低氧預處理改善腸道缺血再灌流所引發之細菌轉移和屏障受損現象:其保護機轉之探討

Intestinal Ischemia/Reperfusion-Induced Bacterial Translocation and Barrier Defects are Ameliorated by Hypoxic Preconditioning

盧彥臻(Yen Zhen Lu)

指導教授 ： 余佳慧

國立臺灣大學/醫學院/生理學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02006

摘要

腸道缺血再灌流（ischemai/reperfusion, I/R）之傷害常見於心肺手術、小腸移植手術以及外傷性休克病人身上。由於在正常生理環境中，腸道上皮細胞之間會緊密結合而形成腸道屏障，可阻擋腸管中大量的細菌進入身體內部。但缺血再灌流之刺激引發腸道上皮細胞死亡及屏障功能缺損，使得大量細菌進入體內，進而引發全身性發炎反應和多重器官之衰竭。過去研究顯示低氧預處理（hypoxic preconditioning, HPC）可減緩心臟和腦部缺血再灌流刺激所導致的損傷程度，然而低氧預處理是否對於腸道有保護作用目前並不清楚。本篇研究目的主要探討低氧預處理是否可以改善腸道缺血再灌流所引發的黏膜損傷和細菌轉移的現象。

本篇研究首先將實驗大鼠分為常氧（normoxia）和低氧預處理兩大組。常氧鼠飼養於大氣壓為760毫米汞柱之環境中，即一般空氣下。低氧預處理鼠飼養於低壓低氧艙（380毫米汞柱），每天17小時持續21天。每一大組又依手術處理方式分為正常對照組（normal control, con）、假手術組（sham-operated control, sham）和缺血再灌流組，其中缺血再灌流組的大鼠以動脈夾夾擊上腸繫膜動脈（superior mesenteric artery, SMA）20分鐘之後，移除動脈夾恢復血流再灌流1小時。在手術處理完畢後，取出腸道組織作組織染色、嗜中性白血（neutrophils）球染色、骨髓過氧化酵素（myeloperoxidase, MPO）分析和嗜中性白血球趨化吸引分子-1（cytokine-induced neutrophil chemoattractant, CINC-1）蛋白表現量評估。此外拿取肝臟、脾臟和腸繫膜淋巴結（mesenteric lymph nodes, MLNs）之組織均質液培養於新鮮血液培養基（fresh blood agar plate）和馬康氏培養基（MacConkey agat plate）作細菌轉移之腸道屏障功能分析。而為了測量腸道上皮細胞在缺血再灌流後刺激下通透性的改變，在大鼠之空腸腸道綁環之管腔中注入顯影劑釓偶醯胺（gadodiamide），再利用核磁共振影像（magnetic resonance imaging, MRI）技術擷取肝臟和腎臟之影像強度作分析。

實驗結果顯示，常氧鼠在受到腸道缺血再灌流的刺激之後，空腸（jejunum）和迴腸（ileum）絨毛長度明顯短縮，絨毛之頂端上皮細胞脫落。而缺血再灌流後，腸道屏障功能之喪失可藉由肝臟和脾臟組織液中菌落形成單位（colony forming unit, CFU）數目增加及利用核磁共振所偵測到之肝臟影像強度增加趨勢所得知。此外缺血再灌流刺激會造成空腸之骨髓過氧化酵素活性和嗜中性白血球趨化吸引分子-1表現量蛋白顯著性地增加。而當實驗大鼠預先處以低氧預處理後，缺血再灌流所導致的腸道黏膜層受損情況及細菌轉移之數目皆減緩，顯示低氧預處理對腸道有保護作用。最後實驗結果發現低氧預處理大鼠受缺血再灌流刺激後，空腸和迴腸組織中骨髓過氧化酵素活性和嗜中性白血球趨化吸引分子-1表現較常氧鼠來得高，然而從核磁共振之肝臟影像的結果來看，低氧預處理無法降低缺血再灌流所引發的腸道通透性增加之現象。

