The mechanism of intestinal stem cells differentiation after ischemia-reperfusion injury in a rat model.

PURPOSE: Notch and Wnt/ß-catenin signaling are responsible for regulation of intestinal stem cells (ISCs) proliferation and differentiation. The purpose of the study was to evaluate Wnt/ß-catenin and Notch signaling roles in regulation of ISC differentiation following ischemia-reperfusion (IR) injury in a rat. METHODS: Rats were assigned into two groups: Sham rats underwent laparotomy without vascular intervention and IR rats underwent occlusion of SMA and portal vein for 20 min followed by 48 h of reperfusion. Wnt/ß-catenin and Notch-related gene expression were determined using Real-Time PCR. Enterocyte proliferation, differentiation and Wnt-related proteins were determined by immunohistochemistry. RESULTS: IR rats demonstrated a significant decrease in ß-catenin gene expression, a decrease in cyclin D1 and ß-catenin positive cells in jejunum and ileum compared to Sham rats. IR rats demonstrated a significant increase in Notch-related gene expression in jejunum and ileum compared to Sham rats. The number of secretory cells was higher mainly in the jejunum and number of absorptive cells was significantly lower in jejunum and lower in ileum in IR rats compared to Sham rats. CONCLUSIONS: Intestinal stem-cell differentiation is toward secretory cells 48 h after IR injury; however, Wnt/ß-catenin pathway inhibition and Notch-related gene expression stimulation suggest crosstalk between pathways.

The Mechanisms of the Anti-Inflammatory and Anti-Apoptotic Effects of Omega-3 Polyunsaturated Fatty Acids during Methotrexate-Induced Intestinal Damage in Cell Line and in a Rat Model.

Background: The aim of this study was to examine the anti-inflammatory and anti-apoptotic patterns of omega-3 polyunsaturated fatty acids (n-3 PUFAs) during methotrexate (MTX) induced intestinal damage in cell culture and in a rat model. Methods: Non-treated and treated with MTX HT 29 and HCT116cells were exposed to increasing doses of n-3 PUFAs and cell viability was evaluated using PrestoBlue® assay. Male Sprague-Dawley rats were divided into 4 experimental groups: Control rats, CONTR+n-3 PUFA rats that were treated with oral n-3 PUFA, MTX rats were treated with MTX given IP, and MTX+n-3 PUFA rats were treated with oral n-3 PUFA before and following injection of MTX. Intestinal mucosal parameters and mucosal inflammation, enterocyte proliferation and apoptosis, TNF-α in mucosal tissue and plasma (ELISA), NF-κB, COX-2, TNF-α, Fas, FasL, Fadd, Bid, Bax and Bcl-2gene and protein levels were determined 72 h following MTX injection. Results: Exposure of HT 29 and HCT116cells to n-3 PUFA attenuated inhibiting effects of MTX on cell viability. MTX-n-3 PUFA rats demonstrated a lower intestinal injury score and enhanced intestinal repair. A significant decrease in enterocyte apoptosis in MTX+n-3 PUFA rats was accompanied by decreased TNF-α, FAS, FasL, FADD and BID mRNA levels. Decreased NF-κB, COX-2 and TNF-α levels in mucosa was accompanied by a decreased number of IELs and macrophages. Conclusions: n-3 PUFAs inhibit NF-κB/COX-2 induced production of pro-inflammatory cytokines and inhibit cell apoptosis mainly by extrinsic pathway in rats with MTX-induced intestinal damage.

Cell death induction (extrinsic versus intrinsic apoptotic pathway) by intestinal ischemia-reperfusion injury in rats is time-depended.

PURPOSE: We investigate the mechanism of intestinal cell apoptosis and its relation to the time of reperfusion in a rat model of intestinal ischemia-reperfusion (IR). METHODS: Rats were divided into 4 groups: Sham-24 and Sham-48 rats underwent laparotomy without an intentional ischemic intervention and were sacrificed 24 or 48 h hours later; IR-24 and IR-48 rats underwent occlusion of SMA and portal vein for 20 min followed by 24 or 48 h of reperfusion, respectively. Park's injury score, cell proliferation and apoptosis were determined at sacrifice. Proliferation and apoptosis-related gene and protein expression were determined using Real-Time PCR, Western Blot and Immunohistochemistry. RESULTS: IR-24 rats demonstrated a strong increase in cell apoptosis along with an elevated Bax and decreased Bcl-2 expression and a decrease in cell proliferation (vs Sham-24). IR-48 group showed an increase in cell proliferation and a decrease in cell apoptosis compared to IR-24 animals. IR-48 rats demonstrated an increase in apoptotic rate that was accompanied by greater TNF-α mRNA, Fas mRNA and FasL mRNA compared to Sham-48 animals. CONCLUSION: While cell apoptosis in IR-24 rats is regulated mainly by intrinsic apoptotic pathway, 48 h followed ischemia extrinsic apoptotic pathway is responsible for pro-apoptotic effects of IR injury.

