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Role of Transcription Factors in Small Intestinal Ischemia-Reperfusion Injury and Tolerance Induced by Ischemic Preconditioning
Role of Transcription Factors in Small Intestinal Ischemia-Reperfusion Injury and Tolerance Induced by Ischemic Preconditioning M. Takeshita, T. Tani, S. Harada, H. Hayashi, H. Itoh, H. Tajima, I. Ohnishi, H. Takamura, S. Fushida, and M. Kayahara ABSTRACT Background. Small intestinal ischemia-reperfusion (I/R) injury, a clinically important condition, induces severe organ damage. Ischemic preconditioning (IPC) produces tolerance to long-term I/R by inducing a short-term I/R. Herein, we have examined the reduction in the extent of injury by IPC. Methods. Small intestinal I/R injury was induced in rats by clamping the superior mesenteric artery (SMA) for 30 minutes followed by reperfusion for various 30 minutes. The IPC ⫹ I/R group underwent a short-term I/R (IPC) prior to long-term I/R. Nuclear factor-B (NF-B) activity was analyzed by an electrophoretic mobility shift assay and cytokine mRNA levels, by reverse transcription-polymerase chain reaction. Apoptosisrelated genes were analyzed by Western blotting and immunohistochemistry, and apoptotic cells, by TUNEL staining. Results. The animals were subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion. NF-B activity increased in the I/R group and decreased in the IPC ⫹ I/R group. The IPC ⫹ I/R group showed decreased cytokine in mRNA levels. Expression of the proapoptotic gene caspase-3 was increased in the I/R and decreased in the IPC ⫹ I/R group. Expression of the antiapoptotic gene Bcl-xL was increased in the IPC ⫹ I/R group. The number of apoptotic cells was increased in the I/R and decreased in the IPC ⫹ I/R group. Conclusion. Small intestinal I/R injury was reduced by IPC produced by clamping the SMA; thus, IPC may have potential clinical applications in the future. RANSPLANTATION as a treatment for organ dysfunction uses immunosuppression, which demands exquisite perioperative management, and infectious disease management. Small intestinal transplantation is widely performed in Europe and the United States. In Japan, however, transplantation is performed to a small extent. The small intestine is less tolerant to ischemia-reperfusion (I/R) injury, graft rejection, and infectious diseases than other organs. As a result, there has been no drastic improvement in treatment results. The vulnerability of the transplanted small intestine to warm ischemia, cold preservation, and reperfusion injury is possibly associated with impaired immediate posttransplant graft function. After I/R injury, bacterial translocation due to small intestinal obstruction may develop into a systemic inflammatory response syndrome (SIRS) and subsequently multiple organ failure.1,2 I/R injury during surgery is a phenomenon of tissue damage
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due to cessation of the blood flow and furthermore to anoxic damage aggravated by reperfused blood flow.3,4 Treatment results can certainly be improved by reducing the extent of this injury. From the Department of Gastroenterologic Surgery (M.T., T.T., H.H., H.I., H.T., I.O., H.T., S.F., M.K.), Division of Cancer Medicine, Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan, the Department of Surgery (M.T.), Toyama Rosai Hospital, Toyama, Japan, and the Center for Biomedical Research and Education (S.I.H.), Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan. Takashi Tani, Shin-ichi Harada, Hironori Hayashi, Hidehiro Tajima, Ichirou Ohnishi, Hiroyuki Takamura, Sachio Fushida, and Masato Kayahara contributed new reagents or analytic tools and participated in data analysis. Address reprint requests to Masaki Takeshita, Department of Surgery, Toyama Rousail Hospital, Toyama, Japan.
0041-1345/10/$–see front matter doi:10.1016/j.transproceed.2010.06.038
© 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
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In 1986, Murry et al5 observed that ischemic preconditioning (IPC) for a short duration before long-term I/R reduced the severity of the subsequent I/R injury. This phenomenon has subsequently been documented to occur in a variety of organs. Because it is easy, efficient, and cost-effective, it has been applied to clinical medicine. The duration of I/R necessary to acquire tolerance to ischemia differs for every internal organ; the effect of IPC on the small intestine is unknown to date. Nuclear factor-B (NF-B) plays a key role in the regulation of genes that function in immune and inflammatory responses.6 Itoh et al7 have suggest that overexpression of transcription factors in the small intestine after I/R correlates with programmed cell death and subsequent cellular regeneration. Among the epithelia of the internal organs, that of the small intestine has the most rapid cell turnover rate. The cell turnover is balanced by active shedding and regeneration of apoptotic elements even under physiological conditions. Apoptosis plays an important role in I/R injury;8 therefore, there is the possibility that the extent of I/R injury can be reduced by controlling apoptosis.9 In addition, Hunaki et al10 have reported that attenuation of NF-B activation with subsequent reduction in tumor necrosis factor-␣ (TNF-␣) mRNA expression after sustained ischemia plays an important role in the protective mechanisms of IPC against hepatic I/R injury. I/R injury of the liver activates NF-B and inflammatory cytokines, thus inhibition of NF-B may attenuate expression of inflammatory cytokines.11,12 We investigated the protective effects of IPC against I/R injury in rat small intestine by pathological examination, by biological examination of the DNA binding activity of NF-B, and by evaluation of the expression levels of apoptosis-related genes and of cytokine genes encoding TNF-␣, intercellular adhesion molecule-1 (ICAM-1), and interleukin-1 (IL-1). MATERIALS AND METHODS Experimental Model After male Lewis rats weighing 200 to 250 g were fasted for 24 hours, they were anesthetized with ether and their abdomens opened via a median incision. The superior mesenteric artery was occluded for 30 minutes with an atraumatic microvascular clamp. At the end of the ischemic period, the clamp was removed and the intestinal segment allowed to reperfuse for defined times. To block collateral blood supply, we used the procedure developed by Megison et al.13 The animals were injected subcutaneously with 50 U/kg of heparin before the experiment and randomly assigned to the following 3 groups: (1) the I/R group, which included 30-minute ischemia followed by defined times of reperfusion without IPC; (2) IPC ⫹ I/R group, which included animals subjected to 30 minutes IPC, namely, 5 minutes ischemia and 5 minutes reperfusion followed by ischemia and defined times of reperfusion; and (3) a sham group, which included animals subjected to anesthesia and laparotomy without ischemia. The jejunum was divided into 4 equal parts; we removed a quarter of the segment in the second part starting from the ligament of Treitz. The tissue was cut longitudinally, rinsed in physiological saline, and immediately frozen in
3407 liquid nitrogen, and was stored at ⫺80°C until use. All experiments were performed in accordance with our university guidelines for the care and use of animals.
