时间：2022-07-07 01:36:44
BACKGROUND： Hyperbaric oxygen （HBO） is an effective adjuvant therapy for ischemia- reperfusion （I/R） injury of the brain， small intestine and testis in addition to crushing injury. Studies have shown that HBO increases the activity of villi of the ileum 30 minutes after I/R injury. The present study aimed to observe the effect of HBO on apoptosis of epithelial cells in the small intestine during different periods of I/R and to elucidate the potential mechanisms.
METHODS： Rats were subjected to 60-minute ischemia by clamping the superior mesenteric artery and 60-minute reperfusion by removal of clamping. The rats were randomly divided into four groups： I/R group， HBO precondition or HBO treatment before ischemia （HBO-P）， HBO treatment during ischemia period （HBO-I）， and HBO treatment during reperfusion （HBO-R）. After 60-minute reperfusion， samples of the small intestine were prepared to measure the level of ATP by using the colorimetric method and immunochemical expression of caspase-3. The levels of TNF-α in intestinal tissue were measured using the enzyme-linked immunosorbent assay method （Elisa）.
RESULTS： TNF-α levels were significantly lower in the HBO-I group than in the HBO-P （P0.05）. The expression of caspas-3 was significantly lower in the HBO-I group than in the HBO-P group （P
CONCLUSIONS： There is an association between HBO， small intestinal I/R injury， and mucosa apoptosis. HBO maintains ATP and aerobic metabolism， inhibites TNF-α production， and thus prevents intestinal mucosa from apoptosis. Best results can be obtained when HBO is administered to patients in the period of ischemia， and no side effects are produced when HBO is given during the period of reperfusion.
KEY WORDS： Hyperbaric oxygen； Ischemia-reperfusion injury； Apoptosis
World J Emerg Med 2012；3（2）：135-140
DOI： 10.5847/ wjem.j.1920-8642.2012.02.010
INTRODUCTION
Ischemia-reperfusion （I/R） injury is common in tissues or/and organs.[1，2] Bowel villi are extremely sensitive to I/R injury.[3] The intestine is very active in metabolism and has a unique immune function， and it is also the largest body of bacterial libraries. Intestinal I/R injury causes local intestinal injury and damage of the liver， lung， kidney and other organs.[2，3] Intestinal I/R injury may induce multiple organ dysfunction syndrome （MODS）， which is associated with damage of oxygen radicals， energy metabolism， overload of intracellular calcium， leukocyte adhesion， and endothelial cell injury.[4]
During reperfusion， oxygen reaches the tissue and produces a large amount of reactive oxygen species （ROS）， resulting in peroxidation of cell membrane and ultimately apoptosis due to exacerbation of necrosis and triggering of a series of biochemical reactions.[4-6] Thus many methods have been used to prevent or reduce oxidative stress damage， including the use of low temperature， anti-oxidants， drugs such as vitamin C， allopurinol， free radical scavengers for reducing the active oxygen production， and ischemic pretreatment.[5，7-9]
Hyperbaric oxygen （HBO） shows promising results in some models， which can reduce ischemic injury.[3] HBO is used as an effective adjuvant therapy for the I/R injury of the brain， small intestine， and testis in addition to crushing injury. HBO increases the activity of villi of the ileum in 30-minute I/R injury and is effective in the treatment of I/R injury to other organs.[10，11] This study was undertaken to observe the effect of HBO on apoptosis of epithelial cells in the small intestine in different periods of I/R and to elucidate its potential mechanisms.
METHODS
Animals and grouping
Forty healthy male SD rats， weighing 250-300 g， from the Experimental Animal Center of China Medical University， were randomly divided into four groups： I/R group， 1 hour intestinal ischemia by clamping the SMA followed by 1 hour reperfusion； HBO-P （HBO preconditioning） group， 1 hour HBO pre-treatment before ischemia； HBO-I （HBO during ischemia） group， 1 hour HBO treatment during ischemia； and HBO-R （HBO during reperfusion） group， 1 hour HBO treatment during reperfusion.
Intestinal ischemia/reperfusion injury model
An intestinal I/R injury model was established by the reported method.[12] Male SD rats were anesthetized with chloral hydrate （0.2 mL/kg， i.p.） and fixed in a supine position. Subsequently， they were subjected to laparotomy， and the main branches of the superior mesenteric artery were clamped. Their abdominal cavities were closed. After 60 minutes， the clamp was removed and the abdominal cavity closed. After 60 minutes of reperfusion， the abdominal cavity of the rat was opened and the small bowel was removed. The I/R group underwent 1-hour ischemia and 1-hour reperfusion； the HBO-P group was given 1-hour HBO treatment and then underwent intestinal I/R； the HBO-I group was given 1-hour HBO treatment during ischemia； and the HBO-R group was given 1-hour HBO treatment during reperfusion.
