靶點(diǎn)。腸道菌群由上萬(wàn)億個(gè)共生微生物組成,以人體內(nèi)的營(yíng)養(yǎng)成分維持生存和代謝,并與人體共同對(duì)外界的環(huán)境因素做出反應(yīng),進(jìn)行代謝和免疫活動(dòng)維持人體健康。研究證實(shí),腸道菌群一方面通過(guò)代謝膳食卵磷脂產(chǎn)生三甲胺(trimethylamine,TMA),而后TMA經(jīng)肝臟黃素單加氧酶(flavin monooxygenases,FMO)活化生成一種新的獨(dú)立的AS危險(xiǎn)因子氧化三甲胺(trimethylamine-N-oxide,TMAO),而誘發(fā)AS發(fā)生;另一方面腸道菌群可通過(guò)減少膽汁酸在腸道的水解,從而抑制肝臟膽汁酸的合成,引起膽固醇的代謝紊亂進(jìn)而促進(jìn)AS的進(jìn)程。此外,越來(lái)越多的證據(jù)表明包括RSV在內(nèi)的多種植物化學(xué)物能夠通過(guò)影響和重塑腸道菌群進(jìn)而發(fā)揮健康功效。因此,基于國(guó)內(nèi)外研究進(jìn)展,我們提出如下假說(shuō):RSV可能通過(guò)影響腸道菌群從而分別減少TMAO生成和調(diào)節(jié)膽汁酸代謝,進(jìn)而降低AS發(fā)生風(fēng)險(xiǎn)。為了驗(yàn)證該假說(shuō),本課題以腸道菌群為靶點(diǎn),從腸道菌群依賴的TMAO生成途徑及膽汁酸代謝途徑兩個(gè)關(guān)鍵環(huán)節(jié)入手,以C57BL/6J和ApoE-/-小鼠為研究對(duì)象,利用腸道菌群研究的主要技術(shù)方法(16Sr RNA高通量測(cè)序、q PCR、細(xì)菌培養(yǎng))和分子營(yíng)養(yǎng)學(xué)技術(shù)方法(免疫印跡、qPCR、LC/MS)等,深入揭示腸道菌群在RSV抗AS中的作用及潛在的分子機(jī)制。本研究的主要實(shí)驗(yàn)結(jié)果和結(jié)論如下:1、rsv能顯著抑制模型動(dòng)物as斑塊形成,腸道菌群在其中有重要作用。高膽堿膳食可顯著促進(jìn)apoe-/-小鼠as斑塊的形成(p0.01),而加入rsv或抗生素后,可明顯減輕高膽堿膳食誘導(dǎo)的as斑塊形成(p0.01);同時(shí),采用抗生素抑制腸道菌群后,可顯著削弱rsv對(duì)高膽堿膳食誘導(dǎo)的as的改善作用(p0.05)。此外,rsv單獨(dú)處理亦可顯著抑制as的形成(p0.01)。2、rsv能夠通過(guò)調(diào)控腸道菌群抑制tmao生成。(1)rsv可顯著抑制tmao的生成。rsv可顯著抑制單次或長(zhǎng)期高膽堿膳食引起的血漿tma和tmao的生成(p0.01);同時(shí),與普食組比較,rsv單獨(dú)處理亦可顯著降低小鼠血漿tma和tmao的含量(p0.01)。(2)rsv可顯著改變腸道菌群結(jié)構(gòu),抑制腸tma的生成。rsv可明顯增加擬桿菌(bacteroides)豐度而降低普雷沃氏菌(prevotella)豐度(p0.05);經(jīng)相關(guān)性分析和多重比較發(fā)現(xiàn),普雷沃氏菌(prevotella)和擬桿菌(bacteroides)的豐度與血漿tma和tmao水平具有顯著的相關(guān)性(p0.05);同時(shí),rsv可顯著抑制腸道細(xì)菌代謝膽堿產(chǎn)生tma(p0.01)。此外,同一批小鼠在rsv干預(yù)一個(gè)月后,再利用普通膳食洗脫一個(gè)月,rsv對(duì)單次膽堿灌胃引起的血漿tma和tmao生成的抑制作用被顯著的減弱(p0.01)。結(jié)果表明,腸道菌群在rsv抑制tmao生成中發(fā)揮關(guān)鍵作用。3、rsv能夠通過(guò)調(diào)控腸道菌群影響膽汁酸代謝。(1)rsv能顯著影響膽汁酸代謝,促進(jìn)肝臟膽汁酸合成,維持膽固醇的代謝平衡。rsv可降低小鼠肝臟膽固醇的含量、增加膽汁酸池大小、促進(jìn)糞便膽汁酸的外排、減少小腸腸壁組織中膽汁酸的含量、增加膽汁和腸道內(nèi)膽汁酸的含量(p0.01),而對(duì)血漿和肝臟組織中的膽汁酸水平?jīng)]有顯著影響(p0.05);同時(shí),rsv還能增加膽汁tca/tβmca的比值、促進(jìn)肝臟cyp7a1的表達(dá)(p0.01)。(2)rsv可顯著改變腸道菌群結(jié)構(gòu),促進(jìn)肝臟膽汁酸合成。rsv可顯著增加乳酸桿菌(lactobacillus)和雙歧桿菌(bifidobacterium)的豐度,從而增加腸道膽汁酸水解酶活性,以促進(jìn)膽汁酸在腸道的分解代謝,進(jìn)而促進(jìn)糞便膽汁酸的外排,最終促進(jìn)膽汁酸在肝臟的合成。同時(shí),使用抗生素抑制腸道菌群后,rsv對(duì)肝臟膽汁酸合成的促進(jìn)作用被顯著削弱(p0.05)。結(jié)果表明,腸道菌群在rsv調(diào)節(jié)膽汁酸代謝中發(fā)揮關(guān)鍵作用。4、RSV通過(guò)抑制腸肝FXR/FGF15軸促進(jìn)肝臟膽汁酸合成。RSV能顯著抑制腸道FGF15的表達(dá)(p0.01),而對(duì)腸FXR和肝FXR/小異二聚體伴侶(SHP)的表達(dá)沒(méi)有顯著作用(p0.05)。FXR抑制劑作用后,RSV不能進(jìn)一步的抑制FGF15表達(dá)和促進(jìn)肝CYP7A1表達(dá);而FXR激動(dòng)劑可逆轉(zhuǎn)RSV引起的FGF15表達(dá)減少和CYP7A1表達(dá)增加。這些結(jié)果提示,腸肝FXR/FGF15軸在RSV調(diào)節(jié)肝臟膽汁酸合成中發(fā)揮重要作用。綜上所述,RSV能夠通過(guò)改變腸道菌群結(jié)構(gòu)減少TMAO的生成;同時(shí),通過(guò)影響腸道菌群結(jié)構(gòu),抑制腸肝FXR/FGF15軸,促進(jìn)肝臟膽汁酸的合成,進(jìn)而實(shí)現(xiàn)其抗AS的作用。該結(jié)果一方面為RSV的臨床應(yīng)用提供了新的科學(xué)試驗(yàn)依據(jù),另一方面也將為CVD膳食營(yíng)養(yǎng)防治提供新的策略和指導(dǎo)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R543.5
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