Histochemistry We obtained 10% formalin-fixed paraffin-embedded tissue blocks of small intestinal tissue to stain with hematoxylin-eosin (HE). The intestinal mucosal injury was graded using the Park scores.14
Electrophoretic Mobility Shift Assay Nuclear proteins from the frozen small intestinal tissue were extracted using nuclear and cytoplasmic extraction reagents (NE-PER; Pierce, Rockford, Ill, USA) according to the manufacturer’s instructions. Protein concentrations were determined using a protein assay reagent (Advanced Protein Assay Reagent; Cytoskeleton, Denver, Colo, USA) with bovine serum albumin as the reference standard. Double-stranded NF-B consensus oligonucleotides (5=-AGTGAGGGGACTTTCCCAGGC-3= and 3=-TCAACTCCCCTGAA AGGGTCCG-5=; Promega, Madison, Wis, USA) were end-labeled with [␥-32P] adenosine triphosphate (3000 Ci/ mmol at 10 mCi/mL; NEN Life Science Products Inc., Boston, Mass, USA) using T4 polynucleotide kinase (TaKaRa, Kyoto, Japan). Binding reactions were performed by preincubation of 10 g of nuclear protein extracts for 30 minutes at room temperature in binding buffer (10 mM HEPES [pH 7.6], 50 mM KCl, 1 mM EDTA, 0.4% Ficoll, 1 mM DTT, 0.125 mM phenylmethylsulfonyl fluoride, and 0.05 mg/mL poly[dI-dC]; Amersham Pharmacia Biotech, Princeton, NJ, USA) followed by a 30 minutes incubation at room temperature after addition of 1 ⫻ 105 cpm of the oligonucleotide probe. Reaction products separated on 4% polyacrylamide gels in 0.5⫻ TBE buffer at 150 V for 2 hours were analyzed by autoradiography.
RNA Isolation and Reverse Transcription–Polymerase Chain Reaction Total RNA from the frozen small intestinal tissue was extracted using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions. The RNA concentration was determined by ultraviolet spectrophotometry at 260 nm. In all samples, the 260/280 nm ratio of absorbance was ⬎1.8. Total RNA (2 g) reverse transcribed to cDNA at 42°C for 60 minutes using oligo(dT) primers was inactivated by treatment with MULV RNA reverse transcriptase at 95°C for 5 minutes. Reverse-transcribed cDNA corresponding to 60 ng of total RNA was amplified using gene-specific primers and probe sets for the target cDNA, followed by quantitative detection using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif, USA). The polymerase chain reaction (PCR) was performed using the following cycling parameters: 50°C for 2 minutes and 95°C for 5 minutes, followed by 45 cycles each at 95°C for 15 second and at 60°C for 1 minute. The Taqman gene expression assay mix used for reverse transcription (RT)-PCR was TNF-␣ primer and probe mix (Rn00562055, Taqman Gene Expression Assays: Applied Biosystems), ICAM-1 primer and probe mix (Rn00564227, Taqman Gene Expression Assays: Applied Biosystems), IL-1 primer and probe mix (Rn00580432, Taqman Gene Expression Assays: Applied Biosystems), and GAPDH primer and probe mix (Taqman Gene Expression Assays: Applied Biosystems). Data were analyzed using Sequence Detection Software (Applied Biosystems) as described in User Bulletin 2.
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Western Blot Analysis Cellular protein was extracted from the frozen small intestinal tissue in a lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.1% sodium deoxycholate, 5 mM EDTA, 1% P-40 substitute [Nonidet], 1% polyethylene glycol tert-octylphenyl ether [Triton X-100], and 0.2% protease inhibitor cocktail [Sigma Chemical Co., St. Louis, Mo, USA]). Lysis buffersolubilized protein (10 g) was denatured in a loading buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 5% SDS, 5% -mercaptoethanol, and 0.01% bromophenol blue) at 95°C for 5 minutes, electrophoresed on a precast 10% SDS-polyacrylamide gel (SDSPAGE) (ATTO, Tokyo, Japan), and transferred onto PVDF membrane filters (Immobilon; Millipore, Bedford, Mass, USA) by semidry electroblotting. Nonspecific binding was blocked by incubation in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) and 5% skimmed milk. The transferred membranes were incubated for 2 hours at room temperature with anti-Bcl-xL and anti-caspase-3 antibodies (dilution 1:1000; Cell Signaling Technology, Beverly, Mass, USA) in PBS-T containing 0.5% bovine serum albumin (PBS-T-BSA). The membranes washed 4 times in PBS-T were incubated with horseradish peroxidase-conjugated antirabbit immunoglobulin G antibody (dilution 1:2000; GE Healthcare Bio-Science Corp., Piscataway, NJ, USA) in PBS-TBSA for 1 hour at room temperature. After 4 additional washes with PBS-T, the signals were visualized by enhanced chemiluminescence (ECL; GE Healthcare Bio-Science Corp.) according to the manufacturer’s instructions.
Immunohistochemical Detection of Apoptosis-Related Genes and ss-DNA Three 5-m-thick sections cut from each paraffin block, deparaffinized with graded xylene and alcohol, were immersed in absolute methanol containing 0.3% H2O2 to block endogenous peroxidase activity. After rinsing with PBS, the sections were incubated with 10% normal goat serum for 30 minutes at room temperature to block nonspecific binding. Next, the slides were incubated with anti-Bcl-xL antibody (dilution 1:100; Cell Signaling Technology), anti-caspase-3 antibody (dilution 1:100; Cell Signaling Technology), and anti-ss-DNA antibody (dilution 1:100; DAKO Japan) overnight at 4°C. The sections were then treated with biotinylated goat anti-mouse IgG (Dakopatts, Copenhagen, Denmark) for 30 minutes and with peroxidase-labeled streptavidin (Dakopatts) for 30 minutes at room temperature. The reaction products were developed by immersing the sections in 3,3=-diaminobenzidine tetrahydrochloride (DAB) solution containing 0.1% H2O2.
TAKESHITA, TANI, HARADA ET AL minutes were washed with PBS for subsequent incubation with peroxidase-labeled antidigoxigenin antibody for 30 minutes and staining with diaminobenzidine-H2O2 solution. The sections were then counterstained with DAB solution containing 0.1% H2O2.
Statistical Analysis Data are presented as mean values ⫾ SEM. Statistical assessments were performed using Mann-Whitney U tests with statistical significance accepted at P ⬍ .05.
RESULTS Activation of NF-B During Small Intestinal I/R Injury
NF-B activation during I/R injury was determined in the small intestinal nuclear extracts at 5 time points by performing electrophoretic mobility shift assay. NF-B activation started within 10 minutes after the initiation of reperfusion reaching a maximal level at 30 minutes. NF-B binding activity decreased after 60 minutes of reperfusion (Fig 1A). Activation of TNF-␣ mRNA During Small Intestinal I/R Injury
TNF-␣ activation during small intestinal I/R injury was determined in the small intestinal RNA extracts at 5 times by performing RT-PCR. It started immediately after the initiation of reperfusion and decreased after 60 minutes (Fig 1B). Histochemistry
On HE staining at 30 minutes reperfusion (Fig 2A), the I/R group showed lifting of the epithelium along the villi in the
Immunohistochemical Detection of Apoptosis in Cells DNA breaks were detected by TUNEL staining, which is based on the specific binding of the terminal deoxynucleotidyl transferase to the 3=-OH ends of DNA, to yield a polydeoxynucleotide polymer. In brief, the paraffin sections were dewaxed, rehydrated through a graded alcohol series, washed in PBS, and digested with 20 g/mL proteinase K at room temperature. The duration varied from 0 to 60 min; digestion was stopped by washing the sections under running tap water. They were then treated with 2% H2O2 solution and washed with distilled water. The washed sections were then equilibrated in Apoptag equilibration buffer containing digoxigeninuridine 5=-triphosphate (Oncor Inc., Gaithersburg, Md, USA) for 2 minutes, followed by incubation with terminal deoxynucleotidyl transferase from the same kit for 60 min at 37°C in a humid chamber. Next, samples washed in stop/wash buffer at 37°C for 30
Fig 1. (A) Electrophoretic mobility shift assay for activation of NF-B during small intestinal I/R injury. (B) Reverse transcription–polymerase chain reaction for determining the activation of TNF-␣ during small intestinal I/R injury.
