Departments of 1Internal Medicine and 2Pathology, Saga Medical School, Saga 849-8501, Japan
Submitted 4 August 2003 ; accepted in final form 24 September 2003
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ABSTRACT |
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mitochondria; reactive oxygen species; reduced glutathione; cytochrome c; caspase-6
ROS, which are generated as a result of respiration as well as in response to a variety of extracellular stimuli including cytokines and peptide growth factors, serve as intracellular messengers at low concentrations but induce cell death at higher concentrations (8). The mitochondrial respiratory chain is one of the most important sites of ROS production under physiological conditions (17, 36). It has been long suspected that mitochondria-derived ROS are vital not only because mitochondrial respiratory chain components are present in almost all eukaryotic cells but also because the ROS produced in mitochondria can readily influence mitochondrial function without having to cope with long diffusion times from the cytosol. Two sites in the respiratory chain, complex I and complex III, have been suggested to be the major ROS sources (21). Reduced glutathione (GSH) is known to function as an antioxidant and a physiological reservoir for cysteine and is involved in DNA synthesis, protein synthesis regulation, and detoxification, etc. Cellular GSH deficiency affects the mitochondrial GSH pool and the cytosolic GSH pool. Mitochondrial GSH is important for the detoxification of ROS generated by the respiratory chain, conjugation of xenobiotics, maintenance of thiol-containing proteins, and regulation of the mitochondrial membrane potential (MMP) (14, 38). Mitochondria play a central role in cell life and death by regulating the MMP, because reduction of the MMP enhances the opening of mitochondrial permeability pores leading to the release of cytochrome c and apoptosis-inducing factor into the cytosol and thus triggering the apoptotic cascade (7, 21). ROS may interact with cellular biomolecules, such as DNA, leading to oxidative DNA damage (5). However, it is unclear whether ROS induces oxidative mitochondrial damage resulting in mitochondrial apoptotic signaling via reduced MMP.
Ischemic preconditioning (IPC) refers to a phenomenon in which a tissue is rendered resistant to the deleterious effects of prolonged ischemia-reperfusion (I/R) by prior exposure to brief periods of vascular occlusion. The phenomenon provides a way of protecting organs from damage inflicted by I/R. It was first demonstrated in the heart in 1986 (20) and has been the subject of intensive investigation since then in organs including the intestine (31), liver (43), brain (26), skeletal muscle (24), spinal cord (28), retina (27), and kidney (10). Recent studies (3, 25, 29, 34) have suggested that IPC can reduce I/R-induced injury by reducing oxidant stress, conversion of xanthine dehydrogenase to xanthine oxidase, leukocyte adhesion, intracellular Na+ accumulation, and opening mitochondrial ATP-dependent potassium channels. However, it is poorly understood whether IPC suppresses I/R-induced small intestinal mucosal apoptosis by downregulating mitochondrial apoptotic signaling.
The aims of this study were to determine 1) whether ROS induced peroxidative mitochondrial damage resulting in mitochondrial apoptotic signaling via reduced MMP after I/R and 2) whether IPC attenuated mucosal apoptosis after prolonged I/R by inhibiting the ROS-mediated mitochondria-dependent pathway in the rat small intestine.
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MATERIALS AND METHODS |
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Experimental design. To investigate mucosal apoptosis in the small intestine after ischemia followed by reperfusion, the SMA was occluded for different times followed by different times of reperfusion. Six rats were studied in each group. In sham-operated rats, the SMA was isolated in a similar fashion but was not occluded.
To evaluate the effect of IPC on mucosal apoptosis in the small intestine after prolonged I/R, the SMA was occluded for 5 min, 10 min, 15 min, 20 min, and 25 min followed by 5-min or 10-min reperfusion; and the SMA was again occluded for 60 min followed by 60-min reperfusion. Additionally, the SMA was occluded for 5 min followed by 5-min reperfusion (24 bouts), and the SMA was then again occluded for 60 min followed by 60-min reperfusion. Six rats were studied in each group.
DNA fragmentation assay. Mucosal scrapings were processed immediately after collection to minimize nonspecific DNA fragmentation. The amount of fragmented DNA was determined as previously described (22, 41). Mucosal scrapings of the different intestinal segments were homogenized in a lysis buffer (pH 8.0) consisting of 5 mM Tris·HCl, 20 mM EDTA (Sigma, St. Louis, MO), and 0.5% (wt/vol) Triton X-100 (Sigma). Aliquots of 1 ml of each sample were centrifuged at 27,000 g for 20 min at 4°C to separate the intact chromatin (pellet) from the fragmented DNA (supernatant). The supernatant was decanted and saved, and the pellet was resuspended in 1 ml of Tris buffer (pH 8.0) consisting of 10 mM Tris·HCl and 1 mM EDTA. The pellet and supernatant fractions were assayed for DNA content by using diphenylamine reaction. Results were expressed as percentage of fragmented DNA divided by the total DNA.
