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Effect of Chelerythrine on Intestinal Cell Turnover following Intestinal Ischemia-Reperfusion Injury in a Rat Model
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Igor Sukhotnik, Sivan Bitterman, Yoav Ben Shahar, Yulia Pollak, Nir Bitterman, Salim Halabi, Arnold Coran, Arie Bitterman
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Igor Sukhotnik, Department of Pediatric Surgery, Bnai Zion Medical Center, Technion-Israel Institute of Technology, The Bruce Rappaport Faculty of Medicine, Laboratory of Intestinal Adaptation and Recovery, 47 Golomb St., P.O.B. 4940, Haifa
[email protected] 31048, Israel
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Original Article
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Effect of Chelerythrine on Intestinal Cell Turnover following Intestinal IschemiaReperfusion Injury in a Rat Model Igor Sukhotnik1 Sivan Bitterman2 Arnold Coran5 Arie Bitterman3
Yoav Ben Shahar3
1 Department of Pediatric Surgery, Bnai Zion Medical Center,
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Technion-Israel Institute of Technology, Haifa, Israel 2 Technion-Israel Institute of Technology, Haifa, Israel Q2 3 Department of Surgery, Carmel Medical Center, Technion-Israel Institute of Technology, Haifa, Israel 4 Department of Emergency Medicine, Carmal Medical Center, Technion-Israel Institute of Technology, Haifa, Israel 5 Section of Pediatric Surgery, C.S. Mott Children’s Hospital, Ann Arbor, Michigan, United States
Yulia Pollak2
Nir Bitterman2
Salim Halabi4
Address for correspondence Igor Sukhotnik, Q3 Department of Pediatric Surgery, Bnai Zion Medical Center, Technion-Israel Institute of Technology, The Bruce Rappaport Faculty of Medicine, Laboratory of Intestinal Adaptation and Recovery, 47 Golomb St., P.O.B. 4940, Haifa 31048, Israel (e-mail:
[email protected]).
Eur J Pediatr Surg 2016;00:1–8.
Abstract
Keywords
► ► ► ►
ischemia-reperfusion intestine chelerythrine apoptosis
received May 14, 2016 accepted June 24, 2016
Background Chelerythrine (CHE) is a benzophenanthridine alkaloid that is a potent, selective, and cell-permeable protein kinase C inhibitor. The purpose of the present study was to examine the effect of CHE on intestinal recovery and enterocyte turnover after intestinal ischemia-reperfusion (IR) injury in rats. Methods Male Sprague-Dawley rats were divided into four experimental groups: (1) sham rats underwent laparotomy, (2) sham-CHE rats underwent laparotomy and were treated with intraperitoneal CHE; (3) IR-rats underwent occlusion of both superior mesenteric artery and portal vein for 30 minutes followed by 48 hours of reperfusion, and (4) IR-CHE rats underwent IR and were treated with intraperitoneal CHE immediately before abdominal closure. Intestinal structural changes, Park injury score, enterocyte proliferation, and enterocyte apoptosis were determined 24 hours following IR. The expression of Bax, Bcl-2, p-ERK, and caspase-3 in the intestinal mucosa was determined using real Western blot and immunohistochemistry. Results Treatment with CHE resulted in a significant decrease in Park injury score in jejunum (threefold decrease) and ileum (twofold decrease), and parallel increase in mucosal weight in jejunum and ileum, villus height in jejunum and ileum, and crypt depth in ileum compared with IR animals. IR-CHE rats also experienced a significantly lower apoptotic index in jejunum and ileum, which was accompanied by a lower Bax/ Bcl2 ratio compared with IR animals. Conclusions Treatment with CHE inhibits programmed cell death and prevents intestinal mucosal damage following intestinal IR in a rat.
© 2016 Georg Thieme Verlag KG Stuttgart · New York
DOI http://dx.doi.org/ 10.1055/s-0036-1587588. ISSN 0939-7248.