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Fig 2. (A) Effect of IPC for small intestinal I/R injury examined by HE staining. Small intestine tissue samples harvested after ischemia and 30 minutes reperfusion with or without IPC were analyzed by RT-PCR. (B) Small intestine mucosal injury score was graded using the Park score (Park classification). Values are represented as mean ⫾ SEM; n ⫽ 10 in each group.
completely denuded parts. However, the IPC ⫹ I/R group showed only an extended subepithelial space. In the IPC ⫹ I/R group, the small intestinal mucosal score (Park classification) was significantly lower than that among the I/R group (P ⬍ .05; Fig 2B).
shown in Fig 5A. Bcl-xL protein expression increased in the IPC ⫹ I/R group, whereas caspase-3 protein expression increased in the I/R group and decreased in the IPC ⫹ I/R group.
IPC for Small Intestinal I/R Injury Attenuates NF-B Activation
NF-B activation induced by small intestinal I/R injury was maximal at 30 minutes of reperfusion. Therefore, we assessed whether IPC reduced the extent of NFB activation at 30 minutes of reperfusion in the small intestine. NF-B activity was clearly decreased in the IPC ⫹ I/R group (Fig 3). IPC for Small Intestinal I/R Injury Attenuates the Activation of TNF-␣, IL-1, and ICAM-1 mRNA
To study the effects of IPC on small intestinal I/R injury, RT-PCR for inflammatory genes was performed at 30 minutes reperfusion. The expression levels of TNF-␣, IL1, and ICAM-1 mRNAs in the sham group were designated as 1. The levels of TNF-␣, IL-1, and ICAM-1 mRNAs were significantly decreased in the IPC ⫹ I/R group (P ⬍ .05; Fig 4A, 4B, 4C). Changes in Protein Expression of the Apoptosis-Related Genes After IPC for Small Intestinal I/R Injury
The protein levels of caspase-3 and Bcl-xL at 30 minutes reperfusion after IPC in small intestinal I/R injury are
Fig 3. Effect of IPC on small intestinal I/R-induced NF-B activation. Nuclear translocation of NF-B was assessed in nuclear extracts of small intestine tissue samples harvested after ischemia and 30 minutes reperfusion with or without IPC. Small intestine tissue samples from the sham group served as the control.
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Assessment of Apoptosis After IPC for Small Intestinal I/R Injury
To confirm the presence of apoptotic events after IPC for small intestinal I/R injury, we performed TUNEL and ss-DNA assays, both of which showed that the number of apoptotic cells at 30 minutes reperfusion was increased in the I/R and decreased in the IPC ⫹ I/R group, respectively (Figs 6A, 6B). DISCUSSION
Fig 4. Effect of IPC on TNF-␣ (A), IL-1 (B), and ICAM-1 (C) mRNA expression in the small intestine. Small intestine RNA extracts harvested after ischemia and 30 minutes reperfusion with or without IPC were analyzed by RT-PCR. Values are represented as mean ⫾ SEM; n ⫽ 10 in each group.
Changes in Immunohistochemical Activation of the Apoptosis-Related Genes After IPC for Small Intestinal I/R Injury
Immunohistochemical analysis of tissues was performed at 30 minutes of reperfusion after IPC. Bcl-xL immunostaining increased in the IPC ⫹ I/R group (Fig 5B). However, caspase-3 immunostaining level increased in the I/R and decreased in the IPC ⫹ I/R group, respectively (Fig 5C).
Small intestinal transplantation is performed for patients with intestinal tract malfunctions, including short bowel syndrome, who have been stabilized by total parental nutrition (TPN). The small intestine is more vulnerable than other organs to I/R injury, infection, and graft rejection. Tissue damage results from ischemia, which leads to the release of reactive oxygen species (ROS). Upon reperfusion, these ROS cause further injury due to the sudden increase in oxygen load, the I/R injury.3,4 The onset of ischemia immediately causes structural damage to the small intestinal mucosa: mucosal edema, bleeding, ulceration, and cell shedding from the villi. Although the mucosa is susceptible to ischemic damage, it possesses remarkable regenerative ability. If the villi are severely injured but normalcy of the crypts is maintained, early histological damage is ameliorated. The mucosal regeneration process starts by rolling of the absorptive epithelial cells to cover the mucous-deficient site with a simultaneous increase in active mitosis and cell multiplication in the crypts, which play an important role in regeneration after I/R. The mechanism of I/R injury involves: (1) generation of ROS, (2) activation of transcription factors, (3) cytokine production, (4) expression of inducible nitric oxide synthase, (5) expression of adhesion molecules, (6) eicosanoid production, (7) injury to blood vessel endothelium, (8) neutrophil infiltration, (9) platelet adhesion, (10) microscopic circulating incompletion, and (11) apoptosis. The hypothesis that leukocyte–vessel wall interactions occur after I/R injury has attracted attention as the major step in the mechanism of I/R injury. It is assumed that microcirculation injury produces mediators such as ROS that are released from activated neutrophils. Under the influence of inflammatory cytokines, neutrophils roll, adapt, and migrate outside the blood vessels after adhering to the endothelium via adhesion molecules, thereby leading to organ injury.15–17 TNF-␣, an inflammatory cytokine, mediates the expression of other inflammatory cytokines such as IL-1 and IL-8 and of adhesion molecules such as ICAM-1, activates neutrophils,18,19 stimulates production of plateletactivating factor (PAF) and endothelin, and causes platelet thrombus formation and blood vessel contraction.20,21 The result is the no-reflow phenomenon that induces organ damage. IL-1 promotes the expression of adhesion molecules in the blood vessel endothelium that facilitates neutrophil binding. The extent of small intestinal I/R injury can be reduced by administering an anti-TNF-␣ antibody.22
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Fig 5. (A) Western blotting analysis of apoptosis-related genes. (B, C) Immunohistochemical analysis of the activation of apoptosis-related genes. The expression levels of the antiapoptotic gene Bcl-xL and the proapoptotic gene caspase-3 were analyzed after ischemia and 30 minutes reperfusion with or without IPC.
