Intestinal Intestinal Ischemia-reperfusion Research Articles

Analysis of lipid metabolites derived from gut microbiota in ischemia-reperfusion model.

Disruption of intestinal barrier caused by intestinal ischemia due to hemorrhagic shock is associated with the pathogenesis of multiple organ dysfunction (MOD) after severe trauma. Mesenteric lymph (ML) plays an important role as a route for transporting inflammatory mediators, including lipids. Postbiotics, such as 10-hydroxy-cis-12-octadecenoic acid (HYA), have received much attention as a treatment option. However, the relationship between postbiotics and MOD has yet to be clarified. The aim of the present study was to analyze lipid metabolites derived from gut microbiota in the intestinal ischemia-reperfusion (IR) rat model. Male Sprague-Dawley rats underwent laparotomy, and their ML duct and superior mesenteric artery were exposed. The superior mesenteric artery was clamped for 60 minutes, followed by 120 minutes of reperfusion. The ML and the plasma were collected before and after intestinal IR. Lipids were extracted from plasma and ML, and liquid chromatography-tandem mass spectrometry was performed. The concentration of linoleic acid in plasma samples was not different before and after IR; however, the linoleic acid concentration in the ML samples increased after intestinal IR. Eicosapentaenoic acids and docosahexaenoic related to linoleic acids showed similar changes with IR-induced increase in the ML. The concentration of HYA, a linoleic acid-derived bioactive metabolite produced by gut bacteria, was high in ML samples, while that in plasma samples was low. The relative increase rate of HYA in ML samples after IR was higher than that of the plasma samples (the ML samples: relative increase, 3.23 ± 1.36; the plasma samples: relative increase, 0.95 ± 0.35; n = 3, p = 0.048). The present study demonstrated increased linoleic acids and high concentrations of HYA, lipid metabolites derived from gut bacteria in the ML after intestinal IR. These findings may contribute to clarifying the relation between gut microbiota and MOD after severe trauma.

Microfluidic intestinal organoid-on-a-chip uncovers therapeutic targets by recapitulating oxygen dynamics of intestinal IR injury.

Increasing evidence demonstrates that mammals have different reactions to hypoxia with varied oxygen dynamic patterns. It takes ∼24h for tri-gas incubator to achieve steady cell hypoxia, which fails to recapitulate ultrafast oxygen dynamics of intestinal ischemia/reperfusion (IR) injury. Inspired from the structure of native intestinal villi, we engineered an intestinal organoid chip embedded with engineered artificial microvessels based on co-axial microfluidic technology by using pH-responsive ZIF-8/sodium alginate scaffold. The chip was featured on: (i) eight times the oxygen exchange efficiency compared with the conventional device, tri-gas incubator, (ii) implantation of intestinal organoid reproducing all types of intestinal epithelial cells, and (iii) bio-responsiveness to hypoxia and reoxygenation (HR) by presenting metabolism disorder, inflammatory reaction, and cell apoptosis. Strikingly, it was found for the first time that Olfactomedin 4 (Olfm4) was the most significantly down-regulated gene under a rapid HR condition by sequencing the RNA from the organoids. Mechanistically, OLFM4 played protective functions on HR-induced cell inflammation and tissue damage by inhibiting the NF-kappa B signaling activation, thus it could be used as a therapeutic target. Altogether, this study overcomes the issue of mismatched oxygen dynamics between in vitro and in vivo, and sets an example of next-generation multisystem-interactive organoid chip for finding precise therapeutic targets of IR injury.

Qingchang Mixture Prevents the Intestinal Ischemia-reperfusion Injury through TLR4/NF-kB Pathway.

