Equol, a soybean metabolite with estrogen-like functions, decreases lipopolysaccharide-induced human neutrophil extracellular traps in vitro.

OBJECTIVE: Neutrophil extracellular traps (NETs) defend against acute infections. However, their overexpression causes organ failure during sepsis. Control of NET formation may improve the outcomes of patients with sepsis. Equol, a soybean isoflavone, is a female hormone analog, which prevents inflammation. We evaluated the effects of equol on NET formation in human neutrophils during inflammatory stimulation in vitro. METHODS: Healthy volunteers provided blood samples. An enzyme-linked immunosorbent assay (ELISA) assessed serum equol concentrations. NET formation in neutrophils was induced by lipopolysaccharide (LPS) treatment. ELISA quantified DNA-binding elastase, and immunostaining assessed NET formation. Reverse-transcription quantitative PCR and western blotting detected G-protein-coupled receptor 30 (GPR30) or peptidyl arginine deiminase 4 (PAD4) expression. Flow cytometry assessed neutrophil phagocytic ability with inactivated Escherichia coli. RESULTS: In neutrophils derived from males with low-serum equol levels (low-serum equol group), equol significantly decreased DNA-binding elastase levels and NET formation. Equol did not decrease NETs in neutrophils from males with high-serum equol levels. GPR30 expression of neutrophils was higher in the low-serum than in the high-serum equol group. PAD4 mRNA levels and nuclear PAD4 protein expression also decreased than the vehicle control in the low-serum equol group. Equol did not alter the phagocytic ability of neutrophils. In neutrophils from young females, equol had no inhibitory effect on NET formation. CONCLUSIONS: Equol decreases LPS-induced NET formation in neutrophils from males via inhibition of PAD4 expression. Our findings provide a rationale for investigating a new therapeutic approach using equol to control neutrophil activity during sepsis.

The role of death receptor signaling pathways in mouse Sertoli cell avoidance of apoptosis during LPS- and IL-18-induced inflammatory conditions.

Lipopolysaccharide (LPS) triggers infectious acute inflammation, and interleukin (IL)-18 is an inflammasome-mediated cytokine. We previously demonstrated that endogenous IL-18 induces testicular germ cell apoptosis during acute inflammation when plasma IL-18 levels are high. Additionally, high-dose recombinant IL-18 (rIL-18) induced Leydig cell apoptosis. The blood-testis barrier formed by Sertoli cells protects testicular germ cells from both exogenous and endogenous harmful substances. However, the impact of LPS and IL-18 on Sertoli cells remained unclear. We stimulated TM4 cells, a mouse Sertoli cell line, with LPS (200 or 1000 ng/mL) or rIL-18 (0.1-100 ng/mL) at levels that induced Leydig cell apoptosis in our previous study and assessed caspase 3 cleavage and the mRNA expression of inflammatory cytokines and markers of apoptotic pathways (Tnfr1, Fasl, Fas, Fadd) after stimulation. Il6 mRNA was increased by LPS stimulation. Tnfα mRNA was increased by 200 ng/mL LPS but not 1000 ng/mL LPS. Fas was increased, but Fasl was decreased, by LPS. LPS had little influence on Tnfr1 or Fadd mRNA expression and did not induce apoptosis. Il18 mRNA was not increased, and Il18r1 was significantly decreased following LPS treatment. Treatment with rIL-18 increased Il18r1 mRNA and induced inflammation, but decreased Tnfr1 and had little influence on apoptosis, as indicated by Tnfα, Fasl, Fas, Fadd and cleaved caspase 3. These results suggested that Sertoli cells do not easily undergo apoptosis despite strong inflammatory stimuli. Additionally, Sertoli cells may resist inflammation and play a larger role in protecting testicular homeostasis than other component cells of the testis.

High Plasma tPAPAI-1C Levels May Be Related to a Poor Prognosis in Patients with Severe or Critical COVID-19: A Single-Center Retrospective Study.