Anti-TNF-α Therapy Exerts Intestinal Anti-inflammatory and Anti-apoptotic Effects After Massive Bowel Resection in a Rat.

OBJECTIVES: The aim of this study was to examine the effect of massive small bowel resection on proinflammatory cytokine intestinal expression and the effect of anti-TNF-α antibodies (ATA) on intestinal inflammation, epithelial cell turnover, and intestinal adaptation after bowel resection in rats. METHODS: Male Sprague-Dawley rats were divided into 4 experimental groups: Sham-rats underwent bowel transection; Sham-ATA rats underwent bowel transection and were treated with ATA; SBS-animals underwent 75% bowel resection; and SBS-ATA rats underwent bowel resection and were treated with ATA similarly to Group B. Parameters of intestinal adaptation, enterocyte proliferation, and apoptosis were determined at sacrifice. TNF-α and apoptosis-related gene and protein levels were determined by Illumina's Digital Gene Expression (DGE) analysis, Real Time PCR, Western blotting, and immunohistochemistry. RESULTS: From 25 genes related to TNF-α signalling that were investigated, 8 genes in the jejunum and 10 genes in the ileum were found to be up-regulated in resected versus sham animals. SBS rats demonstrated a significant increase in tissue and plasma TNF-α, IL-6 levels, intestinal mucosal TNF-α related gene expression, and microscopic parameters of inflammation. Treatment of resected animals with ATA resulted in a significant decrease in TNF-α levels, intestinal mucosal TNF-α-related gene expression, decreased number of intraepithelial lymphocytes and macrophages, and lower apoptotic index compared with SBS animals. CONCLUSIONS: In a rat model of SBS, ATA decreased plasma and tissue TNF-α levels, diminished mucosal inflammation, and inhibited cell apoptosis. Anti-apoptotic effects of ATA appear to be associated with an inhibited extrinsic apoptotic pathway.

Accelerated Intestinal Epithelial Cell Turnover Correlates with Stimulated BMP Signaling Cascade following Intestinal Ischemia-Reperfusion in a Rat.

INTRODUCTION: Bone morphogenetic proteins (BMPs) are a family of proteins that regulate proliferation and differentiation of intestinal epithelial cells. The purpose of this study was to evaluate the role of BMP signaling following intestinal ischemia-reperfusion (IR) in a rat model. MATERIALS AND METHODS: Male Sprague-Dawley rats were divided into four experimental groups: Sham-24 and Sham-48 rats underwent laparotomy and were sacrificed 24 or 48 hours later, respectively; IR-24 and IR-48 rats underwent occlusion of superior mesenteric artery and portal vein for 30 minutes followed by 24 or 48 hours of reperfusion, respectively. Enterocyte proliferation and apoptosis were determined at sacrifice. BMP-related genes and protein expression were determined using real-time polymerase chain reaction, Western blot, and immunohistochemistry for 48 hours followed by IR. RESULTS: IR rats demonstrated a significant increase in BMP2 (twofold increase, p < 0.05), BMP4 (sevenfold increase), STAT3 (70% increase), BMPR1 (70% increase) messenger ribonucleic acid levels in jejunum and was accompanied by a significant increase in BMP2 and BMP4 protein levels in jejunum (sixfold increase) (Western blot) and upward increase in the number of BMP-positive cells (by immunohistochemistry) in jejunal (48% increase) and ileal (56% increase) villi compared with Sham-48 animals. Elevation in BMP2 and BMP4 levels was associated with increased rates of cell proliferation and increased cell apoptosis. CONCLUSION: Forty-eight hours following intestinal IR in rats, BMP signaling pathway was stimulated. The increase in BMP signaling pathway activity correlates with accelerated cell turnover.