This study aims to determine the protective effect and molecular responses of the traditional Chinese medicine Qingchang mixture on intestinal ischemia-reperfusion (IR) injury. The rat intestinal IR model was prepared. The intestinal ischemic injury was evaluated by HE staining, biochemical assay and western blot. In addition, a human hypoxia-reoxygenation (HR) in vitro model was prepared using intestinal epithelial cells (IEC-6). The viability and apoptosis of IEC-6 cells were measured by CCK8 and apoptosis detection. TAK242 or PDTC was used as a small molecule inhibitor of TLR4 or NF-κB, respectively. Compared with the IR group, the pretreatment of the Qingchang mixture reduced the morphological damage, oxidative stress, inflammatory response, and barrier function damage of the small intestine tissue. IR significantly increased the expression of TLR4 and NF-κB, while the pretreatment of the Qingchang mixture inhibited the expression of TLR4 and NF-κB. Furthermore, the pretreatment of Qingchang mixture, TAK242, or PDTC effectively improved the viability and hindered apoptosis of the HR-induced IEC-6 cells. Traditional Chinese medicine Qingchang mixture prevents intestinal IR injury through TLR4/NF-kB pathway.

Transplanted hair follicle mesenchymal stem cells alleviated small intestinal ischemia–reperfusion injury via intrinsic and paracrine mechanisms in a rat model

Background: Small intestinal ischemia-reperfusion (IR) injury is a common intestinal disease with high morbidity and mortality. Mesenchymal stem cells (MSCs) have been increasingly used in various intestinal diseases. This study aimed to evaluate the therapeutic effect of hair follicle MSCs (HFMSCs) on small intestinal IR injury. Methods: We divided Sprague–Dawley rats into three groups: the sham group, IR group and IR + HFMSCs group. A small intestinal IR injury rat model was established by clamping of the superior mesenteric artery (SMA) for 30 min and reperfusion for 2 h. HFMSCs were cultured in vitro and injected into the rats through the tail vein. Seven days after treatment, the intrinsic homing and differentiation characteristics of the HFMSCs were observed by immunofluorescence and immunohistochemical staining, and the paracrine mechanism of HFMSCs was assessed by Western blotting and enzyme-linked immunosorbent assay (ELISA). Results: A small intestinal IR injury model was successfully established. HFMSCs could home to damaged sites, express proliferating cell nuclear antigen (PCNA) and intestinal stem cell (ISC) markers, and promote small intestinal ISC marker expression. The expression levels of angiopoietin-1 (ANG1), vascular endothelial growth factor (VEGF) and insulin growth factor-1 (IGF1) in the IR + HFMSCs group were higher than those in the IR group. HFMSCs could prevent IR-induced apoptosis by increasing B-cell lymphoma-2 (Bcl-2) expression and decreasing Bcl-2 homologous antagonist/killer (Bax) expression. Oxidative stress level detection showed that the malondialdehyde (MDA) content was decreased, while the superoxide dismutase (SOD) content was increased in the IR + HFMSCs group compared to the IR group. An elevated diamine oxidase (DAO) level reflected the potential protective effect of HFMSCs on the intestinal mucosal barrier. Conclusion: HFMSCs are beneficial to alleviate small intestinal IR injury through intrinsic homing to the small intestine and by differentiating into ISCs, via a paracrine mechanism to promote angiogenesis, reduce apoptosis, regulate the oxidative stress response, and protect intestinal mucosal function potentially. Therefore, this study suggests that HFMSCs serve as a new option for the treatment of small intestinal IR injury.

Role and Potential Mechanism of Heme Oxygenase-1 in Intestinal Ischemia-Reperfusion Injury.

Intestinal ischemia-reperfusion (IR) injury is a complex, multifactorial, and pathophysiological condition with high morbidity and mortality, leading to serious difficulties in treatment, especially in humans. Heme oxygenase (HO) is the rate-limiting enzyme involved in heme catabolism. HO-1 (an inducible form) confers cytoprotection by inhibiting inflammation and oxidation. Furthermore, nuclear factor-erythroid 2-related factor 2 (Nrf2) positively regulates HO-1 transcription, whereas BTB and CNC homolog 1 (Bach1) competes with Nrf2 and represses its transcription. We investigated the role and potential mechanism of action of HO-1 in intestinal IR injury. Intestinal ischemia was induced for 45 min followed by 4 h of reperfusion in wild-type, Bach1-deficient, and Nrf2-deficient mice, and a carbon monoxide (CO)-releasing molecule (CORM)-3 was administered. An increase in inflammatory marker levels, nuclear factor-κB (NF-κB) activation, and morphological impairments were observed in the IR-induced intestines of wild-type mice. These inflammatory changes were significantly attenuated in Bach1-deficient mice or those treated with CORM-3, and significantly exacerbated in Nrf2-deficient mice. Treatment with an HO-1 inhibitor reversed this attenuation in IR-induced Bach1-deficient mice. Bach1 deficiency and treatment with CORM-3 resulted in the downregulation of NF-κB activation and suppression of adhesion molecules. Together, Bach1, Nrf2, and CO are valuable therapeutic targets for intestinal IR injury.