Severe novel coronavirus disease 2019 (COVID-19) patients have a high incidence of thrombotic complications and mortality. The pathophysiology of coagulopathy involves fibrinolytic system impairment and vascular endothelial damage. This study examined coagulation and fibrinolytic markers as outcome predictors. In an observational study of 164 COVID-19 patients admitted to our emergency intensive care unit, hematological parameters on days 1, 3, 5, and 7 were retrospectively compared between survivors and nonsurvivors. Nonsurvivors had a higher APACHE II score, SOFA score, and age than survivors. Nonsurvivors also had a significantly lower platelet count and significantly higher plasmin/α2plasmin inhibitor complex (PIC), tissue plasminogen activator/plasminogen activator inhibitor-1 complex (tPAPAI-1C), D-dimer, and fibrin/fibrinogen degradation product (FDP) levels than survivors throughout the measurement period. The 7-day maximum or minimum values of the tPAPAI-1C, FDP, and D-dimer levels were significantly higher in nonsurvivors. A multivariate logistic regression analysis showed that the maximum tPAPAI-1C (OR = 1.034; 95% CI,1.014-1.061; p = 0.0041) was an independent factor affecting mortality, with an area under the curve (AUC) of 0.713 (optimum cut-off of 51 ng/mL; sensitivity, 69.2%; and specificity, 68.4%). COVID-19 patients with poor outcomes exhibit exacerbated coagulopathy with fibrinolysis inhibition and endothelial damage. Consequently, plasma tPAPAI-1C might be a useful predictor of the prognosis in patients with severe or critical COVID-19.

The effects of inhaling hydrogen gas on macrophage polarization, fibrosis, and lung function in mice with bleomycin-induced lung injury.

BACKGROUND: Acute respiratory distress syndrome, which is caused by acute lung injury, is a destructive respiratory disorder caused by a systemic inflammatory response. Persistent inflammation results in irreversible alveolar fibrosis. Because hydrogen gas possesses anti-inflammatory properties, we hypothesized that daily repeated inhalation of hydrogen gas could suppress persistent lung inflammation by inducing functional changes in macrophages, and consequently inhibit lung fibrosis during late-phase lung injury. METHODS: To test this hypothesis, lung injury was induced in mice by intratracheal administration of bleomycin (1.0 mg/kg). Mice were exposed to control gas (air) or hydrogen (3.2% in air) for 6 h every day for 7 or 21 days. Respiratory physiology, tissue pathology, markers of inflammation, and macrophage phenotypes were examined. RESULTS: Mice with bleomycin-induced lung injury that received daily hydrogen therapy for 21 days (BH group) exhibited higher static compliance (0.056 mL/cmH2O, 95% CI 0.047-0.064) than mice with bleomycin-induced lung injury exposed only to air (BA group; 0.042 mL/cmH2O, 95% CI 0.031-0.053, p = 0.02) and lower static elastance (BH 18.8 cmH2O/mL, [95% CI 15.4-22.2] vs. BA 26.7 cmH2O/mL [95% CI 19.6-33.8], p = 0.02). When the mRNA levels of pro-inflammatory cytokines were examined 7 days after bleomycin administration, interleukin (IL)-6, IL-4 and IL-13 were significantly lower in the BH group than in the BA group. There were significantly fewer M2-biased macrophages in the alveolar interstitium of the BH group than in the BA group (3.1% [95% CI 1.6-4.5%] vs. 1.1% [95% CI 0.3-1.8%], p = 0.008). CONCLUSIONS: The results suggest that hydrogen inhalation inhibits the deterioration of respiratory physiological function and alveolar fibrosis in this model of lung injury.

Interleukin-18 levels and mouse Leydig cell apoptosis during lipopolysaccharide-induced acute inflammatory conditions.