Purification of mucosal DNA and agarose gel electrophoresis. Total DNA from the jejunum and ileum was extracted sequentially with a phenol/chloroform/isoamyl alcohol mixture (25:24:1, vol/vol/vol) to remove the proteins and then purified as previously described (41). Resolving agarose gel electrophoresis was performed with 1.5% gel strength containing 1.0 µg/ml ethidium bromide. Depending on the experiment, 20 µg DNA per well was loaded. DNA standards (0.5 µg/well) were included to identify the size of the DNA fragments. Electrophoresis was performed for 2 h at 70 V, and the DNA was observed under ultraviolet fluorescent light.
Terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick-end labeling staining and apoptotic index analysis. Tissue samples were removed from the jejunum and ileum and were immediately fixed in 10% neutral-buffered formalin. The samples were then embedded in paraffin and sectioned. Fragmented DNA was stained by the terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick-end labeling (TUNEL) method (41) by using an Apop Tag Kit (Oncor, Gaithersburg, MD). The apoptotic index was calculated randomly in a minimum of 20 crypts and analyzed in three separate samples. The apoptotic index was determined by dividing the number of apoptotic cells by the total number of cells in the crypt column and multiplying by 100.
Measurement of intestinal mucosal cellular mitochondrial respiration. Mucosal cellular mitochondrial respiration was studied by measuring the mitochondrial dehydrogenase-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT; Sigma) to its formazan derivative (MTT-FZ) (18, 37). Under halothane anesthesia, a 10-cm segment of ileum was isolated and both ends were tied off. A plastic tube was inserted into the lumen, and MTT dissolved in physiological saline (5 mg/5 ml) was placed in the lumen. After 30 min, 10 ml of cold physiological saline was used to wash out the remaining MTT solution from the ileal segment. The segment was opened, and the mucosal layer was collected. Mucosal MTT-FZ and total DNA were determined as previously described (41). Results were expressed as the ratio of MTT-FZ to DNA absorbance per millimeter aliquot of tissue homogenate.
Measurement of intestinal mucosal cellular ROS. Oxidant formation was measured by using the oxidant-sensitive nonfluorescent probe dihydrorhodamine 123 (DHR) (Molecular Probes, Eugene, OR). DHR was prepared as a 25 mM stock solution in nitrogenpurged dimethylformamide (DMF) and stored in the dark at -20°C. On the day of each experiment, the stock DHR was freshly diluted with DMF to a final concentration of 5 µM. Under halothane anesthesia, a 10-cm segment of ileum was isolated and both ends were tied off. A plastic tube was inserted into the lumen, and the DHR solution (5 ml) was placed in the lumen 30 min before the reperfusion ended. After I/R, 10 ml of cold PBS, pH 7.4, was used to wash out the remaining DHR solution from the ileal segment. The segment was opened, and the mucosal layer was collected and placed into 3 ml of cold PBS (pH 7.4). After centrifugation (2,500 g for 10 min), the supernatant was removed, and the pellet was homogenized in 3 ml of PBS (pH 7.4). Aliquots of the tissue homogenate, 1 ml each, were transferred into two Eppendorf tubes (1 to be used for the rhodamine 123 assay and the other for a total protein assay). Aliquots were sonicated (Typ T25-S1 Sonic Membrator). Rhodamine 123 accumulation was quantified at excitation and emission wavelengths of 500 and 536 nm by using a spectrofluorophotometer (Shimadzu RF-5000). Total protein was determined by using a commercial kit (Bio-Rad, Hercules, CA). The results were expressed as relative fluorescence units per milligram protein.
