Lee-Wei Chen

Kupffer cells protect liver from ischemia-reperfusion injury by an inducible nitric oxide synthase-dependent mechanism

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.

TLR LIGAND DECREASES MESENTERIC ISCHEMIA AND REPERFUSION INJURY-INDUCED GUT DAMAGE THROUGH TNF-α SIGNALING

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 &agr; (TNF-&agr;) 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 &mgr;g/&mgr;L) 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 &kgr;B (NF-&kgr;B) and AP-1 DNA-binding activity; Bcl-w, TNF-&agr;, 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-&kgr;B and AP-1 DNA-binding activity, HSP70 protein expression, and decreased Bcl-w and TNF-&agr; 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-&kgr;B activity of the intestine is abolished in Tnfrsf1a−/− mice, we conclude that TLR ligand decreases mesenteric I/R injury-induced gut damage through TNF-&agr; signaling.

Commensal microflora contribute to host defense against Escherichia coli pneumonia through Toll-like receptors.

The influence of the gut-lung axis on the lung immunity, although appreciated, remains undefined mechanically. This study was designed to investigate whether commensal microflora in gut increase host defense against subsequent pneumonia through toll-like receptor (TLR) signaling and if oral TLR4 ligand supplementation enhances lung defense against bacterial challenge. We found that commensal gut depletion by antibiotic pretreatment before Escherichia coli pneumonia challenge induced a 15-fold and a 3-fold increase in bacterial counts in blood and lung, respectively, and a 30% increase of mortality when compared with the E. coli group. Commensal depletion also induced a suppression of cytokines expression as well as nuclear factor &kgr;B activity in intestine. Furthermore, LPS supplementation during antibiotic pretreatment reversed these effects. Commensal depletion also decreased bacterial killing activity of alveolar macrophages and increased IL-6 as well as IL-1&bgr; levels and keratinocyte-derived chemokine, macrophage inflammatory protein 2, and IL-1&bgr; expression of lung, and LPS supplementation reversed them. In conclusion, commensal gut microflora in the intestinal tract appear to be critical in inducing TLR4 expression as well as nuclear factor &kgr;B activation of intestine and lung innate defense against E. coli pneumonia.

Peroxynitrite is an important mediator in thermal injury-induced lung damage.

ObjectiveIntestinal 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. DesignProspective, experimental study. SettingResearch laboratory at a university hospital. SubjectsThermal injury models in the rat. InterventionsIn 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 ResultsThe 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. ConclusionsThermal 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.

Inhibition of inducible nitric oxide synthase (iNOS) prevents lung neutrophil deposition and damage in burned rats.

This study was designed to investigate the role of NO and effect of iNOS inhibitor on the lung neutrophil deposition and damage after burn. In Experiment 1, specific pathogen-free (SPF) Sprague-Dawley rats underwent 35% total body surface area (TBSA) burn. On the 4th, 8th, 16th, and 24th h after burn, blood was collected for peroxynitrite-mediated dihydrorhodamine 123 (DHR 123) oxidation assay, and lung tissues were harvested for myeloperoxidase (MPO) test and histologic study. Pulmonary microvascular dysfunction was quantitated by measuring the extravasation of Evans blue dye (EBD). In Experiment 2, S-methylisothiourea (SMT) was given (7.5 mg/kg, intraperitoneal immediately post-burn) to suppress iNOS activity. On the 8th h after burn, the effect of SMT on blood DHR 123 oxidation, lung MPO, lung damage, and lung iNOS expression were evaluated. Lung MPO activity increased up to a maximum of 2-fold 8 h after burn. Blood DHR 123 oxidation increased up to a maximum of 2-fold 8 h after burn. Lung permeability increased up to a maximum of 2.5-fold 4 h after burn. SMT significantly decreased lung MPO activity, blood DHR 123 oxidation, and lung permeability by 31%, 41%, and 54%, respectively. SMT markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and iNOS staining in bronchiolar epithelium, endothelial cells, and perivascular and interstitial inflammatory cells. In conclusion, thermal injury induces blood DHR 123 oxidation, lung neutrophil deposition, lung iNOS expression, and lung damage. Peroxynitrite might play an important role in thermal injury-induced lung neutrophil deposition and damage. Specific inhibition of lung iNOS expression and blood DHR 123 oxidation with SMT on thermal injury not only attenuated the lung neutrophil deposition, but also reduced lung damage.

Interleukin-1 mediates thermal injury-induced lung damage through C-Jun NH2-terminal kinase signaling.

