Yen-Zhen Lu

Anti-apoptotic PI3K/Akt signaling by sodium/glucose transporter 1 reduces epithelial barrier damage and bacterial translocation in intestinal ischemia

Intestinal ischemia/reperfusion (I/R) causes mucosal barrier damage and bacterial translocation (BT), leading to septic complications. Previous in vitro studies showed that activation of sodium/glucose transporter 1 (SGLT1) prevented the epithelial apoptosis and permeability rise induced by microbial products. Our aim was to investigate whether luminal glucose uptake by SGLT1 protects against ischemia-induced epithelial cell death and barrier dysfunction, and to explore the glucose-mediated cellular survival pathways in vivo. Rat jejunum was luminally instilled with either vehicle, a pancaspase inhibitor ZVAD, or glucose prior to I/R challenge (occlusion of the superior mesenteric artery for 20 min and reperfusion for 60 min). Histopathology and apoptosis in the jejunum were examined by TUNEL staining and caspase-3 cleavage. Intestinal permeability was evaluated using in vivo assays measuring luminal-to-blood passage of fluorescein-dextran and portal drainage of enterally administered gadodiamide by magnetic resonance imaging. BT was determined by culturing liver and spleen homogenates. Immunofluorescent analysis and kinase assay were used to study PI3K/Akt signaling pathways. Intestinal I/R caused enterocyte apoptosis and villous destruction. Intestinal infusion with ZVAD decreased the I/R-triggered gut permeability rise and BT, suggesting that the barrier damage was partly dependent on cell apoptosis. Enteral instillation of glucose attenuated the epithelial apoptosis, barrier damage, and mucosal inflammation caused by I/R. Phloridzin (a SGLT1 inhibitor) reduced the protective effect of glucose in a dose-dependent manner. Enteral glucose increased the mucosal Akt kinase activity as evidenced by the augmented phosphorylation of exogenous GSK3. Enhanced membrane translocation and phosphorylation of Akt in epithelial cells were associated with elevated phosphorylation of mTOR, Bad, and FoxO1/3a following glucose uptake. Inhibition of PI3K/Akt signaling by LY294002 and wortmannin partially blocked the glucose-mediated rescue of cell apoptosis and barrier damage. In conclusion, SGLT1 glucose uptake alleviated I/R-induced barrier dysfunction and BT, partly by inhibiting epithelial apoptosis via activation of PI3K/Akt signaling.

Persistent gut barrier damage and commensal bacterial influx following eradication of Giardia infection in mice

BackgroundRecent studies of Giardia lamblia outbreaks have indicated that 40–80% of infected patients experience long-lasting functional gastrointestinal disorders after parasitic clearance. Our aim was to assess changes in the intestinal barrier and spatial distribution of commensal bacteria in the post-clearance phase of Giardia infection.MethodsMice were orogastrically inoculated with G. lamblia trophozoites (strain GS/M) or pair-fed with saline and were sacrificed on post-infective (PI) days 7 (colonization phase) and 35 (post-clearance phase). Gut epithelial barrier function was assessed by Western blotting for occludin cleavage and luminal-to-serosal macromolecular permeability. Gut-associated, superficial adherent, and mucosal endocytosed bacteria were measured by agar culturing and were examined by fluorescence in situ hybridization. Intracellular bacteria cultured from isolated mucosal cells were characterized by 16S rDNA sequencing. Neutrophil-specific esterase staining, a myeloperoxidase activity assay, and enzyme-linked immunosorbent assays for cytokine concentrations were used to verify intestinal tissue inflammation.ResultsTight junctional damage was detected in the intestinal mucosa of Giardia-infected mice on PI days 7 and 35. Although intestinal bacterial overgrowth was evident only during parasite colonization (PI day 7), enhanced mucosal adherence and endocytosis of bacteria were observed on PI days 7 and 35. Multiple bacterial strains, including Bacillus, Lactobacillus, Staphylococcus, and Phenylobacterium, penetrated the gut mucosa in the post-infective phase. The mucosal influx of bacteria coincided with increases in neutrophil infiltration and myeloperoxidase activity on PI days 7 and 35. Elevated intestinal IFNγ, TNFα, and IL-1β levels also were detected on PI day 35.ConclusionsGiardia-infected mice showed persistent tight junctional damage and bacterial penetration, accompanied by mucosal inflammation, after parasite clearance. These novel findings suggest that the host’s unresolved immune reactions toward its own microbiota, due to an impaired epithelial barrier, may partly contribute to the development of post-infective gut disorders.

Epithelial inducible nitric oxide synthase causes bacterial translocation by impairment of enterocytic tight junctions via intracellular signals of Rho-associated kinase and protein kinase C zeta.