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Effect of Chelerythrine on Intestinal Cell Turnover following Intestinal Injury
Introduction Intestinal ischemia-reperfusion (IR) injury is a potentially life-threatening condition with high morbidity and mortality that commonly occurs in critically ill patients and may lead to systemic inflammation and multiple-organ failure. The causes of mesenteric ischemia are well known and its major complication, gangrenous necrosis of portions of the gastrointestinal tract following necrotizing enterocolitis, midgut volvulus, and incarcerated hernia, is well recognized by all pediatric surgeons.1,2 In addition, many life-threatening illnesses such as multiple trauma, shock, and sepsis may impair intestinal blood flow. Common to most critically ill patients is preservation of blood flow to vital organs, which occurs at the expense of blood flow to the intestine. Intestinal IR remains a clinically challenging problem despite decades of research in this area.3 Ischemia may cause tissue injury directly as a result of decreased oxygen delivery, depletion of cellular energy stores, and accumulation of toxic metabolites, resulting in cell necrosis. However, reperfusion of ischemic tissue, although necessary as a reparative mechanism, has been shown to exacerbate acute ischemic injury through the generation of damage-associated molecular pattern molecules, including oxygen radical species, cytokines, and chemokines.4,5 A production of these agents initiates a cascade of events, including activation of mast cells and neutrophils and production of systemic inflammatory mediators and cytokines, which lead to neutrophils increased bowel permeability, increased bacterial translocation, a systemic inflammatory reaction, and multiple organ failure.6,7 Therapy of IR injury includes the pharmacological agents that can prevent ischemic injury as well as the agents that can prevent production of reactive oxygen species and the agents that can improve intestinal recovery following the IR event. The benzophenanthridine alkaloid chelerythrine (CHE) is a potent, selective antagonist of the Ca2þ/phospholipid-dependent protein kinase C (PKC), which shows a wide range of biological activities including antitumor, anti-inflammatory and antiproliferative agents against many cancers.8 The effect of CHE during IR is controversial. Several experimental studies have shown that CHE exacerbates tissue damage during IR. CHE blocks the renal and cardiac protective effects of postconditioning.9,10 In other models, CHE has a potent protective effect against IR injury through the direct antioxidative stress and anti-inflammatory mechanisms. The effect of CHE has never been investigated in intestinal IR injury. The purpose of the present study was to evaluate the effect of CHE on structural mucosal changes in the small bowel induced by IR injury in a rat and to evaluate the mechanisms by which CHE influences intestinal recovery including its effect on enterocyte proliferation and death via apoptosis.
Materials and Methods Animals Surgical procedures and animal care were conducted in compliance with the guidelines established by the “Guide for the Care and Use of Laboratory Animals,” Rappaport European Journal of Pediatric Surgery
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Faculty of Medicine, Technion (Haifa, Israel). Male SpragueDawley rats, weighing 250 to 280 g, were acclimatized at 21°C on 12-hour day and night cycles for a minimum of 3 days before the experiment. The rats had free access to water and were fed with standard chow. Rats were fasted for 24 hours before the experiment.
Experimental Design Male Sprague-Dawley rats were divided into four experimental groups: (1) sham rats underwent laparotomy, (2) shamCHE rats underwent laparotomy and were treated with intraperitoneal (IP) CHE (5mg/kg); (3) IR-rats underwent occlusion of both the superior mesenteric artery and portal vein for 30 minutes followed by 48 hours of reperfusion, and (4) IR-CHE rats underwent IR and were treated with IP CHE (5 mg/kg) immediately before abdominal closure.
Surgical Procedure As previously described in detail,11,12 a midline laparotomy of 5 cm in length was performed. Then the SMA and PVQ4 were isolated and occluded with a microsurgical clip. After occlusion for 30 minutes, the clip was removed, and intestinal perfusion was reestablished. In all animals the abdomen was closed in two layers with a running suture of 3–0 Dexon (Davis & Geck, Inc, NY). Postoperatively, animals were allowed water ad libitum immediately after operation and normal chow at the beginning of the first postoperative day.
Intestinal Mucosal Parameters All animals were sacrificed 48 hours following the ischemia. The small intestine from the pylorus to the ileocecal valve was removed. Portions of intestine 10 cm distal to the ligament of Treitz and 10 cm proximal to the ileocecal region were split on the antimesenteric border, washed with cold saline, dried, and weighed. The mucosa was scarped from the underlying tissue with a glass slide. Mucosal samples were homogenized with TRIzol reagent. DNA and protein were extracted by the method of Chromsczynski 13 and were expressed as micrograms per centimeter of bowel per 100 g of body weight.