Interleukin (IL)-18 is an inflammasome-mediated cytokine produced by germ cells, Leydig cells, and resident macrophages that is indispensable in the maintenance of homeostasis in the testis. We previously demonstrated that endogenous IL-18 induces testicular germ cell apoptosis during acute inflammation when plasma IL-18 levels are very high. However, the impact of acute inflammation and IL-18 on Leydig cells remained unclear. TM3 cells, a mouse Leydig cell line, and RAW264.7 cells, a mouse macrophage cell line, were stimulated with lipopolysaccharide (LPS) or recombinant IL-18 (rIL-18). We assessed the expression of inflammatory cytokines, caspase cleavage, and markers of apoptotic pathways. In Leydig cells, caspase 3 cleavage was increased and death-receptor-mediated apoptotic pathways were activated after LPS stimulation. However, LPS stimulation did not increase IL-18 expression in the Leydig cell line. When high-dose rIL-18 was administered to the Leydig cell line to mimic levels seem after inflammation, rIL-18 upregulated Tnf-α mRNA, Fadd mRNA, and Fas protein, promoted cleavage of caspase-8 and caspase-3, and induced apoptosis. Low-dose rIL-18 did not stimulate apoptosis. To determine if the high level of IL-18 seen in the testes after inflammation was derived from immune cells, we examined IL-18 protein expression in a macrophage cell line, RAW264.7. In contrast to the TM3 cells, IL-18 was significantly increased in RAW264.7 cells after LPS stimulation. These results suggest that high-dose IL-18 derived from macrophages is harmful to Leydig cells. Reducing the overexpression of IL-18 could be a new therapeutic approach to prevent Leydig cell apoptosis as a result of acute inflammation.

Inhibitory Effects of Short-Chain Fatty Acids and ω-3 Polyunsaturated Fatty Acids on Profibrotic Factors in Dermal Fibroblasts.

Objective: Dermal fibroproliferative disorders impair patients' quality of life. Although several therapeutic approaches exist for treatment of dermal scars, the development of effective ointments with few adverse effects could improve these therapeutic methods. Short-chain and ω-3 polyunsaturated fatty acids are reported to be immunomodulators with anti-inflammatory properties. Our aim was to evaluate anti-inflammatory and antifibrogenic effects of these fatty acids in human dermal fibroblasts. Methods: Cells were incubated with short-chain fatty acids (butyrate or propionate; 0-16 mM) and/or ω-3 polyunsaturated fatty acids (docosahexaenoic acid or eicosapentaenoic acid; 0-100 µM) for 24 hours to evaluate antifibrogenic effects and for 3 or 48 hours to evaluate anti-inflammatory effects after stimulation with lipopolysaccharide or without stimulation. Expression levels of α-smooth muscle actin, collagen I, collagen III, and IL-6 were evaluated, as were cell proliferation, stress fiber formation, and histone acetylation. Results: In the lipopolysaccharide-unstimulated group, butyrate inhibited mRNA expression of α-smooth muscle actin and collagen III more effectively than propionate and increased histone acetylation. Docosahexaenoic acid inhibited mRNA expression of α-smooth muscle actin and collagen III, whereas eicosapentaenoic acid did not. Combining butyrate with docosahexaenoic acid had stronger effects, downregulating α-smooth muscle actin, collagen I, and collagen III mRNA. As for cell proliferation and stress fiber formation, butyrate acted as a stronger inhibitor than docosahexaenoic acid and the combined administration had stronger effects. In the lipopolysaccharide-stimulated group, butyrate and docosahexaenoic acid attenuated IL-6 mRNA upregulation by lipopolysaccharide. Conclusion: Butyrate and docosahexaenoic acid may be a novel therapeutic approach to treatment of dermal fibroproliferative disorders.

Inhaled hydrogen ameliorates endotoxin-induced bowel dysfunction.

Aim: Gastrointestinal dysmotility frequently occurs during sepsis and multiple organ failure, remaining a major cause of morbidity and mortality in critically ill patients. Previous studies have shown that hydrogen, a new therapeutic gas, can improve various organ damage associated with sepsis. In this study, we investigated the protective efficacies of inhaled hydrogen against lipopolysaccharide (LPS)-induced ileus. Methods: Sepsis was induced in rats and mice by a single i.p. injection of LPS at 15 mg/kg for mice and 5 mg/kg for rats. Four groups of rats and mice including sham/air, sham/hydrogen, LPS/air, and LPS/hydrogen were analyzed. Hydrogen (1.3%) was inhaled for 25 h beginning at 1 h prior to LPS treatment. Gastrointestinal transit was quantified and cytokine levels, as well as neutrophil extravasation, in the intestinal muscularis propria were determined. Results: Lipopolysaccharide challenge remarkably delayed gastrointestinal transit of non-absorbable dextran, associated with increased leukocyte recruitment and upregulation of pro-inflammatory cytokine mRNA expressions in the muscularis propria. Hydrogen significantly prevented LPS-induced bowel dysmotility and reduced leukocyte extravasation, as well as inhibition of inflammatory cytokine expression. In vitro analysis of cytokine levels after LPS treatment of cultured macrophages showed an increase of interleukin-10 by hydrogen regardless of the presence of nitric oxide. Conclusions: This study showed the protective effects of hydrogen inhalation on LPS-induced septic ileus through inhibition of inflammation in the muscularis propria. These inhibitory effects on the pro-inflammatory response may be partially derived from anti-inflammatory cytokine interleukin-10 induction.

