ORIGINAL ARTICLE
Conjugated bile acids are nutritionally re-programmable antihypertensive metabolites
アブストラクト
背景
高血圧は、世界の死亡率に影響を与える最大の危険因子である。薬物療法があるにもかかわらず、コントロールできない高血圧が増加しており、新規かつ持続可能な治療法の開発が急務となっています。現在、腸内細菌叢は血圧調節において重要な存在であると認識されており、そのような新しい方法の1つは、宿主と微生物叢の相互作用を介して代謝物が伝達される腸-肝臓軸を標的とすることです。腸-肝臓軸のどの代謝物が血圧を調節しているかについては、ほとんど知られていません。
方法です：
そこで、ヒトと高血圧ラットモデルの両方の胆汁酸プロファイルを解析し、ヒトとラットで共役胆汁酸が血圧と逆相関することを報告した。
結果
高血圧ラットにおいて、タウリンを介在させることで胆汁酸の抱合を抑制し、血圧を低下させることができた。その後、アンターゲットメタボロミクスにより、胆汁酸抱合に伴うエネルギー代謝の低下が高血圧を緩和するメカニズムであることが判明しました。
結論
本研究により、共役胆汁酸が栄養学的に再プログラム可能な抗高血圧代謝産物であることが明らかになりました。
イントロダクション
胆汁は、古代文明にさかのぼる医学的な系譜を持ち、いくつかの有機および無機成分で包装された義務的な生体流体である[1-4]。脊椎動物では、胆汁酸塩は胆汁中に最も多く含まれる有機溶質であり、おそらく腸肝脂肪の吸収という生理的な役割を担っているためであろう。宿主の肝細胞は、コレステロールを胆汁酸に異化し、アミノ酸の側鎖と結合させ、最終的に両親媒性の胆汁酸塩を生成することにより、一次胆汁酸塩を合成する。胆汁酸は、肝胆道と小腸を通過する際に、腸内細菌によって酵素的に二次胆汁酸に変換される。胆汁酸の効率的な腸肝循環は、何十年も前から腸肝軸の一部として確立されていたにもかかわらず、胆汁、ひいては胆汁酸が腸と肝臓の健康に影響を与える重要な因子であるという認識は、より最近のことです [5-9]。
私たちや他の研究者の最近の研究から、腸-肝臓軸は、心血管の健康を支配する主要な、これまであまり認識されていなかった要因であることが示されています[10-15]。具体的には、健康の証の一つである血圧の恒常性は、短鎖脂肪酸などの腸内細菌叢由来の代謝物だけでなく、ケトン体であるβ-ヒドロキシブチレートなどの肝臓由来の宿主代謝物によっても調節されている[10,16-20]。これらの研究は、腸-肝臓軸を通過する代謝物のごく一部に過ぎず、これらの宿主および腸内細菌叢の共代謝物が血圧調節に及ぼすと考えられる影響に関する知識のギャップが極めて小さいことを意味する。そこで、胆汁酸組成の異常が血圧調節に悪影響を及ぼし、高血圧を促進するという仮説を検証することにした。
胆汁酸抱合に着目した理由は、胆汁酸代謝の重要な末端工程であり、唯一、宿主と微生物相の両方を必要とするからである。胆汁酸抱合には、タウリン抱合とグリシン抱合の2つのタイプがある。ヒトではグリシン抱合型が顕著であるが、げっ歯類モデルではタウリン抱合型が顕著である[21]。そこで、まずヒトとラットにおけるグリシンとタウリンの抱合に関する関連性を評価し、次に栄養補助食品の降圧代謝物としてのタウリンとタウロコール酸を評価しました。
METHODS
実験モデルおよび参加者の詳細：ヒト試験
研究参加者
アメリカの黒人および白人の成人における心血管疾患（CVD）リスク因子に関する進行中の前向きコホート研究であるCARDIA（coronary artery risk development in young adults）研究のデータが用いられた。CARDIAは、成人期における心血管疾患（CVD）リスクの進化を研究するために設計されました。CARDIAの参加者は、1985年から1986年にかけて、米国の4つの都市部（アラバマ州バーミンガム、イリノイ州シカゴ、ミネソタ州ミネアポリス、カリフォルニア州オークランド）から集められた（n = 5115、18～30歳） [1,22].CARDIA参加者のデータは、ベースライン時およびフォローアップ時（時変）に、有効な調査機器と臨床評価を用いた標準的な手順で収集された。生存している参加者の大半は、30年目（2015-2016年）の8回目のフォローアップで71％を含むフォローアップ検査に参加しています。本分析では、30年目の試験に参加したCARDIA参加者のサンプル（n = 240）のデータが含まれています（表2、Supplemental Digital Content、https://links.lww.com/HJH/C166）。試験特有のプロトコルの詳細は、CARDIAの中央ウェブサイトに掲載されています： https://www.cardia.dopm.uab.edu/scientific-resources-landing-page2.本研究は、関連するすべての倫理規定を遵守しています。
測定方法
CARDIA参加者のデータは、ベースライン時および追跡調査時（時変）に、標準化された手順、有効な調査機器および臨床評価により収集された。参加者は、年齢、人種、性別、学歴、脂質低下薬および降圧薬の使用状況を自己申告した。訓練された臨床スタッフが、肘と前腕を椅子の肘掛けに置いた座位で、参加者の安静時収縮期血圧と拡張期血圧を測定しました。血圧値は、ランダムゼロ血圧計に校正されたオシロメーター（OmROn HEM907XL自動/オシロメーター血圧計）で測定した3回のうち2番目と3番目の平均値として計算されました。腕のカフブラダーのサイズは、腕の周囲径に基づいて以下のように設定された：17.0～22.5cmの腕には9cmのカフ、22.6～32.5cmの腕には12.0cm、32.6～42.5cmの腕には15.0cm、42.6～50.0cmの腕には17.5cmカフ。腕周りが50cmを超える人には、OmROn 108MLアネロイド/手動血圧計と一緒に大腿カフを使用しました[23]。