HBO treatment
In this study， the animal chamber （DWC400A/0.2-1， Huaxin Chamber Company， Weifang， Shandong） was washed with pure oxygen 5 minutes at first so that the oxygen concentration reached 90% in the chamber. Then the pressure inside the chamber increased to 2.4 ATA with a constant speed over 15 minutes and the oxygen concentration was higher than 95%. The pressure and oxygen concentration were maintained for 60 minutes. Finally， the pressure decreased with a constant speed over 15 minutes. When the pressure in the chamber was 1 ATA， the door was opened and the rats were taken out.
Measurement of intestinal TNF-α
Intestinal tissue of 1000 mg was taken from each rat and washed by saline at 37 °C at different time points， then kept at -70 °C for the preparation of frozen tissue. Intestinal tissue of 300 mg was rinsed in cold saline to remove blood and dried by filter paper. Tissue homogenate was prepared with normal saline， and cells were ruptured by an ultrasonic generator [14 μm amplitude ultrasonic treatment （Soniprep150， Torbeo， USA）] for 30 seconds， and centrifuged by 10% prepared homogenates at low temperature and a low speed of 3000 r/min for 15 minutes. The supernatant was used for detection of intestinal tissue TNF-α by enzyme-linked immunosorbent assay.
Caspase-3 expression and quantitative analysis in intestinal mucosa
Expression of aspartate-specific cysteine protease （caspase-3） in rat small intestinal mucosa was detected by immunohistochemistry. Samples from the distal small bowel （5 cm to the ileocum） were fixed with 10% formalin， paraffin-embedded after dehydration and cut into sections of 4 μm thickness.
SABC method
The sections were dewaxed conventionally， incubated in 30 mL/L H2O2 methanol solution at room temperature for 15 minutes， and washed with PBS for 10 minutes. Then antigens were repaired for 15 minutes in 0.01 mol/L citrate buffer （pH 6.0）， heated to 92-96 °C， closed by dropping of normal rabbit serum 10 minutes after cooling， and then incubated with first anti-caspase-3 body overnight at 4 °C. The sections were washed with PBS solution next day， and then incubated with biotinylated secondary antibody， avidin-chain enzyme， and at last stained with DAB， re-stained with hematoxylin. The sections were dehydrated conventionally with ethanol， cleared with xylene， and mounted with neutral gum. ERK1 expression was observed and analyzed under a light microscope （1：200 dilution of caspase-3 antibody， PBS as negative control instead of primary antibody）.
Image analysis
Image analysis was made with an automatic medical image HPIAS21000 color image analysis system quantitatively； five horizons in each section were randomly selected. The average value of absorbance A of caspase-3 of intestinal tissue was determined before and after treatment. Caspase-3 protein expression was detected by an immunohistochemical method and morphological changes of apoptotic cells in rat intestinal epithelial tissue were observed under a light microscope.
Measurement of intestinal ATP enzyme
Similar to the measurement of intestinal TNF-a， 1000 mg intestinal tissue was washed with saline at 37 °C at different time points. The tissue was frozen at -70 °C and 300 mg of them was rinsed in cold saline to remove blood， and dried by filter paper. Tissue homogenate was prepared with normal saline. Cells were treated with 14 μm amplitude ultrasound in an ultrasonic generator （Soniprep150， Torbeo， US）， 10% of prepared homogenates were centrifuged at low temperature and a low speed of 3000 r/min for 15 minutes. The supernatant was obtained for target detection including ATP.
