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Evaluation of gut-blood barrier dysfunction in various models of trauma, hemorrhagic shock, and burn injury. J Trauma Acute Care Surg 2017; 83:944-953. [DOI: 10.1097/ta.0000000000001654] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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Cheng Y, Wei Y, Yang W, Cai Y, Chen B, Yang G, Shang H, Zhao W. Ghrelin Attenuates Intestinal Barrier Dysfunction Following Intracerebral Hemorrhage in Mice. Int J Mol Sci 2016; 17:ijms17122032. [PMID: 27929421 PMCID: PMC5187832 DOI: 10.3390/ijms17122032] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2016] [Revised: 11/10/2016] [Accepted: 11/28/2016] [Indexed: 02/07/2023] Open
Abstract
Intestinal barrier dysfunction remains a critical problem in patients with intracerebral hemorrhage (ICH) and is associated with poor prognosis. Ghrelin, a brain-gut peptide, has been shown to exert protection in animal models of gastrointestinal injury. However, the effect of ghrelin on intestinal barrier dysfunction post-ICH and its possible underlying mechanisms are still unknown. This study was designed to investigate whether ghrelin administration attenuates intestinal barrier dysfunction in experimental ICH using an intrastriatal autologous blood infusion mouse model. Our data showed that treatment with ghrelin markedly attenuated intestinal mucosal injury at both histomorphometric and ultrastructural levels post-ICH. Ghrelin reduced ICH-induced intestinal permeability according to fluorescein isothiocyanate conjugated-dextran (FITC-D) and Evans blue extravasation assays. Concomitantly, the intestinal tight junction-related protein markers, Zonula occludens-1 (ZO-1) and claudin-5 were upregulated by ghrelin post-ICH. Additionally, ghrelin reduced intestinal intercellular adhesion molecule-1 (ICAM-1) expression at the mRNA and protein levels following ICH. Furthermore, ghrelin suppressed the translocation of intestinal endotoxin post-ICH. These changes were accompanied by improved survival rates and an attenuation of body weight loss post-ICH. In conclusion, our results suggest that ghrelin reduced intestinal barrier dysfunction, thereby reducing mortality and weight loss, indicating that ghrelin is a potential therapeutic agent in ICH-induced intestinal barrier dysfunction therapy.
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Affiliation(s)
- Yijun Cheng
- Department of Neurosurgery, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
| | - Yongxu Wei
- Department of Neurosurgery, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
| | - Wenlei Yang
- Department of Neurosurgery, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
| | - Yu Cai
- Department of Neurosurgery, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
| | - Bin Chen
- Department of Neurosurgery, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
| | - Guoyuan Yang
- Department of Neurology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
- Neuroscience and Neuroengineering Research Center, Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, China.
| | - Hanbing Shang
- Department of Neurosurgery, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
| | - Weiguo Zhao
- Department of Neurosurgery, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
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Rae L, Fidler P, Gibran N. The Physiologic Basis of Burn Shock and the Need for Aggressive Fluid Resuscitation. Crit Care Clin 2016; 32:491-505. [PMID: 27600122 DOI: 10.1016/j.ccc.2016.06.001] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Burn trauma in the current age of medical care still portends a 3% to 8% mortality. Of patients who die from their burn injuries, 58% of deaths occur in the first 72 hours after injury, indicating death from the initial burn shock is still a major cause of burn mortality. Significant thermal injury incites an inflammatory response, which distinguishes burns from other trauma. This article focuses on the current understanding of the pathophysiology of burn shock, the inflammatory response, and the direction of research and targeted therapies to improve resuscitation, morbidity, and mortality.
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Affiliation(s)
- Lisa Rae
- Department of Trauma, Surgical Critical Care and Emergency General Surgery, Vanderbilt University Medical Center, 1211 21st Avenue South, MAB 404, Nashville, TN 37212, USA.
| | - Philip Fidler
- Swedish Hospital, 601 E. Hampden Avenue, Englewood, CO 80113, USA
| | - Nicole Gibran
- UW Burn Center, 325 9th Avenue, Seattle, WA 98104, USA
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Singh BK, Kumar V, Chauhan AK, Dwivedi A, Singh S, Kumar A, Singh D, Patel DK, Ray RS, Jain SK, Singh C. Neuronal Nitric Oxide Synthase Negatively Regulates Zinc-Induced Nigrostriatal Dopaminergic Neurodegeneration. Mol Neurobiol 2016; 54:2685-2696. [PMID: 26995406 DOI: 10.1007/s12035-016-9857-7] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2016] [Accepted: 03/11/2016] [Indexed: 12/21/2022]
Abstract
The study aimed to investigate the role of NO and neuronal NO synthase (nNOS) in Zn-induced neurodegeneration. Animals were treated with zinc sulfate (20 mg/kg), twice a week, for 2-12 weeks along with control. In a few sets, animals were also treated with/without a NO donor, sodium nitroprusside (SNP), or S-nitroso-N-acetyl penicillamine (SNAP) for 12 weeks. Moreover, human neuroblastoma (SH-SY-5Y) cells were also employed to investigate the role of nNOS in Zn-induced toxicity in in vitro in the presence/absence of nNOS inhibitor, 7-nitroindazole (7-NI). Zn caused time-dependent reduction in nitrite content and total/nNOS activity/expression. SNP/SNAP discernibly alleviated Zn-induced neurobehavioral impairments, dopaminergic neurodegeneration, tyrosine hydroxylase (TH) expression, and striatal dopamine depletion. NO donors also salvage from Zn-induced increase in lipid peroxidation (LPO), mitochondrial cytochrome c release, and caspase-3 activation. While Zn elevated LPO content, it attenuated nitrite content, nNOS activity, and glutathione level along with the expression of TH and nNOS in SH-SY-5Y cells. 7-NI further augmented Zn-induced changes in the cell viability, oxidative stress, and expression of TH and nNOS. The results obtained thus demonstrate that Zn inhibits nNOS that partially contributes to an increase in oxidative stress, which subsequently leads to the nigrostriatal dopaminergic neurodegeneration.
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Affiliation(s)
- Brajesh Kumar Singh
- Developmental Toxicology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226 001, Uttar Pradesh, India
| | - Vinod Kumar
- Developmental Toxicology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226 001, Uttar Pradesh, India
- Academy of Scientific and Innovative Research, CSIR-IITR Campus, Lucknow, 226 001, Uttar Pradesh, India
| | - Amit Kumar Chauhan
- Developmental Toxicology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226 001, Uttar Pradesh, India
- Academy of Scientific and Innovative Research, CSIR-IITR Campus, Lucknow, 226 001, Uttar Pradesh, India
| | - Ashish Dwivedi
- Phototoxicology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR-IITR, Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226 001, Uttar Pradesh, India
| | - Shweta Singh
- Developmental Toxicology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226 001, Uttar Pradesh, India
| | - Ashutosh Kumar
- Developmental Toxicology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226 001, Uttar Pradesh, India
| | - Deepali Singh
- Developmental Toxicology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226 001, Uttar Pradesh, India
- Academy of Scientific and Innovative Research, CSIR-IITR Campus, Lucknow, 226 001, Uttar Pradesh, India
| | - Devendra Kumar Patel
- Analytical Chemistry Laboratory, Regulatory Toxicology Group, CSIR-IITR, Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226 001, Uttar Pradesh, India
| | - Ratan Singh Ray
- Phototoxicology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR-IITR, Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226 001, Uttar Pradesh, India
| | - Swatantra Kumar Jain
- Department of Biotechnology, Jamia Hamdard Deemed University, New Delhi, 110 062, Delhi, India
| | - Chetna Singh
- Developmental Toxicology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226 001, Uttar Pradesh, India.
- Academy of Scientific and Innovative Research, CSIR-IITR Campus, Lucknow, 226 001, Uttar Pradesh, India.
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Pan H, Chen D, Liu B, Xie X, Zhang J, Yang G. Effects of sodium hydrosulfide on intestinal mucosal injury in a rat model of cardiac arrest and cardiopulmonary resuscitation. Life Sci 2013; 93:24-29. [PMID: 23727354 DOI: 10.1016/j.lfs.2013.05.012] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2013] [Revised: 04/19/2013] [Accepted: 05/15/2013] [Indexed: 12/11/2022]
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Wu QJ, Zhou YM, Wu YN, Zhang LL, Wang T. The effects of natural and modified clinoptilolite on intestinal barrier function and immune response to LPS in broiler chickens. Vet Immunol Immunopathol 2013; 153:70-6. [PMID: 23453767 DOI: 10.1016/j.vetimm.2013.02.006] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2012] [Revised: 01/30/2013] [Accepted: 02/07/2013] [Indexed: 12/13/2022]
Abstract
The protection of intestinal barrier function and the anti-inflammatory effects of natural clinoptilolite (NCLI) and modified clinoptilolite (MCLI) were investigated in broilers that were repeatedly challenged with lipopolysaccharide (LPS). A total of 288 1-d-old broiler chicks were divided equally into three treatment groups: control, NCLI-treated (2%) and MCLI-treated (2%). Half of the birds from each treatment group were challenged with 0.9% NaCl solution or LPS (250μg/kg body weight, administered orally) at 16, 18 and 21d of age. The results indicated that, prior to LPS challenge, the diet had no effect on bird growth performance (P>0.05). The oral administration of LPS was also not associated with any significant changes in poultry performance (P>0.05). In LPS-challenged birds that were pretreated with NCLI (2%) or MCLI (2%), the LPS-induced increases in the plasma and intestinal mucosa concentrations of TNF-α, IL-1β, IL-2, IL-6, IL-4 and IL-10 were dramatically attenuated. Additionally, significant decreases in the plasma d-lactic acid and diamine oxidase (DAO) levels were found in birds that were pretreated with NCLI or MCLI. Furthermore, both NCLI and MCLI reduced the sICAM-1 concentration in the intestinal mucosa. In conclusion, NCLI and MCLI are able to prevent the LPS-induced intestinal mucosa damage and inflammatory response in vivo. These beneficial effects suggest that NCLI and MCLI act as anti-inflammatory agents in part by inhibiting neutrophil infiltration and hyperactivation and by suppressing the secretion of various plasma and intestinal mucosa inflammatory mediators.