Histology Intestinal samples from the proximal jejunum and distal ileum were fixed in 10% formalin, dehydrated in progressive concentrations of ethanol, cleared in xylene, and embedded in paraffin wax. Deparaffinized 5-μm sections were stained with haematoxylin and eosin. The villus height and crypt depth for each specimen were measured using an objective mounted micrometer (100 magnification) and an optical microscope (10 100 magnification). Villus height and crypt depth data are from six rats, and each measurement consists of the mean of 10 villi and crypts. The mucosal damage of the small bowel was graded using the Park score14: 0: normal mucosa, 1: subepithelial space at villus tip, 2: more extended subepithelial space, 3: epithelial lifting along villus sides, 4: denuded villi, 5: loss of villus tissue, 6: crypt layer infarction, 7: transmucosal infarction, and 8: transmural infarction.
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Enterocyte Proliferation and Apoptosis Crypt cell proliferation was assessed using 5-bromodeoxyuridine (5-BrdU). Standard BrdU labeling reagent (Zymed Laboratories, Inc, San Francisco, CA) was injected intraperitoneally at a concentration of 1ml/100 g body weight 2 hours before sacrifice. Tissue slices (5 μm) were stained with a biotinylated monoclonal anti-BrdU antibody system provided in a kit form (Zymed Laboratories, Inc, San Francisco, CA). An index of proliferation was determined as the ratio of crypt cells staining positively for BrdU per 10 crypts. Additional 5-μm thick sections were prepared to establish the degree of enterocyte apoptosis. Immunohistochemistry for caspase-3 (caspase-3 cleaved concentrated polyclonal antibody; dilution 1:100; Biocare Medical, Walnut Greek, CA) was performed for identification of apoptotic cells using a combination of the streptavidin–biotin–peroxidase method and microwave antigen retrieval on formalin-fixed, paraffinembedded tissues according to the manufacturer’s protocols. The apoptotic index (AI) was defined as the number of apoptotic cells per 10 villi. A qualified pathologist blinded as to the source of intestinal tissue performed all measurements.
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Tissue was homogenized in RIPAQ5 lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 2 mM EDTA, and supplemented with a cocktail of protease and phosphatase inhibitors. Protein concentrations were determined by Bradford reagent according to the manufacturer’s instructions. Samples containing equal amounts of total protein (30 μg) were resolved by SDS-PAGEQ6 under reducing conditions. After electrophoresis, proteins were transferred to a PVDFQ7 membrane and probed with various primary antibodies to anti-caspase-3 antibody (1:1000 dilution, sc-7148), anti-phospho-ERK antibody (1:2500 dilution, sc-7383), and anti-tubulin antibody (1:1000 dilution, sc-56899). Horseradish-peroxidase-conjugated secondary antibody was purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA) and an enhanced chemiluminescent substrate from Biological Industries (Kibbutz Beth HaEmek, Israel). The optical density of the specific protein bands was quantified by using a densitometer (Vilber Lourmat, Lion, France).
Fig. 1 Effect of ischemia-reperfusion and treatment with CHE on bowel and mucosal weight. Values are mean SEM. IR, ischemiareperfusion; CHE, chelerythrine. p < 0.05 IR vs. Sham rats, † p < 0.05 IR-CHE vs. IR rats.
statistically significant (►Fig. 1). IR rats (Group C) demonstrated a significant decrease in bowel weight in ileum (17.6.3 0.4 vs. 19.7 1.1 mg/cm length/100 g body weight, p < 0.05), mucosal weight in jejunum (7 0.4 vs. 9 0.4 mg/cm length/100 g body weight, p < 0.05) and ileum (6 0.6 vs. 8.3 0.4 mg/cm length/100 g body weight, p < 0.05) (►Fig. 1), and mucosal DNA content in jejunum (60 12 vs. 93 15 µg/cm length/100 g body weight, p < 0.05) and ileum (79 9 vs. 102 13 µg/cm length/100 g body weight, p < 0.05) (►Fig. 2) compared with sham animals (Group A). Following IP CHE administration (Group D), IR-rats demonstrated a significant increase in jejunal (21.7 0.9 vs. 19.2 0.6 mg/cm/100 g, p < 0.05) and ileal (18.4 0.3 vs. 17.6 0.4 mg/cm/100 g, p < 0.05) bowel weight, as well as in jejunal (8.8 0.5 vs. 7 0.4 mg/cm/100 g, p < 05) mucosal weight, jejunal (twofold increase, p < 0.05) and ileal (28% increase, p < 0.05) mucosal DNA content, and jejunal protein content (60%, p < 0.05) (►Fig. 2) compared with IR- animals (Group C).