Suppression of fibrosis in human pterygium fibroblasts by butyrate and phenylbutyrate.

AIM: To evaluate the antifibrogenic effects of butyrate or phenylbutyrate, a chemical derivative of butyrate, in human pterygium fibroblasts. METHODS: Human pterygium fibroblasts obtained from patient pterygium tissue were treated with butyrate or phenylbutyrate for 48h. Expression of α-smooth muscle actin, collagen I, collagen III and matrix metalloproteinase-1 mRNA was measured by quantitative real-time reverse transcription polymerase chain reaction, and acetylated histone was evaluated by Western blotting. RESULTS: Butyrate inhibited α-smooth muscle actin, type III collagen and matrix metalloproteinase-1 expressions, and phenylbutyrate inhibited types I and III collagen and matrix metalloproteinase-1 expressions without changing cell viability as well as both of these increased histone acetylation. These results suggested that butyrate and phenylbutyrate suppress fibrosis through a mechanism involving histone deacetylase inhibitor. CONCLUSION: This indicates that butyrate or phenylbutyrate have antifibrogenic effects in human pterygium fibroblasts and could be novel types of prophylactic and/or therapeutic drugs for pterygium, especially phenylbutyrate, which does not have the unpleasant smell associated with butyrate.

Combination therapy with butyrate and docosahexaenoic acid for keloid fibrogenesis: an in vitro study.

BACKGROUND:: A single, effective therapeutic regimen for keloids has not been established yet, and the development of novel therapeutic approaches is expected. Butyrate, a short-chain fatty acid, and docosahexaenoic acid (DHA), a ω-3 polyunsaturated fatty acid, play multiple anti-inflammatory and anticancer roles via their respective mechanisms of action. OBJECTIVE:: In this study, we evaluated the antifibrogenic effects of their single and combined use on keloid fibroblasts. METHODS:: Keloid fibroblasts were treated with butyrate (0-16 mM) and/or DHA (0-100 µM) for 48 or 96 h. RESULTS:: Butyrate inhibited cell proliferation, and α-smooth muscle actin (α-SMA) and type III collagen expressions, with inhibition of the transforming growth factor (TGF)-ß1 and TGF-ß type I receptor expressions and increased prostaglandin E2 with upregulation of cyclooxygenase-1 expression with induction of histone acetylation. DHA inhibited α-SMA, type III collagen, and TGF-ß type I receptor expressions. Then, the butyrate/DHA combination augmented the antifibrogenic effects, resulting in additional inhibition of α-SMA, type I and III collagen expressions, with strong disruption of stress fiber and apoptosis induction. Moreover, the butyrate/DHA combination inhibited the cyclooxygenase-2 expression, suggesting stronger anti-inflammatory effect than each monotherapy. STUDY LIMITATIONS:: Activation in keloid tissue is affected not only by fibroblasts but also by epithelial cells and immune cells. Evaluation of the effects by butyrate and DHA in these cells or in an in vivo study is required. CONCLUSION:: This study demonstrated that butyrate and docosahexaenoic acid have antifibrogenic effects on keloid fibroblasts and that these may exert therapeutic effects for keloid.

Combination therapy with butyrate and docosahexaenoic acid for keloid fibrogenesis: an in vitro study