The protective effect of α7-nACh receptor and its interaction with 5-HT1B/1D receptors in acute intestinal ischemia-reperfusion injury in rats.

Over the past decades, great attention has been given to the nervous system modulating effects on the immune response in inflammation-associated injuries, such as acute intestinal ischemia-reperfusion (IR). Recently, we proved the anti-inflammatory and antioxidant effects of 5-hydroxytryptamine (5-HT)1B/1D receptors in intestinal IR injury in rats. Also, the alpha7 nicotinic acetylcholine (α7-nACh) receptor has anti-inflammatory effects in different inflammation-associated injuries. Starting from these premises, we aimed to examine the function of the α7-nACh receptors and the functional interactions between the anti-inflammatory and antioxidant effects of α7-nACh and 5-HT1B/1D receptors in acute intestinal IR injury. To confirm the expression and localization of α7-nACh receptors on the ileum nerves, an immunofluorescence-based method was applied. Then, intestinal IR injury was induced by 30-min occlusion of superior mesenteric artery and reperfusion for 2h in rats. Acute systemic administration of α7-nACh receptor agonist PNU-282987 and antagonist methyllycaconitine, and 5-HT1B/1D receptors agonist (sumatriptan) and antagonist (GR127, 935) were used in the model of intestinal IR injury. Finally, biochemical and histological parameters were assessed. Α7-nACh receptors were expressed by 9% on the ileum nerves. Likewise, activation of the α7-nACh receptor showed anti-inflammatory and antioxidant effects in intestinal IR injury but not as well as 5-HT1B/1D receptors. Interestingly, 5-HT1B/1D receptors via attenuation of glutamate (Glu) release indirectly activated the α7-nACh receptor and its protective effects against inflammation and oxidative stress. The protective effect of the α7-nACh receptor on intestinal IR injury was activated indirectly through the 5-HT1B/1D receptors' modulatory impact on Glu release.

Complement Initiation Varies by Sex in Intestinal Ischemia Reperfusion Injury.

Intestinal ischemia reperfusion (IR)-induced tissue injury represents an acute inflammatory response with significant morbidity and mortality. The mechanism of IR-induced injury is not fully elucidated, but recent studies suggest a critical role for complement activation and for differences between sexes. To test the hypothesis that complement initiation differs by sex in intestinal IR, we performed intestinal IR on male and female WT C57B6L/, C1q-/-, MBL-/-, or properdin (P)-/- mice. Intestinal injury, C3b and C5a production and ex vivo secretions were analyzed. Initial studies demonstrated a difference in complement mRNA and protein in male and female WT mice. In response to IR, male C1q-, MBL- and P-deficient mice sustained less injury than male WT mice. In contrast, only female MBL-/- mice sustained significantly less injury than female wildtype mice. Importantly, wildtype, C1q-/- and P-/- female mice sustained significant less injury than the corresponding male mice. In addition, both C1q and MBL expression and deposition increased in WT male mice, while only elevated MBL expression and deposition occurred in WT female mice. These data suggested that males use both C1q and MBL pathways, while females tend to depend on lectin pathway during intestinal IR. Females produced significantly less serum C5a in MBL-/- and P-/- mice. Our findings suggested that complement activation plays a critical role in intestinal IR in a sex-dependent manner.

Absence of the C5a Receptor C5aR2 Worsens Ischemic Tissue Injury by Increasing C5aR1-Mediated Neutrophil Infiltration.