Statistical Analysis The data are expressed as the mean SEM. Statistical analysis of parameters of adaptation, enterocyte proliferation, and apoptosis was performed using the nonparametric Kruskal– Wallis analysis of variance test, followed by the corrected Mann–Whitney test, with p less than 0.05 considered statistically significant.
Results Intestinal Mucosal Parameters Treatment of sham animals with CHE (Group B) resulted in a small but significant decrease in jejunal mucosal weight (16%, p ¼ 0.02), as well as in a trend toward decrease in bowel weight in jejunum and ileum; however, this trend was not
Fig. 2 Effect of ischemia-reperfusion and treatment with CHE on mucosal DNA and protein content. Values are mean SEM. IR, ischemia-reperfusion; CHE, chelerythrine. p < 0.05 IR vs. Sham rats, † p < 0.05 IR-CHE vs. IR rats. European Journal of Pediatric Surgery
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Effect of Chelerythrine on Intestinal Cell Turnover following Intestinal Injury Microscopic Bowel Appearance In the small bowel segments from sham rats, the mean intestinal injury grade ranged from 0.1 to 2 in jejunum and from 0.1 to 1 in ileum without significant variation (►Fig. 3). Treatment with CHE of sham animals (Group B) did not change significantly intestinal injury score compared with sham animals. IR injury (Group C) resulted in a threefold increase in the mean intestinal injury grade in both jejunum (p < 0.05) and ileum (p < 0.05) compared with sham animals (►Fig. 3). Treatment with CHE attenuated this effect. IR-CHE (Group D) rats demonstrated a threefold decrease in the mean intestinal injury grade in both jejunum (p < 0.05) and ileum (p < 0.05) compared with IR (Group C) animals. IR rats (Group B) demonstrated a lower villus height in jejunum (574 39 vs. 748 27 µm, p < 0.05) and ileum (437 27 vs. 518 19 µm, p < 0.05), as well as crypt depth in jejunum (149 11 vs. 173 10 µm, p < 0.05) and ileum (152 11 vs. 201 18 µm, p < 0.05) compared with sham animals (►Fig. 3). IR-CHE rats (Group D) showed greater villus height in jejunum (712 16 vs. 574 39 µm, p < 0.05) and ileum (534 50 vs. 437 27 µm, p < 0.05), as well as crypt depth in jejunum (170 8 vs. 149 11 µm, p < 0.05) compared with IR animals (Group C).
Cellular Proliferation and Apoptosis Treatment with CHE of sham animals (Group B) resulted in a trend toward decrease in cell proliferation in jejunum; however, this trend was not statistically significant (►Fig. 4). A significant decrease in enterocyte proliferation occurred following IR injury (Group C) in jejunum (11%, p < 0.05) and ileum (10%, p < 0.05) when compared with sham animals.
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CHE-treated animals (Group D) did not change significantly cell proliferation rates compared with IR-animals (Group C). Sham-CHE (Group B) rats demonstrated a significant increase in cell apoptosis in jejunum (3-fold increase, p < 0.05) and ileum (4-fold increase, p < 0.05) compared with sham animals (Group A) (►Fig. 5). The AI increased after the IR insult in jejunum (8-fold increase, p < 0.05) and ileum (15-fold increase, p < 0.05) compared with sham animals. The frequency of apoptotic cells was decreased following CHE administration (Group D) in jejunum (twofold decrease, p < 0.05) and ileum (sevenfold decrease, p < 0.05) compared with IR-untreated animals.
Western Blotting Treatment with CHE of sham animals (Group B) did not change significantly (compared with control animals) proliferation- and apoptosis-related protein levels. (►Fig. 6). Decreased cell proliferation rates in IR animals (Group C) were accompanied by decreased levels of p-ERK protein levels. Treatment of sham animals with CHE (Group B) resulted in a significant increase in caspase-3 protein levels compared with animals that correlated with increased cell apoptosis in this group. An increased cell apoptosis in IR rats was accompanied by increased caspase-3 protein levels. Treatment of ischemic rats with CHE (Group D) resulted in a significant decrease in caspase-3 protein levels compared with IR. These findings correlate with decreased cell apoptosis in this group.