Abstract: Background: A single, effective therapeutic regimen for keloids has not been established yet, and the development of novel therapeutic approaches is expected. Butyrate, a short-chain fatty acid, and docosahexaenoic acid (DHA), a ω-3 polyunsaturated fatty acid, play multiple anti-inflammatory and anticancer roles via their respective mechanisms of action. Objective: In this study, we evaluated the antifibrogenic effects of their single and combined use on keloid fibroblasts. Methods: Keloid fibroblasts were treated with butyrate (0-16 mM) and/or DHA (0-100 µM) for 48 or 96 h. Results: Butyrate inhibited cell proliferation, and α-smooth muscle actin (α-SMA) and type III collagen expressions, with inhibition of the transforming growth factor (TGF)-β1 and TGF-β type I receptor expressions and increased prostaglandin E2 with upregulation of cyclooxygenase-1 expression with induction of histone acetylation. DHA inhibited α-SMA, type III collagen, and TGF-β type I receptor expressions. Then, the butyrate/DHA combination augmented the antifibrogenic effects, resulting in additional inhibition of α-SMA, type I and III collagen expressions, with strong disruption of stress fiber and apoptosis induction. Moreover, the butyrate/DHA combination inhibited the cyclooxygenase-2 expression, suggesting stronger anti-inflammatory effect than each monotherapy. Study limitations: Activation in keloid tissue is affected not only by fibroblasts but also by epithelial cells and immune cells. Evaluation of the effects by butyrate and DHA in these cells or in an in vivo study is required. Conclusion: This study demonstrated that butyrate and docosahexaenoic acid have antifibrogenic effects on keloid fibroblasts and that these may exert therapeutic effects for keloid.

Low-Intensity Ultrasound Enhances Histone Acetylation and Inhibition of Interleukin 6 Messenger RNA Expression by the Histone Deacetylase Inhibitor Sodium Butyrate in Fibroblasts.

OBJECTIVES: Sodium butyrate, an inhibitor of histone deacetylase, has several therapeutic actions, including anti-inflammation. These actions depend on the concentration of sodium butyrate. In addition, lower concentrations have shown no effect on inflammation. Sonoporation by ultrasound can modify the permeability of the cell plasma membrane. Thus, the effects of sodium butyrate may be enhanced by the ultrasonic acoustics. Therefore, the facilitative effects of low-intensity ultrasound on histone acetylation and interleukin 6 (IL-6) regulation by sodium butyrate were investigated in this study. METHODS: Human dermal fibroblasts were treated with 1-mM sodium butyrate for 3 hours with 20 minutes of 0.1-W/cm2 pulsed or continuous ultrasound irradiation at the beginning of the sodium butyrate treatments. RESULTS: The combination of treatments with sodium butyrate and ultrasound significantly increased histone acetylation in fibroblasts (P < .05), whereas sodium butyrate could not increase histone acetylation. In addition, this combined treatment significantly suppressed the IL-6 messenger RNA expression level with lipopolysaccharide stimulation for 1 hour (P < .05). Meanwhile, the treatment with sodium butyrate alone could not suppress IL-6 messenger RNA expression in fibroblasts. These effects were achieved with both 20% pulsed and continuous ultrasound but not observed with ultrasound treatment alone. CONCLUSIONS: These results suggest that low-intensity ultrasound treatment promotes the physiologic actions of sodium butyrate as a histone deacetylase inhibitor.

Antithrombin III improved neutrophil extracellular traps in lung after the onset of endotoxemia.

BACKGROUND: Coagulation and inflammation are closely linked during acute inflammatory conditions, such as sepsis. Antithrombin (AT) is an anticoagulant that also has anti-inflammatory activities. The effects of therapeutically administering AT III after the onset of endotoxemia or sepsis were not clear. Here, we studied the effects of administering AT III after inducing lethal endotoxemia in mice. METHODS: Mice were injected intraperitoneally with lipopolysaccharide (LPS) to induce endotoxemia. AT III was administered 3 h later. We assessed survival and the severity of endotoxemia and quantified plasma cytokine levels and biochemical markers of liver and kidney function. In the lungs, we examined neutrophil accumulation, neutrophil extracellular traps, alveolar wall thickness, and chemokine (C-X-C motif) ligand 1 (cxcl-1), cxcl-2, and high mobility group box 1 expression. RESULTS: Administering AT III reduced the severity and mortality of LPS-induced endotoxemia as indicated by 24-h survival of 84% of the mice that received LPS + AT III and only 53% of mice given LPS alone (P < 0.05). AT III treatment attenuated several changes induced in the lungs by endotoxemia including cxcl-2 mRNA expression, high mobility group box 1 protein expression, neutrophil accumulation, alveolar septal thickening, and neutrophil extracellular trap formation. AT III did not decrease plasma cytokine levels or plasma urea nitrogen levels that were upregulated as a result of LPS-induced endotoxemia. CONCLUSIONS: Administration of AT III after the onset of endotoxemia improved outcomes in a mouse model. The attenuation of lung inflammation may have a large impact on mortality and morbidity. Because lung inflammation increases the likelihood of mortality from sepsis, AT III could be a useful agent in septic patients.