由上述實驗結果顯示，腸道的缺血再灌流刺激會導致黏膜組織受損和管腔內細菌轉移至體內的現象，而低氧預處理則可降低缺血再灌流所引起的腸道型態上的受損及維持腸道的屏障功能，其保護機轉可能是透過嗜中性白血球的趨化和活化來達到毒殺細菌的效果以降低細菌轉移的現象，而非經由腸道上皮通透性之調節。

關鍵字

缺血再灌流 ； 低氧預處理 ； 細菌轉移 ； 嗜中性白血球 ； 骨髓過氧化酵素

並列摘要

Intestinal ischemia/reperfusion (I/R) injuries were reported in cardiovascular diseases, abdominal surgeries, as well as septic and traumatic shock. The intestinal epithelial cells connected by tight junction act as a barrier against enteric microbes. Intestinal barrier defects associated with bacterial translocation were documented in I/R injuries and may lead to multiple organ failure. Hypoxic preconditioning (HPC was shown to protect brain and heart tissues against ischemic insults. The aim of this study was to assess the effect of HPC in preventing intestinal I/R-induced mucosal injuries and bacterial translocation.

Wistar rats were either raised in normoxic atmosphere at 760 mmHg (Groups 1, 2, and 3）or in a hypobaric chamber (380 mmHg) for 21 consecutive days with 17 hrs/day for HPC (Groups 4, 5 and 6). These normoxic or HPC rats were randomly divided into three groups: normal controls, sham-operated controls and I/R treatment groups. I/R rats received occlusion of superior mesenteric artery（SMA）for ischemia occlusion for 20 minutes of ischemia and one hour of reperfusion. After the surgeries, intestinal tissues were collected for the assessment of morphology, neutrophils staining, myeloperoxidase (MPO) activity, and cytokine-unduced induced neutrophil chemoattractant-1（CINC-1） protein level. Homogenized spleen and liver were cultured on McConkey and fresh blood agar plates for analysis of bacterial translocation as an indicator of barrier defects. To measure intestinal transepithelial permeability, magnetic resonance imaging （MRI） techniques were utilized to detect the transport of luminally administered contrasting agent, gadodiamide, into the liver and kidney in rats following I/R treatment.

In rats raised in normoxic air, I/R treatment induced significant shortening of the length of intestinal villi, and epithelial cells at the villi tip were sloughed off in the jejunum and ileum. Intestinal barrier defects and epithelial permeability changes following I/R were evidenced by increased bacterial colony forming unit（CFU）counts in the liver and spleen, as well as heightened MRI intensity in liver, compared to those in sham controls. Moreover, jejunal MPO activity and CINC-1 protein level were increased suggesting neutrophil recruitment and activation as a response to I/R. Rats that were exposed to HPC prior to I/R showed a reduction in structural damages in the intestinal mucosa and lowered bacterial counts in internal organs compared to those in normoxic I/R groups. Therefor, these findings suggest partial protection against I/R-induced intestinal injuries by HPC. Moreover, elevated MPO activity and CINC-1 level in the intestine were found in the HPC I/R rats compared to those in normoxic I/R group. However, the enhanced transport of gadodiamide from intestinal lumen to liver induced by I/R was not ameliorated by HPC.

In summary, intestinal I/R resulted in mucosal damages and epithelail barrier defects associated with increased bacterial translocation in rats, and these I/R-induced intestinal structural and functional abnormalities were ameliorated by prior exposure of animals to HPC. This protective mechanism exerted by HPC may be attributed to enhanced neutrophil infiltration and bactericidal effect against the translocation bacterial, but not by modulation the transepithelial permeability of the intestine.

並列關鍵字

ischemia/reperfusion ； hypoxic preconditioning ； bacteria translocation ； neutrophil ； myeloperoxidase

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