Discussion The exact cellular signaling involved in oxidative-stress-mediated intestinal epithelial cell damage following intestinal IR
Fig. 3 Effect of intestinal IR and CHE administration on microscopic intestinal appearance (Park injury score, villus height, and crypt depth). As expected, Sham rats demonstrated a normal histologic architecture. IR rats showed extended subepithelial space and epithelial lifting (arrows) along the villus sides (Park score 2–3). IR-CHE rats exhibited a less marked subepithelial space at the villus tips (Park score 1–2). Values are mean SEM. IR, ischemia-reperfusion; CHE, chelerythrine. p < 0.05 IR vs. Sham rats, †p < 0.05 IR-CHE vs. IR rats. European Journal of Pediatric Surgery
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Effect of Chelerythrine on Intestinal Cell Turnover following Intestinal Injury
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Fig. 4 Effect of intestinal IR and CHE on crypt cell proliferation. 5-BrdU incorporation into proliferating jejunal and ileal crypt cells was detected with a goat anti-BrdU antibody. Values are mean SEM. IR, ischemia-reperfusion; CHE, chelerythrine. p < 0.05 IR vs. Sham rats, †p < 0.05 IR-CHE vs. IR rats.
has not been clearly defined. Intestinal ischemia induces intestinal mucosal cell death, which is attributed mainly to a reduction of oxygen supply relative to metabolic demands, depletion of cellular energy stores, and accumulation of toxic metabolites. Reperfusion phase may significantly exacerbate ischemia-induced mucosal injury via the formation of reactive oxygen species and reactive nitrogen species.15,16 Although necrosis is responsible for the intestinal cell death during ischemic phase, apoptosis or “programmed cell death” has recently been recognized to be a key phenomenon in enterocyte turnover and gut barrier function following an IR insult17 We have shown previously that intestinal IR results in decreased enterocyte proliferation and increased cell death via apoptosis, leading to inhibited cell turnover.11,12 Oxidative stress is known to induce apoptosis in a variety of cell types by activating intracellular cell death signaling cascades.18 On the other hand, oxidative stress can also trigger the activation of certain signaling pathways that protect against cell death.19,20 Existing literature suggests that inhibition of PKC results in activation of apoptotic cascades in nucleated mammalian cells through a distinct phase of their development. PKC has been shown to activate end effectors such as mitochondrial adenosine triphosphate (ATP) -sensitive potassium channels and maintains the mitochondrial
permeability transition pore in a closed state, which is a pivotal mechanism that ultimately determines cell salvage, or pursuit of a necrotic or an apoptotic pathway to cell death.21 Whereas pharmacological inhibition of PKC activity triggers apoptosis in most mammalian cells, cell line, and tissue differences in sensitivities to these inhibitors remain. The isoquinoline alkaloid CHE is described as a selective inhibitor of group A and B PKC isoforms. CHE has exhibited cytotoxic activity against nine human tumor cell lines tested in vitro. We designed the present study to evaluate the effects of CHE in preventing mucosal damage caused by IR and in stimulating intestinal recovery following the IR event. Similar to our previous experiments,11,12 the present study shows that intestinal IR causes a direct intestinal mucosal injury. We observed an obvious increase in Park intestinal injury score in IR rats, suggesting intestinal mucosal damage. Additionally, gut IR leads to mucosal hypoplasia. The observed decreased bowel and mucosal weight, decreased mucosal DNA and protein, and decreased villus height and crypt depth in this model support this conclusion. This damaging effect was accompanied by a decrease in enterocyte production and an increase in enterocyte loss via apoptosis. Both mechanisms may be responsible for decreased enterocyte turnover and decreased cell mass. Decreased villus height is presumably European Journal of Pediatric Surgery
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Fig. 5 Effect of intestinal IR and treatment with CHE on enterocyte apoptosis in jejunum and ileum. Immunochemistry for caspase-3 was used to determine enterocyte apoptosis. Values are mean SEM. IR, ischemia-reperfusion; CHE, chelerythrine. p < 0.05 IR vs. Sham rats, †p < 0.05 IRCHE vs. IR rats.