Interleukin-18 Reduces Blood Glucose and Modulates Plasma Corticosterone in a Septic Mouse Model.

BACKGROUND: Dysregulation of glucose metabolism, including hyperglycemia with insulin resistance, is commonly observed in critically ill patients. Interleukin-18 (IL-18) improves the insulin resistance associated with obesity, but the relationship between IL-18 and glucose metabolism in sepsis was unclear. The purpose of this study was to investigate the influence of IL-18 on hyperglycemia during sepsis. METHODS: Sepsis was induced using cecal ligation and puncture (CLP) in wild-type (WT) mice, IL-18 knockout (KO) mice, and IL-18 KO mice pretreated with recombinant IL-18. Blood glucose and plasma insulin, glucagon, and corticosterone were measured. The mRNAs for gluconeogenic enzymes (g6pc, pck1) and activation of insulin signaling were also analyzed. RESULTS: In both WT and IL-18 KO mice, CLP operation led to hyperglycemia that lasted longer (18 h) than after sham operation (6 h). Blood glucose levels in IL-18 KO mice were significantly higher than in WT mice, without alteration of insulin or glucagon levels. In IL-18 KO mice, insulin signaling in the liver and skeletal muscle was decreased during hyperglycemia as compared with WT mice without suppression of hepatic glucose production enzymes. Pretreatment with recombinant IL-18 reduced blood glucose levels after CLP. Additionally, corticosterone levels were higher after CLP in the presence of either endogenous or exogenous IL-18. CONCLUSION: IL-18 may reduce blood glucose by modulating insulin signaling in the liver during sepsis-induced hyperglycemia. IL-18 is an important factor associated with alterations in blood glucose during sepsis.

Donor pretreatment with carbon monoxide prevents ischemia/reperfusion injury following heart transplantation in rats.

Because inhaled carbon monoxide (CO) provides potent anti-inflammatory and antioxidant effects against ischemia reperfusion injury, we hypothesized that treatment of organ donors with inhaled CO would decrease graft injury after heart transplantation. Hearts were heterotopically transplanted into syngeneic Lewis rats after 8 hours of cold preservation in University of Wisconsin solution. Donor rats were exposed to CO at a concentration of 250 parts per million for 24 hours via a gas-exposure chamber. Severity of myocardial injury was determined by total serum creatine phosphokinase and troponin I levels at three hours after reperfusion. In addition, Affymetrix gene array analysis of mRNA transcripts was performed on the heart graft tissue prior to implantation. Recipients of grafts from CO-exposed donors had lower levels of serum troponin I and creatine phosphokinase; less upregulation of mRNA for interleukin-6, intercellular adhesion molecule-1, and tumor necrosis factor-α; and fewer infiltrating cells. Although donor pretreatment with CO altered the expression of 49 genes expressly represented on the array, we could not obtain meaningful data to explain the mechanisms by which CO potentiated the protective effects. Pretreatment with CO gas before organ procurement effectively protected cardiac grafts from ischemia reperfusion-induced injury in a rat heterotopic cardiac transplant model. A clinical report review indicated that CO-poisoned organ donors may be comparable to non-poisoned donors.

Intraperitoneally administered, hydrogen-rich physiologic solution protects against postoperative ileus and is associated with reduced nitric oxide production.