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Affiliation(s)
- Qiu Jue Wu
- College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, Henan, PR China
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Oppeltz RF, Rani M, Zhang Q, Schwacha MG. Gamma delta (γδ) T-cells are critical in the up-regulation of inducible nitric oxide synthase at the burn wound site. Cytokine 2012; 60:528-34. [PMID: 22831879 DOI: 10.1016/j.cyto.2012.07.003] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2012] [Accepted: 07/02/2012] [Indexed: 11/16/2022]
Abstract
BACKGROUND The high incidence of morbidity and mortality following major burn can in part be attributed to immune derangements and wound healing complications. Inflammation plays an important role in wound healing, of which inducible nitric oxide synthase (iNOS) derived nitric oxide is a central mediator. T-cells of the γδ TCR lineage have also been shown to be important in healing of the burn wound site. Nonetheless, the role of γδ T-cells in the regulation of the burn wound iNOS expression is unknown. METHODS Wildtype (WT) and δ TCR(-/-) male C57BL/6 mice were subjected to burn (3rd degree, 12.5% TBSA) or sham treatment. Three days after injury, skin samples from non-injured and the burn wound were collected and analyzed for the expression of iNOS and cytokines and chemokine levels. In a second series of experiments, WT mice were subjected to burn and left untreated or treated with the iNOS inhibitor, L-Nil. Skin cytokine and chemokine levels were assessed 3days thereafter. RESULTS Burn induced an 18-fold increase in iNOS expression at the wound site as compared to the uninjured skin of WT sham mice. In δ TCR(-/-) mice iNOS expression at the wound site was significantly lower than that of the WT group. Burn also induced increased levels of IL-1β, IL-6, G-CSF, TNF-α, KC, MCP-1, MIP-1α and MIP-1β at the wound site in WT and δ TCR(-/-) mice, but G-CSF, TNF-α, and MIP-1β levels were greater in δ TCR(-/-) mice. Inhibition of iNOS activity in WT mice with L-Nil suppressed burn wound levels of IL-1β, G-CSF, and MIP-1α, whereas IL-6, TNF-α, KC, MCP-1 and MIP-1β were unaffected. CONCLUSIONS T-cells of the γδ TCR lineage significantly contribute to the up-regulation of iNOS expression which contributes to wound inflammation.
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Affiliation(s)
- Richard F Oppeltz
- Department of Surgery, The University of Texas Health Science Center, San Antonio, TX 78229, USA.
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Diao L, Mei Q, Xu JM, Liu XC, Hu J, Jin J, Yao Q, Chen ML. Rebamipide suppresses diclofenac-induced intestinal permeability via mitochondrial protection in mice. World J Gastroenterol 2012; 18:1059-66. [PMID: 22416180 PMCID: PMC3296979 DOI: 10.3748/wjg.v18.i10.1059] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/02/2011] [Revised: 06/22/2011] [Accepted: 07/08/2011] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the protective effect and mechanism of rebamipide on small intestinal permeability induced by diclofenac in mice.
METHODS: Diclofenac (2.5 mg/kg) was administered once daily for 3 d orally. A control group received the vehicle by gavage. Rebamipide (100 mg/kg, 200 mg/kg, 400 mg/kg) was administered intragastrically once a day for 3 d 4 h after diclofenac administration. Intestinal permeability was evaluated by Evans blue and the FITC-dextran method. The ultrastructure of the mucosal barrier was evaluated by transmission electron microscopy (TEM). Mitochondrial function including mitochondrial swelling, mitochondrial membrane potential, mitochondrial nicotinamide adenine dinucleotide-reduced (NADH) levels, succinate dehydrogenase (SDH) and ATPase activities were measured. Small intestinal mucosa was collected for assessment of malondialdehyde (MDA) content and myeloperoxidase (MPO) activity.
RESULTS: Compared with the control group, intestinal permeability was significantly increased in the diclofenac group, which was accompanied by broken tight junctions, and significant increases in MDA content and MPO activity. Rebamipide significantly reduced intestinal permeability, improved inter-cellular tight junctions, and was associated with decreases in intestinal MDA content and MPO activity. At the mitochondrial level, rebamipide increased SDH and ATPase activities, NADH level and decreased mitochondrial swelling.
CONCLUSION: Increased intestinal permeability induced by diclofenac can be attenuated by rebamipide, which partially contributed to the protection of mitochondrial function.
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Higashimori H, Whetzel TP, Carlsen RC. Inhibition of inducible nitric oxide synthase reduces an acute peripheral motor neuropathy produced by dermal burn injury in mice. J Peripher Nerv Syst 2009; 13:289-98. [PMID: 19192069 DOI: 10.1111/j.1529-8027.2008.00195.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
The systemic inflammatory response produced by a full-thickness dermal burn injury is associated with a peripheral motor neuropathy. We previously reported that a 20% body surface area (BSA) full-thickness dermal burn in C57BL6 mice produced structural and functional deficits in motor axons at a distance from the burn site. The etiology of the neuropathy, however, is not well characterized. Burn injury leads to an increase in production of a number of proinflammatory mediators, including nitric oxide (NO). We tested the hypothesis that dermal burn-induced motor neuropathy is mediated by increased production of NO. NO synthase (NOS) activity was inhibited following a 20% BSA full-thickness burn by injection of non-specific NOS inhibitor, nitro-L-arginine methyl ester or inducible NOS (iNOS) inhibitors, L-N6-(1-iminoethyl) lysine, and aminoguanidine. NOS inhibitors also prevented the reduction in ventral roots mean axon caliber and the decrease in a motor nerve conduction velocity (MCV) following burn. iNOS knockout mice prevented MCV decrease in the first 3 days post-burn, but iNOS knockout MCV was significantly reduced at 7-14 days post-burn. These results suggest that an increase in NO production generated by systemic inflammatory response pathways after burn injury contributes to the development of structural and functional deficits in peripheral motor axons.
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Affiliation(s)
- Haruki Higashimori
- Department of Neurobiology and Center for Glial Biology in Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
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TLR ligand decreases mesenteric ischemia and reperfusion injury-induced gut damage through TNF-alpha signaling. Shock 2009; 30:563-70. [PMID: 18317407 DOI: 10.1097/shk.0b013e31816a3458] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Ischemic gut contributes to the development of sepsis and organ failure in critically ill patients. Toll-like receptors (TLRs) have been reported to mediate the pathophysiology of organ damage following ischemia/reperfusion (I/R) injury. We hypothesize that LPS, a ligand for TLR4, decreases mesenteric I/R injury-induced gut damage through tumor necrosis factor alpha (TNF-alpha) signaling. First, wild-type (WT) mice were fed with oral antibiotics for 4 weeks to deplete the intestinal commensal microflora. At week 3, drinking water was supplemented with LPS (10 microg/microL) to trigger TLRs. The intestinal mucosa was harvested for TLR4 protein, caspase 3 activity, and terminal deoxynucleotide transferase labeling assay. Second, WT and Tnfrsf1a mice received 30-min ischemia and 30-min reperfusion (30I-30R) or 30I-180R of the intestine; intestinal permeability and lipid peroxidation of the intestine were examined. Third, WT and Tnfrsf1a mice were fed with oral antibiotics with or without LPS and received 30I-180R of the intestine. The intestinal mucosa was harvested for lipid peroxidation; glutathione (GSH) level; nuclear factor kappaB (NF-kappaB) and AP-1 DNA-binding activity; Bcl-w, TNF-alpha, and CXCR2 mRNA expression; and HSP70 protein assay. Commensal depletion increased caspase 3 activity as well as villi apoptosis and decreased TLR4 expression of the intestinal mucosa. LPS increased TLR4 expression and decreased villi apoptosis. Commensal depletion augmented 30I-180R-induced intestine permeability as well as lipid peroxidation and decreased GSH level in WT mice but not in Tnfrsf1a mice. LPS decreased 30I-180R-induced intestinal permeability as well as lipid peroxidation and increased GSH level of the intestinal mucosa in WT mice but not in Tnfrsf1a mice. Commensal depletion with 30I-180R increased NF-kappaB and AP-1 DNA-binding activity, HSP70 protein expression, and decreased Bcl-w and TNF-alpha mRNA expression of the intestinal mucosa in WT mice but not in Tnfrsf1a mice. Collectively, commensal microflora induces TLR4 expression and decreases apoptosis of the intestinal mucosa. Commensal depletion enhances I/R-induced gut damage. LPS prevents I/R-induced intestinal permeability, lipid peroxidation, and decrease in GSH level. Given that the preventive effect of LPS on I/R-induced gut damage and NF-kappaB activity of the intestine is abolished in Tnfrsf1a mice, we conclude that TLR ligand decreases mesenteric I/R injury-induced gut damage through TNF-alpha signaling.
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Gammadelta T-cells: potential regulators of the post-burn inflammatory response. Burns 2008; 35:318-26. [PMID: 18951718 DOI: 10.1016/j.burns.2008.08.002] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2008] [Accepted: 08/12/2008] [Indexed: 02/02/2023]
Abstract
Severe burn induces an immunopathological response that contributes to the development of a systemic inflammatory response (SIRS) and subsequent multiple organ failure. While, multiple immune cells type (T-cells, macrophages, neutrophils) are involved in this response, recent evidence suggests that a unique T-cell subset, gammadelta T-cells are central in the response to injury. While gammadelta T-cells represent only a small percentage of the total T-cell population, they display specific functional characteristics that uniquely position them in the immune/inflammatory axis to influence a number of important aspects of the body's response to burn. This review will focus on the potential regulator role of gammadelta T-cells in immunopathological response following burn and thereby their potential as therapeutic targets for affecting inflammation and healing.
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Chen LW, Chen PH, Chang WJ, Wang JS, Karin M, Hsu CM. IκB-kinase/nuclear factor-κB signaling prevents thermal injury–induced gut damage by inhibiting c-Jun NH2-terminal kinase activation*. Crit Care Med 2007; 35:1332-40. [PMID: 17414734 DOI: 10.1097/01.ccm.0000261891.30360.f0] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
OBJECTIVE The molecular mechanism of major burn-induced gut damage is not clear. This study is to determine whether IkappaB-kinase (IKK)/nuclear factor-kappaB signaling in intestinal mucosa maintains gut function through the regulation of the c-Jun NH2-terminal kinase (JNK) and p38 phosphorylation. DESIGN Prospective, experimental study. SETTING Research laboratory at a university hospital. SUBJECTS Thermal injury models in mice. INTERVENTIONS Conditional intestinal epithelial cell IKKbeta knockout (Vil-Cre/Ikkbeta(F/Delta) mice and control (Ikkbeta(F/Delta) mice were subjected to 30% total body surface area third-degree burn. JNK inhibitor (SP600125) or p38 inhibitor (SB203580) was given to mice immediately after burn injury. MEASUREMENTS AND MAIN RESULTS Thermal injury induced a significant increase of intestinal permeability, nuclear factor-kappaB DNA-binding activity, phosphorylated JNK, phosphorylated p38, and caspase 3 expression of intestinal mucosa in Vil-Cre/Ikkbeta(F/Delta) mice compared with those of Ikkbeta(F/Delta) mice. BCL-xL and cellular FLICE inhibitory protein, but not GADD45beta (growth arrest and DNA damage-inducing protein beta), cellular inhibitor of apoptosis 1, Bfl-1, or TRAIL, messenger RNA expression was significantly decreased in Vil-Cre/Ikkbeta(F/Delta) mice compared with that of Ikkbeta(F/Delta) mice. SP600125 decreased intestinal permeability and increased phosphorylated p38 and tumor necrosis factor receptor-associated factor 2 expression of intestinal mucosa in Vil-Cre/Ikkbeta(F/Delta) mice. SB203580 treatment enhanced thermal injury-induced gut damage in Vil-Cre/Ikkbeta(F/Delta) mice. CONCLUSIONS Thermal injury induces nuclear factor-kappaB activation of intestinal mucosa and IKK protects intestinal mucosa from thermal injury-induced gut damage. IKK blocks caspase 3 expression by up-regulating BCL-xL and cellular FLICE inhibitory protein expression. IKK inhibits JNK and p38 but not p44/42 phosphorylation of intestinal mucosa. JNK inhibition increases p38 and tumor necrosis factor receptor-associated factor 2 expression and decreases thermal injury-induced gut damage. Taken together with the enhanced thermal injury-induced gut damage by p38 inhibition, we conclude that IKK maintains gut function by inhibiting JNK phosphorylation, which suppresses p38 phosphorylation and induces gut damage.