Neutrophil infiltration to ischemic tissues following reperfusion worsens injury. A key driver of neutrophil recruitment and activation is the complement factor C5a, which signals through two receptors, C5aR1 and C5aR2. In this study, we used a neutrophil-dependent mouse model of intestinal ischemia-reperfusion (IR) injury to investigate the underexplored role of C5aR2 in neutrophil mobilization, recruitment, and disease outcomes. We show that intestinal IR induces rapid neutrophil mobilization along with a concomitant reduction in plasma C5a levels that is driven by both C5aR1 and C5aR2. Intestinal IR in C5aR2-/- mice led to worsened intestinal damage and increased neutrophil infiltration. Inhibition of C5aR1 signaling in C5aR2-/- mice with PMX53 prevented neutrophil accumulation and reduced IR pathology, suggesting a key requirement for enhanced neutrophil C5aR1 activation in the absence of C5aR2 signaling. Interestingly, C5aR2 deficiency also reduced circulating neutrophil numbers after IR, as well as following G-CSF-mediated bone marrow mobilization, which was independent of C5aR1, demonstrating that C5aR2 has unique and distinct functions from C5aR1 in neutrophil egress. Despite enhanced tissue injury in C5aR2-/- IR mice, there were significant reductions in intestinal proinflammatory cytokines, highlighting complicated dual protective/pathogenic roles for C5aR2 in pathophysiology. Collectively, we show that C5aR2 is protective in intestinal IR by inhibiting C5aR1-mediated neutrophil recruitment to the ischemic tissue. This is despite the potentially local pathogenic effects of C5aR2 in increasing intestinal proinflammatory cytokines and enhancing circulating neutrophil numbers in response to mobilizing signals. Our data therefore suggest that this balance between the dual pro- and anti-inflammatory roles of C5aR2 ultimately dictates disease outcomes.

The effect of pre- and post-remote ischemic conditioning reduces the injury associated with intestinal ischemia/reperfusion.

Midgut volvulus is associated with intestinal ischemia/reperfusion (IR) injury and can progress to severe intestinal damage. Remote ischemic conditioning (RIC) reduces IR-induced injury in distant organs. The aim of this study was to investigate whether RIC protects the intestine from IR injury. We investigated intestinal IR injury in 3weeks old SD rats. Animals underwent: (i) sham laparotomy, (ii) intestinal IR injury, (iii) intestinal IR + RIC during ischemia, or (iv) intestinal IR + RIC after reperfusion. Intestinal IR injury was achieved by 45min occlusion of superior mesenteric artery followed by de-occlusion. RIC was administered via four cycles of 5min of hind limb ischemia followed by 5min reperfusion. Animals were sacrificed 24h after surgery and the ileum was harvested for evaluation. Intestinal injury was present after IR. However, this injury was reduced in both IR + RIC groups. Expression of inflammatory cytokine IL6 was lower in IR + RIC groups compared to IR alone. Carbonyl protein was also significantly lower in IR + RIC compared to IR, indicating lower oxidative stress in both IR + RIC groups. Remote ischemic conditioning attenuated intestinal injury, inflammation, and oxidative stress in experimental intestinal ischemia/reperfusion injury. Remote ischemic conditioning may be useful in children with midgut volvulus to reduce the intestinal injury. Experimental study. Animal experiment.

Complement initiation and regulation differ by sex in intestinal ischemia reperfusion injury

Abstract Males and females frequently sustain distinct symptoms during ischemic events and subsequent inflammatory events. The inflammatory events include significant complement activation, neutrophil infiltration and significant cytokine secretions. The past literature suggests that complement activation may include a sex-linked protein. Therefore, we hypothesized that complement activation may vary by sex during intestinal ischemia/reperfusion (IR) injury. We subjected male and female mice to 30 min intestinal ischemia followed by 15, 30, 60 or 120 min reperfusion. Additional mice of each sex was subjected to sham surgery. Intestinal sections were stained and complement initiation pathways and time points of deposition determined. Complement regulatory molecules were also examined. Initial results suggest that males expressed more C1q compared to females, while females expressed more MBL than males. In addition, complement regulators varied by sex. Our data demonstrate that complement initiation and regulation differ between sexes during intestinal IR. These response differences in intestinal IR provide insight into the sexual dimorphism of ischemic intestine diseases and may aid in the development of sex-specific therapies that reduce intestinal injury.

The Potential Role of Protein Tyrosine Phosphatase, Receptor Type C (CD45) in the Intestinal Ischemia-Reperfusion Injury.