BACKGROUND: Postoperative ileus, a transient impairment of bowel motility initiated by intestinal inflammation, is common after an abdominal operation and leads to increased hospital stays and costs. Hydrogen has potent anti-inflammatory and antioxidant properties and potential therapeutic value. Solubilized hydrogen may be a portable and practical means of administering therapeutic hydrogen gas. We hypothesized that intraperitoneal administration of hydrogen-rich saline would ameliorate postoperative ileus. METHODS: Ileus was induced via surgical manipulation in mice and rats. The peritoneal cavity was filled with 1.0 mL saline or hydrogen-rich saline (≥1.5-2.0 ppm) before closure of the abdominal incision. Intestinal transit was assessed 24 hours postoperatively. Inflammation was examined by quantitation of neutrophil extravasation and expression of proinflammatory markers. Nitric oxide production was assessed in cultured muscularis propria. RESULTS: Surgical manipulation resulted in a marked delay in intestinal transit and was associated with upregulation of proinflammatory cytokines and increased neutrophil extravasation. Bowel dysmotility, induced by surgical manipulation and inflammatory events, was significantly attenuated by intra-abdominal administration of hydrogen-rich saline. Nitric oxide production in the muscle layers of the bowel was inhibited by hydrogen treatment. CONCLUSION: A single intraperitoneal dose of hydrogen-rich saline ameliorates postoperative ileus by inhibiting the inflammatory response and suppressing nitric oxide production.

Endogenous interleukin 18 regulates testicular germ cell apoptosis during endotoxemia.

Orchitis (testicular swelling) often occurs during systemic inflammatory conditions, such as sepsis. Interleukin 18 (IL18) is a proinflammatory cytokine and is an apoptotic mediator during endotoxemia, but the role of IL18 in response to inflammation in the testes was unclear. WT and IL18 knockout (KO) mice were injected lipopolysaccharide (LPS) to induce endotoxemia and examined 12 and 48  h after LPS administration to model the acute and recovery phases of endotoxemia. Caspase activation was assessed using immunohistochemistry. Protein and mRNA expression were examined by western blot and quantitative real-time RT-PCR respectively. During the acute phase of endotoxemia, apoptosis (as indicated by caspase-3 cleavage) was increased in WT mice but not in IL18 KO mice. The death receptor-mediated and mitochondrial-mediated apoptotic pathways were both activated in the WT mice but not in the KO mice. During the recovery phase of endotoxemia, apoptosis was observed in the IL18 KO mice but not in the WT mice. Activation of the death-receptor mediated apoptotic pathway could be seen in the IL18 KO mice but not the WT mice. These results suggested that endogenous IL18 induces germ cell apoptosis via death receptor mediated- and mitochondrial-mediated pathways during the acute phase of endotoxemia and suppresses germ cell apoptosis via death-receptor mediated pathways during recovery from endotoxemia. Taken together, IL18 could be a new therapeutic target to prevent orchitis during endotoxemia.

Hydrogen inhalation protects against acute lung injury induced by hemorrhagic shock and resuscitation.

INTRODUCTION: Hemorrhagic shock followed by fluid resuscitation (HS/R) triggers an inflammatory response and causes pulmonary inflammation that can lead to acute lung injury (ALI). Hydrogen, a therapeutic gas, has potent cytoprotective, antiinflammatory, and antioxidant effects. This study examined the effects of inhaled hydrogen on ALI caused by HS/R. METHODS: Rats were subjected to hemorrhagic shock by withdrawing blood to lower blood pressure followed by resuscitation with shed blood and saline to restore blood pressure. After HS/R, the rats were maintained in a control gas of similar composition to room air or exposed to 1.3% hydrogen. RESULTS: HS/R induced ALI, as demonstrated by significantly impaired gas exchange, congestion, edema, cellular infiltration, and hemorrhage in the lungs. Hydrogen inhalation mitigated lung injury after HS/R, as indicated by significantly improved gas exchange and reduced cellular infiltration and hemorrhage. Hydrogen inhalation did not affect hemodynamic status during HS/R. Exposure to 1.3% hydrogen significantly attenuated the upregulation of the messenger RNAs for several proinflammatory mediators induced by HS/R. Lipid peroxidation was reduced significantly in the presence of hydrogen, indicating antioxidant effects. CONCLUSION: Hydrogen, administered through inhalation, may exert potent therapeutic effects against ALI induced by HS/R and attenuate the activation of inflammatory cascades.

Endogenous interleukin 18 suppresses hyperglycemia and hyperinsulinemia during the acute phase of endotoxemia in mice.

