後制約訓練對大鼠小腸缺血再灌流傷害的保護效應與機制

The Protective Effects of Ischemic Postconditioning on Intestinal Ischemia-Reperfusion Injury among Rats

鄭敬學(Ching-Hsueh Cheng)

指導教授 ： 賴逸儒

國立臺灣大學/醫學院/解剖學暨細胞生物學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.00430

摘要

前言：
小腸缺血再灌流傷害 (intestinal ischemia-reperfusion injury，簡稱IIR injury)為臨床上常見病理現象，造成傷害的原因包括急性的上腸系膜動脈阻塞以及小腸的移植手術等情況。IIR injury會導致全身性的發炎反應 (systemic inflammatory response，簡稱SIRS)及多重器官的衰竭 (multiorgan dysfunction response，簡稱MODS)，病情嚴重時，甚至造成患者的死亡。本研究使用大鼠模式，探討缺血後制約訓練 (ischemic postconditioning，簡稱IPoC)，一種改變缺血後再灌流(reperfusion)的程序，以間歇、短暫回復上腸系膜動脈血流的方法，降低組織缺血再灌流後的傷害。並且探究IPoC保護機制是否透過調節粒線體通透過渡性孔洞(mitochondrial permeability transition，簡稱mPTP)的開闔達成。

材料與方法：
將雄性Wistar大鼠 (重量約200~300 g)的上腸系膜動脈 (superior mensenteric artery，簡稱SMA)以血管夾阻斷血流30分鐘 (缺血期)，之後移除血管夾以恢復小腸血流60分鐘 (再灌流期)，此為IR組。後制約訓練係在小腸恢復血流前，給予連續3次短暫30秒的恢復灌流及接著30秒的缺血循環。為探究後制約訓練的保護效果是否與mPTP的開闔有關，某些組於缺血前5分鐘及缺血後15分鐘分別給予carboxy-atractyloside (CATR，造成mPTP的開啟)以及NIM811 (抑制mPTP的行成)。實驗分為五個組別，依序是Sham組，I/R組，IPoC組，I/R+NIM811組以及IPoC+CATR組。個組別於術前術後採集血液樣本以及空腸做分析，包括評估血清中乳酸脫氫酶 (lactate dehydrogenase，簡稱LDH)及小腸脂肪酸鍵結蛋白 (intestinal fatty acid binding protein，簡稱I-FABP)的含量、蘇木紫-伊紅染色 (Hematoxylin-Eosin staining)、細胞凋亡的評估 (TUNEL assay)、小腸黏膜中cytochrome c、cleaved-caspase 3及丙二醛 (malonialdehyde，簡稱MDA)的表現量。

實驗結果：
I/R組不論在術後血清LDH數值，血清I-FABP的表現量，Chiu’s傷害指數，crypt/villus ratio (C/V ratio)，細胞凋亡指數 (apoptotic index)以及黏膜細胞中cytochrome c、cleaved-caspase 3及MDA的表現量都明顯的高於Sham組，達到顯著差異 (p<0.05) (LDH：1273.67±277.43 U/L v.s 3427±236.81 U/L，Chiu’s score：0 v.s 4，C/V ratio：0.38±0.02 v.s 0.72±0.12，apoptotic index：0 v.s 59.5±4.56 %，MDA：5.43±0.27 v.s 8.68±0.36)。在IPoC組，上述所有傷害評估指標都有減少的現象 (LDH：1190.5±36.67 U/L，Chiu’s score：0.2±0.2，C/V ratio：0.39±0.03，apoptotic index：15.7±15.7 % ，MDA：5.58±0.27)。在給予NIM811的組別中，各項傷害評估指標也與IPoC組一樣有減少的現象。反觀於給予CATR，各項傷害指標皆與IR組相同，高於Sham組、IPoC組以及I/R+NIM811組。以上結果顯示缺血後制約訓練可以減少再灌流後血清LDH與I-FABP的增加，減少小腸黏膜傷害程度以及細胞凋亡的程度，這些保護效應與施予NIM811一致，並因施予CATR而減少。顯示缺血後制約訓練的保護機制可藉由抑制mPTP的開啟以減少小腸缺血再灌流傷害。

結論：
本實驗顯示缺血後制約訓練可以減少小腸缺血再灌流所造成的細胞傷害，其機制可能是透過調節降低粒線體膜通透性來達到保護效果。

關鍵字

小腸缺血再灌流傷害 ； 粒線體通透過渡性孔洞 ； 小腸脂肪酸鍵結蛋白質

並列摘要

Introduction：Intestinal ischemia-reperfusion (IIR)injury is an important clinical problem occurring in, for example, mesenteric arterial obstruction and intestinal transplantation. IIR injury leads to systemic inflammatory response (SIRS), multiorgan dysfunction response and even mortality. In this study, we explored the protective effect and mechanism of ischemic postconditioning (IPoC), a modulation of reperfusion maneuver, on IIR injury in a rat models.

