低氧預處理引起之腸道上皮細胞緊密連接蛋白組合和屏障功能加強的訊息傳遞路徑探討

Signaling pathways for tight junctional assembly and barrier enhancement in intestinal epithelial cells induced by hypoxic preconditioning

黃怡禎(Yi-Chen Huang)

指導教授 ： 余佳慧

國立臺灣大學/醫學院/生理學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.01258

摘要

腸道缺血再灌流 (ischemia/reperfusion，I/R) 會導致黏膜層受損、上皮細胞間緊密連接構造破壞及屏障功能失調等。過去許多研究指出，低氧預處理可減低腦、心臟、肺及腎臟等器官受缺血刺激造成的傷害。本實驗室過去研究結果顯示，低氧預處理 (hypoxic preconditioning，HPC) 會啟動嗜中性白血球之預活化(neutrophil priming)，達到抑制腸道I/R誘發細菌轉移之功效; 然而目前低氧對腸道上皮細胞的保護機制尚未釐清。因此本研究目的為探討低氧對腸道上皮細胞屏障功能的直接影響及其中所牽涉的訊息傳遞路徑。實驗方式為給予Wistar 大鼠常氧 (normoxia) 或低氧預處理 (HPC) 3天、7天或21天後，再進行腸繫膜動脈缺血再灌流的手術處理。結果顯示，大鼠在HPC處理7天及21天的血容比和嗜中性白血球活性相較於常氧組有顯著性的上升，此結果可確認大鼠於生理上有感受到低氧環境。低氧預處理7天和21天可減緩I/R引發的腸道絨毛構造變形、腺窩部位增生量降低及腸道組織通透性上升的現象。為了進一步探討低氧直接對腸道上皮細胞的影響，實驗中將人類腸道上皮Caco-2細胞培養至過滿並確立緊密連接(tight junction)已形成狀態，給予常氧(5% CO2 和95 % 空氣)及低氧(5% CO2 和95 % N2)多重時間點的處理。當給予Caco-2細胞1、2及4小時的低氧處理會使跨上皮細胞電阻(transepithelial electrical resistance，TER)上升，但在48小時低氧處理則會使TER顯著性下降。相似的結果也發現在以模擬低氧藥物(例如oligomycin A，OA和Rotenone)處理的Caco-2細胞中，2至18小時的OA和rotenone處理皆會促使Caco-2細胞TER升高，而OA和rotenone作用48小時則使TER大幅下降。此外，低氧1小時會使NP-40不可溶蛋白部分的occludin、ZO-1、claudin-1及JAM1蛋白表現量上升，顯示緊密連接蛋白由細胞質部分轉移至細胞骨架鄰近細胞膜部位。PI3K抑制劑 (如Wortmannin及LY-294002)和PKC抑制劑(如PKCζ pseudosubstrate)會減緩低氧1小時所造成的TER升高，其他抑制劑如Akt和p38 MAPK抑制劑則不影響低氧造成的上皮電阻改變。實驗亦觀察到低氧1小時會引起細胞質之PI3K和PKCζ蛋白轉移到細胞膜的情形。此外, Akt及其下游訊息傳遞分子的磷酸化如mTOR、FoxO1/O3a或GSK3蛋白在低氧1小時後則是顯著降低。綜論之，低氧可透過PI3K和PKCζ依賴性的訊息傳遞路徑，促進腸道上皮細胞緊密連接蛋白的組合及跨上皮細胞電阻值的提升，此保護機制或許有參與避免腸道缺血再灌流引發之屏障失常。

關鍵字

腸道缺血再灌流 ； 低氧預處理 ； 緊密連接蛋白 ； 跨上皮細胞電阻 ； 訊息傳遞路徑 ； PI3K ； PKCζ ； Akt

並列摘要

Intestinal ischemia/reperfusion (I/R) causes mucosal histopathology, epithelial tight junctional disruption and barrier damage. Hypoxic preconditioning (HPC) was shown to attenuate ischemic injury in brain, heart, lung and kidney. Our previous study demonstrated that HPC induced neutrophil priming and prevented intestinal I/R-induced bacterial translocation. Nevertheless, the protective mechanism of HPC on intestinal epithelial cells remains unknown. Our aim is to characterize the effect of hypoxia on enterocytes and the underlying signaling pathways for tight junctional assembly and barrier enhancement. Rats were raised in normoxia or kept in a hypoxic hypobaric chamber (380 mmHg) 17 hrs/day for 3, 7 or 21 days, and then subjected to mesenteric I/R. Higher levels of hematocrit and neutrophil respiratory burst activity were seen in HPC rats after 7 or 21 days, confirming the preconditioning status. I/R-induced villous deformation, loss of proliferating cell nuclear antigen staining in crypts, and increase of transmural macromolecular flux in intestinal tissues were partly attenuated by HPC-21d and HPC-7d. To examine whether hypoxia modify epithelial barrier function, post-confluent human intestinal epithelial Caco-2 cells that established tight junctions were exposed to normoxia (5% CO2 and 95 % air) or hypoxia (5 % CO2 and 95 % N2) for various time points. Cells exposed to 1, 2 and 4 hrs of hypoxia showed increase in transepithelial electrical resistance (TER), whereas 48 hrs of hypoxia caused TER to drop. Similarly, elevated TER was also seen in Caco-2 cells after treatment with hypoxic-mimetic agent (i.e. oligomycin A and rotenone) for 2-18 hrs, whereas TER decreased after treatment for 48 hrs. Furthermore, increased levels of occludin, ZO-1, claudin-1 and JAM1 proteins were seen in NP40-insoluble fractions of cellular extract after one hour of hypoxia, suggesting translocation of tight junctional proteins from cytosol to cytoskeleton-associated membranous region. The hypoxia-induced TER increase was diminished by pretreatment with PI3K inhibitors (i.e. Wortmannin and LY-294002) and PKCζ inhibitor (i.e. PKCζ pseudosubstrate), but not inhibitors to Akt and p38 MAPK. Membrane translocation of cytosolic PI3K and PKCζ was found after hypoxia for 1 hr. Besides, hypoxia for 1 hr reduced the phosphorylation of Akt and downstream signaling molecules such as mTOR, FoxO1/O3a and GSK3 in Caco-2 cells. In conclusion, HPC increases tight junctional assembly and enhances epithelial resistance through PI3K- and PKCζ-dependent signaling pathway, which may contribute to the protection against I/R-induced barrier dysfunction.

並列關鍵字

intestinal ischemia/reperfusion ； hypoxic preconditioning ； tight junction proteins ； signaling pathway ； PI3K ； PKCζ ； Akt

參考文獻

2. Tanaka S, Kamiike W, Kosaka H, et al. Detection of nitric oxide production and its role in pancreatic ischemia-reperfusion in rats. Am J Physiol 1996; 271: G405-409.

3. Schnitzlein HN. Regulation of blood flow through the stomach of the rat. Anat Rec 1957; 127: 735-753.

4. Leung FW, Su KC, Pique JM, et al. Superior mesenteric artery is more important than inferior mesenteric artery in maintaining colonic mucosal perfusion and integrity in rats. Dig Dis Sci 1992; 37: 1329-1335.

5. Fujimoto K, Price VH, Granger DN, et al. Effect of ischemia-reperfusion on lipid digestion and absorption in rat intestine. Am J Physiol 1991; 260: G595-602.

6. Moore KA, Lemischka IR. Stem cells and their niches. Science 2006; 311: 1880-1885.