微生物肠道血屏障及肝脏代谢的体内评价方法
Summary
营养物质、微生物代谢物和药物进入血液循环的途径由肠道血屏障 (GBB) 控制。我们描述了一种直接测量体内 GBB 通透性的方法, 与常用的间接方法相比, 它几乎不受肝肾功能的影响。
Abstract
肠道血屏障 (GBB) 控制养分、细菌代谢物和药物从肠道腔到血液的通道。GBB 完整性在胃肠道、心血管和代谢疾病中受到干扰, 这可能导致生物活性化合物 (如肠道细菌代谢物) 更容易进入血液。因此, GBB 的通透性可能是肠道和肠外疾病的标志物。此外, 细菌代谢物的渗透率增加可能影响整个机体的功能。
研究 GBB 通透性的常用方法是在体内进行。这些方法的准确性是有限的, 因为 GBB 的功能取决于肠道血流。另一方面, 通常使用的活体方法可能有偏见的肝脏和肾脏的表现, 因为这些方法是基于评估尿液或/和外周血浓度的外源标记。在这里, 我们提出了一种直接测量大鼠 GBB 通透性的方法, 采用基于门静脉采血的活体法, 可保持肠道血流, 几乎不受肝肾功能的影响。
聚氨酯导管插入门静脉和下腔静脉刚好高于肝静脉汇合处。血液在基线和被选择的标记的管理后被取样到胃肠道的所需部分。在这里, 我们提出了一些应用的方法, 包括 (1) 结肠通透性对 tma, 肠道细菌代谢物的评价, (2) tma 肝清除率的评价, 和 (3) 肠道门静脉血-肝-外周血通路的评价细菌衍生的短链脂肪酸。此外, 该协议还可用于跟踪药物的肠道吸收和肝脏代谢或门静脉血压测量。
Introduction
肠道血屏障 (GBB), 也被称为肠道屏障, 是一个复杂的多层系统, 分离肠道腔与血液, 以限制有害化合物的通过, 同时允许营养素的吸收1。它由三主要层组成: 粘液层、上皮和固有。
许多因素可能影响 GBB 的完整性和功能2。已经表明, GBB 在胃肠道和肠外疾病, 包括心血管和代谢疾病3, 这可能导致肠道细菌代谢物的通过增加到血液4的紊乱。肠道细菌代谢物的渗透增加可能影响整个机体的功能。例如, 最近的研究显示, 细菌代谢产物, 如吲哚, H2S, 短链脂肪酸 (欢迎) 和三甲胺 n-氧化物, 在循环系统功能5,6,7 显著影响,8,9。最后, 有人建议, 增加的 GBB 渗透率可以作为心血管和代谢疾病的标志, 与形态学和功能改变在肠道10。因此, 跟踪细菌代谢物的肠道门血-肝系统血液通路可能对基础和临床科学有兴趣。
常用的实验方法对 GBB 通透性进行体外检测, 使用切除肠段、粘膜片段或人工膜11、12。这些方法的准确性受到损害的事实, GBB 的正常运作需要恒定的肠道血流。另一方面, 可用的活体方法是基于对外源标记13的尿液或外周血浓度的评估。但是外源性化合物的外周血和尿液浓度受肾脏功能的影响,即肾小球滤过率和管状排泄, 以及肝脏代谢,即第一次代谢。两个参数在独立于 GBB 函数的研究对象之间可能有显著差异。
本文介绍了用门静脉采血法直接测定大鼠 GBB 通透性的方法。这种在体内的方法保留肠道血流, 几乎不受肝脏和肾脏功能的影响。所描述的方法并不常用, 可能是因为一些方法上的困难。我们详细描述了门静脉导管和下腔静脉在肝静脉汇合上方的插管。从门静脉和下腔静脉的血液取样允许评估 GBB 通透性和肝脏清除, 以及跟踪胃肠门血-肝系统血液通路的兴趣分子, 如肠道细菌代谢物或药物。我们还介绍了在我们的实验室测试的方法的几个应用。这些包括对 tma 的结肠通透性的评估, 肠道细菌代谢物, tma 的肝清除率的评价, 和欢迎的肠门血-肝-系统血通路的评价。
为了评估肠血屏障通透性, 应遵循以下协议步骤, 按顺序: 1 (插入 intraintestinal 管理线), 3 (门静脉导管), 4 (门静脉血取样), 6 (肠道通透性标记的管理), 4。
为了评估肝脏清除和肠道门血-肝系统的血液通路, 应遵循以下协议步骤, 按顺序: 1 (插入 intraintestinal 行政部门的行), 2 (下腔静脉导尿), 3 (门静脉导管), 4 (门静脉血采样), 5 (下腔静脉采血), 6 (胃肠通透性标记的管理), 4, 5, 7 (肝间隙的计算).