Objective:The molecular mechanisms of lung damage following thermal injury are not clear. The purpose of this study was to determine whether interleukin (IL)-1 mediates burn-induced inducible nitric oxide synthase (iNOS) expression, peroxynitrite production, and lung damage through c-Jun NH2-terminal kinase (JNK) signaling. Design:Prospective, experimental study. Setting:Research laboratory at a university hospital. Subjects:Thermal injury models in the mice. Interventions:IL-1 receptor type 1 (IL-1R1)−/− mice, Tnfrsf1a−/− mice, and wild-type (WT) mice were subjected to 30% total body surface area third-degree burn. The JNK inhibitor, SP600125, was given to mice to study the involvement of the JNK pathway in thermal injury-induced lung damage. WT → WT, WT → IL-1R1−/−, and IL-1R1−/− → WT chimeric mice were generated to determine the role of hematopoietic cells in IL-1-mediated lung damage. Neutrophils were harvested and treated in vitro with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Measurements and Main Results:IL-1R1−/− mice rather than Tnfrsf1a−/− mice showed less thermal injury-induced lung damage. IL-1R1−/− mice displayed less lung JNK activity; intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), chemokine receptor 2 (CXCR2), and macrophage inflammatory protein-2 (MIP2), messenger RNA expression; myeloperoxidase activity; and neutrophil p38 mitogen-activated protein kinase (MAPK) phosphorylation after thermal injury. SP600125 significantly reduced thermal injury-induced blood dihydrorhodamine (DHR) 123 oxidation, iNOS expression, and lung permeability in WT mice but not in IL-1R1−/− mice. IL-1R1−/− → WT chimeric mice rather than WT → IL-1R1−/− chimeric mice showed less thermal injury-induced lung damage. fMLP increased reactive oxygen species (ROS) production of neutrophils in WT mice but not in IL-1R1−/− mice. SP600125 decreased ROS production of neutrophils in WT mice but not in IL-1R1−/− mice. Conclusions:Thermal injury-induced lung JNK activation; lung ICAM, VCAM, CXCR2, and MIP2 expression; and DHR 123 oxidation are IL-1 dependent. JNK inhibition decreases IL-1-mediated thermal injury-induced lung damage. Given that the IL-1 receptor is critical in thermal injury-induced p38 MAPK phosphorylation and ROS production of neutrophils, we conclude that IL-1 mediates thermal injury-induced iNOS expression and lung damage through the JNK signaling pathway.

Burn-induced lung damage in rat is mediated by a nitric oxide/cGMP system.

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, 1 H-[1,2,4] oxadiazolo [4,3-&agr;] quinoxalin-1-one (ODQ) was given (20 mg/kg ip) 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 &mgr;M, 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.

Inducible nitric oxide synthase inhibitor reverses exacerbating effects of hypertonic saline on lung injury in burn.

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.

Hypertonic saline-enhanced postburn gut barrier failure is reversed by inducible nitric oxide synthase inhibition

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.

Hypertonic saline enhances host defense and reduces apoptosis in burn mice by increasing toll-like receptors.

Hypertonic saline (HTS) is useful in the management of intracranial hypertension and shock patients. The aim of this study was to investigate whether HTS enhances host defense in burn mice through the increase of Toll-like receptors (TLRs) and nuclear factor &kgr;B (NF-&kgr;B) activation. C57BL/6, TLR4−/−, C3H/HeN, and C3H/HeJ (nonfunctional TLR4 mutant) mice underwent burn and were given 10 mL/kg HTS (7.5% NaCl, 1.28 mol/L), 10 mL/kg saline (154 mmol/L), or 80 mL/kg saline (154 mmol/L) at 8 h after burn. At 24 h after burn, mesenteric lymph nodes were harvested and assayed for bacterial translocation (BT). Next, animals received i.p. Escherichia coli challenge, and bacterial clearance was measured. Finally, peritoneal cells were isolated for assay of bacterial killing activity, phagocytic activity, apoptotic ratio, NF-&kgr;B DNA binding activity, and expression of TLR4, MyD88, p-Akt, pp38, macrophage inflammatory protein 2, and Bcl-xL. Hypertonic saline decreased BT in C57BL/6 and C3H/HeN mice but not in TLR4 mutant mice. Also, HTS increased bacterial clearance and bacterial killing activity and decreased apoptotic ratio of peritoneal cells from C57BL/6 and C3H/HeN mice but not TLR4−/− or C3H/HeJ mice. Finally, HTS increased NF-&kgr;B activity and expression of TLR4, MyD88, p-Akt, pp38, macrophage inflammatory protein 2, and Bcl-xL in C57BL/6 but not in TLR4−/− mice. Hypertonic saline increases bacterial clearance and bacterial killing activity and decreases thermal injury-induced BT in wild-type but not in TLR4 mutant mice. Given that HTS induces NF-&kgr;B activity and TLR4, MyD88, and pp38 expression but decreases the apoptosis of inflammatory cells, we conclude that HTS resuscitation enhances host defense against bacterial challenge and reduces apoptosis of inflammatory cells in burn mice by increasing TLR4 expression and NF-&kgr;B activation.