Activation of the transcription factor NF-B is required for the expression of cytokines or adhesion molecules. NF-B plays an important role in the immune and inflammatory responses.6 In the cytoplasm, NF-B is bound to inhibitor B (IB) when inactive; however, when cells are stimulated, NF-B translocates to the nucleus by separating from IB, particularly IB-␣, functioning as a transcription activity factor for the target gene. Tyrosinal phosphorylation is required for separation from IB-␣.23 Upon external stimulation, one of the control systems reacts through a biochemical change in an existing factor without requiring new protein synthesis for NF-B activation. NF-B is activated after early reperfusion in hepatic I/R injury.6,11,12 It is assumed that neutrophil-mediated tissue injury progresses because of
activation of inflammatory cytokines and adhesion molecules.24,25 In this study, activation of NF-B in the small intestinal nuclear extracts increased at 30 minutes after reperfusion with consequently increased expression of TNF-␣. Reduction in the extent of I/R injury prevents posttransplantation graft dysfunction. A reduction in NF-B activity during I/R decreases the expression of cytokines and thus prevents mucosal membrane damage. The small intestine, which is involved in purine catabolism, contains xanthine oxidase,26 a factor responsible for the abundant release of ROS during hypoxic states. Therefore, it is assumed that the small intestine is poorly tolerant of I/R injury.27 However, if crypt integrity is maintained, mucosal ischemic injury becomes reversible, and the nuclear division is acti-
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Fig 6. Effect of IPC for small intestinal I/R injury on the number of apoptotic cells evaluated by TUNEL staining (A) and immunohistochemical detection of ss-DNA (B) after ischemia and 30 minutes reperfusion with or without IPC.
vated in the crypt domain. The mucosa regenerates to cover the injured part.28 IPC has been considered to be a precautionary measure for various organs. Murry et al5 reported that short durations of ischemia and reperfusion rendered myocardium resistant to a subsequent sustained ischemic insult. This manuever is considered to reinforce the capacity to overcome the same or a different stress when the organization receives stress once.29 However, there are few studies on small intestinal IPC. On the basis of the time required for acquisition of a protective effect, preconditioning is classified into early (1 or 2 hours) and delayed (approximately 24 hours). In addition, nitric oxide (NO) mainly participates in early preconditioning and heat-shock proteins (HSPs) in delayed preconditioning.30,31 In this study, we examined the effect of early preconditioning on the small intestine at the cellular level determining the mRNA levels of TNF-␣, IL-1, and ICAM-1. The mRNA levels of these cytokines increased after 30 minutes of reperfusion and decreased after IPC. Therefore, it appeared that leukocyte–vessel wall interactions were reduced. In addition, decreased expression of NF-B, which is required to cause I/R injury, is believed to be one of the important mechanisms in the acquisition of a protective effect during the early stage of small intestinal IPC. In addition, histopathological evaluation of the mucosal membrane injury by the Park score revealed that IPC reduced its extent.
Apoptosis plays an important role to cause I/R injury;8 therefore, it is possible that the extent of I/R injury may be reduced by controlling apoptosis, which is characterized by cell size reduction, nuclear chromatin condensation, cell fragmentation, and removal of the nucleus. It is distinguished from necrosis, which is characterized by cellular swelling.32 Apoptosis-related genes, such as those of the Bcl-2 or caspase families, are known components of the signaling pathway controlling apoptosis. The Bcl-2 oncogene was isolated from a human follicular B-cell lymphoma harboring the chromosomal translocation.14,18 It is an important gene family controlling the main pathway of apoptosis. Bcl-2 and Bcl-xL of the Bcl-2 family suppress apoptosis, whereas Bax induces apoptosis. Apoptosis is controlled by the balance between induction and suppression.33 Caspase, a cysteine protease homologous to the nematode cell death gene ced-3, functions as an important mediator of apoptosis. Apoptosis is mediated by cleavage of activated caspase-3 protein, which leads to a change in its conformation.34 Caspase-3 is activated by intracytoplasmic release of mitochondrial proteins such as cytochrome c and AIF, which is restrained by Bcl-2 and Bcl-xL upstream of caspase.35,36 Furthermore, the expression of antiapoptotic genes such as Bcl-2 or Bcl-xL is activated by the suppression of the transcription factor NF-B, which restrains apoptosis.37 This study revealed reduced NF-B and apoptosisrelated gene activation after IPC. Under the influence of
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IPC, the protein expression and immunostaining levels revealed increased Bcl-xL expression and decreased caspase-3 expression. In addition, apoptosis was restrained by IPC, as revealed by TUNEL staining and ss-DNA detection. In conclusion, transcription factors and cytokines play roles in small intestinal I/R injury. IPC produced by clamping of the superior mesenteric artery attenuated apoptosis and cell injury occurring during small intestinal I/R, documenting a beneficial effect that was due to attenuation of signal transmission via the transcription factor NF-B and the cytokine TNF-␣. REFERENCES 1. Deitch EA: Multiple organ failure: pathology and potential future therapy. Ann Surg 216:117, 1992 2. Meakins JL, Marshall JC: The gastrointestinal tract: the motor of multiple organ failure. Arch Surg 121:197, 1986 3. Corner HD, Gao W, Nukina S, et al: Evidence that free radicals are involved in graft failure following orthotopic liver transplantation in the rat-an electron paramagnetic resonance spin tapping study. Transplantation 54:199, 1992 4. Ploeg RJ, D’Alessandro AM, Knechtle SJ, et al: Risk factors for primary dysfunction after liver transplantation: a multivariate analysis. Transplantation 55:807, 1993 5. Murry CE, Jennings RB, Reimer KA, et al: Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74:1124, 1986 6. Baeuerle PA, Henkel T: Function and activation of NF-B in the immune system. Annu Rev Immunol 12:141, 1994 7. Itoh H, Yagi M, Fusida S, et al: Activation of immediate early gene, c-fos, and c-jun in the rat small intestine after ischemiareperfusion. Transplantation 69:598, 2000 8. Noda T, Iwakiri R, Fujimoto K, et al: Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa. Am J Physiol 274:G270, 1998 9. Shah KA, Shurey S, Green CJ: Apoptosis after intestinal ischemia-reperfusion injury; a morphological study. Transplantation 64:1393, 1997 10. Funaki H, Shimizu K, Harada S, et al: Essential role for nuclear factor B in ischemic preconditioning for ischemiareperfusion injury of the mouse liver. Transplantation 74:551, 2002 11. Zwacka RM, Zhang Y, Zhou W, et al: Ischemia/reperfusion injury in the liver of BALB/c mice activates AP-1 and nuclear factor B independently of IB degradation. Hepatology 28:1022, 1998 12. Yoshidome H, Kato A, Edwards MJ, et al: Interleukin-10 suppresses hepatic ischemia/ reperfusion injury in mice: implication of a central role for nuclear factor B. Hepatology 30:203, 1999 13. Megison SM, Horton JW, Chao H, et al: A new model for intestinal ischemia in the rat. J Surg Res 49:168, 1990 14. Park PO, Haglund U, Bulkley GB, et al: The sequence of development of intestinal tissue injury after strangulation ischemia and reperfusion. Surgery 107:574, 1990 15. Kubes P: The role of shear forces in ischemia/reperfusioninduced neutrophil rolling and adhesion. J Leukoc Biol 62:458, 1997 16. Beuk RJ, Heineman E, Tangelder GJ, et al: Total warm ischemia and reperfusion impairs flow in all rat gut layers but