Protocol
Representative Results
Discussion
直接,在体内, 测量 GBB 渗透率的方法保持胃肠系统中的 closetophysiological 条件 (保留肠道血流), 几乎不受肝肾功能的影响。
这项技术的关键步骤是插入门导管。这必须在同一时间轻轻和果断地完成。轻微, 短出血可能发生从正确执行的门静脉穿刺;但是, 当针头插入容器时, 它会停止。持续出血表明门静脉穿孔。为便于导管插入, 门静脉应充分暴露。exteriorizing 肠后, 肠系膜?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了波兰科学和高等教育部的支持, 钻石赠款号: DI2017 009247。
Materials
| Needle OD: 9 mm | Becton Dickinson S.A. | 301300 | |
| Polyethylene catheter ID: 0.025", OD: 0.040" | Scientific Commodities, Inc. | #BB520-40 | |
| Polyethylene catheter ID: 0.012", OD: 0.025" | Scientific Commodities, Inc. | #BB520-25 | |
| C-Flex Tubing,Opaque White 1/50"ID x 1/12 " OD | Cole-Parmer Instrument Co. | 06424-59 | |
| Pediatric Foley catheter (size 10F or 8F) | Sigmed | 0000 80305 | |
| Surgical ligatures 3/0 | Yavo Sp. Z o.o. | P48JE | |
| Absorbable surgical sutures – Polyglactine 910 4/0 | KRUUSE Polska Sp. Zo.o. | 152336 | |
| Tissue glue – Loctite 454Cyanoacrylate Adhesive | Loctite | 1370127 | |
| Povidone iodine | EGIS Pharmaceuticals PLC | 4449 11 | |
| Heparin – Heparinium WZF | WZF Polfa S.A. | 02BK0417 | Dilute 10 times with physiological saline |
| Glycerin 86% | Laboratorium Farmaceutyczne Avena | 5.90999E+12 | Serves as a lubricant in colon catheterization |
| Xylocaine 2% | AstraZenca | 9941342 | |
| Urethane | Sigma-Aldrich (Merck) | U2500-500G | |
| Trimethylamine solution 45% | Sigma-Aldrich (Merck) | 92262-1L | |
| Syringes 2 mL | B.Braun Melsungen AG | 4606027V | |
| Saline 250 mL | Fraesenius Kabi Polska Sp. Z o.o. | 15LL707WL | |
| Surgical scissors, straight, length 115 mm, 4 1/2 "blunt ends | Braun | NS-010-115-PKM | |
| Artery forceps type Micro-Adson bent, length 140 mm 5 1/2 " | Braun | KN-008-140-ZMK | |
| Anatomic forceps, lenght 95 mm, 3 3/4" sharp 0.7×0.55 | Braun | PO-001-007-ZMK | |
| Micro Scissors type Vannas, straight, lenght 85 mm, 3 3/8 " the length of the blades 6 mm | Braun | NO-010-085-PMK | |
| Towel clamps type Backhouse, lenght 130 mm, 5 1/8" | Braun | HO-128-130-PMK | |
| Needle holders, lenght 150 mm, 6" t=0.4 1/2 | Braun | IM-927-150-PZMK | |
| Delicate Scissors, lenght 110 mm , straight, 4 3/8” sharp | Braun | NO-052-110-PMK | |
| Anatomic forceps, lenght 95 mm, 3 3/4" sharp | Braun | PO-022-001-PMK | |
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