Bile and circulating HMGB1 contributes to systemic inflammation in obstructive jaundice.

BACKGROUND: Obstructive jaundice (OJ) patients with cholangitis are prone to sepsis; however, the underlying mechanisms are still not clear and need to be clarified. METHODS: Analyzing all available published data related to the title of this article. RESULTS: OJ leads to absence of gut luminal bile and accumulation of hepatic and circulating bile acids. Absence of gut luminal bile deprives the gut from its antiinflammatory, endotoxin-binding, bacteriostatic, mucosal-trophic, epithelial tight-junction maintaining, and gut motility-regulating effects, leading to gut bacterial overgrowth, mucosal atrophy, mucosal tight-junction loss, and gut motility dysfunction. These alterations promote intestinal endotoxin and bacterial translocation (BT) into portal and systemic circulation. Gut BT triggers systemic inflammation, which can lead to multiple organ dysfunctions in OJ. The accumulation of hepatic and circulating bile acids kills/damages hepatocyte and Kupffer cells, and it also significantly decreases the number of liver natural killer T-cells in OJ. This results in impaired hepatic and systemic immune function, which facilitates BT. In addition, neutralizing bile HMGB1 can reverse endotoxemic bile-induced gut BT and mucosal injury in mice, suggesting that bile HMGB1 in OJ patients can be responsible for internal drainage-related clinical complications. Moreover, the elevated circulating HMGB1 level may contribute to multiple organ injuries, and it might also mediate gut BT in OJ. CONCLUSIONS: HMGB1 may significantly contribute to systemic inflammation and multiple organ dysfunctions in OJ.

HMGB1 and Extracellular Histones Significantly Contribute to Systemic Inflammation and Multiple Organ Failure in Acute Liver Failure.

Acute liver failure (ALF) is the culmination of severe liver cell injury from a variety of causes. ALF occurs when the extent of hepatocyte death exceeds the hepatic regenerative capacity. ALF has a high mortality that is associated with multiple organ failure (MOF) and sepsis; however, the underlying mechanisms are still not clear. Emerging evidence shows that ALF patients/animals have high concentrations of circulating HMGB1, which can contribute to multiple organ injuries and mediate gut bacterial translocation (BT). BT triggers/induces systemic inflammatory responses syndrome (SIRS), which can lead to MOF in ALF. Blockade of HMGB1 significantly decreases BT and improves hepatocyte regeneration in experimental acute fatal liver injury. Therefore, HMGB1 seems to be an important factor that links BT and systemic inflammation in ALF. ALF patients/animals also have high levels of circulating histones, which might be the major mediators of systemic inflammation in patients with ALF. Extracellular histones kill endothelial cells and elicit immunostimulatory effect to induce multiple organ injuries. Neutralization of histones can attenuate acute liver, lung, and brain injuries. In conclusion, HMGB1 and histones play a significant role in inducing systemic inflammation and MOF in ALF.

HMGB1 neutralization is associated with bacterial translocation during acetaminophen hepatotoxicity.

BACKGROUND: Acetaminophen (APAP) hepatotoxicity is associated with a high rate of gram-negative enteric bacterial infection; however, the underlying mechanism is still unknown. APAP overdose induces massive hepatocyte necrosis, necrotic tissue releases high mobility group B1 (HMGB1) and exogenous HMGB1 is able to induce gut bacterial translocation (BT) in normal mice; therefore, it is possible that HMGB1 mediates gut BT in APAP hepatotoxicity. This study aims to test this hypothesis by using anti-HMGB1 neutralizing antibody to treat APAP overdose for 24-48 hours. METHODS: Male C57BL/6 mice were intraperitoneally (i.p.) injected with a single dose of APAP (350 mg/kg dissolved in 1 mL sterile saline). 2 hrs after APAP injection, the APAP challenged mice were randomized to receive treatment with either anti-HMGB1 antibody (400 µg per dose) or non-immune (sham) IgG every 24 h for a total of 2 doses. RESULTS: 24 and 48 hrs after APAP challenge, anti-HMGB1 treatment instead of sham IgG therapy significantly decreased serum HMGB1 concentrations and reduced BT by 85%; serum HMGB1 levels were positively correlated with the amount of BT; anti-HMGB1 therapy decreased hepatic BT at 48 h, which was associated with better recovered liver structure and better restored hepatic immune system that was shown by enhanced hepatic mRNA expression of TNF-α, IL-6 and extensive proliferation of inflammatory and reticuloendothelial cells; however, anti-HMGB1 treatment did not decrease gut mucosal permeability as compared to the sham IgG therapy at either 24 or 48 hrs. CONCLUSION: HMGB1 neutralization is associated with bacterial translocation during APAP hepatotoxicity.