Intestinal mucosal cellular MMP analysis. MMP was analyzed by using a MMP detection kit (BioCarta, Carlsbad, CA). Via a midline laparotomy under halothane anesthesia, the distal end of a 10-cm segment of ileum was isolated, and both ends were tied off. A plastic tube was inserted into the lumen and 1x 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (5 ml) was placed in the lumen 15 min before the reperfusion ended. After I/R, the segment was opened, and the mucosal layer was collected into 5 ml cold PBS (pH 7.4). After centrifugation, the supernatant was removed, and the pellet was resuspended in 5 ml 1x assay buffer and homogenized. The red fluorescence (excitation 550 nm, emission 600 nm) and green fluorescence (excitation 485 nm, emission 535 nm) were measured in aliquots of tissue homogenate by using a spectrofluorophotometer (Shimadzu RF-5000). Results were expressed as the ratio of red fluorescence divided by green fluorescence.
Purification of mitochondria and cytosol. The ileal mucosal scrapings were immediately washed twice with ice-cold PBS (pH 7.4) and centrifuged at 1,000 g for 5 min at 4°C. The pellets were then resuspended with two volumes of buffer A [in mM: 250 sucrose (Sigma), 20 HEPES (Sigma)/KOH pH 7.5, 10 KCl, 1.5 MgCl2, 1 EDTA, 1 EGTA, 1 dithiothreitol, and 1 phenylmethylsulfonyl fluoride with 10 µg/ml aprotinin, 10 µg/ml leupetin, and 1.8 mg/ml iodoacetamide] and lysed at 4°C for 30 min (42, 44). Homogenates were centrifuged at 1,000 g for 10 min at 4°C to remove the nuclei. Supernatants were centrifuged at 10,000 g for 15 min at 4°C, and the pellets were the mitochondrial fractions. Pellet fractions were resuspended in buffer A. The supernatants of the 10,000-g spin were further centrifuged at 100,000 g for 1 h at 4°C, and the resulting supernatants were the soluble cytosolic fractions. Cytosolic fractions and mitochondrial fractions were divided into multiple samples and frozen at -80°C. Protein concentrations were determined.
Determination of mitochondrial GSH, oxidized GSH, and malondialdehyde. Mitochondrial GSH and oxidized GSH (GSSG) were measured by using a microtiter plate assay as previously described (2). Malondialdehyde (MDA), an established indicator of lipid peroxidation, was measured by the thiobarbituric acid assay.
Western blot analysis. Cytochrome c was analyzed in both the mitochondrial and cytosolic fractions. Caspase-9 and caspase-6 were determined in the cytosolic fraction. Equal quantities (40 µg) of lysates were electrophoresed in a SDS-PAGE and then electroblotted onto a nitrocellulose membrane (Trans-Blot; Bio-Rad). After blocking with PBS containing 0.1% polyoxyethylene sorbitan monolaurate (Tween 20; Sigma) and 5% skim milk at 4°C overnight, the membrane was incubated with a rabbit polyclonal anti-cytochrome c antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), a rabbit polyclonal anti-caspase-9 antibody (1:1,000; Santa Cruz Biotechnology), and a rabbit polyclonal anti-caspase-6 antibody (1:500; Santa Cruz Biotechnology) for 1 h. Antigen-antibody complexes were detected with horseradish peroxidase-conjugated anti-rabbit IgG (1:1,000; Santa Cruz Biotechnology). Chemiluminescence detection was carried out by using enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech).
Statistical analysis. Results are expressed as means ± SE. Data were evaluated by one-way ANOVA in which multiple comparisons were performed by using the method of least significant difference. Differences were considered significant if the probability of the difference occurring by chance was <5 in 100 (P < 0.05).
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RESULTS |
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Resolving agarose gel electrophoresis was performed to evaluate the fragmented DNA in the small intestinal mucosa, and the result of the ileal total DNA electrophoresis is shown in Fig. 1A. The 60 I/60 R significantly induced fragmentation of the mucosal DNA resulting in an increase in DNA ladders. These were clearly evident and characteristic of apoptosis. Pretreatment with 20-min ischemia followed by 5-min reperfusion (20 I/5 R) significantly reduced the ladder formation induced by the prolonged 60 I/60 R. In the jejunum, results of the total DNA electrophoresis were similar to those seen in the ileum (data not shown).
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Results of TUNEL staining in the small intestine showed that few apoptotic cells were observed in the sham-operated rats (data not shown). Compared with the sham-operated rats, marked destruction of the structure in the small intestine with mucosal erosion and edema were seen in the 60 I/60 R rats. Pretreatment with 20 I/5 R significantly decreased the number of apoptotic cells and reduced the destruction of the structure in the intestinal mucosa. The ileal mucosal apoptotic index in each group is shown in Fig. 1B. Pretreatment with 20 I/5 R significantly attenuated the apoptotic index after the prolonged 60 I/60 R. In the jejunum, results were similar to those seen in the ileum (data not shown).