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Affiliation(s)
- Lee-Wei Chen
- Department of Surgery, Kaohsiung Veterans General Hospital, National Yang-Ming Medical University, Taipei, Taiwan.
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Al-Qattan MM. 'Late' multiorgan failure in major burns: a "three-event" construct rather than a "two-event" construct. Burns 2007; 33:268-70. [PMID: 17239541 DOI: 10.1016/j.burns.2006.07.008] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2006] [Accepted: 07/28/2006] [Indexed: 10/23/2022]
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Abstract
The systemic pathophysiologic changes following thermal injuries affect multiple organs and body systems leading to clinical manifestations including shock, intestinal alterations, respiratory and renal failure, immunosuppression and others. Recent advances in the comprehension of mechanisms underlying systemic complications of thermal injuries have contributed to uncover part of the cellular and molecular basis that underlie such changes. Recently, programmed cell death (apoptosis) has been considered playing an important role in the development of such pathological events. Therefore, investigators utilizing animal models and clinical studies involving human primates have produced a large body of information suggesting that apoptosis is associated with most of the tissue damages triggered by severe thermal injuries. In order to draw the attention on the important role of apoptosis on systemic complications of thermal injuries, in this review we describe most of these studies, discuss possible cellular and molecular mechanisms and indicate ways to utilize them for the development of therapeutic strategies by which apoptosis may be prevented or counteracted.
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Affiliation(s)
- G Gravante
- Department of Surgery, University of Rome Tor Vergata, Via U Maddalena 40/a 00043, Ciampino, Rome, Italy.
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Chen LW, Huang HL, Lee IT, Hsu CM, Lu PJ. THERMAL INJURY-INDUCED PRIMING EFFECT OF NEUTROPHIL IS TNF-α AND P38 DEPENDENT. Shock 2006; 26:69-76. [PMID: 16783201 DOI: 10.1097/01.shk0000209531.38188.18] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Priming response of neutrophil in clinical-related conditions and its mechanism has not been clarified. This study is to determine if thermal injury-induced priming effect of neutrophil is TNF-alpha and p38 dependent. In Experiment 1, bone marrow neutrophil of wild-type (WT) mice and TNF receptor superfamily, member 1A (Tnfrsf1a-/-) mice were harvested and treated with TNF-alpha, platelet activating factor (PAF) first, then with or without N-formyl-Met-Leu-Phe (fMLP). Reactive oxygen species (ROS) production and p38 phosphorylation were evaluated. In Experiment 2, ROS of neutrophil from WT and Tnfrsf1a-/- mice at 3 or 15 h after thermal injury with or without fMLP treatment were assayed. In Experiment 3, p38 and p44/42 phosphorylation, CXCR2 and macrophage inflammatory protein-2 expression, apoptotic ratio, and activating protein-1 (AP-1) and nuclear factor-kappa B (NF-kappaB) activation of neutrophil from WT and Tnfrsf1a-/- mice at 3 h after thermal injury were tested. FMLP treatment after TNF-alpha or PAF incubation of neutrophil increased ROS of PAF-treated but not TNF-alpha-treated neutrophil. PAF treatment increased ROS of neutrophil in WT and Tnfrsf1a-/- mice. FMLP increased ROS of neutrophil of WT mice at 3 h after thermal but not that of Tnfrsf1a-/- mice. TNF-alpha and PAF increased p38 phosphorylation of neutrophil in WT but not that in Tnfrsf1a-/- mice. Thermal injury increased p38 phosphorylation, NF-kappaB activation, and decreased apoptosis of neutrophil at 3 h after thermal injury in WT but not in Tnfrsf1a-/- mice. Thermal injury also induced AP-1 activation and ROS production on neutrophil at 3 and 15 h after thermal injury, respectively, in WT and Tnfrsf1a-/- mice. Collectively, fMLP stimulates ROS of neutrophil through TNF-alpha signaling; PAF stimulates that of neutrophil through both TNF-alpha-dependent and TNF-alpha-independent pathway. Thermal injury induces a TNF-alpha-dependent priming effect and a TNF-alpha-independent activation effect on neutrophil at 3 and 15 h after thermal injury, respectively. NF-kappaB signaling pathway plays an important role in neutrophil activation. Thermal injury also induces TNF-alpha-dependent delay apoptosis and TNF-alpha-independent AP-1 activation of neutrophil at 3 h after thermal injury. Taken together with the TNF-alpha-dependent p38 and NF-kappaB activation in primed neutrophil, we conclude that thermal injury-induced priming effect of polymorphonuclear neutrophil is TNF-alpha and p38 dependent.
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Affiliation(s)
- Lee-Wei Chen
- Department of Surgery, Kaohsiung Veterans General Hospital, National Yang-Ming Medical University, Taipei, Taiwan, ROC
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Chen LW, Huang HL, Lee IT, Hsu CM, Lu PJ. Hypertonic saline enhances host defense to bacterial challenge by augmenting Toll-like receptors*. Crit Care Med 2006; 34:1758-68. [PMID: 16625117 DOI: 10.1097/01.ccm.0000218810.66485.01] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
OBJECTIVE To determine whether hypertonic saline infusion modulates thermal injury-induced bacterial translocation and host response to bacterial challenge through the augmentation of Toll-like receptors (TLRs). DESIGN Prospective, experimental study. SETTING Research laboratory at a university hospital. SUBJECTS Thermal injury models in the mice. INTERVENTIONS In experiment 1, mice underwent burn were given with 10 mL/kg hypertonic saline (7.5% NaCl), 10 mg/kg saline (N/S1), or 80 mL/kg saline (N/S2) at 4 or 8 hrs after burn. At 24 hrs after burn, mesenteric lymph nodes were harvested for bacterial translocation assay. In experiment 2, mice receiving hypertonic saline or saline after thermal injury received peritoneal challenge with Escherichia coli, and bacterial clearance was measured. In experiment 3, peritoneal cells from mice receiving hypertonic saline or saline after thermal injury were incubated with E. coli, and bacterial count, TLR2, TLR4, MIP2, CXCR2, pp38, and ERK expression were evaluated. In experiment 4, reactive oxygen species production, CXCR2, MIP2, TLR2, and TLR4 expression of bone marrow neutrophil from mice receiving hypertonic saline or saline treatment after thermal injury were evaluated. In experiment 5, neutrophil were cultured with hypertonic saline or N/S and incubated with E. coli. TLR2 and TLR4 expression and bacterial count were evaluated. In experiment 6, mice were fed with oral antibiotics with or without lipopolysaccharide, a TLR ligand, supplements. At 24 hrs after burn, mesenteric lymph nodes were harvested for bacterial translocation assay, and neutrophils were harvested for TLR2 and TLR4 protein assay. MEASUREMENTS AND MAIN RESULTS Hypertonic saline decreased thermal injury-induced bacterial translocation. Hypertonic saline increased bacterial clearance, phagocytic activity, and TLR2, TLR4, CXCR2, pp38, and p44/42 expression of peritoneal cells. Hypertonic saline treatment at 4 or 8 hrs after thermal injury decreased reactive oxygen species production of neutrophil. Hypertonic saline injection increased TLR2, TLR4, and pp38 expression of neutrophil. In vitro treatment of neutrophil with hypertonic saline increased phagocytic activity and TLR2 and TLR4 expression. Commensal depletion with oral antibiotics decreased TLR2 and TLR4 expression of neutrophil; lipopolysaccharide increased TLR4 expression of neutrophil and decreased thermal injury-induced bacterial translocation. CONCLUSIONS Restoration of extracellular fluid in burn shock with hypertonic saline decreased thermal injury-induced bacterial translocation. Hypertonic saline increased the phagocytic activity and TLR2, TLR4, CXCR2, pp38, and P44/42 expression of peritoneal cells. Hypertonic saline decreased reactive oxygen species but increased TLR2, TLR4, and pp38 expression and phagocytic activity of bone marrow neutrophil. Stimulation of the TLRs with lipopolysaccharide in commensal depleted mice increased TLRs expression of neutrophil and decreased thermal injury-induced bacterial translocation. Taken together with the fact that stimulation of TLRs with hypertonic saline increases phagocytic activity of systemic inflammatory cells, we conclude that TLRs play a critical role in the innate immunity by recognizing bacteria and that hypertonic saline enhances host response to bacterial challenge by increasing TLRs of inflammatory cells.
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Affiliation(s)
- Lee-Wei Chen
- Department of Surgery, Kaohsiung Veterans General Hospital, National Yang-Ming Medical University, Taipei (L-WC, I-TL), R.O.C
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17
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Abstract
Immediate and early trauma death rates are determined by "first hits" such as hypoxia, hypotension and organ injury, while late mortality correlates closely with "second hits" such as infection. An imbalance between the early systemic inflammatory response (SIRS), and the later compensatory counter-inflammatory response (CARS), is considered to be responsible for much post-traumatic morbidity and mortality. From a clinical perspective, this remains a significant healthcare problem, which has stimulated decades of experimental and clinical research aimed at understanding the functional effects of injury on the immune system. This review describes the impact of injury on the innate and adaptive immune systems. Though it is worth noting that the features of the immune response to injury overlap in many areas with immune dysregulation in sepsis, we attempt here to elucidate the mechanism by which injury predisposes to infection rather than to describe the alterations in host immunity consequent to established sepsis.
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Affiliation(s)
- N Ni Choileain
- Department of Surgery, Cork University Hospital, Ireland.