The urine protein NGAL predicts renal replacement therapy, but not acute kidney injury or 90-day mortality in critically ill adult patients.

BACKGROUND: Urine neutrophil gelatinase-associated lipocalin (uNGAL) is increasingly used as a biomarker for acute kidney injury (AKI). However, the clinical value of uNGAL with respect to AKI, renal replacement therapy (RRT), or 90-day mortality in critically ill patients is unclear. Accordingly, we tested the hypothesis that uNGAL is a clinically relevant biomarker for these end points in a large, nonselected cohort of critically ill adult patients. METHODS: We prospectively obtained urine samples from 1042 adult patients admitted to 15 Finnish intensive care units. We analyzed 3 samples (on admission, at 12 hours, and at 24 hours) with NGAL ELISA Rapid Kits (BioPorto® Diagnostics, Gentofte, Denmark). We chose the highest uNGAL (uNGAL24) for statistical analyses. We calculated the areas under receiver operating characteristics curves (AUC) with 95% confidence intervals (95% CIs), the best cutoff points with the Youden index, positive likelihood ratios (LR+), continuous net reclassification improvement (NRI), and the integrated discrimination improvement (IDI). We performed sensitivity analyses excluding patients with AKI or RRT on day 1, sepsis, or with missing baseline serum creatinine concentration. RESULTS: In this study population, the AUC of uNGAL24 (95% CI) for development of AKI (defined by the Kidney Disease: Improving Global Outcomes [KDIGO] criteria) was 0.733 (0.701-0.765), and the continuous NRI for AKI was 56.9%. For RRT, the AUC of uNGAL24 (95% CI) was 0.839 (0.797-0.880), and NRI 56.3%. For 90-day mortality, the AUC of uNGAL24 (95% CI) was 0.634 (0.593 to 0.675), and NRI 15.3%. The LR+ (95% CI) for RRT was 3.81 (3.26-4.47). CONCLUSION: In this study, we found that uNGAL associated well with the initiation of RRT but did not provide additional predictive value regarding AKI or 90-day mortality in critically ill patients.

High mobility group B1 impairs hepatocyte regeneration in acetaminophen hepatotoxicity.

BACKGROUND: Acetaminophen (APAP) overdose induces massive hepatocyte necrosis. Necrotic tissue releases high mobility group B1 (HMGB1), and HMGB1 contributes to liver injury. Even though blockade of HMGB1 does not protect against APAP-induced acute liver injury (ALI) at 9 h time point, the later time points are not studied and the role of HMGB1 in APAP overdose is unknown, it is possible that neutralization of HMGB1 might improve hepatocyte regeneration. This study aims to test whether blockade of HMGB1 improves hepatocyte regeneration after APAP overdose. METHODS: Male C57BL/6 mice were treated with a single dose of APAP (350 mg/kg). 2 hrs after APAP administration, the APAP challenged mice were randomized to receive treatment with either anti-HMGB1 antibody (400 µg per dose) or non-immune (sham) IgG every 24 hours for a total of 2 doses. RESULTS: 24 hrs after APAP injection, anti-HMGB1 therapy instead of sham IgG therapy significantly improved hepatocyte regeneration microscopically; 48 hrs after APAP challenge, the sham IgG treated mice showed 14.6% hepatic necrosis; in contrast, blockade of HMGB1 significantly decreased serum transaminases (ALT and AST), markedly reduced the number of hepatic inflammatory cells infiltration and restored liver structure to nearly normal; this beneficial effect was associated with enhanced hepatic NF-κB DNA binding and increased the expression of cyclin D1, two important factors related to hepatocyte regeneration. CONCLUSION: HMGB1 impairs hepatocyte regeneration after APAP overdose; Blockade of HMGB1 enhances liver recovery and may present a novel therapy to treat APAP overdose.

Ethyl pyruvate reduces liver injury at early phase but impairs regeneration at late phase in acetaminophen overdose.

INTRODUCTION: Inflammation may critically affect mechanisms of liver injury in acetaminophen (APAP) hepatotoxicity. Kupffer cells (KC) play important roles in inflammation, and KC depletion confers protection at early time points after APAP treatment but can lead to more severe injury at a later time point. It is possible that some inflammatory factors might contribute to liver damage at an early injurious phase but facilitate liver regeneration at a late time point. Therefore, we tested this hypothesis by using ethyl pyruvate (EP), an anti-inflammatory agent, to treat APAP overdose for 24-48 hours. METHODS: C57BL/6 male mice were intraperitoneally injected with a single dose of APAP (350 mg/kg dissolved in 1 mL sterile saline). Following 2 hours of APAP challenge, the mice were given 0.5 mL EP (40 mg/kg) or saline treatment every 8 hours for a total of 24 or 48 hours. RESULTS: Twenty-four hours after APAP challenge, compared to the saline-treated group, EP treatment significantly lowered serum transaminases (ALT/AST) and reduced liver injury seen in histopathology; however, at the 48-hour time point, compared to the saline therapy, EP therapy impaired hepatocyte regeneration and increased serum AST; this late detrimental effect was associated with reduced serum TNF-α concentration and decreased expression of cell cycle protein cyclin D1, two important factors in liver regeneration. CONCLUSIONS: Inflammation likely contributes to liver damage at an early injurious phase but improves hepatocyte regeneration at a late time point, and prolonged anti-inflammation therapy at a late phase is not beneficial.