Materials and methods：Male Wistar rats weighing 200 ~300 g were used. IIR injury was induced by clamping superior mesenteric artery (SMA) for 30 minutes and de-clamping for 60 minutes (I/R group). Three cycles of 30 seconds of reperfusion followed by 30 seconds of ischemia was performed just before reperfusion began in IPoC group. Carboxy- atractyloside (CATR, a mitochondria permeability transition pore activator)was injected at 5 minutes before intestinal ischemia in IPoC+CATR group. NIM811 (a mitochondria permeability transition pore inhibitor)was injected 15 minutes after ischemia began in I/R+ NIM811 group. The blood samples and jejunum were collected at indicated time for analysis including serum levels of lactate dehydrogenase (LDH), the expression of small intestinal fatty acid binding protein (I-FABP)in portal blood , hematoxylin-Eosin staining and TUNEL assay of intestinal sections, the expressions of cytochrome c cleaved-of caspase 3 and malonialdehyde (MDA)in the small intestine mucosa.

Results： Compared to the sham group, serum LDH values, I-FABP expression, Chiu's injury score, crypt/villus ratio (C/V ratio), the apoptotic index , expressions of cytochrome c, cleaved-caspase 3 and MDA are significantly higher than in the IR group (p<0.05) (LDH: 1273.67 277.43 U/L v.s. 3427±236.81 U/L , Chiu's score: 0 v.s. 4, C/V ratio: 0.38±0.02 v.s. 0.72±0.12, apoptotic index: 0 v.s. 59.5±4.56 %, MDA: 5.43±0.27 v.s. 8.68±0.36). The IIR injury was lessened in the IPoC group and I/R+ NIM811 group , when compared with those of the I/R group(LDH: 1190.5±36.67 U/L & 1399.33±295.64 U/L, Chiu's score: 0.2±0.2 & 0.4±0.24, C/V ratio: 0.39±0.03 & 0.37±0.02, apoptotic index: 15.7±15.7 % & 3.51±3.51 %, MDA: 5.58±0.27 & 6.45±0.13). On the other hand, the administration of CATR mitigated the protection offered by IPoC. (LDH: 2002±370.89 U/L, I-FABP: 182.09±70.43, Chiu's score: 4.2±0.2, C/V ratio: 0.80±0.08, apoptotic index: 67.07±9.33 %, MDA: 10.03±0.23)
Conclusion：This study demonstrated ischemic postconditioning can reduce cell damage caused by intestinal ischemia and reperfusion injury, and the protective mechanism of IPoC was related with the modulation of mitochondrial permeability transition.

並列關鍵字

intestinal ischemia-reperfusion injury ； mitochondrial permeability transition pore ； intestinal fatty acid binding protein

參考文獻

1. Orsenigo, M.N., et al., Effects of creatine in a rat intestinal model of ischemia/reperfusion injury. Eur J Nutr, 2012. 51(3): p. 375-84.

2. Daneman, R. and M. Rescigno, The gut immune barrier and the blood-brain barrier: are they so different? Immunity, 2009. 31(5): p. 722-35.

3. Lipkin, M., B. Bell, and P. Sherlock, Cell Proliferation Kinetics in the Gastrointestinal Tract of Man. I. Cell Renewal in Colon and Rectum. J Clin Invest, 1963. 42(6): p. 767-76.

4. Nusrat, A., C. Delp, and J.L. Madara, Intestinal epithelial restitution. Characterization of a cell culture model and mapping of cytoskeletal elements in migrating cells. J Clin Invest, 1992. 89(5): p. 1501-11.

5. Grootjans, J., et al., Rapid lamina propria retraction and zipper-like constriction of the epithelium preserves the epithelial lining in human small intestine exposed to ischaemia-reperfusion. J Pathol, 2011. 224(3): p. 411-9.