IPC ameliorated I/R-induced mitochondrial respiratory dysfunction. With the use of an assay on the basis of mitochondrial dehydrogenase-dependent conversion of MTT to its formazan derivative (MTT-FZ), we measured the mitochondrial respiratory function. Figure 2 shows that the mucosal mitochondrial respiratory function was significantly impaired by 60 I/60 R in the small intestine compared with the sham-operated rats. This mitochondrial respiratory dysfunction was significantly ameliorated by pretreatment with 20 I/5 R.
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IPC improved ROS-mediated reduction of MMP in the small intestinal mucosa after prolonged I/R. We quantified ROS generation by measuring the fluorescence of rhodamine 123 after DHR oxidation. Figure 3A shows that the 60 I/60 R significantly induced mucosal ROS generation in the small intestine. This increase in ROS was suppressed by pretreatment with 20 I/5 R. Furthermore, we evaluated the small intestinal mucosal MMP by using a MMP detection kit. Figure 3B shows that 60 I/60 R significantly reduced the MMP in the small intestinal mucosa. However, pretreatment with 20 I/5 R significantly improved this MMP after the prolonged 60 I/60 R.
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IPC suppressed mitochondrial lipid peroxidation by inhibiting mitochondrial GSH oxidation in the small intestinal mucosa after prolonged I/R. To study the effect of GSH oxidation on MMP, we measured the mitochondrial GSH and GSSG and an indicator of lipid peroxidation MDA. Results are shown in Fig. 4. Compared with the sham-operated rats, 20 I/5 R did not decrease the mitochondrial GSH or GSH/GSSG ratio or increase the mitochondrial MDA. However, 60 I/60 R significantly decreased the mitochondrial GSH and GSH/GSSG ratio and increased the mitochondrial MDA level. Pretreatment with 20 I/5 R significantly suppressed this decrease in the mitochondrial GSH and GSH/GSSG ratio as well as the increase in the mitochondrial MDA after the prolonged 60 I/60 R.
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IPC prevented the release of cytochrome c from mitochondria and inhibited the activation of caspase-9 and caspase-6 in the small intestinal mucosa after prolonged I/R. Results are shown in Fig. 5. In the sham-operated rats and 20 I/5 R rats, the majority of cytochrome c was seen in the mitochondrial fractions, and only small amounts of cytochrome c were detected in the cytosolic fractions. In contrast, the amounts of cytochrome c released into the cytosol from mitochondria as well as expressions of cleaved activated caspase-9 and caspase-6 significantly increased after 60 I/60 R compared with the sham-operated rats. However, this release of cytochrome c from the mitochondria to the cytosol was significantly prevented, and the expressions of cleaved activated caspase-9 and caspase-6 were significantly inhibited by pretreatment with 20 I/5 R.
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DISCUSSION |
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Two candidates for early mediators of I/R are inducible nitric oxide synthase-derived nitric oxide and ROS (3). Subsequent studies indicated relevant extracellular ROS formation by neutrophils and that intracellular mitochondria-derived ROS could be modified when the mitochondrial respiratory chain was interrupted. It has been suggested that ROS are involved in apoptosis and that this apoptosis was inhibited by antioxidants (2, 4, 9, 11, 13). Two sites in the mitochondrial respiratory chain, complex I and complex III, have been suggested to be the major ROS sources (14). It has been demonstrated that the ability of the mitochondrial respiratory chain complex I inhibitor rotenone to induce apoptosis is closely related to its ability to induce mitochondrial ROS production (15). Induction of mitochondrial ROS production by rotenone has frequently been attributed to the ability of rotenone to block mitochondrial respiratory chain complex I, thereby increasing the formation of ubisemiquinone, the primary electron donor in mitochondrial superoxide generation. Recently, it has been shown that mitochondrial respiratory chain complex II plays an essential role in hypoxia-induced ROS generation (23). Using an assay based on the mitochondrial dehydrogenase-dependent conversion of MTT to its formazan derivative (MTT-FZ), we were able to show that the small intestinal mucosal mitochondrial respiratory function was impaired by I/R and that this I/R-induced mitochondrial respiratory dysfunction promoted ROS generation. IPC attenuated prolonged I/R-induced apoptosis is time sensitive; we found that only 1520 min of the IPC created protective effects from small intestinal mucosal apoptosis induced by prolonged I/R. In the present study, the 5- to 20-min ischemia followed by 5-min reperfusion did not impair mitochondrial respiratory function, but the 25-min ischemia followed by 5-min reperfusion impaired mitochondrial respiratory function (data not shown). The 510 min of IPC could not ameliorate the prolonged I/R-induced mitochondrial respiratory dysfunction, resulting in an increase in ROS generation (data not shown). However, the 15 or 20 min of IPC significantly ameliorated the I/R-induced mitochondrial respiratory dysfunction, resulting in a reduction in ROS generation. Mitochondrial dehydrogenases, such as succinate dehydrogenase and glycerolphosphate dehydrogenase, are key enzymes in electron transport chain and are oxygen sensitive. Inhibition of the dehydrogenase activity leads to mitochondrial respiratory dysfunction; a suitable IPC may offer its hypooxygen resistance after prolonged I/R. We (12) previously demonstrated that oxidative stress with production of ROS was related to I/R-induced mucosal apoptosis and that the exogenous antioxidative agent GSH reduced I/R-induced mucosal apoptosis in rat small intestine. In this study, we found that ROS generation significantly increased mitochondrial GSSG, decreased the mitochondrial GSH and GSH/GSSG ratio, and promoted mitochondrial lipid peroxidation after I/R in the small intestine.