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Chen LW, Chang WJ, Wang JS, Hsu CM. Thermal injury-induced peroxynitrite production and pulmonary inducible nitric oxide synthase expression depend on JNK/AP-1 signaling. Crit Care Med 2006; 34:142-50. [PMID: 16374168 DOI: 10.1097/01.ccm.0000190621.48720.8c] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
OBJECTIVE To determine whether burn-induced peroxynitrite production and expression of lung inducible nitric oxide synthase (iNOS), intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, CXCR2, macrophage inflammatory protein (MIP)-2, and neutrophil chemokine (KC) are mediated by the c-Jun NH2-terminal kinase (JNK). DESIGN Prospective, experimental study. SETTING Research laboratory at a university hospital. SUBJECTS Thermal injury models in the mice. INTERVENTIONS In experiment 1, specific pathogen-free C57/BL6 mice were subjected to 30% total body surface area third-degree burn over shaved back. At 0 hr, 2 hrs, 4 hrs, and 6 hrs after burn, lung tissues of those mice were harvested for JNK activity assay, AP-1 DNA-binding activity, and pJNK immunohistochemistry. In experiment 2, a specific JNK inhibitor, SP600125, was given (30 mg/kg intraperitoneally) to mice immediately postburn to suppress the JNK activity. At 8 hrs after burn, blood was assayed for the peroxynitrite-mediated dihydrorhodamine (DHR) 123 oxidation. Lung tissues were harvested for myeloperoxidase (MPO) determination, ICAM-1, VCAM-1, CXCR2, KC, MIP-2, interleukin-1beta, and interleukin-6 messenger RNA expression; iNOS immunohistochemical staining; and histologic studies. Pulmonary microvascular dysfunction was quantified by measuring the extravasations of Evans blue dye. MEASUREMENTS AND MAIN RESULTS The JNK activity and AP-1 DNA-binding activity of lung tissue significantly increased to a peak at 2 hrs and 4 hrs, respectively, after thermal injury. Immunohistochemical study demonstrated that the increase of the pJNK was mostly from the bronchiole epithelial cells. This increase of MPO activity in lung, blood DHR 123 oxidation level, and lung permeability increased six-fold, nine-fold, and four-fold after burn. SP600125 administration obliterated the thermal injury-induced JNK activity, AP-1 DNA-binding activity, and iNOS expression in lung tissue. SP600125 treatment also significantly decreased MPO activity, blood DHR 123 oxidation, and lung permeability by 54%, 8%, and 47%, respectively, and markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and septum edema. Furthermore, SP600125 abolished thermal injury-induced ICAM-1, VCAM-1, CXCR2, MIP-2, and KC but not interleukin-1beta and interleukin-6 messenger RNA levels of lung tissues. CONCLUSIONS Thermal injury induces lung tissue JNK activation and AP-1 DNA-binding activity mainly from airway epithelial cells. Thermal injury-induced peroxynitrite production and lung iNOS, ICAM-1, and VCAM-1 expression are mediated by the JNK signaling. JNK inhibition decreases thermal injury-induced lung neutrophil infiltration and subsequently pulmonary hyperpermeability.
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Affiliation(s)
- Lee-Wei Chen
- Department of Surgery, Kaohsiung Veterans General Hospital, National Yang-Ming Medical University, Taipei, Taiwan
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19
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Ueno S, Aoki D, Kubo F, Hiwatashi K, Matsushita K, Oyama T, Maruyama I, Aikou T. Roxithromycin inhibits constitutive activation of nuclear factor {kappa}B by diminishing oxidative stress in a rat model of hepatocellular carcinoma. Clin Cancer Res 2006; 11:5645-50. [PMID: 16061884 DOI: 10.1158/1078-0432.ccr-05-0652] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE Recently, 14-member macrolide antibiotics such as clarithromycin and roxithromycin have been shown to have anticancer and antiangiogenic effects. We investigated the suppressive effect of roxithromycin on accelerated hepatocellular carcinoma growth in a rat hepatocarcinogenetic model and compared results with effects from TNP-470. EXPERIMENTAL DESIGN Tumor was induced by oral diethylnitrosamine administration for 17 weeks. Normal saline, TNP-470 (50 mg/kg), or roxithromycin (40 or 100 mg/kg) was administered i.p. thrice per week from week 10 to 17. RESULTS Carcinomatous tissue growing outside dysplastic nodules and a marked expression of placental glutathione S-transferase were detected in rats with induced carcinogenesis. Tumor growth was accompanied by augmented expression of inducible nitric oxide synthase, activation of nuclear factor kappaB, and increased lipid peroxidation level. All these effects were absent in animals that received roxithromycin or TNP-470. The inhibitory effect of roxithromycin was dose dependent and no clear differences were noted between groups given roxithromycin 100 mg/kg and TNP-470 50 mg/kg. CONCLUSIONS Our results indicate that roxithromycin inhibits oxidative stress, nitric oxide production, and nuclear factor kappaB activation induced by experimental hepatocarcinogenesis. The data provide additional evidence for the potential use of roxithromycin in treatment of hepatocellular carcinoma prevention.
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Affiliation(s)
- Shinichi Ueno
- Department of Surgical Oncology and Digestive Surgery, School of Medicine, Kagoshima University, Kagoshima, Japan.
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Daniel T, Alexander M, Hubbard WJ, Chaudry IH, Choudhry MA, Schwacha MG. Nitric oxide contributes to the development of a post-injury Th2 T-cell phenotype and immune dysfunction. J Cell Physiol 2006; 208:418-27. [PMID: 16642464 DOI: 10.1002/jcp.20677] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Severe injury induces immune dysfunction resulting in increased susceptibility to opportunistic infections. Previous studies from our laboratory have demonstrated that post-burn immunosuppression is mediated by nitric oxide (NO) due to the increased expression of macrophage inducible nitric oxide synthase (iNOS). In contrast, others suggest that injury causes a phenotypic imbalance in the regulation of Th1- and Th2 immune responses. It is unclear whether or not these apparently divergent mediators of immunosuppression are interrelated. To study this, C57BL/6 mice were subjected to major burn injury and splenocytes were isolated 7 days later and stimulated with antiCD3. Burn injury induced NO-mediated suppression of proliferative responses that was reversed in the presence of the NOS inhibitor L-monomethyl-L-arginine and subsequently mimicked by the addition of the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP). SNAP also dose-dependently suppressed IFN-gamma and IL-2 (Th1), but not IL-4 and IL-10 (Th2) production. Delaying the addition of SNAP to the cultures by 24 h prevented the suppression of IFN-gamma production. The Th2 shift in immune phenotype was independent of cGMP and apoptosis. The addition of SNAP to cell cultures also induced apoptosis, attenuated mitochondrial oxidative metabolism and induced mitochondrial membrane depolarization. However, these detrimental cellular effects of NO were observed only at supra-physiologic concentrations (>250 microM). In conclusion, these findings support the concept that NO induces suppression of cell-mediated immune responses by selective action on Th1 T cells, thereby promoting a Th2 response.
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Affiliation(s)
- Tanjanika Daniel
- Department of Surgery, Center for Surgical Research, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA
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21
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22
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Chen LW, Hwang YC, Wang JS, Chen JS, Hsu CM. Inhibition of nitric oxide synthase reverses the effect of albumin on lung damage in burn. J Am Coll Surg 2005; 200:574-83. [PMID: 15804472 DOI: 10.1016/j.jamcollsurg.2004.11.010] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2004] [Revised: 09/24/2004] [Accepted: 11/02/2004] [Indexed: 11/25/2022]
Abstract
BACKGROUND Early colloid resuscitation in major burn patients has been stopped because of its deteriorating effect on thermal injury-induced vascular hyperpermeability. We hypothesized that inhibition of inducible nitric oxide synthase (iNOS) to stabilize endothelial permeability and to retain colloid solution in the vascular space will reverse its effect on lung damage. STUDY DESIGN In experiment 1, specific pathogen free rats underwent 35% total-body surface area burn or sham burn and were given equal volumes (7.5 mL/kg) of normal saline or albumin from femoral veins for fluid resuscitation immediately after burn. In experiment 2, S-methylisothiourea (SMT, 7.5 mg/kg, IP) was given immediately after burn to rats from different groups, as in experiment 1. At 8 hours after burn, blood was assayed for peroxynitrite-mediated dihydrorhodamine 123 (DHR 123) oxidation, and lung tissues were harvested for myeloperoxidase (MPO) determination and histologic studies. Pulmonary microvascular dysfunction was quantified by measuring the extravasations of Evans blue dye. RESULTS Blood peroxynitrite level and iNOS expression, MPO activity, permeability, and inflammatory cell infiltration of lungs were significantly induced after thermal injury. Albumin resuscitation after burn without iNOS inhibition enhanced thermal injury-induced lung damage with 10%, 14%, and 5% increases in blood DHR oxidation level, lung MPO activity, and lung permeability, respectively, compared with saline injection. In contrast, burn + SMT rats with albumin injection showed significant, 23%, 37%, and 20%, decreases, respectively, in blood DHR 123 oxidation level, lung MPO activity, and lung permeability compared with burn + SMT + saline rats. CONCLUSIONS Thermal injury induced lung damage. Restoration of extracellular fluid in early burn shock with albumin markedly augmented the lung neutrophil deposition, lung permeability increase, and blood peroxynitrite level. Inhibition of iNOS before albumin supplementation reversed its damaging effects on thermal injury-induced lung dysfunction to beneficial ones.
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Affiliation(s)
- Lee-Wei Chen
- Department of Surgery, Kaohsiung Veterans General Hospital, National Yang-Ming University, Taipei, Taiwan
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23
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Abstract
During the last 50 years, our understanding of the role of the gastrointestinal tract as a first-line defense against the development of postburn sepsis has increased dramatically. Starting with the concept of that gut-derived bacteria cause distant injury, investigators have delineated a complex series of physical changes in the barrier of the gastrointestinal tract. Along with an understanding of these physical changes has come an appreciation of the role of the immune system in modulating postburn organ failure. Importantly, recent investigations into the role of mesenteric lymph have fundamentally changed the paradigm of organ failure and have implicated the gut as a cytokine-secreting organ. This article traces the development of key concepts in the study of burn sepsis and their clinical implications.
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Affiliation(s)
- Ankush Gosain
- Burn and Shock Trauma Institute, Department of Surgery, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA
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Chen LW, Hwang B, Wang JS, Chen JS, Hsu CM. Hypertonic saline-enhanced postburn gut barrier failure is reversed by inducible nitric oxide synthase inhibition. Crit Care Med 2005; 32:2476-84. [PMID: 15599154 DOI: 10.1097/01.ccm.0000147831.07329.32] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
OBJECTIVE To determine whether inhibition of inducible nitric oxide synthase to stabilize endothelial permeability and to retain hypertonic saline in the vascular space will ameliorate burn-induced gut barrier dysfunction. DESIGN Prospective, experimental study. SETTING Research laboratory at a university hospital. SUBJECTS Thermal injury models in the rat. INTERVENTIONS In experiment 1, specific pathogen free rats underwent 3% total body surface area burn or sham burn and were given 7.5 mL/kg hypertonic saline (7.5% NaCl), 7.5 mg/kg saline, or 50 mL/kg saline (nearly equal sodium load with hypertonic saline) in the right femoral vein for 15 mins for fluid resuscitation at 0, 4, or 8 hrs after burn. In experiment 2, S-methylisothiourea (7.5 mg/kg, intraperitoneally), a specific inducible nitric oxide synthase inhibitor, was given immediately after burn to rats from different groups as in experiment 1. At 24 hrs after burn, the intestinal mucosa was assayed for myeloperoxidase activity and lipid peroxidation, the distribution of fluorescein isothiocyanate-dextran across the lumen of the small intestine was determined, and bacterial translocation to the mesenteric lymph nodes and ileum histology were also examined. MEASUREMENTS AND MAIN RESULTS Burn induced significant increases in intestinal mucosa inducible nitric oxide synthase expression, myeloperoxidase activity, lipid peroxidation, intestinal permeability, bacterial translocation to mesenteric lymph nodes, and villi sloughing in rats. Hypertonic saline administration at 0 or 4 hrs after burn worsened intestinal mucosa lipid peroxidation, neutrophil sequestration, intestinal permeability, and villi sloughing compared with those of burn + 7.5 mg/kg saline and burn + 50 mL/kg saline rats. To the contrary, burn + S-methylisothiourea rats with hypertonic saline injection at 4 or 8 hrs after burn showed an improvement of gut barrier function compared with burn + S-methylisothiourea + 7.5 mg/kg saline and burn + S-methylisothiourea + 50 mL/kg saline rats. Administration of hypertonic saline at 8 hrs after burn and S-methylisothiourea injection also significantly attenuated the bacterial translocation to mesenteric lymph nodes and villi sloughing. CONCLUSIONS Using hypertonic saline as a resuscitation fluid in early burn shock markedly augmented the thermal injury-induced intestinal mucosa neutrophil deposition, lipid peroxidation, and intestinal hyperpermeability. Inhibition of inducible nitric oxide synthase not only significantly attenuated neutrophil deposition and mucosa lipid peroxidation but also reversed the deteriorating effects of hypertonic saline on thermal injury-induced gut barrier dysfunction and bacterial translocation.