Statistical analysis
Each section was placed under a microscope and computerized with a DIPAS2200 type cells image analysis system. Ten was selected on the screen for a positive rate of statistics. The data were processed using SPSS 13.0 for Windows software. They were presented as mean ± standard deviation. After single factor analysis of variance， independent samples were used to compare the results of the SNK-q test （P
RESULTS
Changes of TNF-α level in I/R intestinal tissue
The TNF-α level in the HBO-I group was significantly lower than in the HBO-P group （P
Changes of ATP enzyme in I/R intestinal tissue
There was an increasing tendency in ATP enzyme content in different groups. APT enzyme content was significantly higher in the HBO-I group than in the HBO-P group （P
Bcl-2 protein was mainly expressed in the cytoplasm or membrane of cardiomyocyte. The positive expression of Bcl-2 protein in the myocardium increased significantly in groups B， C and D compared with group A （P
Bcl-2/Bax ratio in the myocardium decreased significantly in groups B， C and D compared with group A （P
Caspase-3 expression in I/R intestinal tissue
Specimens of the small bowel from the I/R group showed loss of intestinal villi， interstitial edema， infiltration of inflammatory cells， and brown areas of expression of caspase-3 in mucosa （Figure 1）. Histological examination of the small bowel showed loss of intestinal villi and brown areas of expression of caspase-3 in mucosa （Figure 1）. In the HBO-R group， the above changes also appeared， but they were slighter than those in the I/R group. In the HBO-P group， specimens of the small bowel showed normal structure of villi and mucosa， mild interstitial edema， infiltration of a small amount of inflammatory cells， and brown areas of expression of caspase-3 in the mucosa at the top （Figure 1）. In the HBO-I group， the structure of intestinal villi was normal， light brown areas of caspase-3 expression was seen in the mucosa at the top， but there were no interstitial edema and inflammatory cell infiltration （Figure 1）.
There was a decreasing tendency for caspase-3 expression in different groups. The expression of caspase-3 was significantly lower in the HBO-I group than in the HBO-P group （P
DISCUSSION
The intestine is a continuously updated organ， which can be promoted by apoptosis， programmed cell death. Regulated by the physiological mechanism， apoptosis can eliminate the dead， old or damaged cells， and play an important role in embryogenesis， environmental balance， lymphocyte development and tumor regression.[13] In the pathogenesis， apoptosis is associated with loss of intestinal villi at the top， intestinal adenocarcinoma， inflammatory bowel disease， radiation injury， and I/R injury.[13，14] Thus， apoptosis plays an important role in normal and stable gastrointestinal tract environment， but it can cause gastrointestinal disease. The intestinal cell apoptosis can also regulate the balance between cell proliferation and death.[15] When cells are subjected to death-inducing stimulation， apoptosis occurs.[15] This change starts in the initial phase （Fas-R， cyt c， Bcl-2）， and is followed by the signal phase （caspase-2， 8， 9 and 10） and the implementation phase （caspase-3， 6 and 7）， eventually leading to cleavage of DNA and activation of chromatin endonuclease.[14，15] Animal studies have confirmed that I/R can trigger apoptosis， suggesting that I/R of intestinal epithelial cells trigger apoptosis.[16，17] Cell death results in damage to the intestinal barrier.
The occurrence of apoptosis is related to mitochondrial function. The direct interference with mitochondrial function can induce apoptosis. Meanwhile， the potential （Δ ψm） of mitochondrial transmembrane is decreased in cell apoptosis. Once Δ ψm is declined， apoptosis of cells will inevitably take place. Thus the inhibition of Δ ψm decline can prevent apoptosis. This finding indicates that Δ ψm decline is an early feature of apoptosis. In addition， cytochrome C， a composition of mitochondria could induce the characteristic apoptotic nuclear changes. As an essential component of the respiratory chain， mitochondrial cytochrome C can activate protease cPP32， making DNA fragmented. In the apoptosis induced by staurosporine in a variety of cells， cytosolic cytochrome C is increased significantly.[18，19]
Pathological hypoxia in cells is an acquired intrinsic defect of cellular respiration. Despite oxygen perfusion is increased or overload oxygen is released into the organization， anaerobic metabolism is escalated.[20] The mechanism of cell respiration disorder is related to various cytokines （IL-1β， IL-6， and TNF-α）， NO， ADP-ribose-polymerase， and mitochondrial damage induced by oxidative stress.[21-23] During ischemia， ATP consumption and hypoxanthine accumulation occur. Reperfusion can lead to production of a large number of oxygen free radicals.[24] Furthermore， intestinal I/R triggers accumulation of cytokines， platelet activating factor， IL-1β， IL-6 and NO.[25，26] Previous studies[27，28] found mitochondrial damage in the intestinal I/R model， confirming that impaired intestinal mitochondria result in morphological changes and changes of respiratory function. The administration of oxygen saturated solution to the intestine may partially restore mitochondrial function.