This study was designed to identify several key genes and their functions in preventing or ameliorating intestinal ischemia-reperfusion (IR) injury, which could provide rationale for further exploring the regulatory mechanisms or clinical treatment for intestinal IR injury. The microarray GSE37013 of human intestinal IR injury was downloaded from Gene Expression Omnibus database. The differentially expressed genes (DEGs) with changes of reperfusion time were screened using Short Time-series Expression Miner, followed by function enrichment analysis, protein-protein interaction (PPI) network, and module construction. Subsequently, the key DEGs were identified with VEEN analysis based on the significant results of function enrichment analysis and PPI module. Finally, the gene-drug interactions were predicted using DGIdb 2.0. The DEGs of intestinal IR injury were significantly divided into three clusters with changes of reperfusion time. The genes in the three clusters were mainly enriched in transmembrane transport, defense responses, and cellular component assembly related pathways, respectively. There were 121 nodes and 281 interactions in PPI network, including one significant submodule. Protein tyrosine phosphatase, receptor type C (PTPRC) was a hub code both in PPI network and in submodule. A total of eight key DEGs were identified but only PTPRC was predicted to be interacted with eight drugs, such as infliximab. Totally, eight key genes associated with intestinal IR were identified; PTPRC especially was the most prominent potential drug target. These findings provided several potential therapeutic targets or potential breakthrough area in the study of intestinal IR injury.

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

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. 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. 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. 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.

The olfactomedin-4 positive neutrophil has a role in murine intestinal ischemia/reperfusion injury.

Olfactomedin-4 (OLFM4) identifies a subset of neutrophils conserved in both mouse and man, associated with worse outcomes in several inflammatory conditions. We investigated the role of OLFM4-positive neutrophils in murine intestinal ischemia/reperfusion (IR) injury. Wild-type (WT) C57Bl/6 and OLFM4 null mice were subjected to intestinal IR injury and then monitored for survival or tissues harvested for further analyses. In vivo intestinal barrier function was determined via functional assay of permeability to FITC-dextran. OLFM4 null mice had a significant 7-d survival benefit and less intestinal barrier dysfunction compared with WT. Early after IR, WT mice had worse mucosal damage on histologic examination. Experiments involving adoptive transfer of bone marrow demonstrated that the mortality phenotype associated with OLFM4-positive neutrophils was transferrable to OLFM4 null mice. After IR injury, WT mice also had increased intestinal tissue activation of NFκB and expression of iNOS, 2 signaling pathways previously demonstrated to be involved in intestinal IR injury. In combination, these experiments show that OLFM4-positive neutrophils are centrally involved in the pathologic pathway leading to intestinal damage and mortality after IR injury. This may provide a therapeutic target for mitigation of intestinal IR injury in a variety of common clinical situations.-Levinsky, N. C., Mallela, J., Opoka, A., Harmon, K., Lewis, H. V., Zingarelli, B., Wong, H. R., Alder, M. N. The olfactomedin-4 positive neutrophil has a role in murine intestinal ischemia/reperfusion injury.

Accelerated cell turnover 48h after intestinal ischemia is NOTCH independent.

Notch signaling plays important roles in maintaining intestinal epithelial homeostasis. When Notch signaling is blocked, proliferation ceases and epithelial cells become secretory. The purpose of the present study was to evaluate the role of Notch signaling pathway following intestinal ischemia-reperfusion (IR) injury in a rat model. Male Sprague-Dawley rats were randomly divided into four experimental groups: Sham-24 and Sham-48 rats underwent laparotomy and were killed 24 or 48h later, respectively; IR-24 and IR-48 rats underwent occlusion of SMA and portal vein for 30min followed by 24 or 48h of reperfusion, respectively. Enterocyte proliferation and enterocyte apoptosis were determined at killing. Notch-related gene and protein expression were determined using Real Time PCR, Western blotting and immunohistochemistry 48h followed IR. IR-48 rats demonstrated significantly increased rates of cell proliferation and increased cell apoptosis in both jejunum and ileum compared to Sham rats. IR-48 rats exhibited a significant decrease in Notch-1 protein expression (Western blot) that was coincided with a significant decrease in the number of Notch-1 positive cells (immunohistochemistry) in jejunum (35% decrease, p < 0.05) and ileum (twofold decrease, p < 0.05) as well as Hes-1 positive cells in jejunum (28% decrease, p < 0.05) and ileum (31% decrease, p < 0.05) compared to Sham-48 rats. Forty-eight hours following intestinal IR in rats, accelerated cell turnover was associated by inhibited Notch signaling pathway. Intestinal stem cells differentiation toward secretory progenitors rather than differentiation toward absorptive cells is important at this phase of intestinal recovery.