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Affiliation(s)
- Lee-Wei Chen
- Department of Surgery, Kaohsiung Veterans General Hospital, National Yang-Ming Medical University, Taipei, Taiwan
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25
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Gan HT, Chen JDZ. Roles of nitric oxide and prostaglandins in pathogenesis of delayed colonic transit after burn injury in rats. Am J Physiol Regul Integr Comp Physiol 2005; 288:R1316-24. [PMID: 15650120 DOI: 10.1152/ajpregu.00733.2004] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Burn injury has been shown to impair gut transit, but the exact mechanism remains unknown. The present study investigated whether nitric oxide synthase (NOS) and cyclooxygenase (COX) mediated changes in burn-induced colonic transit. After rats underwent 30% total body surface area burn injury, they were injected with S-methylisothiourea (SMT, selective inducible NOS inhibitor), 7-nitronidazole (7-NI, selective neuronal NOS inhibitor), and nimesulide (NIM, selective COX-2 inhibitor), respectively. The protein and mRNA of NOS and COX-2 were measured by Western blot analysis and real-time RT-RCR, and localization of NOS and COX-2 protein was determined by immunohistochemistry. Our results showed that colonic transit assessed by the geometric center was delayed from 3.47+/-0.28 in controls to 2.21+/-0.18 after burn (P<0.009). SMT and NIM significantly improved colonic transit in burned rats but had no effect in sham-operated rats. 7-NI failed to modify delayed transit in burned rats but significantly delayed colonic transit in sham-operated rats. Both protein and mRNA of inducible NOS and COX-2 increased significantly but not neuronal NOS in burned rats. Inducible NOS protein expression was noted not only in epithelial cells but also in neurons of the myenteric ganglia in burned rats. These findings suggest that nitric oxide (NO) produced by neuronal NOS plays an important role in mediating colonic transit under the physiological condition. NO produced by inducible NOS and prostaglandins synthesized by COX-2 are both involved in the pathogenesis of delayed colonic transit after burn injury. Inducible NOS expression in neurons of the myenteric ganglia may contribute to dysmotility with burn injury.
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Affiliation(s)
- Hua Tian Gan
- Division of Gastroenterology, University of Texas Medical Branch, Galveston, TX 77555-0632, USA
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Chen LW, Hwang B, Chang WJ, Wang JS, Chen JS, Hsu CM. INDUCIBLE NITRIC OXIDE SYNTHASE INHIBITOR REVERSES EXACERBATING EFFECTS OF HYPERTONIC SALINE ON LUNG INJURY IN BURN. Shock 2004; 22:472-7. [PMID: 15489641 DOI: 10.1097/01.shk.0000140304.71215.f7] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
The use of hypertonic saline (HTS) resuscitation in major trauma patients is still controversial. The objective of this study is to determine if inhibition of inducible nitric oxide synthase (iNOS) to stabilize the endothelial permeability and to retain HTS in the vascular space will reverse its exacerbating effect on burn-induced lung damage. In Experiment 1, specific pathogen-free (SPF) rats underwent 35% total body surface area (TBSA) burn and were resuscitated with 7.5 mL/kg HTS (7.5% NaCl), 7.5 mL/kg saline, or 50 mL/kg saline (nearly equal sodium load as HTS) via femoral veins for 15 min immediately after the burn. In Experiment 2, S-methylisothiourea (SMT) (7.5 mg/kg, i.p.) was given immediately after the burn to rats from the different groups of Experiment 1. At 8 h after the burn, the permeability and myeloperoxidase (MPO) activity of lung tissues were determined, and plasma samples were assayed for peroxynitrite levels. Burn significantly induced lung MPO activity, lung permeability, and blood dihydrorhodamine 123 (DHR 123) oxidation in rats. HTS administration after burn significantly increased the blood DHR 123 oxidation level, lung MPO activity, lung permeability, and inflammatory cell infiltration in comparison with those of burn plus 7.5 mg/kg saline and burn plus 50 mL/kg saline rats. In contrast, burn plus SMT rats with HTS injection showed significant 54%, 11%, and 35% decreases in blood DHR 123 oxidation level, lung MPO activity, and lung permeability, respectively, in comparison with burn plus SMT plus 7.5 mg/kg saline rats. In conclusion, restoration of extracellular fluid in early burn shock with HTS supplementation significantly exacerbates burn-induced lung neutrophil deposition, lung hyperpermeability, and blood peroxynitrite production. Inhibition of iNOS before HTS supplementation reverses the deteriorating effects of HTS on thermal injury-induced lung damage to beneficial ones. Using HTS in thermal injury resuscitation without the inhibition of iNOS is dangerous.
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Affiliation(s)
- Lee-Wei Chen
- Department of Surgery, Kaohsiung Veterans General Hospital, National Yang-Ming Medical University, Taipei, Taiwan
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Sakoda M, Ueno S, Kihara K, Arikawa K, Dogomori H, Nuruki K, Takao S, Aikou T. A potential role of hyperbaric oxygen exposure through intestinal nuclear factor-kappaB. Crit Care Med 2004; 32:1722-9. [PMID: 15286550 DOI: 10.1097/01.ccm.0000132898.27101.6c] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
OBJECTIVE Recent studies have demonstrated the therapeutic effectiveness and pharmacologic mechanisms of hyperbaric oxygen therapy (HBOT) in the treatment of a systemic shock state. To elucidate the in vivo role of HBOT during sepsis, we evaluated the effects of HBOT on intestinal mucosal injury and bacterial translocation after lipopolysaccharide challenge. DESIGN Experimental study. SETTING First Department of Surgery and Division of Emergency Care, Kagoshima University School of Medicine, Kagoshima, Japan. SUBJECTS : Male rats were treated with lipopolysaccharide by an intraperitoneal route or with lipopolysaccharide and HBOT. INTERVENTIONS The survival rate, small intestinal tissue damage, and bacterial translocation in the HBOT-treated group were compared with those in the untreated group. Moreover, plasma tumor necrosis factor-alpha and nitrite/nitrate concentrations, inducible nitric oxide synthase and myeloperoxidase activities, and nuclear factor-kappaB in ileal mucosa were investigated. HBOT was initiated 3 hrs after lipopolysaccharide challenge and administered as 100% oxygen, at 2.53 x 10 kPa (2.5 atm absolute), for 60 mins. MEASUREMENTS AND MAIN RESULTS When a sublethal dose of lipopolysaccharide (24 mg/kg) was given, the survival rate was much better in the HBOT-treated group (75%) than in the untreated group (33%). HBOT given 3 hrs after lipopolysaccharide injection (10 mg/kg) also lessened the histologic tissue damage of the terminal ileum and the incidence and magnitude of bacterial translocation to mesenteric lymph nodes at 24 hrs after the lipopolysaccharide injection. Moreover, HBOT was able to reduce mucosal inducible nitric oxide synthase and myeloperoxidase activities and plasma nitrite/nitrate concentrations but not serum tumor necrosis factor-alpha concentrations. Immunohistochemical examination revealed that HBOT specifically modified the mucosal nuclear factor-kappaB activation within 4-6 hrs after the injection. CONCLUSIONS HBOT performed 3 hrs after lipopolysaccharide challenge alleviates intestinal barrier dysfunction and improves survival rates. Herein, we propose one possible mechanism for these beneficial effects: HBOT can modify the nuclear factor-kappaB activation in the intestinal mucosa and attenuate the sequential nitric oxide overproduction and myeloperoxidase activation. Consequently, bacterial translocation could be potentially decreased. We believe that the present study should lead to an improved understanding of HBOT's potential role in sepsis.
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Affiliation(s)
- Masahiko Sakoda
- First Department of Surgery, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan
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Al-Ghoul WM, Khan M, Fazal N, Sayeed MM. Mechanisms of postburn intestinal barrier dysfunction in the rat: Roles of epithelial cell renewal, E-cadherin, and neutrophil extravasation*. Crit Care Med 2004; 32:1730-9. [PMID: 15286551 DOI: 10.1097/01.ccm.0000132896.62368.01] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
OBJECTIVE Our group has previously shown that the intestinal epithelium exhibits increased postburn barrier permeability and bacterial translocation associated with deranged neutrophil activity. The purpose of this investigation is to explore possible underlying intestinal structural mechanisms, leading to those functional changes with emphasis on (1) neutrophil influx and extravasation in the intestinal lamina propria 1-3 days after burn and (2) enterocyte proliferation, migration, apoptosis, and E-cadherin junctional epithelium levels 3 days after burn. DESIGN Freshly isolated ileum specimens were quick frozen, then cut by a cryostat into 30-micron-thick sections. Sections from day 1 postburn rats were immunostained with (1) anti-granulocyte or anti-elastase antibodies to assess neutrophil influx or (2) combined anti-granulocyte and anti-von Willebrand factor double immunolabeling to compare levels of neutrophil extravasation. Sections from day 3 postburn rats were immunostained with (1) bromodeoxyuridine immunohistochemistry 1, 3, 6, or 18 hrs after bromodeoxyuridine injection to assess enterocyte proliferation and migration, (2) cytokeratin-18 M30-immunohistochemistry to compare levels of enterocyte apoptosis, and (3) E-cadherin immunohistochemistry to compare junctional E-cadherin integrity. Ileal myeloperoxidase activity and bacterial translocation of Enterococcus faecalis were assessed biochemically and by E. faecalis-specific bacterial cultures, respectively, in day 3 postburn rats. SETTING : Research laboratories in a medical center and an academic institution. SUBJECTS Male Sprague-Dawley rats given sham treatment or treatment as a burn model with full-thickness skin scald over 30% total body surface area. CONCLUSIONS We report (1) increased levels of neutrophil influx and extravasation in villi lamina propriae, including elastase-positive cells (postburn day 1), (2) heightened levels of intestinal myeloperoxidase activity (postburn day 3), (3) decreased levels of epithelial cell proliferation, migration, and E-cadherin (postburn day 3), and (4) increased enterocyte apoptosis and E. faecalis bacterial translocation (postburn day 3). Based on these structural and functional abnormalities, we propose a mechanism for burn injury-related intestinal barrier dysfunction that includes increased trans- and para-cellular leakage caused by impaired enterocyte renewal and decreased junctional E-cadherin levels subsequent to increased neutrophil influx and extravasation within the villus lamina propria microenvironment.