Effect of N-Acetylserotonin on TLR-4 and MyD88 Expression during Intestinal Ischemia-Reperfusion in a Rat Model.

BACKGROUND: Accumulating evidence indicates that changes in intestinal toll-like receptors (TLRs) precede histological injury in a rodent model of necrotizing enterocolitis. N-acetylserotonin (NAS) is a naturally occurring chemical intermediate in the biosynthesis of melatonin. A recent study has shown that treatment with NAS prevents gut mucosal damage and inhibits programmed cell death following intestinal ischemia-reperfusion (IR). The objective of this study was to determine the effects of NAS on TLR-4, myeloid differentiation factor 88 (Myd88), and TNF-α receptor-associated factor 6 (TRAF6) expression in intestinal mucosa following intestinal IR in a rat. MATERIALS AND METHODS: Male Sprague-Dawley rats were randomly assigned to one of the four experimental groups: 1) Sham rats underwent laparotomy; 2) Sham-NAS rats underwent laparotomy and were treated with intraperitoneal (IP) NAS (20 mg/kg); 3) IR rats underwent occlusion of both superior mesenteric artery and portal vein for 20 minutes followed by 48 hours of reperfusion; and 4) IR-NAS rats underwent IR and were treated with IP NAS immediately before abdominal closure. Intestinal structural changes, mucosal TLR-4, MyD88, and TRAF6 mucosal gene, and protein expression were examined using real-time PCR, Western blot, and immunohistochemistry. RESULTS: Significant mucosal damage in IR rats was accompanied by a significant upregulation of TLR-4, MyD88, and TRAF6 gene and protein expression in intestinal mucosa compared with control animals. The administration of NAS decreased the intestinal injury score, inhibited cell apoptosis, and significantly reduced the expression of TLR-4, MyD88, and TRAF6. CONCLUSION: Treatment with NAS is associated with downregulation of TLR-4, MyD88, and TRAF6 expression along with a concomitant decrease in intestinal mucosal injury caused by intestinal IR in a rat.

Sonic hedgehog signaling controls gut epithelium homeostasis following intestinal ischemia-reperfusion in a rat.

PURPOSE: One of the major regulators of gastrointestinal tract development is the hedgehog signaling pathway. The purpose of this study was to evaluate the role of sonic hedgehog (SHh) signaling 24 and 48 h following intestinal ischemia-reperfusion (IR) in a rat. MATERIALS AND METHODS: Male rats were divided into four experimental groups: (1) Sham-24 h rats underwent laparotomy and were sacrificed after 24 h, (2) Sham-48h rats underwent laparotomy and were sacrificed after 48 h, (3) IR-24h rats underwent occlusion of both superior mesenteric artery and portal vein for 20 min followed by 24 h of reperfusion, and (4) IR-48 h rats underwent ischemia for 20 min followed by 48 h of reperfusion. Intestinal structural changes, enterocyte proliferation and enterocyte apoptosis were determined by immunohistochemistry 24 and 48 h following IR. SHh-related genes and protein expression were determined using real-time PCR, Western blot and immunohistochemistry. RESULTS: IR-24 rats demonstrated a significant decrease in Shh, Ihh, GIL and Ptch2 mRNA in jejunum and ileum compared to Sham-24 animals that was accompanied by a significant decrease in the number of SHH-positive cells (Immunohistochemistry) in jejunum (2.5-fold decrease) and ileum (37%). After 48 h, IR rats demonstrated a significant increase in Dhh, Ihh, Gil and PTCH2 mRNA in jejunum as well as in Dhh, Ihh, SMO, GIL, PTCH2 mRNA in ileum compared to IR-24 animals that was coincided with increased number of SHH-positive cells in jejunum (2.6-fold increase) and ileum (1.4-fold increase). CONCLUSIONS: 24 h following intestinal IR, inhibited cell turnover was associated with inhibited SHh signaling pathway. Signs of intestinal recovery appeared 48 h after IR and were correlated with increase in SHh signaling pathway activity.

Quercetin prevents small intestinal damage and enhances intestinal recovery during methotrexate-induced intestinal mucositis of rats.