The protective effects of urapidil on lung tissue after intestinal ischemia-reperfusion injury

Abstract Objective The effects of urapidil in intestinal ischemia-reperfusion (IR) model were investigated using histopathological and biochemical methods. Materials and methods Forty Wistar albino rats were subjected to sham operation (Group 1), IR (Group 2), IR+dimethyl sulfoxide (Group 3), IR+urapidil 0.5 mg/kg (Group 4), and IR+urapidil 5 mg/kg (Group 5). Levels of MDA, TAS, TOS, SOD, MPO, NF-κB, caspase-3, and LC3B were measured. Results and discussion The groups 2 and 3 had significantly higher TOS and MPO levels than the sham group had (p &lt; 0.001), whereas the TAS and SOD levels were significantly lower in Group 2 than in the sham group. In treatment groups, TAS and SOD levels increased, whereas TOS, MPO, and MDA levels decreased compared to Group 2. Caspase-3 and LC3B immunopositivities were seen at severe levels in Group 2 and 3. However, Group 4 and 5 were found to have lower levels of immunopositivity. Immunopositivity was observed in interstitial areas, peribronchial region, and bronchial epithelial cells. A moderate level of NF-κB immunopositivity was seen in Group 2 and 3. Conclusion Our results show that urapidil is one of the antioxidant agents and protects lung tissue from oxidant effects of intestinal IR injury.

Histopathology of human small intestinal and colonic ischemia-reperfusion: Experiences from human IR-models.

Intestinal ischemia-reperfusion (IR) injury is a frequent, but potentially life-threatening condition. Although much has been learned about its pathophysiology from animal IR models, the translation to the human setting is imperative for better understanding of its etiology. This could provide us with new insight into development of early detection and potential new therapeutic strategies. Over the past decade, we have studied the pathophysiology of human small intestinal and colonic ischemia-reperfusion (IR) in newly developed human in vivo IR models. In this review, we give an overview of new insights on the sequelae of human intestinal IR, with particular attention for the differences in histopathology between small intestinal and colonic IR.

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

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 48h following intestinal ischemia-reperfusion (IR) in a rat. Male rats were divided into four experimental groups: (1) Sham-24h rats underwent laparotomy and were sacrificed after 24h, (2) Sham-48h rats underwent laparotomy and were sacrificed after 48h, (3) IR-24h rats underwent occlusion of both superior mesenteric artery and portal vein for 20min followed by 24h of reperfusion, and (4) IR-48h rats underwent ischemia for 20min followed by 48h of reperfusion. Intestinal structural changes, enterocyte proliferation and enterocyte apoptosis were determined by immunohistochemistry 24 and 48h following IR. SHh-related genes and protein expression were determined using real-time PCR, Western blot and immunohistochemistry. 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 48h, 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). 24h following intestinal IR, inhibited cell turnover was associated with inhibited SHh signaling pathway. Signs of intestinal recovery appeared 48h after IR and were correlated with increase in SHh signaling pathway activity.

Effect of ambroxol on experimentally induced acute oxidative stress in the heart, kidney, and intestine in rats