ROS are generated from mitochondria that have been suggested to be an apoptotic regulator (19). Our previous studies (40, 41) showed that mitochondria are deeply involved in I/R-induced mucosal apoptosis in the small intestine. In apoptosis, coincident with the permeabilization of the outer mitochondrial membrane, there is typically a rapid reduction in the MMP. Mitochondria manifest signs of outer membrane and/or inner membrane permeability on exposure to a variety of proapoptotic second messengers. Thus cytochrome c, which is normally confined to the mitochondrial intermembrane space, is found in the cytosol of cells undergoing apoptosis (44). Loss of the mitochondrial transmembrane electrical potential, part of a process described as MMP, precedes the gross morphological changes associated with apoptosis. The present data suggest that ROS generation promotes mitochondrial lipid peroxidation by increasing GSH oxidation resulting in the reduction of the MMP after I/R in the small intestinal mucosa.
GSH is the major cellular reductant for the GSH-peroxidase-catalyzed elimination of organic and lipid peroxides (1). A decrease in mitochondrial GSH enhances mitochondrial lipid peroxidation, and this mitochondrial lipid peroxidation reduces the membrane stability by reducing the MMP. Reduction in the MMP enhances the release of cytochrome c from the mitochondria to the cytosol. Cytosolic cytochrome c forms a complex with Apaf-1 and caspase-9, resulting in the activation of caspase-9, which then processes and activates other caspases to orchestrate the biochemical execution of apoptosis (16). We (40, 41) previously suggested that I/R promotes a release of cytochrome c from the mitochondria to the cytosol to activate caspase-9 but that this active caspase-9 does not activate caspase-3. In the present study, our results showed that the active caspase-9 activated caspase-6, and the active caspase-6 participated in the execution phase of apoptosis. IPC significantly ameliorated mitochondrial respiratory dysfunction, reduced ROS generation, suppressed an increase in GSH oxidation, improved the MMP, prevented a release of cytochrome c from the mitochondria, and inhibited activation of caspase-9 and caspase-6, resulting in inhibition of apoptosis after prolonged I/R in the small intestinal mucosa.
In summary, I/R induced mitochondrial respiratory dysfunction resulting in an increase in ROS generation, and these ROS promoted mitochondrial GSH oxidation and lipid peroxidation in the small intestinal mucosa. Mitochondrial lipid peroxidation reduced the MMP, resulting in a release of cytochrome c from the mitochondria to the cytosol to activate caspase-9 and caspase-6 in the small intestinal mucosa. However, IPC significantly ameliorated the mitochondrial respiratory dysfunction, reduced ROS generation, suppressed mitochondrial GSH oxidation, improved the MMP, prevented the release of cytochrome c from the mitochondria, and inhibited the activation of caspase-6, resulting in the inhibition of apoptosis after prolonged I/R in the small intestinal mucosa. The results suggest that IPC attenuates I/R-induced mucosal apoptosis partly by inhibiting the ROS-mediated mitochondria-dependent pathway in the small intestine.
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ACKNOWLEDGMENTS |
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This work was supported, in part, by Ministry of Education, Science, and Culture in Japan for Scientific Research Grants-in-Aid 15590658 (K. Fujimoto) and 14570478 (R. Iwakiri).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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