BACKGROUND: Gastrointestinal mucositis occurs as a consequence of cytotoxic treatment. Quercetin (QCT) is a bioflavonoid that exerts significant antioxidant activity and anti-inflammatory as well as anti-malignancy properties. OBJECTIVE: To evaluate the effects of oral QCT consumption in preventing intestinal mucosal damage and stimulating intestinal recovery following methotrexate (MTX)-induced intestinal damage in a rat model. DESIGN: Male Sprague-Dawley rats were divided into four groups: Control Group A (CONTR) - rats were treated with 2 cc of saline given by gavage for 6 days. Group B (CONTR-QCT) - rats were treated with QCT (100 mg/kg in 2 ml saline) given by gavage 3 days before and 3 days after intraperitoneal (IP) injection of saline. Group C (MTX) - rats were injected a single dose (25 mg/kg) of MTX IP. Group D (MTX-QCT) rats were treated with QCT (similar to Group B) 3 days before and 3 days after IP MTX injection. Intestinal mucosal parameters (bowel and mucosal weight, mucosal DNA and protein content, and villus height and crypt depth), enterocytes proliferation, and enterocyte apoptosis degree were investigated at sacrifice on the 4th day after MTX or saline injection. RESULTS: Administration of QCT to MTX-treated rats resulted in: (1) significant decrease in intestinal injury score, (2) significant increase in intestinal and mucosal weight in jejunum and ileum, (3) increase on the protein content of the ileum, (4) increase in the villus height in the ileum, (5) increase of crypt depth of jejunum and ileum, and (6) increase in cell proliferation in the jejunum and ileum compared to MTX-nontreated group. CONCLUSIONS: Administration of QCT prevents intestinal damage and improves intestinal recovery following MTX-induced intestinal damage in a rat. We surmise that the effect of QCT is based on induction of cell proliferation in the crypt rather than inhibition of apoptosis.

Effect of Pomegranate Juice on Intestinal Recovery Following Methotrexate-Induced Intestinal Damage in a Rat Model.

BACKGROUND/AIMS: Several studies have demonstrated the antimicrobial, antihelminthic, and antioxidant potential of the active ingredients of pomegranate (PMG) extracts, suggesting their preventive and curative role in several gastrointestinal disorders. In the present study, the authors evaluated the effects of oral PMG supplementation on intestinal structural changes, enterocyte proliferation, and apoptosis during methotrexate (MTX)-induced intestinal damage in a rat. METHODS: Male rats were divided into 4 experimental groups: control rats; CONTR-PMG rats were treated with oral PMG given by gavage once a day 72 hours before and 72 hours following vehicle injection; MTX rats were treated with single dose of methotrexate; and MTX-PMG rats were treated with oral PMG following injection of MTX. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation, and enterocyte apoptosis were determined 72 hours following MTX injection. Western blotting was used to determine phosphorylated extracellular signal-regulated kinase (p-ERK) and caspase 3 protein levels. RESULTS: MTX-PMG rats demonstrated greater jejunal and ileal bowel and mucosal weights, greater jejunal and ileal mucosal DNA and protein levels, greater villus height in jejunum and ileum and crypt depth in ileum, compared with MTX animals. A significant decrease in enterocyte apoptosis in ileum of MTX-PMG rats (vs MTX) was associated with a decrease in caspase 3 protein expression as well as increased cell proliferation, which was correlated with elevated p-ERK protein levels. CONCLUSIONS: Treatment with oral PMG prevents mucosal injury and improves intestinal recovery following MTX injury in the rat.

Effect of Chelerythrine on Intestinal Cell Turnover following Intestinal Ischemia-Reperfusion Injury in a Rat Model.

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.

The Effect of Elevated Intra-Abdominal Pressure on TLR4 Signaling in Intestinal Mucosa and on Intestinal Bacterial Translocation in a Rat.