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Affiliation(s)
- Walid M Al-Ghoul
- Trauma/Critical Care Labs, Department of Surgery, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA.
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Toth B, Alexander M, Daniel T, Chaudry IH, Hubbard WJ, Schwacha MG. The role of γδ T cells in the regulation of neutrophil-mediated tissue damage after thermal injury. J Leukoc Biol 2004; 76:545-52. [PMID: 15197233 DOI: 10.1189/jlb.0404219] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Thermal injury induces an inflammatory response that contributes to the development of secondary tissue damage. Neutrophil recruitment and activation are in part responsible for this tissue damage. Although gammadelta T cells have been shown to regulate the inflammatory responses in tissues that are prone to neutrophil-mediated injury post-burn, their role in the induction of secondary tissue injury post-burn remains unknown. To study this, gammadelta T cell-deficient (gammadelta TCR-/-) and wild-type (WT) mice were subjected to thermal injury or sham procedure, and tissue samples were isolated 1-24 h thereafter. Burn injury induced neutrophil accumulation in the lung and small intestines of WT mice at 1-3 h post-injury. No such increase in neutrophil tissue content was observed in gammadelta TCR-/- mice. An increase in tissue wet/dry weight ratios was also observed in these organs at 3 h post-burn in WT but not in gammadelta TCR-/- mice. A parallel increase in plasma and small intestine levels of the chemokines macrophage-inflammatory protein-1beta (chemokine ligand 4) and keratinocyte-derived chemokine (CXC chemokine ligand 1) were observed in injured WT mice but not in injured gammadelta TCR-/- mice. Increased activation (CD120b expression) of the circulating gammadelta T cell population was also observed at 3 h post-burn in WT mice. These results indicate the gammadelta T cells, through the production of chemokines, play a central role in the initiation of neutrophil-mediated tissue damage post-burn.
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MESH Headings
- Animals
- Antigens, CD/immunology
- Burns/immunology
- Burns/pathology
- Burns/physiopathology
- Cell Division/genetics
- Cell Division/immunology
- Chemokine CCL4
- Chemotaxis, Leukocyte/genetics
- Chemotaxis, Leukocyte/immunology
- Immunologic Deficiency Syndromes/genetics
- Inflammation/immunology
- Inflammation/pathology
- Inflammation/physiopathology
- Intestine, Small/immunology
- Intestine, Small/physiopathology
- Lung/immunology
- Lung/pathology
- Lung/physiopathology
- Macrophage Inflammatory Proteins/immunology
- Macrophage Inflammatory Proteins/metabolism
- Male
- Mice
- Mice, Inbred C57BL
- Mice, Knockout
- Neutrophils/immunology
- Receptors, Antigen, T-Cell, gamma-delta/deficiency
- Receptors, Antigen, T-Cell, gamma-delta/genetics
- Receptors, Antigen, T-Cell, gamma-delta/immunology
- Receptors, Interleukin-8A/immunology
- Receptors, Tumor Necrosis Factor/immunology
- Receptors, Tumor Necrosis Factor, Type II
- T-Lymphocytes/immunology
- Up-Regulation/immunology
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Affiliation(s)
- Balazs Toth
- Center for Surgical Research, Department of Surgery, G094 Volker Hall, 1670 University Blvd., Birmingham, AL 35294-0019, USA
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Chen LW, Hwang YC, Chen CJ, Wang JS, Chen JS, Hsu CM. Burn-induced lung damage in rat is mediated by a nitric oxide/cGMP system. Shock 2004; 20:369-74. [PMID: 14501952 DOI: 10.1097/01.shk.0000086520.18735.df] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
This study was conducted to demonstrate the burn-induced lung neutrophil deposition and damage in rats is affected by the nitric oxide (NO)-dependent downstream cGMP signaling. In experiment 1, 1H-[1,2,4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ) was given (20 mg/kg i.p.) to specific pathogen-free Sprague-Dawley rats immediately postburn to suppress the guanylate cyclase (GC) activity. At 8 h after burn, blood was assayed for the peroxynitrite-mediated dihydrorhodamine 123 (DHR 123) oxidation and lung tissues were harvested for myeloperoxidase (MPO) determination and histological studies. Pulmonary microvascular dysfunction was quantified by measuring the extravasations of Evans blue dye. In experiment 2, Sodium nitroprusside (SNP) was given (2 mM, i.p.) to elevate cGMP levels and ODQ (20 mg/kg, i.p.) or methylene blue (100 microM, i.p.) or saline was given. The animals were sacrificed 4 h after injection and lung tissues were harvested for iNOS mRNA study. The MPO activity in lung, blood DHR 123 oxidation level, and lung permeability increased up to 2-fold, 4-fold, and 2.5-fold after burn. Inhibition of GC by ODQ administration significantly decreased MPO activity, blood DHR 123 oxidation, and lung permeability by 55%, 66%, and 53%, respectively, and markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and septum edema. The protective effects of ODQ were comparable to the use of selective iNOS inhibitor as demonstrated previously. Furthermore, ODQ decreased the burn or SNP-induced iNOS mRNA levels at 4 h after burn. These findings suggest that burn-induced lung dysfunction is mediated by the NO/cGMP system because it is abolished by application of either iNOS inhibitor or GC inhibitor. Also, the beneficial effect of ODQ is partly due to the attenuation of burn-induced iNOS expression by GC inhibition.
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Affiliation(s)
- Lee-Wei Chen
- Department of Surgery, Kaohsiung Veterans General Hospital, National Yang-Ming Medical University, Taipei, Taiwan
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31
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Abstract
Nitric oxide (NOz.rad;) is a diatomic mediator liberated on oxidation of L-arginine by the nitric oxide synthase (NOS) family of enzymes. It has complex and wide ranging functions in vivo and has been implicated in the development of the profound inflammatory response that occurs as a result of cutaneous burn injury. In addition, dysregulation of NOS activity has been associated with multiple organ failure in human burn patients and may therefore represent a novel therapeutic target in such circumstances. This review focuses on the role of NOz.rad; in inflammation, with particular emphasis on the acute post-burn inflammatory response. Specific areas of discussion include the maintenance of microvascular haemostasis, leukocyte recruitment and remote organ dysfunction following thermal injury.
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Affiliation(s)
- Andrew Rawlingson
- Centre for Cardiovascular Biology & Medicine, King's College London, Guy's Campus, SE1 1UL, London, UK.
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Nayci A, Atis S, Talas DU, Polat A, Bagdatoglu O, Polat G. Bronchoscopy induces intestinal mucosal barrier dysfunction: a possible role for nitric oxide. Int J Pediatr Otorhinolaryngol 2003; 67:957-63. [PMID: 12907050 DOI: 10.1016/s0165-5876(03)00161-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
OBJECTIVE This study investigates the effect of bronchoscopy on intestinal mucosal barrier function and its association with intestinal nitric oxide production. METHODS 30 rats were used. The study group (n=15) underwent rigid bronchoscopy. At 24 h following bronchoscopy, ileal nitrite/nitrate levels were evaluated. The ileum was also examined for mucosal damage, and graded according Chiu's histologic injury scale. RESULTS In the bronchoscopy group, the ileal nitrite/nitrate levels were significantly higher than those of controls (398.5 +/- 85.1 and 44.5 +/- 6.6 nmol/g tissue, respectively, P=0.001). In the bronchoscopy group, the mucosal damage was significant, compared with those of controls (mean ranks, 22.8 and 8.2, P<0.0001). The changes varied from denuded villi and dilated capillaries to significant architectural distortion, lamina propria disintegration, ulceration and hemorrhage. Significant correlation was found between ileal nitrite/nitrate levels and mucosal damage in the bronchoscopy group (rs=0.56, P=0.03). CONCLUSION This study suggests that bronchoscopy induces intestinal mucosal barrier dysfunction in association with excess intestinal nitric oxide production. These events may be involved in mechanisms responsible for bacterial translocation after bronchoscopy.
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Affiliation(s)
- Ali Nayci
- Department of Pediatric Surgery, School of Medicine, Mersin University, Mersin, Turkey.
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Chen LW, Wang JS, Chen HL, Chen JS, Hsu CM. Peroxynitrite is an important mediator in thermal injury-induced lung damage. Crit Care Med 2003; 31:2170-7. [PMID: 12973176 DOI: 10.1097/01.ccm.0000079605.28852.d0] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
OBJECTIVE Intestinal ischemia and reperfusion injury was known to cause postinjury multiple organ failure by neutrophil and unclear nonneutrophil factors. Peroxynitrite formed by the rapid reaction between superoxide and nitric oxide, is a toxic substance that contributes to tissue injury in a number of biological systems. In this study, the role of nitric oxide and neutrophils on lung damage after burn was investigated. DESIGN Prospective, experimental study. SETTING Research laboratory at a university hospital. SUBJECTS Thermal injury models in the rat. INTERVENTIONS In experiment 1, specific pathogen-free Sprague-Dawley rats underwent 35% total body surface area burn. At 4, 8, 16, and 24 hrs after burn, intestinal mucosa and lung tissue were harvested for myeloperoxidase (MPO) assay, blood was collected for measurement of peroxynitrite-mediated oxidation of dihydrorhodamine 123, and pulmonary microvascular dysfunction was quantified by measuring the extravasation of Evans blue dye. In experiment 2, polymorphonuclear granulocyte antibody (0.12 mL/100 g administered intraperitoneally 16 hrs before burn), S-methylisothiourea (7.5 mg/kg, intraperitoneally, immediately after burn), a specific inducible nitric oxide synthase inhibitor, and sterile saline (15 mL/kg, intraperitoneally, immediately after burn) were given to different groups of thermally injured animals individually. The plasma dihydrorhodamine 123 oxidation level, intestinal and lung MPO activity, lung permeability, and lung histology were evaluated at 8 hrs after burn. The cellular localization of nitrotyrosine, a marker for peroxynitrite reactivity, was also examined by immunostaining. In experiment 3, 3-morpholinosydnonimine (10 mM, intraperitoneally), a peroxynitrite donor, was given to nonburned rats to examine the peroxynitrite effect on lung inducible nitric oxide synthase expression. MEASUREMENTS AND MAIN RESULTS The level of MPO activity in intestine and lung, blood dihydrorhodamine 123 oxidation, and lung permeability were increased up to 2-fold, 2.5-fold, 2-fold, and 2-fold of normal, respectively, at 8 hrs after burn. S-methylisothiourea injection significantly decreased (p <.05) 31% of the lung MPO activity, 41% of the blood peroxynitrite level, 54% of the lung permeability, and the lung peroxynitrite production in burned rats. Polymorphonuclear granulocyte antibody pretreatment significantly decreased 60% of the intestinal MPO, 92% of the blood peroxynitrite level, and 56% the lung MPO activity in burned rats, but the lung permeability was only slightly decreased by polymorphonuclear granulocyte antibody pretreatment. Furthermore, 3-morpholinosydnonimine increased the lung inducible nitric oxide synthase messenger RNA levels. CONCLUSIONS Thermal injury induces blood dihydrorhodamine 123 oxidation, intestinal and lung neutrophil deposition, lung nitrotyrosine production, and lung damage. Both specific inhibition of inducible nitric oxide synthase and polymorphonuclear granulocyte antibody pretreatment decrease blood dihydrorhodamine 123 oxidation and intestinal and lung neutrophil deposition, but only inducible nitric oxide synthase inhibition with S-methylisothiourea reduces lung peroxynitrite production and thermal injury-induced lung damage. Nitric oxide and the ensuing peroxynitrite production in lung play a more important role than neutrophil in contributing to thermal injury-induced lung damage.