BACKGROUND: Hyperglycemia associated with insulin resistance is common among critically ill patients. Interleukin (IL)-18 has been linked with hyperglycemia and insulin resistance in chronic disease, but the relation between IL-18 and insulin resistance during critical illness was unexplored. This study investigated whether IL-18 modulates hyperglycemia and insulin resistance during acute inflammation. METHODS: We injected lipopolysaccharide (LPS) 40 mg/kg into wild-type (WT) and IL-18 knockout (KO) mice to induce endotoxemia and examined insulin resistance and insulin-dependent signaling pathways during the acute phase. RESULTS: During the first hour after LPS treatment, IL-18 KO mice showed higher blood glucose and insulin and less insulin receptor substrate-1 and less phosphorylated Akt in the liver compared with WT mice. Interleukin-18 KO mice exhibited better survival after LPS treatment. CONCLUSIONS: The findings suggest that endogenous IL-18 may attenuate hyperglycemia and modulate insulin signaling in liver. Accordingly, IL-18 may modulate glucose tolerance during acute inflammation.

Oral tributyrin prevents endotoxin-induced lipid metabolism disorder.

BACKGROUND & AIMS: Sepsis leads to dysregulation of lipid and lipoprotein metabolism. Butyrate increases peroxisome proliferator-activated receptors (PPARs), which are key nuclear hormone receptors to induce fatty acid oxidation and synthesis. Oral administration of tributyrin, a prodrug of butyrate contained in dairy products, suppresses lipopolysaccharide (LPS)-induced liver injury through attenuating nuclear factor-κB activity with an increased hepatoportal butyrate level. In this study, we elucidated the protective effect of oral administration of tributyrin against LPS-mediated lipid metabolism disorder in rats. METHODS: Male Wistar rats were randomly divided and were administered tributyrin or vehicle orally 1 h before LPS injection and then sacrificed at 0, 1.5, 6, and 24 h after LPS. Liver tissue expressions of nuclear hormone receptors, enzymes associated with fatty acid metabolism, and histone acetylation were analyzed by real-time polymerase chain reaction or western blotting. Plasma lipids levels were measured. RESULTS: Tributyrin enhanced expression of PPARs and histone H3 in the liver at basal levels. Tributyrin suppressed LPS-induced repression of PPARs fatty acid oxidation-associated enzymes: fatty acid transport protein and fatty acid binding protein, and fatty acid synthesis-associated enzyme: sterol regulatory element binding protein-1c. Tributyrin reduced the increase in plasma triglyceride, total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels at 24 h after LPS injection. CONCLUSIONS: Oral tributyrin administration prevented elevation of plasma triglyceride, TC, and LDL-C levels through improved fatty acid oxidation in endotoxemic rats.

Changes of hepatic lipid mediators associated with intake of high-fat diet for 12 weeks in endotoxemic rats using LC-ESI-MS/MS.

BACKGROUND & AIMS: It has recently been reported that anti-inflammatory lipid mediators are increased in the late phase of acute inflammation, whereas proinflammatory lipid mediators are regulated at the initiation of inflammation. The purpose of this study was to evaluate changes of hepatic lipid mediators due to high-fat diet (HFD) feeding in endotoxemic rats. METHODS: Male Wistar rats were fed either HFD or control diet for 12 weeks, and were then killed 0, 1.5, and 6 h after lipopolysaccharide (LPS) injection. Analyses included lipidomics assessment of mediators using liquid chromatography-electrospray ionization/multi-stage mass spectrometry; measuring expression of hepatic polyunsaturated fatty acid (PUFA)-oxidizing enzyme, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and inducible nitric oxide synthase mRNA levels; blood biochemical tests; and liver histology. RESULTS: HFD feeding worsened liver injury, increased expression of TNF-α and IL-6 mRNA, and increased oxidative stress after LPS injection. PUFA-oxidizing enzymes were higher in HFD-fed rats after LPS injection. The proinflammatory prostaglandin (PG)E2 and thromboxane B2 were increased 1.5 h after LPS injection, and had decreased by 6 h in HFD-fed rats. In contrast, potent pro-resolving resolvins derived from eicosapentaenoic acid and docosahexaenoic acid were not detected, but anti-inflammatory epoxyeicosatrienoic acids, lipoxin A4, and 15-deoxy-PGJ2 were increased after LPS injection in HFD-fed rats. CONCLUSIONS: HFD feeding for 12 weeks enhanced proinflammatory lipid mediators 1.5 h after LPS injection suggesting relation to liver injury.