BACKGROUND: Recent evidence suggests that elevated intra-abdominal pressure (IAP) may adversely affect the intestinal barrier function. Toll-like receptor 4 (TLR-4) is responsible for the recognition of bacterial endotoxin or lipopolysaccharide and for initiation of the Gram-negative septic shock syndrome. The objective of the current study was to determine the effects of elevated IAP on intestinal bacterial translocation (BT) and TLR-4 signaling in intestinal mucosa in a rat model. METHODS: Male Sprague-Dawley rats were randomly assigned to one of two experimental groups: sham animals (Sham) and IAP animals who were subjected to a 15 mmHg pressure pneumoperitoneum for 30 minutes. Rats were sacrificed 24 hours later. BT to mesenteric lymph nodes, liver, portal vein blood, and peripheral blood was determined at sacrifice. TLR4-related gene and protein expression (TLR-4; myeloid differentiation factor 88 [Myd88] and TNF-α receptor-associated factor 6 [TRAF6]) expression were determined using real-time PCR, western blotting, and immunohistochemistry. RESULTS: Thirty percent of sham rats developed BT in the mesenteric lymph nodes (level I) and 20% of control rats developed BT in the liver and portal vein (level II). abdominal compartment syndrome (ACS) rats demonstrated an 80% BT in the lymph nodes (Level I) and 40% BT in the liver and portal vein (Level II). Elevated BT was accompanied by a significant increase in TLR-4 immunostaining in jejunum (51%) and ileum (35.9%), and in a number of TRAF6-positive cells in jejunum (2.1%) and ileum (24.01%) compared to control animals. ACS rats demonstrated a significant increase in TLR4 and MYD88 protein levels compared to control animals. CONCLUSIONS: Twenty-four hours after the induction of elevated IAP in a rat model, increased BT rates were associated with increased TLR4 signaling in intestinal mucosa.

Effect of N-Acetylserotonin on Intestinal Recovery Following Intestinal Ischemia-Reperfusion Injury in a Rat.

OBJECTIVE: N-acetylserotonin (NAS) is a naturally occurring chemical intermediate in the biosynthesis of melatonin. Extensive studies in various experimental models have established that treatment with NAS significantly protects heart and kidney injury from ischemia-reperfusion (IR). The purpose of the present study was to examine the effect of NAS on intestinal recovery and enterocyte turnover after intestinal IR injury in rats. METHODS: Male Sprague-Dawley rats were divided into four experimental groups: (1) Sham rats underwent laparotomy, (2) sham-NAS rats underwent laparotomy and were treated with intraperitoneal (IP) NAS (20 mg/kg); (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-NAS rats underwent IR and were treated with IP NAS (20 mg/kg) 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-time polymerase chain reaction, Western blot, and immunohistochemistry. A nonparametric Kruskal-Wallis analysis of variance test was used for statistical analysis with p less than 0.05 considered statistically significant. RESULTS: Treatment with NAS resulted in a significant increase in mucosal weight in jejunum and ileum, villus height in the ileum, and crypt depth in jejunum and ileum compared with IR animals. IR-NAS rats also had a significantly proliferation rates as well as a lower apoptotic index in jejunum and ileum which was accompanied by higher Bcl-2 levels compared with IR animals. CONCLUSIONS: Treatment with NAS prevents gut mucosal damage and inhibits programmed cell death following intestinal IR in a rat.

The role of Wnt/ß-catenin signaling in enterocyte turnover during methotrexate-induced intestinal mucositis in a rat.

BACKGROUND/AIMS: Intestinal mucositis is a common side-effect in patients who receive aggressive chemotherapy. The Wnt signaling pathway is critical for establishing and maintaining the proliferative compartment of the intestine. In the present study, we tested whether Wnt/ß-catenin signaling is involved in methotrexate (MTX)-induced intestinal damage in a rat model. METHODS: Non-pretreated and pretreated with MTX Caco-2 cells were evaluated for cell proliferation and apoptosis using FACS analysis. Adult rats were divided into three experimental groups: Control rats; MTX-2 animals were treated with a single dose of MTX given IP and were sacrificed on day 2, and MTX-4 rats were treated with MTX similar to group B and were sacrificed on day 4. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation, and enterocyte apoptosis were measured at sacrifice. Real Time PCR and Western blot was used to determine the level of Wnt/ß-catenin related genes and protein expression. RESULTS: In the vitro experiment, treatment with MTX resulted in marked decrease in early cell proliferation rates following by a 17-fold increase in late cell proliferation rates compared to early proliferation. Treatment with MTX resulted in a significant increase in early and late apoptosis compared to Caco-2 untreated cells. In the vivo experiment, MTX-2 and MTX-4 rats demonstrated intestinal mucosal hypoplasia. MTX-2 rats demonstrated a significant decrease in FRZ-2, Wnt 3A Wnt 5A, ß-catenin, c-myc mRNA expression and a significant decrease in ß-catenin and Akt protein levels compared to control animals. Four days following MTX administration, rats demonstrated a trend toward a restoration of Wnt/ß-catenin signaling especially in ileum. CONCLUSIONS: Wnt/ß-catenin signaling is involved in enterocyte turnover during MTX-induced intestinal mucositis in a rat.