Yamada et al[29] reported that secretion of TNF-α was inhibited after HBO treatment. In another study， the level of TNF-α increased significantly after recovery from trauma/hemorrhagic shock in rats， whereas HBO significantly reduced TNF-α secretion and inhibited excessive inflammatory response.[30] HBO can block the damage of liver cells by inhibiting the secretion of TNF-α to inhibit apoptosis.[31] Kaelin et al[32] demonstrated that HBO significantly increased SOD activity， thus reducing the oxygen free radical-mediated apoptosis in the mouse.
In our experiment， we applied HBO for I/R intestine in different periods， and found that HBO could inhibit TNF-α secretion significantly during and before ischemia， and maintain ATP enzyme in the intestinal mucosa. In the reperfusion period， however， application of HBO showed no significant difference in the inhibition of TNF-α and can't effectively maintain ATP in the small intestine. Similarly， application of HBO during and before ischemia could inhibit expression of caspase-3 in the intestinal mucosa， but in the reperfusion period it was not effective at all. The possible mechanisms are as follows： 1） The administration of HBO during ischemia can replenish oxygen， resist hypoxia metabolism， maintain ATP level， and suppress excessive production of TNF-α； 2） HBO or preconditioning can increase oxygen reserve in tissue during ischemia， and mitochondria can use oxygen reserves to maintain the level of ATP. But when prolonged ischemia or oxygen reserves is depleted， ATP level will decline.
Funding： None.
Ethical approval： Not needed.
Conflicts of interest： The authors declare that there is no conflict of interest.
Contributors： Zhou SH proposed the study and wrote the paper. All authors contributed to the design and interpretation of the study and to further drafts.
REFERENCES
1 McGhan LJ， Jaroszewski DE. The role of toll-like receptor-4 in the development of multi-organ failure following traumatic haemorrhagic shock and resuscitation. Injury 2011 Jun 18. [Epub ahead of print].
2 Zhi-Yong S， Dong YL， Wang XH. Bacterial translocation and multiple system organ failure in bowel ischemia and reperfusion. J Trauma 1992； 32： 148-153.
3 Abad CL， Kumar A， Safdar N. Antimicrobial therapy of sepsis and septic shock--when are two drugs better than one？. Crit Care Clin 2011； 27： e1-27.
4 Shirasugi N， Wakabayashi G， Shimazu M， Oshima A， Shito M， Kawachi S， et al. Up-regulation of oxygen-derived free radical by interleukin in hepatic ischemia/reperfusion injury. Transplantation 1997； 64： 1398-1403.
5 Conti CR. Oxygen therapy-use and abuse in acute myocardial infarction patients. Clin Cardiol 2009； 32： 480-481.
6 Serra MP， Longhi P. Salvage of upper limb following a severe crushing trauma： immediate reconstruction with a free flap and subsequent hyperbaric oxygen therapy. Case Report Med 2009； 568142. Epub 2009 May 27.
7 Vera-Cruz P， Ferreira M， Zagalo C， dos Santos JM， Aguas AP. Structure of the rat nasal mucosa after acute and chronic hyperbaric oxygen therapy. Undersea Hyperb Med 2010； 37： 125-132.
8 Ames JT， Federle MP. CT hypotension complex （shock bowel） is not always due to traumatic hypovolemic shock. Am J Roentgenol 2009； 192： W230-235.
9 Guo W， Ge D， Wang Q， Xu S， Xue L， Lu C， et al. Diazoxide decreases ischemia-reperfusion injury in a rat model of lung transplantation. Transplant Proc 2011； 43： 2510-2516. Epub 2011 Aug 4.
10 Colletti LM， Green M. Lung and liver injury following hepatic ischemia/reperfusion in the rat is increased by exogenous lipopoly saccharide which also increases hepatic TNF production in vivo and in vitro. Shock 2001； 16： 312-313.
11 Li Y， Chopp M， Jiang N， Zhang ZG， Zaloga C. Induction of DNA fragmentation after 10 to 120 minutes of focal cerebral ischemia in rats. Strock 1995； 26： 1251.
12 Saito T， Unno N， Yamamoto N， Inuzuka K， Sagara D， Suzuki M， et al. Intraperitoneal administration of hyperbarically oxygen perfluorochemical enhances preservation of intestinal mucosa against ischemia/reperfusion injury. Shock 2006； 26： 620-624.
13 Sun Z， Wang X， Wallen R， Deng X， Du X， Hallberg E， et al. The influence of apoptosis on intestinal barrier integrity in rats. Scand J Gastroenterol 1998； 33： 415-422.