ヒト尿中の胆汁酸分析
空腹時スポット尿サンプルは、クリニックで参加者から採取し、直ちに分注して-70℃で保存した。尿中のBAsは、以前に報告された方法に従って測定した[24]。BAsは、超高性能液体クロマトグラフィー-トリプル四重極飛行時間型質量分析計（UPLC-MS）（Waters Corp.、マサチューセッツ州ミルフォード、米国）を使用して定量化した。UPLC-MSから得られた生データは、Target-Lynx version 4.1 applications manager (Waters Corp.)を用いて分析および定量化した。クレアチニンは、Jaffe法を用いて測定した。
統計解析
収縮期血圧と尿中胆汁酸の多変量調整線形回帰を行い、各胆汁酸について別々の回帰を行った。解析のために、BAを一定の和に正規化した（各参加者の測定された胆汁酸の合計を100とする）。モデル1は共変量について未調整、モデル2は年齢、人種、性別、研究施設、学歴、モデル3はモデル2の共変量に脂質低下薬の使用、BMI、尿クレアチニンを加えて調整した。P値は、Benjamini-Hochbergの偽発見率（FDR）を用いて多重比較のために調整された。
動物モデル
すべての手順は、Guide for the Care and Use of Laboratory Animalsに従って行われ、トレド大学医学生命科学部（米国オハイオ州トレド）のInstitutional Animal Care and Use Committeeの審査と承認を受けた。本報告書は、図1、Supplemental Digital Content、https://links.lww.com/HJH/C166 に示す3種類の研究で構成されています。これらの研究の方法は、それぞれの研究ごとに以下に記載されています：
Study 1: Hypertensive and normotensive rat models
The inbred Dahl salt-sensitive (SS/Jr or S) and Dahl salt-resistant (SR/Jr or R) rat strains were used in the study. The S and R rats are a widely used preclinical model selectively bred for hypertension in response to a high salt diet [25–29]. These rats were inbred in our Institution in 1985 and since then have been maintained in-house [29]. Due to their inherent propensity to develop hypertension, the entire S rat colony is bred and maintained on a low salt diet (0.3% NaCl; Harlan Teklad diet TD 7034, Madison, Wisconsin, USA). The Harlan Teklad diet (TD94217) was used for experiments involving a high salt regimen (2% NaCl). All rats in any single experiment were raised by breeding animals concomitantly. Both male and female S and R rats were used, totaling eight groups. Details on the group names, abbreviations and blood pressure indices are provided in the Table 1, Supplemental Digital Content, https://links.lww.com/HJH/C166. Blood pressure index is defined as a relative value that depicts the reported blood pressures of these strains for both systolic and diastolic, recorded for >35 years. The timeline for the study is shown in Figure 1A, Supplemental Digital Content, https://links.lww.com/HJH/C166. Rats were weaned on day 28 and high salt feeding commenced on day 42 for 3–4 weeks. From days 60 to 69, fresh feces were collected and archived at –80°C. At the end of the study, rats were euthanized for archival of serum and tissues.