Objective:Gut barrier dysfunction and bacterial translocation occur in various disorders, including intestinal obstruction. Overexpression of inducible nitric oxide synthase is implicated in the pathogenesis of bacterial translocation, of which the molecular mechanism remains unclear. Epithelial permeability is regulated by tight junction reorganization and myosin light chain phosphorylation. Our aim was to investigate the roles of Rho-associated kinase and protein kinase C &zgr; in epithelial nitric oxide synthase-mediated barrier damage. Design:Animal study and cell cultures. Setting:Research laboratory. Subjects:BALB/c mice. Interventions:Mouse distal small intestine was obstructed in vivo by a 10-cm loop ligation in which vehicle, L-Nil (a nitric oxide synthase inhibitor), or Y27632 (a Rho-associated kinase inhibitor) was luminally administered. After obstruction for 24 hrs, intestinal tissues were mounted on Ussing chambers for macromolecular flux. Liver and spleen tissues were assessed for bacterial counts. Caco-2 cells were exposed to 1 mM S-nitroso-N-acetylpenicillamine (a nitric oxide donor) for 24 hrs, and transepithelial resistance and permeability were evaluated. Measurements and Main Results:Mice with intestinal obstruction displayed epithelial barrier dysfunctions, such as permeability rise and bacterial translocation, associated with tight junction disruption and myosin light chain phosphorylation. Increased inducible nitric oxide synthase and phosphorylated protein kinase C &zgr; were observed in villus epithelium. Enteric instillation of L-Nil and Y27632 attenuated the functional and structural barrier damage caused by intestinal obstruction. L-Nil decreased intestinal obstruction-induced myosin light chain, myosin phosphatase target subunit 1, and protein kinase C &zgr; phosphorylation, suggesting that inducible nitric oxide synthase is upstream of Rho-associated kinase and protein kinase C &zgr; signaling. The intestinal phosphorylated myosin light chain level did not increase in inducible nitric oxide synthase(−/−) mice following intestinal obstruction. In vitro studies showed that S-nitroso-N-acetylpenicillamine-induced transepithelial resistance drop and permeability rise was independent of cell apoptosis. Y27632 inhibited S-nitroso-N-acetylpenicillamine-induced myosin light chain phosphorylation and permeability rise. S-nitroso-N-acetylpenicillamine also triggered phosphorylation and membrane translocation of protein kinase C &zgr;. Inhibitory protein kinase C &zgr; pseudosubstrate blocked S-nitroso-N-acetylpenicillamine-induced tight junction reorganization, but not myosin light chain phosphorylation. Conclusions:Epithelial inducible nitric oxide synthase activates two distinct signals, protein kinase C &zgr; and Rho-associated kinase, to disrupt tight junctions leading to bacterial influx.

Neutrophil priming by hypoxic preconditioning protects against epithelial barrier damage and enteric bacterial translocation in intestinal ischemia/reperfusion

Intestinal ischemia/reperfusion (I/R) induces mucosal barrier dysfunction and bacterial translocation (BT). Neutrophil-derived oxidative free radicals have been incriminated in the pathogenesis of ischemic injury in various organs, but their role in the bacteria-containing intestinal tract is debatable. Primed neutrophils are characterized by a faster and higher respiratory burst activity associated with more robust bactericidal effects on exposure to a second stimulus. Hypoxic preconditioning (HPC) attenuates ischemic injury in brain, heart, lung and kidney; no reports were found in the gut. Our aim is to investigate whether neutrophil priming by HPC protects against intestinal I/R-induced barrier damage and bacterial influx. Rats were raised in normoxia (NM) or kept in a hypobaric hypoxic chamber (380 Torr) 17 h/day for 3 weeks for HPC, followed by sham operation or intestinal I/R. Gut permeability was determined by using an ex vivo macromolecular flux assay and an in vivo magnetic resonance imaging-based method. Liver and spleen homogenates were plated for bacterial culturing. Rats raised in HPC showed diminished levels of BT, and partially improved mucosal histopathology and epithelial barrier function compared with the NM groups after intestinal I/R. Augmented cytokine-induced neutrophil chemoattractant (CINC)-1 and -3 levels and myeloperoxidase activity correlated with enhanced infiltration of neutrophils in intestines of HPC-I/R compared with NM-I/R rats. HPC alone caused blood neutrophil priming, as shown by elevated production of superoxide and hydrogen peroxide on stimulation, increased membrane translocation of cytosolic p47phox and p67phox, as well as augmented bacterial-killing and phagocytotic activities. Neutrophil depletion reversed the mucosal protection by HPC, and aggravated intestinal leakiness and BT following I/R. In conclusion, neutrophil priming by HPC protects against I/R-induced BT via direct antimicrobial activity by oxidative respiratory bursts and through promotion of epithelial barrier integrity for luminal confinement of enteric bacteria.