14 Grossmann J， Walther K， Artinger M， Kiessling S， Scholmerich J. Apoptotic signaling during initiation of detachment-induced apoptosis （"anoikis"） of primary human intestinal epithelial cells. Cell Growth Differ 2001； 12： 147-155.
15 Ramachandran A， Madesh M， Balasubrimanian KA. Apoptosis in the intestinal epithelium： its relevance in normal and pathophysiological conditions. J Gastroenterol Hepatol 2000； 15： 109-120.
16 Ikeda H， Suzuki Y， Suzuki M， Koike M， Tamura J， Tong J， et al. Apoptosis is a major mode of cell death caused by ischaemia and ischaemia/reperfusion injury to the rat intestinal epithelium. Gut 1998； 42： 530-537.
17 Noda T， Iwakiri R， Fujimoto K， Matsuo S， Aw TY. Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa. Am J Physiol 1998； 274： G270- G276.
18 Decaudin D， Geley S， Hirsch T， Castedo M， Marchetti P， Macho A， et al. Bcl-2 and bcl-XL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotheapentic agents. Cancer Res 1997； 57： 62.
19 Zamzami N， Marchetti P， Castedo M， Zanin C， Vayssière JL， Petit PX， et al. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lynphocyte death in vivo. J Exp Med 1995； 181： 1662.
20 Fink MP. Cytopathic hypoxia in sepsis. Acta Anaesthesiol Scand Suppl 1997； 110： 87-95.
21 Szabo C， Cuzzocrea S， Zingarelli B， O'Connor M， Salzman AL. Endothelial dysfunction in a rat model of endotoxic shock. Importance of the activation of poly （ADP-ribose） synthetase by peroxynitrite. J Clin Invest 1997； 100： 723-735.
22 Fink MP. Cytopathic hypoxia. A concept to explain organ dysfunction in sepsis. Minerva Anestesiol 2000； 66： 337-342.
23 Crouser ED. Mitochondrial dysfunction in septic shock and multiple organ dysfunction syndrome. Mitochondrion 2004； 4： 729-741.
24 Parks DA， Bulkley GB， Granger DN， Hamilton SR， McCord JM. Ischemic injury in the cat small intestine： role of superoxide radicals. Gastroenterology 1982； 82： 9-15.
25 Sun Z， Wang X， Lasson A， Borjesson A， Leveau P， Haraldsen P， et al. Roles of platelet- activating factor， interleukin-1beta and interleukin-6 in intestinal barrier dysfunction induced by mesenteric arterial ischemia and reperfusion. J Surg Res 1999； 87： 90-100.
26 Naito Y， Takagi T， Ichikawa H， Tomatsuri N， Kuroda M， Isozaki Y， et al. A novel potent inhibitor of inducible nitric oxide inhibitor， ONO-1714， reduces intestinal ischemia-reperfusion injury in rats. Nitric Oxide 2004； 10： 170-177.
27 Gao C， Xu L， Chai W， Sun X， Zhang H， Zhang G. Amelioration of intestinal ischemia- reperfusion injury with intraluminal hyperoxygenated solution： studies on structural and functional changes of enterocyte mitochondria. J Surg Res 2005； 129： 298-305.
28 Madesh M， Bhaskar L， Balasubramanian KA. Enterocyte viability and mitochondrial function after graded intestinal ischemia and reperfusion in rats. Mol Cell Biochem 1997； 167： 81-87.
29 Yamada T， Taguchi T， Hirata Y， Suita S， Yagi H. The protective effect of hyperbaric oxygenation on the small intestine in ischemia-reperfusion injury. J Pediatr Surg 1995； 30： 786-790.
30 Kannan KB， Colorado I， Reino D， Palange D， Lu Q， Qin X， et al. Hypoxia-inducible factor plays a gut-injurious role in intestinal ischemia reperfusion injury. Am J Physiol Gastrointest Liver Physiol 2011； 300： G853-61. Epub 2010 Dec 23.
31 Luongo C， Imperatore F， Cuzzocrea S. Effects of hyperbaric oxygen exposure on a zymosan-induced shock model. Crit Care Med 1998； 26： 1972-1976.
32 Kaelin CM， Im MJ， Myers RA， Manson PN， Hoopes JE. The effects of hyperbaric oxygen on free flaps in rats. Arch Surg 1990； 125： 607-609.
Received July 28， 2011
Accepted after revision December 19， 2011