Glutamine attenuates the inhibitory effect of methotrexate on TLR signaling during intestinal chemotherapy-induced mucositis in a rat.

Toll-like receptor 4 (TLR-4) is crucial in maintaining intestinal epithelial homeostasis, participates in a vigorous signaling process and heightens inflammatory cytokine output. The objective of this study was to determine the effects of glutamine (GLN) on TLR-4 signaling in intestinal mucosa during methotrexate (MTX)-induced mucositis in a rat. Male Sprague-Dawley rats were randomly assigned to one of four experimental groups of 8 rats each: 1) control rats; 2) CONTR-GLN animals were treated with oral glutamine given in drinking water (2%) 48 hours before and 72 hours following vehicle injection; 3) MTX-rats were treated with a single IP injection of MTX (20 mg/kg); and 4) MTX-GLN rats were pre-treated with oral glutamine similar to group B, 48 hours before and 72 hours after MTX injection. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined 72 hours following MTX injection. The expression of TLR-4, MyD88 and TRAF6 in the intestinal mucosa was determined using real time PCR, Western blot and immunohistochemistry. MTX-GLN rats demonstrated a greater jejunal and ileal mucosal weight and mucosal DNA, greater villus height in ileum and crypt depth and index of proliferation in jejunum and ileum, compared to MTX animals. The expression of TLR-4 and MyD88 mRNA and protein in the mucosa was significantly lower in MTX rats versus controls animals. The administration of GLN increased significantly the expression of TLR-4 and MyD88 (vs the MTX group). In conclusion, treatment with glutamine was associated with up-regulation of TLR-4 and MyD88 expression and a concomitant decrease in intestinal mucosal injury caused by MTX-induced mucositis in a rat.

Effect of ozone on intestinal recovery following intestinal ischemia-reperfusion injury in a rat.

BACKGROUND: Growing evidence suggests that ozone (O3) protects the host against pathological conditions mediated by reactive oxygen species by increasing the activity of antioxidant enzymes. The purpose of the present study was to examine the effect of O3 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-O3 rats underwent laparotomy and were treated with an ozone/oxygen mixture intraperitoneally and intraluminally (50 %/50 %); (3) IR rats underwent occlusion of both superior mesenteric artery and portal vein for 20 min followed by 48 h of reperfusion, and (4) IR-O3 rats underwent IR and were treated with an ozone/oxygen mixture similar to group 2. Intestinal structural changes, Park's injury score, enterocyte proliferation and enterocyte apoptosis were determined 48 h following IR. Western blot was used to determine ERK and Bax protein levels. A non-parametric Kruskal-Wallis ANOVA test was used for statistical analysis with p < 0.05 considered statistically significant. RESULTS: Treatment of IR rats with O3 resulted in a significant increase in mucosal weight in jejunum (70 %) and ileum (32 %), mucosal DNA (twofold increase) and protein (35 %) in ileum, villus height and crypt depth in jejunum (61 and 16 %, correspondingly) and ileum (31 and 43 %, correspondingly) compared to IR animals. IR-O3 rats also had a significantly lower intestinal injury score as well as a lower apoptotic index in jejunum and ileum compared and IR animals. A significant increase in cell proliferation rates in IR-O3 animals was accompanied by increased levels of p-ERK protein. CONCLUSIONS: Treatment with ozone prevents intestinal mucosal damage, stimulates cell proliferation and inhibits programmed cell death following intestinal IR in a rat.

Wnt/ß-catenin signaling cascade down-regulation following massive small bowel resection in a rat.