Role of myosin light chain kinase in intestinal epithelial barrier defects in a rat model of bowel obstruction

BackgroundBowel obstruction is a common cause of abdominal emergency, since the patients are at increased risk of septicemia resulting in high mortality rate. While the compartmentalized changes in enteric microfloral population and augmentation of bacterial translocation (BT) have already been reported using experimental obstruction models, alterations in epithelial permeability of the obstructed guts has not been studied in detail. Myosin light chain kinase (MLCK) is actively involved in the contraction of epithelial perijunctional actinomyosin ring and thereby increases paracellular permeability. In the current study we attempt to investigate the role of MLCK in epithelial barrier defects using a rat model of simple mechanical obstruction.MethodsWistar rats received intraperitoneal injection of ML-7 (a MLCK inhibitor) or vehicle at 24, 12 and 1 hrs before and 12 hrs after intestinal obstruction (IO). The distal small intestine was obstructed with a single ligature placed 10 cm proximal to the ileocecal junction in IO rats for 24 hrs. Sham-operated rats served as controls.ResultsMucosal injury, such as villous blunting and increased crypt/villus ratio, was observed in the distal small intestine of IO rats. Despite massive enterocyte shedding, intestinal villi were covered with a contiguous epithelial layer without cell apoptosis. Increased transmural macromolecular flux was noticed in the distal small intestine and the proximal colon after IO. The bacterial colony forming units in the spleen and liver of IO rats were significantly higher than those of sham controls. Addition of ML-7 ameliorated the IO-triggered epithelial MLC phosphorylation, mucosal injury and macromolecular flux, but not the level of BT.ConclusionsThe results suggest that IO-induced premature enterocytic sloughing and enhanced paracellular antigenic flux were mediated by epithelial MLCK activation. In addition, enteric bacteria may undergo transcytotic routes other than paracellular paths to cross the epithelium.

Magnetic Resonance Imaging Detects Intestinal Barrier Dysfunction in a Rat Model of Acute Mesenteric Ischemia/Reperfusion Injury

Objectives:To develop an in vivo intestinal permeability assay applying magnetic resonance imaging (MRI) to monitor real-time gut barrier defects in animal models of acute mesenteric ischemia/reperfusion (I/R) insult. Materials and Methods:Twenty Wistar rats were divided to 2 groups for I/R challenge or sham controls. I/R rats received occlusion of superior mesenteric artery for 20 minutes and reperfusion for 1 hour. Sham-operation controls received laparotomy without manipulation of artery. To assess gut permeability, a 10-cm jejunal sac was created distal to the ligament of Treitz in both groups of rats after laparotomy, and a contrast agent (gadodiamide) was injected into the lumen of the ligated intestinal sac. The signals produced by gadodamide in the liver, kidney, and plasma before and after the start of reperfusion were examined by 1.5 Tesla MRI (GE Signa Excite), and the increment of signal-to-noise ratio (SNR) in these organs that parallels the luminal-to-serosal flux rate of the probe was used as an indicator of gut permeability. At the end of procedures, jejunal tissues and mucosal scrapings were collected for histologic examination and Western blotting for epithelial tight junctional proteins. Moreover, liver and spleen homogenates were cultured on fresh blood agar plates to measure the bacterial colony-forming units per gram of tissue. Results:In I/R rats, disrupted villous structure and decreased epithelial tight junctional expression were seen in the jejunum associated with massive enteric bacterial translocation to the liver and spleen. The SNR in the liver of I/R rats was higher than sham controls (2.65 ± 0.56 vs. 0.65 ± 0.26, P < 0.01) at 15 minutes postreperfusion. Elevation of SNR in the kidney was also found in I/R rats compared with sham controls (11.61 ± 2.07 vs. 3.06 ± 1.15, P < 0.05). The plasma gadodiamide concentration in I/R rats was significantly increased compared with sham controls (0.220 ± 0.044 vs. 0.006 ± 0.004 mM, P < 0.01) at 15 minutes postreperfusion. Conclusions:This novel MRI-based intestinal permeability assay has shown a significant increase in the signal intensity in liver, kidney, and plasma samples that correlated with mucosal barrier defects in experimental models of acute mesenteric I/R.

Hypoxia‐induced intestinal barrier changes in balloon‐assisted enteroscopy

Balloon‐assisted enteroscopy (BAE) is an emerging standard procedure by utilizing distensible balloons to facilitate deep endoscopy in the small and large intestine. Sporadic cases of bacteraemia were found after BAE. Balloon distension by BAE caused gut tissue hypoxia. The impact of balloon distension‐induced hypoxia on intestinal barriers remains unclear. Murine models of BAE by colonic balloon distension showed that short‐ and long‐term hypoxia evoked opposite effects on epithelial tight junctions (TJs). Short‐term hypoxia fortified TJ integrity, whereas long‐term hypoxia caused damage to barrier function. Our data showed for the first time the molecular mechanisms and signalling pathways of epithelial barrier fortification and TJ reorganization by short‐term hypoxia for the maintenance of gut homeostasis. The findings suggest avoiding prolonged balloon distension during BAE to reduce the risk of hypoxia‐induced gut barrier dysfunction.