3413 increases leukocyte-vessel wall interactions in the submucosa only. Ann Surg 231:96, 2000 17. Boyd AJ, Sherman IA, Saibil FG: Intestinal microcirculation and leukocyte behavior in ischemia-reperfusion injury. Microvasc Res 47:355, 1994 18. Lo SK, Detmers PA, Levin SM, et al: Transient adhesion of neutrophils to endothelium. J Exp Med 169:1779, 1989 19. Berger M, Wetzler EM, Wallis RS: Tumor necrosis factor is the major monocyte product that increases complement receptor expression on mature human neutrophils. Blood 71:151, 1998 20. Goto M, Takei M, Kawano S, et al: Endothelin-1 is involved in the pathogenesis of ischemia/reperfusion liver injury by hepatic microcirculatory disturbances. Hepatology 19:675, 1994 21. Nakamura S, Nishiyama R, Serizawa A, et al: Hepatic release of endothelin-1 after warm ischemia. Reperfusion injury and its hemodynamic effect. Transplantation 59:679, 1995 22. Yeh KY, Yeh M, Glass J, et al: Rapid activation of NF-B and AP-1 and target gene expression in postischemic rat intestine. Gastroenterology 118:525, 2000 23. Imbert V, Rupec RA, Livolsi A, et al: Tyrosine phosphorylation of IB-␣ activates NF-B without proteolytic degradation of IB-␣. Cell 86:787, 1996 24. Fan C, Zwacka RM, Engelhardt JF: Therapeutic approaches for ischemia/reperfusion injury in the liver. J Mol Med 77:577, 1999 25. Lentsch AB, Kato A, Yoshidome H, et al: Inflammatory mechanisms and therapeutic strategies for warm hepatic ischemia/ reperfusion injury. Hepatology 32:169, 2000 26. McCord JM, Roy RS, Schffer SW: Free radicals and myocardial ischemia. The role of xanthine oxidase. Adv Myocardiol 5:183, 1985 27. Battelli MG, Corte ED, Stirpe F: Xanthine oxidase type D (dehydrogenase) in the intestine and other organs of the rat. Biochem J 126:747, 1972 28. Wagner R, Gabert H, Horn P: Ischemia and postischemic regeneration of the small intestinal mucosa, a light microscopic and autoradiographic study. Virchows Arch 31:259, 1994 29. Yellon DM, Baxter GF: A second window of protection or delayed preconditioning phenomenon: future horizons for myocardial protection? J Mol Cell Cardiol 27:1023, 1995 30. Tsuruma T, Yagihashi A, Matsuno T, et al: The heat-shock protein 70 family reduces ischemia/reperfusion injury in small intestine. Transplant Proc 28:1298, 1996 31. Hotter G, Closa D, Prados M, et al: Intestinal preconditioning is mediated by a transient increase in nitric oxide. Biochem Biophys Res Commun 222:27, 1996 32. Kerr JFR, Wyllie AH, Currie AR, et al: Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239, 1972 33. Yang E, Zha J, Jockel J, et al: Bad, a heterodimeric partner for Bcl-xL and Bcl-2, displaces Bax and promotes cell death. Cell 80:285, 1995 34. Kuida K, Zheng TS, Na S, et al: Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384:368, 1996 35. Yang J, Liu X, Bhalla K, et al: Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275:1129, 1997 36. Kluck RM, Bossy WE, Green DR, et al: The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275:1132, 1997 37. Tamatani M, Che YH, Matsuzaki H, et al: Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NF-B activation in primary hippocampal neurons. J Biol Chem 274:8531, 1999
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due to cessation of the blood flow and furthermore to anoxic damage aggravated by reperfused blood flow.3,4 Treatment results can certainly be improved by reducing the extent of this injury. From the Department of Gastroenterologic Surgery (M.T., T.T., H.H., H.I., H.T., I.O., H.T., S.F., M.K.), Division of Cancer Medicine, Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan, the Department of Surgery (M.T.), Toyama Rosai Hospital, Toyama, Japan, and the Center for Biomedical Research and Education (S.I.H.), Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan. Takashi Tani, Shin-ichi Harada, Hironori Hayashi, Hidehiro Tajima, Ichirou Ohnishi, Hiroyuki Takamura, Sachio Fushida, and Masato Kayahara contributed new reagents or analytic tools and participated in data analysis. Address reprint requests to Masaki Takeshita, Department of Surgery, Toyama Rousail Hospital, Toyama, Japan.
0041-1345/10/$–see front matter doi:10.1016/j.transproceed.2010.06.038
© 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
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Transplantation Proceedings, 42, 3406 –3413 (2010)
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In 1986, Murry et al5 observed that ischemic preconditioning (IPC) for a short duration before long-term I/R reduced the severity of the subsequent I/R injury. This phenomenon has subsequently been documented to occur in a variety of organs. Because it is easy, efficient, and cost-effective, it has been applied to clinical medicine. The duration of I/R necessary to acquire tolerance to ischemia differs for every internal organ; the effect of IPC on the small intestine is unknown to date. Nuclear factor-B (NF-B) plays a key role in the regulation of genes that function in immune and inflammatory responses.6 Itoh et al7 have suggest that overexpression of transcription factors in the small intestine after I/R correlates with programmed cell death and subsequent cellular regeneration. Among the epithelia of the internal organs, that of the small intestine has the most rapid cell turnover rate. The cell turnover is balanced by active shedding and regeneration of apoptotic elements even under physiological conditions. Apoptosis plays an important role in I/R injury;8 therefore, there is the possibility that the extent of I/R injury can be reduced by controlling apoptosis.9 In addition, Hunaki et al10 have reported that attenuation of NF-B activation with subsequent reduction in tumor necrosis factor-␣ (TNF-␣) mRNA expression after sustained ischemia plays an important role in the protective mechanisms of IPC against hepatic I/R injury. I/R injury of the liver activates NF-B and inflammatory cytokines, thus inhibition of NF-B may attenuate expression of inflammatory cytokines.11,12 We investigated the protective effects of IPC against I/R injury in rat small intestine by pathological examination, by biological examination of the DNA binding activity of NF-B, and by evaluation of the expression levels of apoptosis-related genes and of cytokine genes encoding TNF-␣, intercellular adhesion molecule-1 (ICAM-1), and interleukin-1 (IL-1). MATERIALS AND METHODS Experimental Model After male Lewis rats weighing 200 to 250 g were fasted for 24 hours, they were anesthetized with ether and their abdomens opened via a median incision. The superior mesenteric artery was occluded for 30 minutes with an atraumatic microvascular clamp. At the end of the ischemic period, the clamp was removed and the intestinal segment allowed to reperfuse for defined times. To block collateral blood supply, we used the procedure developed by Megison et al.13 The animals were injected subcutaneously with 50 U/kg of heparin before the experiment and randomly assigned to the following 3 groups: (1) the I/R group, which included 30-minute ischemia followed by defined times of reperfusion without IPC; (2) IPC ⫹ I/R group, which included animals subjected to 30 minutes IPC, namely, 5 minutes ischemia and 5 minutes reperfusion followed by ischemia and defined times of reperfusion; and (3) a sham group, which included animals subjected to anesthesia and laparotomy without ischemia. The jejunum was divided into 4 equal parts; we removed a quarter of the segment in the second part starting from the ligament of Treitz. The tissue was cut longitudinally, rinsed in physiological saline, and immediately frozen in
3407 liquid nitrogen, and was stored at ⫺80°C until use. All experiments were performed in accordance with our university guidelines for the care and use of animals.