Study 2: Germ-free versus germ-free conventionalized rats
Inbred, male 7-week-old Sprague Dawley (SD) rats from Taconic Biosciences (Rensselaer, New York, USA) that were either germ-free (GF) or germ-free conventionalized (GFC) with microbiota (n = 5–6/group) were used for this study. Conventionalization of GF rats was performed by co- housing GF rats with conventionally raised rats (outbred) for 10 days (1:1 ratio). This allowed GF rats to receive a natural, continuous infusion of microbiota via coprophagy. Upon arrival at the University of Toledo, blood pressure was measured by tail-cuff method and animals were immediately sacrificed and fecal, serum, and tissue samples were collected. The timeline of the study is shown in Figure 1B, Supplemental Digital Content, https://links.lww.com/HJH/C166.
Study 3: Nutritional supplementation of hypertensive S rats with Taurine
Six-week-old rats were raised on a high salt (2% NaCl) diet along with or without 3% taurine (Sigma-Aldrich, St Louis, Missouri, USA) in their drinking water for 6–7 weeks during which blood pressure was monitored (timeline shown in Figure 1C, Supplemental Digital Content, https://links.lww.com/HJH/C166). Rats were surgically implanted with radiotelemetry transmitters as described previously [30]. Postsurgery, rats were housed individually and allowed to recover prior to recording their BLOOD PRESSURE using the DSI software and equipment (https://www.datasci.com/). Systolic, diastolic, and mean arterial pressures were collected at 5-min intervals and analyzed using the Dataquest A.R.T 4.2 Software.
Study 4: Nutritional supplementation of hypertensive S rats with tauro-cholic acid
Eight-week-old rats were raised on a high salt (2% NaCl) diet along with or without 7.5 umol/l tauro cholic acid (Sigma-Aldrich) in their drinking water for 5 weeks during which blood pressure was monitored (timeline shown in Figure 1D, Supplemental Digital Content, https://links.lww.com/HJH/C166). Rats were surgically implanted with radiotelemetry transmitters as described previously [30]. Postsurgery, rats were housed individually and allowed to recover prior to recording their blood pressure using the DSI software and equipment (https://www.datasci.com/). Systolic, diastolic, and mean arterial pressures were collected at 5-min intervals and analyzed using the Dataquest A.R.T 4.2 Software.
Bile acid analysis for studies 1 and 4
Serum samples from rats (n = 5–6/group) were collected and shipped to the West Coast Metabolomics Center at UC Davis (http://metabolomics.ucdavis.edu/) for bile acids profiling by GC-TOF-MS based targeted metabolomics (http://metabolomics.ucdavis.edu/core-services/metabolomics-central-service-core).
Bile acid metabolomic analyses for rat studies 2 and 3
Serum samples from rats were profiled by the Dan L. Duncan Cancer Center's Advanced Technology Core, Baylor College of Medicine. Serum Bile acids were quantified using the Biocrates Bile Acids (Biocrates Life Sciences AG, Innsbruck, Austria) kit in accordance with the user's manual [31]. In brief, 10 μl of the supplied internal standard solution was added to each well on a filter spot of the 96-well extracted plate. Next, 10 μl of each serum sample, quality control samples, blank, zero sample, or calibration standard were added to each well. Sample extract elution was performed with methanol and subjected to liquid chromatography and mass spectrometry (LC–MS/MS). LC–MS analysis were performed based on standard operating procedures provided by Biocrates bile acids kit. Chromatographic separation of the analytes was performed by LC system (Agilent Technology, Santa Clara, California, USA) using a reverse-phase LC and guard column provided by Biocrates then quantified by a calibration curve. Samples were introduced directly into a 6500 QTRAP (Sciex) using negative electrospray ionization operating in the Multiple Reaction Monitoring (MRM) mode and acquisition methods provided in the Biocrates bile acids kit. Data was exported for statistical analysis. The quantified Biocrates data are in absolute concentration (μmol/l) and used directly without further normalization for statistical analysis. The differentially regulated bile acids were identified by performing t-test for adjusted P-value of Padj. <0.25 (FDR, Banjamini–Hochberg procedure).