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Affiliation(s)
- Lee-Wei Chen
- Department of Surgery, Kaohsiung Veterans General Hospital, National Yang-Ming Medical University, Taipei, Taiwan
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Samel S, Keese M, Lanig S, Kleczka M, Gretz N, Hafner M, Sturm J, Post S. Supplementation and inhibition of nitric oxide synthesis influences bacterial transit time during bacterial translocation in rats. Shock 2003; 19:378-82. [PMID: 12688551 DOI: 10.1097/00024382-200304000-00014] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
In the obstructed gut, nitric oxide (NO) may influence intestinal barrier function and translocation of bacteria. By using a novel experimental approach, we investigated the effect of supplementation and inhibition of NO synthesis on the time interval necessary for translocation of green fluorescent protein-transfected Escherichia coli (GFP-uv E. coli) in a rat model of small bowel obstruction. In anesthetized Wistar rats, 4 x 10(8) GFP-uv E. coli were administered into a reservoir of terminal ileum formed by ligature. Animals were randomized to receive either i.v. arginine (10 mg/kg), aminoguanidine (300 mg/kg), L-NAME (25 mg/kg), or saline (control). Translocation of GFP-uv E. coli was assessed using intravital video microscopy. Minimal transit time of translocation was measured as time from injection of GFP-uv E. coli into the gut lumen until bacteria were observed in the lamina submucosa and as time from injection of bacteria into the gut lumen until bacteria were observed in the lamina muscularis propria. Minimal transit times were expressed as mean +/- SD. Bacterial translocation into the submucosa and muscularis propria took 36 +/- 7 min and 81 +/- 9 min, respectively in control animals receiving saline. Aminoguanidine and L-NAME caused a marked delay of minimal transit time into the submucosa (63 +/- 5 min and 61 +/- 7 min, respectively; P < 0.05). Arginine significantly accelerated bacterial translocation into the muscularis propria (61 +/- 9 min, P < 0.05). GFP-uv E. coli were detected on frozen sections of small bowel, mesentery, liver, and spleen 2 h after GFP-uv E. coli administration in all animals. A marked upregulation of inducible NO synthase (NOS) in the obstructed bowel segment was demonstrated on immunohistochemistry. The assessment of a newly defined parameter, minimal bacterial transit time, may serve as an additional functional aspect of intestinal barrier function for pathophysiological and pharmacological studies. Aminoguanidine, L-NAME, and arginine were effective in influencing minimal transit time of E. coli during small bowel obstruction.
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Affiliation(s)
- Stephan Samel
- Department of Surgery, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany
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Abstract
AIM: To investigate the role of tri-iodothyronine supplement in protecting gut barrier in septic rats.
METHODS: Twenty-two rats were randomized into three groups: sham group (n = 6), sepsis group (n = 8), and sepsis plus tri-iodothyronine (T3) group (n = 8). Septic rat model was established through cecal ligation and puncture (CLP). After 5 h, sham and sepsis groups received saline, and the remaining group received T3 intraperitoneally. Twenty-one hours after CLP, intestinal permeability and serum free T3 and T4 were measured with fluorescence spectrophotometer and by radioimmunoassay, respectively. Intestinal ultrastructure and histologic morphology were observed under transmission electron microscopy (TEM) and light microscopy, respectively.
RESULTS: After 21 h, septic symptoms and signs in sepsis plus T3 group were milder than those in sepsis group. Serum FT3 or FT4 concentration in sepsis group was lower than that in sham group (1.59 ± 0.20, 3.41 ± 2.14 pmol/L vs 3.44 ± 1.40,9.53 ± 3.39 pmol/L, P < 0.05), and FT3 concentration in sepsis plus T3 group (3.40 ± 1.65 pmol/L, P < 0.05) was corrected. Portal concentration of fluorescein isothiocyanate-dextran (FITC-D) in sepsis group (2.51 ± 0.56 mg/L) was higher than that in sham group (1.22 ± 0.21 mg/L) (P < 0.01), and in sepsis plus T3 group (1.68 ± 0.38 mg/L) it was decreased significantly (P < 0.01). TEM and light microscopy showed that T3 supplement preserved well ultrastructure and morphology of intetinal mucosa in septic rats.
CONCLUSION: Tri-iodothyronine supplement protects gut barrier in septic rats.
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Affiliation(s)
- Zhi-Li Yang
- Department of Surgery, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
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Abstract
The activation of a pro-inflammatory cascade after burn injury appears to be important in the development of subsequent immune dysfunction, susceptibility to sepsis and multiple organ failure. Macrophages are major producers of pro-inflammatory mediators and their productive capacity for these mediators is markedly enhanced following thermal injury. Thus, macrophage hyperactivity (as defined by increased productive capacity for pro-inflammatory mediators) appears to be of critical importance in the development of post-burn immune dysfunction. This review will focus on the current state of knowledge with regards to the role of macrophages in the development of post-burn immune dysfunction. Particular areas of discussion include: nitric oxide synthase (NOS) and cyclooxygenase (COX) enzyme systems, macrophages and the T-helper (Th)-1/Th-2 cytokine responses, alterations in macrophages signal transduction and a potential role for gamma/delta T-cells in the development of macrophage hyperactivity following thermal injury. A more comprehensive understanding of the relationship between macrophage activity and post-burn immune dysfunction will hopefully provide the basis for improved therapeutic regimes in the treatment of burn patients.
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Affiliation(s)
- Martin G Schwacha
- Center for Surgical Research, University of Alabama at Birmingham, G094 Volker Hall, 1670 University Boulevard, Birmingham, AL 35294-0019, USA.
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Cornish AS, Jijon H, Yachimec C, Madsen KL. Peroxynitrite enhances the ability of Salmonella dublin to invade T84 monolayers. Shock 2002; 18:93-6. [PMID: 12095142 DOI: 10.1097/00024382-200207000-00017] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
In the intestine, epithelial cells continually produce and secrete low levels of nitric oxide (NO). Salmonella sp. invade epithelium by responding to environmental stimuli. The aims of this study were to determine the effect of reactive nitrogen intermediates (RNIs) on S. dublin and S. typhimurium growth and invasion of T84 epithelial monolayers. Intracellular NO formation was inhibited by 7-nitroindazole (7-NI) or N(G)-monomethyl-L-arginine, monoacetate (L-NMMA); extracellular NO and peroxynitrite were scavenged with ferro-hemoglobin or urate. The effect of authentic peroxynitrite (ONOO-); 3-morpholino-sydnonimine (SIN-1), which releases ONOO- via NO and superoxide; spermine NONOate, which releases only NO; or superoxide generated by xanthine oxidase and pterin on S. dublin and S. typhimurium growth and invasion were examined. Inhibition of NO synthesis and scavenging of extracellular NO or peroxynitrite reduced S. dublin invasion into T84 monolayers and enhanced bacterial growth. Pre-exposure of S. dublin to ONOO- and SIN-1 increased subsequent bacterial invasion into T84 monolayers. Conversely, exposure of bacteria to spermine NONOate or superoxide did not affect S. dublin invasion. In contrast, S. typhimurium invasion was not affected by pre-treatment with NO donors. In conclusion, exposure of S. dublin to ONOO- enhances the ability of the bacteria to invade epithelial cells. These results suggest that luminal ONOO- may have a novel role as an extracellular signal between invasive bacteria and epithelial cells.
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Affiliation(s)
- Anthony S Cornish
- Division of Gastroenterology, University of Alberta, Edmonton, Canada
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Hsu CM, Wang JS, Liu CH, Chen LW. Kupffer cells protect liver from ischemia-reperfusion injury by an inducible nitric oxide synthase-dependent mechanism. Shock 2002; 17:280-5. [PMID: 11954827 DOI: 10.1097/00024382-200204000-00007] [Citation(s) in RCA: 65] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
The aim of this study was to investigate the role of nitric oxide (NO) in rat hepatic ischemia-reperfusion (I/R) injury. Animals were divided into four groups: Group I, control; Group II, gadolinium chloride (GdCl3), a Kupffer cell depleting agent, pretreated; Group III, S-methylisothiourea (SMT), a potent inducible NO synthase (iNOS) inhibitor, pretreated; Group IV, pretreated with SMT, then treated with S-Nitroso-N-acetylpenicillamine (SNAP), a NO donor, after ischemia. Sprague-Dawley rats underwent left lateral and median lobe ischemia for 60 min and reperfusion for 120 min. The left lateral and median lobes were used as ischemic lobes, and the right lateral lobe in the same rat was used as a control lobe. The total NOS (tNOS), iNOS, constitutive NOS (cNOS) activity, and liver protein were determined. The liver tissue malonaldehyde (MDA) level was measured as an index of lipid peroxidation. Liver histology was also examined. The liver tNOS activity in ischemic lobes of Group I, II, III, and IV was increased by 214%, 86%, 61%, and 45%, respectively. The increase in tNOS activity is mainly due to the induction of iNOS activity in the ischemic lobes of rat liver. GdCl3 significantly decreased the tNOS by 66% in the ischemic lobes. GdCl3 significantly increased MDA by 39% in the ischemic lobes. SMT significantly decreased tNOS and iNOS activity by 66% and 85% in ischemic lobes. SMT increased MDA by 67% in the ischemic lobes. SMT + SNAP treatment increased iNOS activity by 117% in the ischemic lobes in comparison with the ischemic lobes of the SMT group. SMT + SNAP treatment decreased MDA by 39% in the ischemic lobes. SMT + SNAP treatment also decreased the sinusoidal congestion and spotty necrosis of hepatocytes in the ischemic lobes. iNOS immunostaining showed an obvious increase in sinusodial area of the ischemic lobes where most Kupffer cells were interspersed. In conclusion, in this model of liver I/R injury, I/R increased the activity of tNOS and iNOS, but not the cNOS activity. Kupffer cells might be the major source of the induction of iNOS activity. The iNOS specific inhibitor SMT increased the lipid peroxidation and the tissue damage in hepatic I/R injury. On the contrary, the NO donor SNAP increased the activity of iNOS and decreased the hepatic injury in this study. Kupffer cells could protect liver from I/R injury by an iNOS-dependent mechanism, thus NO production has a beneficial role in hepatic IR injury.