Histochemistry We obtained 10% formalin-fixed paraffin-embedded tissue blocks of small intestinal tissue to stain with hematoxylin-eosin (HE). The intestinal mucosal injury was graded using the Park scores.14
Electrophoretic Mobility Shift Assay Nuclear proteins from the frozen small intestinal tissue were extracted using nuclear and cytoplasmic extraction reagents (NE-PER; Pierce, Rockford, Ill, USA) according to the manufacturer’s instructions. Protein concentrations were determined using a protein assay reagent (Advanced Protein Assay Reagent; Cytoskeleton, Denver, Colo, USA) with bovine serum albumin as the reference standard. Double-stranded NF-B consensus oligonucleotides (5=-AGTGAGGGGACTTTCCCAGGC-3= and 3=-TCAACTCCCCTGAA AGGGTCCG-5=; Promega, Madison, Wis, USA) were end-labeled with [␥-32P] adenosine triphosphate (3000 Ci/ mmol at 10 mCi/mL; NEN Life Science Products Inc., Boston, Mass, USA) using T4 polynucleotide kinase (TaKaRa, Kyoto, Japan). Binding reactions were performed by preincubation of 10 g of nuclear protein extracts for 30 minutes at room temperature in binding buffer (10 mM HEPES [pH 7.6], 50 mM KCl, 1 mM EDTA, 0.4% Ficoll, 1 mM DTT, 0.125 mM phenylmethylsulfonyl fluoride, and 0.05 mg/mL poly[dI-dC]; Amersham Pharmacia Biotech, Princeton, NJ, USA) followed by a 30 minutes incubation at room temperature after addition of 1 ⫻ 105 cpm of the oligonucleotide probe. Reaction products separated on 4% polyacrylamide gels in 0.5⫻ TBE buffer at 150 V for 2 hours were analyzed by autoradiography.
RNA Isolation and Reverse Transcription–Polymerase Chain Reaction Total RNA from the frozen small intestinal tissue was extracted using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions. The RNA concentration was determined by ultraviolet spectrophotometry at 260 nm. In all samples, the 260/280 nm ratio of absorbance was ⬎1.8. Total RNA (2 g) reverse transcribed to cDNA at 42°C for 60 minutes using oligo(dT) primers was inactivated by treatment with MULV RNA reverse transcriptase at 95°C for 5 minutes. Reverse-transcribed cDNA corresponding to 60 ng of total RNA was amplified using gene-specific primers and probe sets for the target cDNA, followed by quantitative detection using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif, USA). The polymerase chain reaction (PCR) was performed using the following cycling parameters: 50°C for 2 minutes and 95°C for 5 minutes, followed by 45 cycles each at 95°C for 15 second and at 60°C for 1 minute. The Taqman gene expression assay mix used for reverse transcription (RT)-PCR was TNF-␣ primer and probe mix (Rn00562055, Taqman Gene Expression Assays: Applied Biosystems), ICAM-1 primer and probe mix (Rn00564227, Taqman Gene Expression Assays: Applied Biosystems), IL-1 primer and probe mix (Rn00580432, Taqman Gene Expression Assays: Applied Biosystems), and GAPDH primer and probe mix (Taqman Gene Expression Assays: Applied Biosystems). Data were analyzed using Sequence Detection Software (Applied Biosystems) as described in User Bulletin 2.
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Western Blot Analysis Cellular protein was extracted from the frozen small intestinal tissue in a lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.1% sodium deoxycholate, 5 mM EDTA, 1% P-40 substitute [Nonidet], 1% polyethylene glycol tert-octylphenyl ether [Triton X-100], and 0.2% protease inhibitor cocktail [Sigma Chemical Co., St. Louis, Mo, USA]). Lysis buffersolubilized protein (10 g) was denatured in a loading buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 5% SDS, 5% -mercaptoethanol, and 0.01% bromophenol blue) at 95°C for 5 minutes, electrophoresed on a precast 10% SDS-polyacrylamide gel (SDSPAGE) (ATTO, Tokyo, Japan), and transferred onto PVDF membrane filters (Immobilon; Millipore, Bedford, Mass, USA) by semidry electroblotting. Nonspecific binding was blocked by incubation in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) and 5% skimmed milk. The transferred membranes were incubated for 2 hours at room temperature with anti-Bcl-xL and anti-caspase-3 antibodies (dilution 1:1000; Cell Signaling Technology, Beverly, Mass, USA) in PBS-T containing 0.5% bovine serum albumin (PBS-T-BSA). The membranes washed 4 times in PBS-T were incubated with horseradish peroxidase-conjugated antirabbit immunoglobulin G antibody (dilution 1:2000; GE Healthcare Bio-Science Corp., Piscataway, NJ, USA) in PBS-TBSA for 1 hour at room temperature. After 4 additional washes with PBS-T, the signals were visualized by enhanced chemiluminescence (ECL; GE Healthcare Bio-Science Corp.) according to the manufacturer’s instructions.
Immunohistochemical Detection of Apoptosis-Related Genes and ss-DNA Three 5-m-thick sections cut from each paraffin block, deparaffinized with graded xylene and alcohol, were immersed in absolute methanol containing 0.3% H2O2 to block endogenous peroxidase activity. After rinsing with PBS, the sections were incubated with 10% normal goat serum for 30 minutes at room temperature to block nonspecific binding. Next, the slides were incubated with anti-Bcl-xL antibody (dilution 1:100; Cell Signaling Technology), anti-caspase-3 antibody (dilution 1:100; Cell Signaling Technology), and anti-ss-DNA antibody (dilution 1:100; DAKO Japan) overnight at 4°C. The sections were then treated with biotinylated goat anti-mouse IgG (Dakopatts, Copenhagen, Denmark) for 30 minutes and with peroxidase-labeled streptavidin (Dakopatts) for 30 minutes at room temperature. The reaction products were developed by immersing the sections in 3,3=-diaminobenzidine tetrahydrochloride (DAB) solution containing 0.1% H2O2.
TAKESHITA, TANI, HARADA ET AL minutes were washed with PBS for subsequent incubation with peroxidase-labeled antidigoxigenin antibody for 30 minutes and staining with diaminobenzidine-H2O2 solution. The sections were then counterstained with DAB solution containing 0.1% H2O2.
Statistical Analysis Data are presented as mean values ⫾ SEM. Statistical assessments were performed using Mann-Whitney U tests with statistical significance accepted at P ⬍ .05.
RESULTS Activation of NF-B During Small Intestinal I/R Injury
NF-B activation during I/R injury was determined in the small intestinal nuclear extracts at 5 time points by performing electrophoretic mobility shift assay. NF-B activation started within 10 minutes after the initiation of reperfusion reaching a maximal level at 30 minutes. NF-B binding activity decreased after 60 minutes of reperfusion (Fig 1A). Activation of TNF-␣ mRNA During Small Intestinal I/R Injury
TNF-␣ activation during small intestinal I/R injury was determined in the small intestinal RNA extracts at 5 times by performing RT-PCR. It started immediately after the initiation of reperfusion and decreased after 60 minutes (Fig 1B). Histochemistry
On HE staining at 30 minutes reperfusion (Fig 2A), the I/R group showed lifting of the epithelium along the villi in the
Immunohistochemical Detection of Apoptosis in Cells DNA breaks were detected by TUNEL staining, which is based on the specific binding of the terminal deoxynucleotidyl transferase to the 3=-OH ends of DNA, to yield a polydeoxynucleotide polymer. In brief, the paraffin sections were dewaxed, rehydrated through a graded alcohol series, washed in PBS, and digested with 20 g/mL proteinase K at room temperature. The duration varied from 0 to 60 min; digestion was stopped by washing the sections under running tap water. They were then treated with 2% H2O2 solution and washed with distilled water. The washed sections were then equilibrated in Apoptag equilibration buffer containing digoxigeninuridine 5=-triphosphate (Oncor Inc., Gaithersburg, Md, USA) for 2 minutes, followed by incubation with terminal deoxynucleotidyl transferase from the same kit for 60 min at 37°C in a humid chamber. Next, samples washed in stop/wash buffer at 37°C for 30
Fig 1. (A) Electrophoretic mobility shift assay for activation of NF-B during small intestinal I/R injury. (B) Reverse transcription–polymerase chain reaction for determining the activation of TNF-␣ during small intestinal I/R injury.