BACKGROUND: Growing evidence suggests that the Wnt/ß-catenin signaling cascade is implicated in the control of stem cell activity, cell proliferation, lineage commitment, and cell survival during normal development and tissue regeneration of the gastrointestinal epithelium. The roles of this signaling cascade in stimulation of cell proliferation after massive small bowel resection are unknown. The purpose of this study was to evaluate the role of Wnt/ß-catenin signaling during late stages of intestinal adaptation in a rat model of short bowel syndrome (SBS). METHODS: Male rats were divided into two groups: sham rats underwent bowel transection and SBS rats underwent a 75 % bowel resection. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined 2 weeks after operation. Illumina's digital gene expression analysis was used to determine Wnt/ß-catenin signaling gene expression profiling. Twelve Wnt/ß-catenin-related genes and ß-catenin protein expression were determined using real-time PCR, western blotting and immunohistochemistry. RESULTS: From the total number of 20,000 probes, 20 genes related to Wnt/ß-catenin signaling were investigated. From these genes, seven genes were found to be up-regulated and eight genes to be down-regulated in SBS vs. sham animals with a relative change in gene expression level of 20 % or more. From 12 genes determined by real-time PCR, nine genes were down-regulated in SBS rats compared to control animals including target gene c-Myc. SBS rats also showed a significant decrease in ß-catenin protein compared to control animals. CONCLUSION: Two weeks following massive bowel resection in rats, Wnt/ß-catenin signaling pathway is inhibited. In addition, it appears that cell differentiation rather than proliferation is most important in the late stages of intestinal adaptation.

Effects of novel neuroprotective and neurorestorative multifunctional drugs on iron chelation and glucose metabolism.

Iron accumulation and iron-related oxidative stress are involved in several pathological conditions and provide a rationale for the development of iron chelators as novel promising therapeutic strategies. Thus, we have recently synthesized multifunctional non-toxic, brain permeable iron chelating compounds, M30 and HLA20, possessing the neuroprotective N-propargyl moiety of the anti-Parkinsonian drug, monoamine oxidase (MAO)-B inhibitor, rasagiline and the antioxidant-iron chelating moiety of an 8-hydroxyquinoline derivative of the iron chelator, VK28. Here, we examined the hepatic regulatory effects of these novel compounds using two experimental approaches: chelation activity and glucose metabolism parameters. The present study demonstrated that M30 and HLA20 significantly decreased intracellular iron content and reduced ferritin expression levels in iron-loaded hepatoma Hep3B cells. In electron microscopy analysis, M30 was shown to reduce the electron-dense deposits of siderosomes by ~30 %, as well as down-regulate cytosolic ferritin particles observed in iron-overloaded cells. In vivo studies demonstrated that M30 administration (1 mg/kg, P.O. three times a week) reduced hepatic ferritin levels; increased hepatic insulin receptor and glucose transporter-1 levels and improved glucose tolerance in C57BL/6 mice and in a mouse model of type-2 diabetes, the ob/ob (leptin(-/-)). The results clearly indicate that the novel multifunctional drugs, especially M30, display significant capacity of chelating intracellular iron and regulating glucose metabolism parameters. Such effects can have therapeutic significance in conditions with abnormal local or systemic iron metabolism, including neurological diseases.

Reversal of severe methotrexate-induced intestinal damage using enteral n-3 fatty acids.

Growing evidence suggests that n-3 PUFA and their specific lipid mediators can reduce the activity of inflammatory processes. In the present study, we evaluated the effects of oral n-3 PUFA supplementation on intestinal structural changes, enterocyte proliferation and apoptosis during methotrexate (MTX)-induced intestinal damage in the rat. A total of thirty-two male rats were divided into four experimental groups: control (CONTR) rats; CONTR-n-3 PUFA rats treated with oral administration of n-3 PUFA at a dose of 300 µg/kg once per d 72 h before and 72 h following vehicle injection; MTX rats treated with a single dose of MTX; MTX-n-3 PUFA rats treated with oral n-3 PUFA following the injection of MTX. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis determined 72 h following MTX injection. Real-time PCR was used to determine B-cell lymphoma 2 (Bcl2)-associated X protein (Bax) and Bcl2 mRNA expression. Western blotting was used to determine phosphorylated extracellular signal-related kinase, ß-catenin, Bax and Bcl2 protein levels. MTX-n-3 PUFA rats demonstrated a greater jejunal and ileal bowel weight, greater ileal mucosal weight, greater ileal mucosal DNA and protein levels, greater villus height in the jejunum and ileum and crypt depth in the ileum, compared with MTX animals. A significant decrease in enterocyte apoptosis in the ileum of MTX-n-3 PUFA rats (v. MTX) was accompanied by decreased Bax mRNA and protein expression and increased Bcl2 mRNA levels. Thus, the treatment with oral n-3 PUFA prevented mucosal injury and improved intestinal recovery following MTX-injury in rats.