Additional targeted metabolomic profiling for studies 2 and 3
Serum metabolites were extracted from using previously described standard procedures for targeted metabolomic profiling using ultra high-performance liquid chromatography/tandem- mass spectrometry [32,33].. The extracted samples were analyzed using high-performance liquid chromatography (HPLC) coupled to Agilent 6495 QQQ mass spectrometry. In ESI positive mode, the HPLC column was waters X-bridge amide 3.5 μm, 4.6 × 100 mm (Waters). Mobile phases A and B were 0.1% formic acid in water and acetonitrile respectively. Gradient flow: 0–3 min 85% B; 3–12 min 30% B, 12–15 min 2% B, 16 min 95% B, followed by re-equilibration till the end of the gradient 23 min to the initial starting condition of 85% B. Flow rate of the solvents used for the analysis is 0.3 ml/min. Data were normalized with internal standards and log2-transformed on a per-sample basis.
16S RNA analysis
DNA extraction and quantification
Fecal DNA was extracted from one fecal pellet (approximately 0.2 g) using QIAampPowerFecalDNA kit (QIAGEN, Hilden , Germany) followed by provided protocol. At elution step, 50 ml of low TE buffer (0.1 mmol/l EDTA, Tris–HCl buffer, 10 mmol/l, pH 8.5) was used instead of AE buffer from the kit. DNA concentration was measured by NanoDrop and diluted to be 5 ng/ml in low TE buffer for PCR library preparation as per the Illumina User Guide: 16S Metagenomic Sequencing Library Preparation-Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System (Part # 15044223 Rev. B).
The inbred Dahl salt-sensitive (SS/Jr or S) and Dahl salt-resistant (SR/Jr or R) rat strains were used in the study. The S and R rats are a widely used preclinical model selectively bred for hypertension in response to a high salt diet [25–29]. These rats were inbred in our Institution in 1985 and since then have been maintained in-house [29]. Due to their inherent propensity to develop hypertension, the entire S rat colony is bred and maintained on a low salt diet (0.3% NaCl; Harlan Teklad diet TD 7034, Madison, Wisconsin, USA). The Harlan Teklad diet (TD94217) was used for experiments involving a high salt regimen (2% NaCl). All rats in any single experiment were raised by breeding animals concomitantly. Both male and female S and R rats were used, totaling eight groups. Details on the group names, abbreviations and blood pressure indices are provided in the Table 1, Supplemental Digital Content, https://links.lww.com/HJH/C166. Blood pressure index is defined as a relative value that depicts the reported blood pressures of these strains for both systolic and diastolic, recorded for >35 years. The timeline for the study is shown in Figure 1A, Supplemental Digital Content, https://links.lww.com/HJH/C166. Rats were weaned on day 28 and high salt feeding commenced on day 42 for 3–4 weeks. From days 60 to 69, fresh feces were collected and archived at –80°C. At the end of the study, rats were euthanized for archival of serum and tissues.
Study 2: Germ-free versus germ-free conventionalized rats
Inbred, male 7-week-old Sprague Dawley (SD) rats from Taconic Biosciences (Rensselaer, New York, USA) that were either germ-free (GF) or germ-free conventionalized (GFC) with microbiota (n = 5–6/group) were used for this study. Conventionalization of GF rats was performed by co- housing GF rats with conventionally raised rats (outbred) for 10 days (1:1 ratio). This allowed GF rats to receive a natural, continuous infusion of microbiota via coprophagy. Upon arrival at the University of Toledo, blood pressure was measured by tail-cuff method and animals were immediately sacrificed and fecal, serum, and tissue samples were collected. The timeline of the study is shown in Figure 1B, Supplemental Digital Content, https://links.lww.com/HJH/C166.
Study 3: Nutritional supplementation of hypertensive S rats with Taurine
Six-week-old rats were raised on a high salt (2% NaCl) diet along with or without 3% taurine (Sigma-Aldrich, St Louis, Missouri, USA) in their drinking water for 6–7 weeks during which blood pressure was monitored (timeline shown in Figure 1C, Supplemental Digital Content, https://links.lww.com/HJH/C166). Rats were surgically implanted with radiotelemetry transmitters as described previously [30]. Postsurgery, rats were housed individually and allowed to recover prior to recording their BLOOD PRESSURE using the DSI software and equipment (https://www.datasci.com/). Systolic, diastolic, and mean arterial pressures were collected at 5-min intervals and analyzed using the Dataquest A.R.T 4.2 Software.
