摘要:腸道缺血再灌流(ischemia/reperfusion, I/R)之傷害常見於腸繫膜動脈栓塞,絞扭性疝氣,新生兒壞死性腸炎,重大心肺和腹部手術,嚴重失血和創傷的病人身上。 I/R 刺激會造成腸道屏障喪失, 引起腸腔細菌移位至血液和其它臟器, 嚴重時會爆發敗血症和多重器官衰竭。低氧前置訓練(hypoxic preconditioning, HPC)是讓活體動物預先接受間斷性低氧處理使之於下次嚴重缺血時受到保護作用。過去研究顯示HPC可減緩心臟, 腦部, 腎臟, 肺臟受I/R刺激時的損傷程度; 然而其保護機轉仍不清楚。目前尚未有HPC對缺血之腸道屏障功能影響之研究。腸道有別於其它無菌臟器, 人體腸道有大量(1012 ~ 1014 )共生細菌存在於空腔中。在生理情況下, 腸道上皮細胞由緊密連結(TJ)構造組成第一道防禦線, 避免細菌侵入。本人研究顯示I/R刺激和病原菌感染皆會促使上皮細胞死亡及緊密連結破損的情形, 導致腸道抗原通透增加或細菌移位的現象(Yu LC et al. (2005) FASEB J 16, 1822; Hsiao JK et al. (2009) Invest Radiol. 44, 329)。腸道黏膜固有層的先天性免疫細胞, 如巨噬細胞和嗜中性球, 於活化時有毒殺細菌的能力, 形成第二道防禦線。傳統觀念上, 在非腸器官(extraintestinal organs)內, 巨噬細胞和嗜中性球的活化被認為是組織傷害之因子。然而在腸道細菌存在的環境中, 免疫細胞的活化卻是避免細菌入侵不可或缺的一環。本計劃研究目的為探討 HPC 是否能防止I/R 刺激造成的腸道細菌入侵和腸道黏膜損害現象, 並深究其保護機轉是否透過: 1)調節上皮細胞死亡和緊密連結構造, 或 2)經由改變免疫細胞(如巨噬細胞和嗜中性球)活化程度。實驗設計將大白鼠置於常氧或低氧環境一段時間後(3, 7, 14, 21天), 利用腸繫膜動脈夾擊作為腸道I/R模式。我們將測試短期或長期HPC, 是否能減緩腸道細菌和產物(如LPS)入侵,黏膜傷害和發炎反應。我們初步實驗結果顯示, HPC預處理能避免I/R刺激引發腸道細菌移位至肝臟和脾臟的現象。我們將利用Ussing Chamber system和實驗室新發展核磁共振造影技術輔助的通透性試驗來監控腸道上皮屏障功能的改變, 並利用免疫染色法觀察TJ的構造。將測試巨噬細胞和嗜中性球的呼吸氧爆作用和殺菌能力,並檢驗趨化激素(chemokine)改變量。此外, 將利用人類腸道上皮細胞培養系統來觀察HIF-1轉錄作用及抗凋亡之下游分子訊息傳遞途徑(如PI3K/Akt/mTOR)在其保護機制中扮演的角色。本研究成果將有助於發展預防性或治療性之療法以避免腸道疾病或手術前後引發之細菌性敗血症。
Abstract: Ischemia/reperfusion (I/R) injury of the intestine is documented in patients with mesenteric artery thrombolism, strangulated hernias, neonatal necrotizing enterocolitis, and those undergoing major abdominal and cardiovascular surgery, as well as severe trauma. Mesenteric I/R results in mucosal barrier dysfunctions associated with enhanced bacterial translocation, leading to the development of sepsis-induced multiple organ failure. Hypoxic preconditioning (HPC) is defined as the phenomenon whereby exposure of the animal to intermittent hypoxia protects them against a sustained period of subsequent ischemia. A number of recent references have documented that HPC protects heart, brain, kidney and lung tissues against I/R damage. However, the precise mechanisms underlying the beneficial effects of HPC are far from clear. Up to date, no reference was reported about the effect of HPC in intestinal I/R injury.The gut lumen is the main reservoir for microbes in the body, and contains over 100 trillion commensal bacteria. The intestinal epithelial cells linked by tight junctions are responsible for separating the luminal microbes from the interstitium, and act as the first line of defense. Our laboratory has demonstrated that I/R challenge or pathogenic exposure stimulates intestinal epithelial cell death and tight junctional disruption, leading to increased permeability to antigens and bacteria (Yu LC et al. (2005) FASEB J 16, 1822; Hsiao JK et al. (2009) Invest Radiol. 44, 329). The underlying immune cells (such as neutrophils and macrophages) display phagocytic and bactericidal activity, and may act as a secondary defense mechanism. In contrast to the majority of sterile extraintestinal organs whereby activation of phagocytic cells is considered harmful to tissues, sufficient activation of neutrophils and macrophages in the gut are crucial for curbing the invasion of enteric bacteria.AIM: To investigate whether HPC protects against I/R-induced mucosal injury and bacterial influx, and to decipher the role of epithelial barrier and phagocytic bactericidal activity in the protective mechanism using rat models and human intestinal epithelial cell cultures.Methods: Rats will be raised in normoxic or hypoxic conditions for 3, 7, 14, or 21 days prior to sham operation or I/R challenge by occlusion of superior mesenteric artery. The level of bacterial translocation will be evaluated by counting the bacterial colony forming units in mesenteric lymph node, liver and spleen homogenates. The amount of endocytosed bacteria in intestinal mucosa will be assessed using a gentamicin resistance assay. The epithelial permeability will be examined by using an in vivo magnetic resonance imaging (MRI)-based assay and an ex vivo Ussing Chamber study. The roles of hypoxia-inducing factor (HIF-1) and PI3K/Akt/mTOR signaling pathways in the epithelial cell survival mechanism will also be assessed using small interference RNA techniques. The respiratory burst and bactericidal activity of macrophages and neutrophils will be determined. The priming state of blood neutrophils, including the phosphorylation and membrane translocation of NAPDH oxidase subunits, i.e. p47phox, p67phox, p40phox and Rac, will be examined by western blotting and immunofluorescent staining.SIGNIFICANCE: Hypoxic preconditioning is a non-medicated strategy that prevents mucosal barrier dysfunction and limits bacterial influx, and may be capable of avoiding overt phagocytic activation and uncontrollable inflammatory response. The understanding of the cellular and molecular mechanism of the protective strategy may shed light to the development of perioperative, prophylactic or therapeutic measures against gut-derived septic complications.