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Fig 2. (A) Effect of IPC for small intestinal I/R injury examined by HE staining. Small intestine tissue samples harvested after ischemia and 30 minutes reperfusion with or without IPC were analyzed by RT-PCR. (B) Small intestine mucosal injury score was graded using the Park score (Park classification). Values are represented as mean ⫾ SEM; n ⫽ 10 in each group.
completely denuded parts. However, the IPC ⫹ I/R group showed only an extended subepithelial space. In the IPC ⫹ I/R group, the small intestinal mucosal score (Park classification) was significantly lower than that among the I/R group (P ⬍ .05; Fig 2B).
shown in Fig 5A. Bcl-xL protein expression increased in the IPC ⫹ I/R group, whereas caspase-3 protein expression increased in the I/R group and decreased in the IPC ⫹ I/R group.
IPC for Small Intestinal I/R Injury Attenuates NF-B Activation
NF-B activation induced by small intestinal I/R injury was maximal at 30 minutes of reperfusion. Therefore, we assessed whether IPC reduced the extent of NFB activation at 30 minutes of reperfusion in the small intestine. NF-B activity was clearly decreased in the IPC ⫹ I/R group (Fig 3). IPC for Small Intestinal I/R Injury Attenuates the Activation of TNF-␣, IL-1, and ICAM-1 mRNA
To study the effects of IPC on small intestinal I/R injury, RT-PCR for inflammatory genes was performed at 30 minutes reperfusion. The expression levels of TNF-␣, IL1, and ICAM-1 mRNAs in the sham group were designated as 1. The levels of TNF-␣, IL-1, and ICAM-1 mRNAs were significantly decreased in the IPC ⫹ I/R group (P ⬍ .05; Fig 4A, 4B, 4C). Changes in Protein Expression of the Apoptosis-Related Genes After IPC for Small Intestinal I/R Injury
The protein levels of caspase-3 and Bcl-xL at 30 minutes reperfusion after IPC in small intestinal I/R injury are
Fig 3. Effect of IPC on small intestinal I/R-induced NF-B activation. Nuclear translocation of NF-B was assessed in nuclear extracts of small intestine tissue samples harvested after ischemia and 30 minutes reperfusion with or without IPC. Small intestine tissue samples from the sham group served as the control.
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Assessment of Apoptosis After IPC for Small Intestinal I/R Injury
To confirm the presence of apoptotic events after IPC for small intestinal I/R injury, we performed TUNEL and ss-DNA assays, both of which showed that the number of apoptotic cells at 30 minutes reperfusion was increased in the I/R and decreased in the IPC ⫹ I/R group, respectively (Figs 6A, 6B). DISCUSSION
Fig 4. Effect of IPC on TNF-␣ (A), IL-1 (B), and ICAM-1 (C) mRNA expression in the small intestine. Small intestine RNA extracts harvested after ischemia and 30 minutes reperfusion with or without IPC were analyzed by RT-PCR. Values are represented as mean ⫾ SEM; n ⫽ 10 in each group.
Changes in Immunohistochemical Activation of the Apoptosis-Related Genes After IPC for Small Intestinal I/R Injury
Immunohistochemical analysis of tissues was performed at 30 minutes of reperfusion after IPC. Bcl-xL immunostaining increased in the IPC ⫹ I/R group (Fig 5B). However, caspase-3 immunostaining level increased in the I/R and decreased in the IPC ⫹ I/R group, respectively (Fig 5C).
Small intestinal transplantation is performed for patients with intestinal tract malfunctions, including short bowel syndrome, who have been stabilized by total parental nutrition (TPN). The small intestine is more vulnerable than other organs to I/R injury, infection, and graft rejection. Tissue damage results from ischemia, which leads to the release of reactive oxygen species (ROS). Upon reperfusion, these ROS cause further injury due to the sudden increase in oxygen load, the I/R injury.3,4 The onset of ischemia immediately causes structural damage to the small intestinal mucosa: mucosal edema, bleeding, ulceration, and cell shedding from the villi. Although the mucosa is susceptible to ischemic damage, it possesses remarkable regenerative ability. If the villi are severely injured but normalcy of the crypts is maintained, early histological damage is ameliorated. The mucosal regeneration process starts by rolling of the absorptive epithelial cells to cover the mucous-deficient site with a simultaneous increase in active mitosis and cell multiplication in the crypts, which play an important role in regeneration after I/R. The mechanism of I/R injury involves: (1) generation of ROS, (2) activation of transcription factors, (3) cytokine production, (4) expression of inducible nitric oxide synthase, (5) expression of adhesion molecules, (6) eicosanoid production, (7) injury to blood vessel endothelium, (8) neutrophil infiltration, (9) platelet adhesion, (10) microscopic circulating incompletion, and (11) apoptosis. The hypothesis that leukocyte–vessel wall interactions occur after I/R injury has attracted attention as the major step in the mechanism of I/R injury. It is assumed that microcirculation injury produces mediators such as ROS that are released from activated neutrophils. Under the influence of inflammatory cytokines, neutrophils roll, adapt, and migrate outside the blood vessels after adhering to the endothelium via adhesion molecules, thereby leading to organ injury.15–17 TNF-␣, an inflammatory cytokine, mediates the expression of other inflammatory cytokines such as IL-1 and IL-8 and of adhesion molecules such as ICAM-1, activates neutrophils,18,19 stimulates production of plateletactivating factor (PAF) and endothelin, and causes platelet thrombus formation and blood vessel contraction.20,21 The result is the no-reflow phenomenon that induces organ damage. IL-1 promotes the expression of adhesion molecules in the blood vessel endothelium that facilitates neutrophil binding. The extent of small intestinal I/R injury can be reduced by administering an anti-TNF-␣ antibody.22
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Fig 5. (A) Western blotting analysis of apoptosis-related genes. (B, C) Immunohistochemical analysis of the activation of apoptosis-related genes. The expression levels of the antiapoptotic gene Bcl-xL and the proapoptotic gene caspase-3 were analyzed after ischemia and 30 minutes reperfusion with or without IPC.