Dietary supplementation with vitamin D stimulates intestinal epithelial cell turnover after massive small bowel resection in rats.

PURPOSE: While the endocrine action of the active metabolite 1,25-dihydroxyvitamin D (VtD) has been well characterized in relation to the maintenance of plasma calcium and phosphate homeostasis through regulation of intestinal absorption, recent research has focused on its autocrine and/or paracrine activities. Such activities have been best characterized in intestine, where VtD regulates cell differentiation and maturation. The purpose of this study was to evaluate the effect of VtD on enterocyte turnover in a rat model of short bowel syndrome (SBS). METHODS: Male rats were divided into four groups: sham rats underwent bowel transection, sham-VtD rats underwent bowel transection and were treated oral VtD, SBS rats underwent a 75 % bowel resection, and SBS-VtD rats underwent bowel resection and were treated with VtD. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined at sacrifice. Illumina's digital gene expression (DGE) analysis was used to determine VtD pathway-related gene expression profiling. VtD receptor (VDR) and its promoter, Bax and Bcl-2 mRNA expression were determined using real-time PCR. Western blotting was used to determine p-ERK, Bax and ß-catenin protein levels. RESULTS: From the total of 20,000 probes, 11 genes related to VtD signaling were investigated. Of these genes, five were found to be up-regulated in SBS versus sham animals with a relative change in gene expression level of 20 %, five remained unchanged, and one was down-regulated. VtD treatment in sham and SBS rats resulted in significant up-regulation of the VDR gene and its promoter's expression. SBS-VtD rats demonstrated a significant increase in all intestinal mucosal parameters compared to SBS animals. A significant increase in cell proliferation in SBS-VtD rats was accompanied by increased ß-catenin protein levels. A significant decrease in cell apoptosis in this group was correlated with lower Bax/Bcl-2 mRNA and protein levels. CONCLUSION: In a rat model of SBS, dietary supplementation with VtD stimulates enterocyte turnover, which correlates with up-regulated VtD receptor expression in the remaining small intestine.

Dietary transforming growth factor-beta 2 (TGF-ß2) supplementation reduces methotrexate-induced intestinal mucosal injury in a rat.

BACKGROUND/AIMS: Dietary supplementation with transforming growth factor-beta (TGF-ß) has been proven to minimize intestinal damage and facilitate regeneration after mucosal injury. In the present study, we evaluated the effects of oral TGF-ß2 supplementation on intestinal structural changes, enterocyte proliferation and apoptosis following methotrexate (MTX)-induced intestinal damage in a rat and in a cell culture model. METHODS: Caco-2 cells were treated with MTX and were incubated with increasing concentrations of TGF-ß2. Cell apoptosis was assessed using FACS analysis by annexin staining and cell viability was monitored using Trypan Blue assay. Male rats were divided into four experimental groups: Control rats, CONTR- TGF-ß rats were treated with diet enriched with TGF-ß2, MTX rats were treated with a single dose of methotrexate, and MTX- TGF-ß rats were treated with diet enriched with TGF-ß2. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined at sacrifice. Real Time PCR and Western blot were used to determine bax and bcl-2 mRNA, p-ERK, ß-catenin, IL-1B and bax protein expression. RESULTS: Treatment of MTX-pretreated Caco-2 cells with TGF-B2 resulted in increased cell viability and decreased cell apoptosis. Treatment of MTX-rats with TGF-ß2 resulted in a significant increase in bowel and mucosal weight, DNA and protein content, villus-height (ileum), crypt-depth (jejunum), decreased intestinal-injury score, decreased level of apoptosis and increased cell proliferation in jejunum and ileum compared to the untreated MTX group. MTX-TGF-ß2 rats demonstrated a lower bax mRNA and protein levels as well as increased bcl-2 mRNA levels in jejunum and ileum compared to MTX group. Treatment with TGF-ß2 also led to increased pERK, IL-1B and ß-catenin protein levels in intestinal mucosa. CONCLUSIONS: Treatment with TGF-ß2 prevents mucosal-injury, enhances p-ERK and ß-catenin induced enterocyte proliferation, inhibits enterocyte apoptosis and improves intestinal recovery following MTX-induced intestinal-mucositis in rats.