due to decreased cell proliferation and migration along the villus axis or to the specific arrangement of the villus microvasculature, which results in an oxygen tension gradient along the villus. Treatment with CHE exerted different effects in normal (sham) and ischemic (IR) intestine. In sham animals, CHE administration resulted in mild inhibition of cell turnover. This is evident from decreased cell proliferation and elevated cell apoptosis that resulted in a mild decrease in bowel and mucosal weight. The most significant effect was observed in
cell apoptosis that increased significantly in nonstressed intestinal mucosa after CHE administration. CHE has been reported to exert cell growth-inhibitory effects via the induction of apoptosis in a variety of cancer cells.22 CHE-mediated apoptosis has been observed in human breast cancer, human uveal melanoma, human neuroblastoma, colon carcinoma cell lines, and neonatal rat cardiac myocytes.23,24 Since the epithelial layer of the small intestine is a rapidly renewing tissue with a high rate of cell turnover, similar to several cytotoxic chemotherapeutic agents, CHE may destroy rapidly
Fig. 6 Effect of intestinal IR and treatment with CHE on expression of p-ERK and caspase-3 protein levels in jejunal and ileal mucosal samples. Western blot was used to determine protein levels. Values are mean SEM. IR, ischemia-reperfusion; CHE, chelerythrine. p < 0.05 IR vs. Sham rats, †p < 0.05 IR-CHE vs. IR rats. European Journal of Pediatric Surgery
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Effect of Chelerythrine on Intestinal Cell Turnover following Intestinal Injury dividing cells, thus making gastrointestinal tract particularly vulnerable. Similar to the tumor target, the DNA of proliferating intestinal stem cells undergoes strand breaks, resulting in direct cellular injury following CHE administration. In contrast, treatment of IR rats with CHE significantly preserved mucosal indices and intestinal histology, suggesting that CHE may be beneficial in attenuating intestinal IR injury and its related systemic effects. Treatment with CHE resulted in a significant decrease in Park injury score, suggesting decreased intestinal damage. Increase in mucosal DNA following CHE administration suggests enhanced cell metabolism, which is consistent with the increased epithelial cell proliferation. Histologically, greater villus height in jejunum and ileum following CHE administration suggests increased absorptive surface area which closely correlates with increased cell mass. This study does not address whether or not impaired structure is associated with altered function. However, in our conventional thinking of intestinal function, it is assumed that an increase in villus height in IR-CHE rats is accompanied by enhanced nutrient absorption. Whereas treatment with CHE did not change significantly cell proliferation; cell apoptosis was inhibited in IR-CHE rats compared with IR-nontreated animals. The effect of CHE on cell apoptosis in IR rats was different from that observed in control animals, suggesting that the effect of CHE on cell apoptosis in ischemic mucosa is different from nonstressed highly proliferated mucosa. Several experiments have shown that inhibition of PKC maximally attenuated IR-induced apoptosis, indicating that IR-mediated cell death requires PKC activation. The involvement of various isoforms of PKC in the regulation of IR-mediated processes has been documented in several cell types25,26; however, its role in the regulation of H2 O2 induced intestinal epithelial cell death is undefined. The activation of selective PKC isoforms, δ and ε, has been implicated in tumor necrosis factor (TNF)-α-induced intestinal epithelial cell death as proapoptotic signals.27 In a recent study, Wang et al have shown that some kind of PKC isoform may play an important role in the progression of secondary injury in the intestine and remote organs (especially lung and liver). The authors have shown that PKCßII suppression by a specific inhibitor, LY333531, significantly attenuated IR-induced histologic damage, inflammatory cell infiltration, oxidative stress, and apoptosis in these organs, and also alleviated systemic inflammation.28 This inhibition of PKC, which resulted in activation of Akt, was found to attenuate the apoptosis induced by H2O2. In conclusion, administration of CHE prevents ischemic damage to intestinal mucosa and accelerates intestinal recovery after intestinal IR. Decreased cell death via apoptosis may be responsible for this positive effect. We have hypothesized that CHE exerts a gut protective effect by the activation of PKC-K(ATP)-independent mechanisms and the direct antioxidative stress mechanisms. Further studies will be directed toward identifying the isoform(s) of PKC and the downstream mediators responsible for intestinal cell death.
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Conflict of Interest None.
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