Activation of the transcription factor NF-B is required for the expression of cytokines or adhesion molecules. NF-B plays an important role in the immune and inflammatory responses.6 In the cytoplasm, NF-B is bound to inhibitor B (IB) when inactive; however, when cells are stimulated, NF-B translocates to the nucleus by separating from IB, particularly IB-␣, functioning as a transcription activity factor for the target gene. Tyrosinal phosphorylation is required for separation from IB-␣.23 Upon external stimulation, one of the control systems reacts through a biochemical change in an existing factor without requiring new protein synthesis for NF-B activation. NF-B is activated after early reperfusion in hepatic I/R injury.6,11,12 It is assumed that neutrophil-mediated tissue injury progresses because of
activation of inflammatory cytokines and adhesion molecules.24,25 In this study, activation of NF-B in the small intestinal nuclear extracts increased at 30 minutes after reperfusion with consequently increased expression of TNF-␣. Reduction in the extent of I/R injury prevents posttransplantation graft dysfunction. A reduction in NF-B activity during I/R decreases the expression of cytokines and thus prevents mucosal membrane damage. The small intestine, which is involved in purine catabolism, contains xanthine oxidase,26 a factor responsible for the abundant release of ROS during hypoxic states. Therefore, it is assumed that the small intestine is poorly tolerant of I/R injury.27 However, if crypt integrity is maintained, mucosal ischemic injury becomes reversible, and the nuclear division is acti-
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Fig 6. Effect of IPC for small intestinal I/R injury on the number of apoptotic cells evaluated by TUNEL staining (A) and immunohistochemical detection of ss-DNA (B) after ischemia and 30 minutes reperfusion with or without IPC.
vated in the crypt domain. The mucosa regenerates to cover the injured part.28 IPC has been considered to be a precautionary measure for various organs. Murry et al5 reported that short durations of ischemia and reperfusion rendered myocardium resistant to a subsequent sustained ischemic insult. This manuever is considered to reinforce the capacity to overcome the same or a different stress when the organization receives stress once.29 However, there are few studies on small intestinal IPC. On the basis of the time required for acquisition of a protective effect, preconditioning is classified into early (1 or 2 hours) and delayed (approximately 24 hours). In addition, nitric oxide (NO) mainly participates in early preconditioning and heat-shock proteins (HSPs) in delayed preconditioning.30,31 In this study, we examined the effect of early preconditioning on the small intestine at the cellular level determining the mRNA levels of TNF-␣, IL-1, and ICAM-1. The mRNA levels of these cytokines increased after 30 minutes of reperfusion and decreased after IPC. Therefore, it appeared that leukocyte–vessel wall interactions were reduced. In addition, decreased expression of NF-B, which is required to cause I/R injury, is believed to be one of the important mechanisms in the acquisition of a protective effect during the early stage of small intestinal IPC. In addition, histopathological evaluation of the mucosal membrane injury by the Park score revealed that IPC reduced its extent.
Apoptosis plays an important role to cause I/R injury;8 therefore, it is possible that the extent of I/R injury may be reduced by controlling apoptosis, which is characterized by cell size reduction, nuclear chromatin condensation, cell fragmentation, and removal of the nucleus. It is distinguished from necrosis, which is characterized by cellular swelling.32 Apoptosis-related genes, such as those of the Bcl-2 or caspase families, are known components of the signaling pathway controlling apoptosis. The Bcl-2 oncogene was isolated from a human follicular B-cell lymphoma harboring the chromosomal translocation.14,18 It is an important gene family controlling the main pathway of apoptosis. Bcl-2 and Bcl-xL of the Bcl-2 family suppress apoptosis, whereas Bax induces apoptosis. Apoptosis is controlled by the balance between induction and suppression.33 Caspase, a cysteine protease homologous to the nematode cell death gene ced-3, functions as an important mediator of apoptosis. Apoptosis is mediated by cleavage of activated caspase-3 protein, which leads to a change in its conformation.34 Caspase-3 is activated by intracytoplasmic release of mitochondrial proteins such as cytochrome c and AIF, which is restrained by Bcl-2 and Bcl-xL upstream of caspase.35,36 Furthermore, the expression of antiapoptotic genes such as Bcl-2 or Bcl-xL is activated by the suppression of the transcription factor NF-B, which restrains apoptosis.37 This study revealed reduced NF-B and apoptosisrelated gene activation after IPC. Under the influence of
ROLE OF TRANSCRIPTION FACTORS
IPC, the protein expression and immunostaining levels revealed increased Bcl-xL expression and decreased caspase-3 expression. In addition, apoptosis was restrained by IPC, as revealed by TUNEL staining and ss-DNA detection. In conclusion, transcription factors and cytokines play roles in small intestinal I/R injury. IPC produced by clamping of the superior mesenteric artery attenuated apoptosis and cell injury occurring during small intestinal I/R, documenting a beneficial effect that was due to attenuation of signal transmission via the transcription factor NF-B and the cytokine TNF-␣. REFERENCES 1. Deitch EA: Multiple organ failure: pathology and potential future therapy. Ann Surg 216:117, 1992 2. Meakins JL, Marshall JC: The gastrointestinal tract: the motor of multiple organ failure. Arch Surg 121:197, 1986 3. Corner HD, Gao W, Nukina S, et al: Evidence that free radicals are involved in graft failure following orthotopic liver transplantation in the rat-an electron paramagnetic resonance spin tapping study. Transplantation 54:199, 1992 4. Ploeg RJ, D’Alessandro AM, Knechtle SJ, et al: Risk factors for primary dysfunction after liver transplantation: a multivariate analysis. Transplantation 55:807, 1993 5. Murry CE, Jennings RB, Reimer KA, et al: Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74:1124, 1986 6. Baeuerle PA, Henkel T: Function and activation of NF-B in the immune system. Annu Rev Immunol 12:141, 1994 7. Itoh H, Yagi M, Fusida S, et al: Activation of immediate early gene, c-fos, and c-jun in the rat small intestine after ischemiareperfusion. Transplantation 69:598, 2000 8. Noda T, Iwakiri R, Fujimoto K, et al: Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa. Am J Physiol 274:G270, 1998 9. Shah KA, Shurey S, Green CJ: Apoptosis after intestinal ischemia-reperfusion injury; a morphological study. Transplantation 64:1393, 1997 10. Funaki H, Shimizu K, Harada S, et al: Essential role for nuclear factor B in ischemic preconditioning for ischemiareperfusion injury of the mouse liver. Transplantation 74:551, 2002 11. Zwacka RM, Zhang Y, Zhou W, et al: Ischemia/reperfusion injury in the liver of BALB/c mice activates AP-1 and nuclear factor B independently of IB degradation. Hepatology 28:1022, 1998 12. Yoshidome H, Kato A, Edwards MJ, et al: Interleukin-10 suppresses hepatic ischemia/ reperfusion injury in mice: implication of a central role for nuclear factor B. Hepatology 30:203, 1999 13. Megison SM, Horton JW, Chao H, et al: A new model for intestinal ischemia in the rat. J Surg Res 49:168, 1990 14. Park PO, Haglund U, Bulkley GB, et al: The sequence of development of intestinal tissue injury after strangulation ischemia and reperfusion. Surgery 107:574, 1990 15. Kubes P: The role of shear forces in ischemia/reperfusioninduced neutrophil rolling and adhesion. J Leukoc Biol 62:458, 1997 16. Beuk RJ, Heineman E, Tangelder GJ, et al: Total warm ischemia and reperfusion impairs flow in all rat gut layers but
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