Background Intestinal ischemia–reperfusion (IR) is a frequently occurring phenomenon during abdominal and thoracic vascular surgery, small bowel transplantation, hemorrhagic shock, and surgery using cardiopulmonary bypass, carrying high morbidity and mortality. Ambroxol hydrochloride is an active N-desmethyl metabolite of bromhexine hydrochloride. Ambroxol is indicated as ‘secretolytic therapy in bronchopulmonary diseases associated with abnormal mucus secretion and impaired mucus transport’. Aim The present study was designed to evaluate the effect of ambroxol on experimentally induced acute organ oxidative stress in the form of remote organ injury including kidney and heart after intestinal IR. Materials and methods Thirty animals were classified into five groups. First group: normal control group, second group: nontreated intestinal IR group (intestinal IR was induced by mesenteric artery ligation), third group: ambroxol pretreated intestinal IR group (35 mg/kg), fourth group: ambroxol pretreated intestinal IR group (70 mg/kg), fifth group: ambroxol pretreated intestinal IR group (140 mg/kg). Results Ambroxol significantly reduced the malondialdehyde level in the heart and the kidney when compared with intestinal IR with no medication group. It also improved cardiac troponin and, kidney functions (urea and creatinine) and histopathological affection when compared with intestinal IR with no medication group. Conclusively, in this study ambroxol could have a protective effect against intestinal IR through its antioxidative and anti-inflammatory effect.

Umbilical mesenchymal stromal cells provide intestinal protection through nitric oxide dependent pathways

BackgroundUmbilical-derived mesenchymal stromal cells (USCs) have shown promise in the protection of ischemic organs. We hypothesized that USCs would improve mesenteric perfusion, preserve intestinal histological architecture, and limit inflammation by nitric oxide–dependent mechanisms following intestinal ischemia/reperfusion (IR) injury. MethodsAdult wild-type C57BL/6J (WT) and endothelial nitric oxide synthase knock out (eNOS KO) mice were used: (1) WT IR + vehicle, (2) WT IR + USC, (3) eNOS KO IR + vehicle, and (4) eNOS KO IR + USC. Mice were anesthetized, and a midline laparotomy was performed. The superior mesenteric artery was clamped with a nonoccluding clamp for 60-min. Following IR, mice were treated with an injection of 250 μL phosphate buffered saline or 2 × 106 USCs suspended in 250-μL phosphate buffered saline solution. Mesenteric perfusion images were acquired using laser Doppler imaging. Perfusion was analyzed as a percentage of baseline. At 24 h, mice were euthanized, and intestines were harvested. Intestines were evaluated for injury, and data were analyzed using the Mann–Whitney or Kruskal–Wallis tests. ResultsIntestinal mesenteric perfusion was significantly improved in WT mice treated with USC therapy compared with eNOS KOs. Intestinal histological architecture was preserved with USC therapy in WT mice. However, in eNOS KO mice, this benefit was abolished. Finally, the presence of several cytokines and growth factors were significantly improved in WT mice compared with eNOS KO mice treated with USCs. ConclusionsThe benefits of USC-mediated therapy following intestinal IR injury likely occur via nitric oxide–dependent pathways. Further studies are required to define the molecular mechanisms by which USCs activate endothelial nitric oxide synthase to bring about their protective effects.

Down-regulation of toll-like receptor 4 alleviates intestinal ischemia reperfusion injury and acute lung injury in mice.

Intestinal ischemia reperfusion (IR) injury is a critical problem, which can cause intestinal injury locally and acute lung injury (ALI) distally by inflammatory responses and oxidative stress. Toll-like receptor 4 (TLR4) is involved in innate immune and inflammatory responses. This study was to determine whether TLR4 mutant can attenuate intestinal and lung injuries after intestinal IR. Wild type (WT) and TLR4 mutant mice were submitted to intestinal IR by occluding the superior mesenteric artery. Histological assessment of the intestine and the lung were conducted by HE staining. The levels of proinflammatory cytokines, oxidative stress markers, apoptotic index and other mediators were measured. In addition, a 24-hour survival study was performed. Histological assessment showed that intestinal IR caused serious injuries in the intestine and the lung, corroborated by increased proinflammatory cytokines in the circulation. TLR4 mutant suppressed the histological injuries as demonstrated by significantly decreased pathological scores. Consistent with the morphological results, the TLR4 mutant mice exhibited remarkably lowered cytokine expressions in the intestine (TNF-α, IL-6, IL-1β, and NF-κB) and the lung (NO, iNOS, MCP-1, MIP-2, NF-κB, and Caspase-3). ALT and creatinine were also significantly dampened in the liver and kidney, respectively. Furthermore, the survival rate over the course of 24 hours was significantly improved. Collectively, the findings reveal that TLR4 mutant significantly abated the intestinal IR injury and ALI at least in part by alleviating the inflammatory response and oxidative stress.