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Affiliation(s)
- Ching-Mei Hsu
- Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan
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Wang Q, Hasselgren PO. Heat shock response reduces intestinal permeability in septic mice: potential role of interleukin-10. Am J Physiol Regul Integr Comp Physiol 2002; 282:R669-76. [PMID: 11832385 DOI: 10.1152/ajpregu.00606.2001] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Sepsis and other critical illnesses are associated with increased permeability of the intestinal mucosa. Loss of mucosal integrity may lead to multiple organ failure in these conditions. We tested the hypothesis that induction of the heat shock response reduces sepsis-induced increase in intestinal permeability. The heat shock response was induced in mice by intraperitoneal injection of 10 mg/kg sodium arsenite. Two hours later, at which time mucosal heat shock protein 72 levels were increased, sepsis was induced by cecal ligation and puncture (CLP) or sham operation was performed. Sixteen hours after sham operation or CLP, intestinal permeability was determined by measuring the appearance in blood of 4.4-kDa fluorescein isothiocyanate-conjugated dextran and 40-kDa horseradish peroxidase administered by gavage. Sepsis resulted in increased mucosal permeability for both markers, and this effect of sepsis was substantially reduced in mice treated with sodium arsenite. Plasma levels of the anti-inflammatory cytokine interleukin (IL)-10 were increased in septic mice pretreated with sodium arsenite, and the protective effect of sodium arsenite on intestinal permeability in septic mice was reversed by treatment with anti-IL-10 antibody. The present results suggest that sepsis-induced increase in mucosal permeability can be reduced by the heat shock response and that increased IL-10 levels may be involved in the protective effects of the heat shock response.
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Affiliation(s)
- Quan Wang
- Department of Surgery, University of Cincinnati, Cincinnati, Ohio 45267, USA
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Forsythe RM, Xu DZ, Lu Q, Deitch EA. Lipopolysaccharide-induced enterocyte-derived nitric oxide induces intestinal monolayer permeability in an autocrine fashion. Shock 2002; 17:180-4. [PMID: 11900335 DOI: 10.1097/00024382-200203000-00004] [Citation(s) in RCA: 64] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Studies indicate that endotoxin (LPS) causes intestinal injury, increases inducible nitric oxide synthase (iNOS) activity, leads to increased NO production, and promotes bacterial translocation (BT). To investigate the mechanism by which LPS causes gut injury and to test the hypothesis that NO produced by enterocytes promotes gut injury in an autocrine fashion, rat intestinal epithelial cell (IEC-6) monolayers were tested. IEC-6 monolayers grown in a bicameral system were incubated with media or with LPS (25 microg/mL) and tested for permeability to phenol red, BT, and nitrate/nitrite (NO2/NO3) production. To determine the direct effect of NO on permeability, monolayers were incubated with the NO donor S-nitroso-acetylpenicillinamide (SNAP; 1 mM) and tested for permeability. Next, the protective effects of two NOS inhibitors (L-NMMA and L-NIL) were tested. Finally, to determine if LPS-induced permeability occurs via a poly (ADP-ribose) synthetase- (PARS) dependent pathway, monolayers incubated with LPS alone or with the PARS inhibitor, INH2BP (100 microM) were tested. LPS significantly increased IEC-6 permeability to phenol red, as well as increased NO2/NO3 by 20-fold (P < 0.001) and increased BT 10-fold (P < 0.001). SNAP mimicked the effect of LPS and significantly increased both permeability to phenol red and BT. Inhibition of iNOS significantly decreased the LPS-induced increase in monolayer permeability and BT (P < 0.05). Monolayers incubated with INH2BP had significantly decreased permeability to phenol red and BT, suggesting that LPS-induced NO production increases monolayer permeability at least in part via a PARS-dependent mechanism. In summary, LPS-induced disruption of monolayer barrier function appears to be related, at least in part, to enterocyte produced NO. This supports the hypothesis that NO produced by LPS-stimulated enterocytes promotes injury in an autocrine fashion and highlights the fact that enterocytes can be a target as well as a producer of NO.
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Affiliation(s)
- Raquel M Forsythe
- Department of Surgery, UMDNJ-New Jersey Medical School, Newark 07103, USA
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Wang Q, Fang CH, Hasselgren PO. Intestinal permeability is reduced and IL-10 levels are increased in septic IL-6 knockout mice. Am J Physiol Regul Integr Comp Physiol 2001; 281:R1013-23. [PMID: 11507020 DOI: 10.1152/ajpregu.2001.281.3.r1013] [Citation(s) in RCA: 96] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Sepsis is associated with increased intestinal permeability, but mediators and mechanisms are not fully understood. We examined the role of interleukin (IL)-6 and IL-10 in sepsis-induced increase in intestinal permeability. Intestinal permeability was measured in IL-6 knockout (IL-6 -/-) and wild-type (IL-6 +/+) mice 16 h after induction of sepsis by cecal ligation and puncture or sham operation. In other experiments, mice or intestinal segments incubated in Ussing chambers were treated with IL-6 or IL-10. Intestinal permeability was assessed by determining the transmucosal transport of the 4.4-kDa marker fluorescein isothiocyanate conjugated dextran and the 40-kDa horseradish peroxidase. Intestinal permeability for both markers was increased in septic IL-6 +/+ mice but not in septic IL-6 -/- mice. Treatment of nonseptic mice or of intestinal segments in Ussing chambers with IL-6 did not influence intestinal permeability. Plasma IL-10 levels were increased in septic IL-6 -/- mice, and treatment of septic mice with IL-10 resulted in reduced intestinal permeability. Increased intestinal permeability during sepsis may be regulated by an interaction between IL-6 and IL-10. Treatment with IL-10 may prevent the increase in mucosal permeability during sepsis.
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Affiliation(s)
- Q Wang
- Department of Surgery, the Shriners Hospitals for Children, Cincinnati, Ohio 45267-0558, USA
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Vento P, Kiviluoto T, Järvinen HJ, Kärkkäinen P, Kivilaakso E, Soinila S. Expression of inducible and endothelial nitric oxide synthases in pouchitis. Inflamm Bowel Dis 2001; 7:120-7. [PMID: 11383584 DOI: 10.1097/00054725-200105000-00007] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
Abstract
To study the induction of nitric oxide synthase (NOS) in different forms of pouchitis, we divided patients in five groups: 1) ulcerative colitis, no pouch; 2) no-pouchitis; 3) chronic asymptomatic pouchitis; 4) chronic active pouchitis; and 5) acute pouchitis. Ileal biopsies were scored for NOS-2 (inducible) and NOS-3 (endothelial) immunoreactivity and acute inflammation. In group 1, most specimens lacked NOS-2 immunoreactivity. In group 2, some specimens showed NOS-2 immunoreactive epithelium. In group 3, areas of NOS-2-immunoreactive epithelium were consistently observed in most specimens. In groups 4 and 5, most specimens showed moderate-to-extensive epithelial NOS-2 staining. NOS-2 immunoreactivity scores of groups 1-5 were 0.25 +/- 0.16, 0.67 +/- 0.19, 1.19 +/- 0.40, 2.0 +/- 0.23, and 2.18 +/- 0.12, respectively. Corresponding acute inflammation scores were 0, 0.53 + 0.17, 1.00 +/- 0.33, 1.80 +/- 0.20, and 1.64 +/- 0.15. NOS-2 score correlated with acute inflammation score (p < 0.0001), indicating that NOS-2 induction correlates with both the clinical degree of pouchitis and the severity of acute inflammation. NOS-3 immunoreactivity increased in all pouchitis groups.
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Affiliation(s)
- P Vento
- Department of Surgery, Helsinki University Central Hospital, Finland
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Sir O, Fazal N, Choudhry MA, Gamelli RL, Sayeed MM. Neutrophil depletion prevents intestinal mucosal permeability alterations in burn-injured rats. Am J Physiol Regul Integr Comp Physiol 2000; 278:R1224-31. [PMID: 10801291 DOI: 10.1152/ajpregu.2000.278.5.r1224] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Cutaneous thermal injury increases intestinal mucosal permeability. The mechanisms of this functional disturbance are not fully understood. We investigated whether accumulation of neutrophils in the intestine contributes to the increase in mucosal permeability. Labeled and unlabeled lactulose and mannitol were infused into a segment of rat ileum or jejunum. Blood concentrations of [(3)H]lactulose and [(14)C]mannitol were measured after 30, 60, and 90 min. On day 1 postburn, lactulose permeability increased fourfold in the ileum and twofold in the jejunum compared with sham-burned rats; mannitol permeability increased twofold in the ileum and 1. 5-fold in the jejunum. A greater increase in permeability occurred on day 3 postburn in the ileum, but not in the jejunum. The depletion of neutrophils in burned rats prevented the increase in permeability in both segments on day 1 postburn. Histological studies of intestines from burned, with or without neutrophil depletion, and sham-burned rats showed similar morphology. However, numerous neutrophils were found in the extravascular compartment in day 1 postburn, but not in neutrophil-depleted and sham-burned rats. These findings support the concept that the burn-induced increase in mucosal permeability is produced during the accumulation of neutrophils in the intestine and can be abrogated by the depletion of neutrophils.
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Affiliation(s)
- O Sir
- Trauma and Critical Care Research Labs, Burn & Shock Trauma Institute, Loyola University Chicago Medical Center, Maywood, Illinois 60153, USA
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Abstract
Simultaneous generation of nitric oxide (NO*) and superoxide (O2-) can lead to the formation of peroxynitrite (ONOO-), a potent oxidant that has been implicated in the pathogenesis of a number of disease states. This study was designed to investigate the possible generation of ONOO- in local cutaneous tissues following thermal injury. Male Wistar rats were anaesthetised in a nonrecovery procedure and subjected to a small (1 cm diameter), abdominal burn of moderate temperature (50 degrees C, 5-15 min). At either the 60 or 180 min time point postburn the animals were killed, and skin sites were removed and homogenised. An ELISA was used to quantify protein bound 3-nitrotyrosine (3NT), a biomarker for ONOO- in the rat skin. In separate experiments the accumulation of [125I]-albumin in thermally injured skin was used to calculate plasma extravasation. Thermal injury (50 degrees C, 10 min) to rat abdominal skin caused a significant increase in both 3NT (p < 0.05) and oedema formation (p < 0.001) when compared to unheated control sites at the 180 min time point postburn. This data is the first to show protein nitration in thermally injured, oedematous skin and strongly suggests that ONOO- is generated in thermally damaged cutaneous tissue.
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Affiliation(s)
- A Rawlingson
- Cardiovascular Biology & Medicie, New Hunt's House, King's College London, UK
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Lazaron V, Dunn DL. NO barrier too tight. Crit Care Med 1999; 27:2318-9. [PMID: 10548243 DOI: 10.1097/00003246-199910000-00058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
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