Study 4: Nutritional supplementation of hypertensive S rats with tauro-cholic acid
Eight-week-old rats were raised on a high salt (2% NaCl) diet along with or without 7.5 umol/l tauro cholic acid (Sigma-Aldrich) in their drinking water for 5 weeks during which blood pressure was monitored (timeline shown in Figure 1D, Supplemental Digital Content, https://links.lww.com/HJH/C166). Rats were surgically implanted with radiotelemetry transmitters as described previously [30]. Postsurgery, rats were housed individually and allowed to recover prior to recording their blood pressure using the DSI software and equipment (https://www.datasci.com/). Systolic, diastolic, and mean arterial pressures were collected at 5-min intervals and analyzed using the Dataquest A.R.T 4.2 Software.
Bile acid analysis for studies 1 and 4
Serum samples from rats (n = 5–6/group) were collected and shipped to the West Coast Metabolomics Center at UC Davis (http://metabolomics.ucdavis.edu/) for bile acids profiling by GC-TOF-MS based targeted metabolomics (http://metabolomics.ucdavis.edu/core-services/metabolomics-central-service-core).
Bile acid metabolomic analyses for rat studies 2 and 3
Serum samples from rats were profiled by the Dan L. Duncan Cancer Center's Advanced Technology Core, Baylor College of Medicine. Serum Bile acids were quantified using the Biocrates Bile Acids (Biocrates Life Sciences AG, Innsbruck, Austria) kit in accordance with the user's manual [31]. In brief, 10 μl of the supplied internal standard solution was added to each well on a filter spot of the 96-well extracted plate. Next, 10 μl of each serum sample, quality control samples, blank, zero sample, or calibration standard were added to each well. Sample extract elution was performed with methanol and subjected to liquid chromatography and mass spectrometry (LC–MS/MS). LC–MS analysis were performed based on standard operating procedures provided by Biocrates bile acids kit. Chromatographic separation of the analytes was performed by LC system (Agilent Technology, Santa Clara, California, USA) using a reverse-phase LC and guard column provided by Biocrates then quantified by a calibration curve. Samples were introduced directly into a 6500 QTRAP (Sciex) using negative electrospray ionization operating in the Multiple Reaction Monitoring (MRM) mode and acquisition methods provided in the Biocrates bile acids kit. Data was exported for statistical analysis. The quantified Biocrates data are in absolute concentration (μmol/l) and used directly without further normalization for statistical analysis. The differentially regulated bile acids were identified by performing t-test for adjusted P-value of Padj. <0.25 (FDR, Banjamini–Hochberg procedure).
Additional targeted metabolomic profiling for studies 2 and 3
Serum metabolites were extracted from using previously described standard procedures for targeted metabolomic profiling using ultra high-performance liquid chromatography/tandem- mass spectrometry [32,33].. The extracted samples were analyzed using high-performance liquid chromatography (HPLC) coupled to Agilent 6495 QQQ mass spectrometry. In ESI positive mode, the HPLC column was waters X-bridge amide 3.5 μm, 4.6 × 100 mm (Waters). Mobile phases A and B were 0.1% formic acid in water and acetonitrile respectively. Gradient flow: 0–3 min 85% B; 3–12 min 30% B, 12–15 min 2% B, 16 min 95% B, followed by re-equilibration till the end of the gradient 23 min to the initial starting condition of 85% B. Flow rate of the solvents used for the analysis is 0.3 ml/min. Data were normalized with internal standards and log2-transformed on a per-sample basis.
16S RNA analysis
DNA extraction and quantification
Fecal DNA was extracted from one fecal pellet (approximately 0.2 g) using QIAampPowerFecalDNA kit (QIAGEN, Hilden , Germany) followed by provided protocol. At elution step, 50 ml of low TE buffer (0.1 mmol/l EDTA, Tris–HCl buffer, 10 mmol/l, pH 8.5) was used instead of AE buffer from the kit. DNA concentration was measured by NanoDrop and diluted to be 5 ng/ml in low TE buffer for PCR library preparation as per the Illumina User Guide: 16S Metagenomic Sequencing Library Preparation-Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System (Part # 15044223 Rev. B).