Ringer's lactate improves liver recovery in a murine model of acetaminophen toxicity.

BACKGROUND: Acetaminophen (APAP) overdose induces massive hepatocyte necrosis. Liver regeneration is a vital process for survival after a toxic insult. Since hepatocytes are mostly in a quiescent state (G0), the regeneration process requires the priming of hepatocytes by cytokines such as TNF-α and IL-6. Ringer's lactate solution (RLS) has been shown to increase serum TNF-α and IL-6 in patients and experimental animals; in addition, RLS also provides lactate, which can be used as an alternative metabolic fuel to meet the higher energy demand by liver regeneration. Therefore, we tested whether RLS therapy improves liver recovery after APAP overdose. METHODS: C57BL/6 male mice were intraperitoneally injected with a single dose of APAP (300 mg/kg dissolved in 1 mL sterile saline). Following 2 hrs of APAP challenge, the mice were given 1 mL RLS or Saline treatment every 12 hours for a total of 72 hours. RESULTS: 72 hrs after APAP challenge, compared to saline-treated group, RLS treatment significantly lowered serum transaminases (ALT/AST) and improved liver recovery seen in histopathology. This beneficial effect was associated with increased hepatic tissue TNF-α concentration, enhanced hepatic NF-κB DNA binding and increased expression of cell cycle protein cyclin D1, three important factors in liver regeneration. CONCLUSION: RLS improves liver recovery from APAP hepatotoxicity.

Bile is a promising gut nutrient that inhibits intestinal bacterial translocation and promotes gut motility via an interleukin-6-related pathway in an animal model of endotoxemia.

OBJECTIVES: People who are critically ill have high rates of endotoxemia that can significantly decrease bile flow and increase bile cytokines, the latter of which might worsen their condition. Bile acids are nutrient-signaling hormones that have a significant impact on gut barrier function and motility, and the gut is considered the origin of systemic inflammation. Therefore, healthy exogenous bile could be a promising gut nutrient for critical illness, so the biomedical role of bile in endotoxemia was investigated in this study. METHODS: Twelve rats were injected with lipopolysaccharide (LPS) and randomized into a group with sham operation) and a group with bile external drainage (n = 6 for each group); six rats with sham operation served as the control group. In addition, interleukin-6 (IL-6) knockout mice and macrophages were treated with LPS. RESULTS: Compared to the control animals, the group with LPS injection and sham operation had significantly increased levels of gut permeability, gut bacterial translocation, gut mucosal tumor necrosis factor α, IL-6 transcripts, and serum tumor necrosis factor α and IL-6. Compared to group with sham operation and LPS injection, bile external drainage (in LPS-challenged rats) increased gut bacterial translocation by 10 times, and this detrimental effect was associated with prolonged intestinal transit time, increased serum IL-6 concentration, and up-regulated gut mucosal IL-6 transcripts. Moreover, bile selectively inhibited LPS-stimulated macrophages in IL-6 release, which can activate gastrointestinal submucosal neurons to promote motility. Knocking out IL-6 significantly reduced gut bacterial translocation in endotoxemic mice. CONCLUSIONS: Bile is a promising gut nutrient that inhibits gut bacterial translocation and promotes gut motility via an IL-6-related pathway in experimental endotoxemia.

Ethyl pyruvate supplemented in drinking water ameliorates experimental nonalcoholic steatohepatitis.

Inflammation and oxidative stress play a significant role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Ethyl pyruvate (EP) is a novel anti-inflammatory agent and a potent reactive oxygen species (ROS) scavenger. Therefore, EP supplemented in drinking water may alleviate experimental NASH in this study (even though 0.3% of EP cannot attenuate the simple non-aggressive fatty liver). The methionine-choline-deficient (MCD) diet was given to the C57BL/6 male mice for 3 weeks to induce NASH. The NASH animals were randomized into 3 treatment groups: animals in the MCD alone group were treated with normal drinking water alone; animals in the delayed EP group were given 3% (v/v) of EP supplemented in normal drinking water, the treatment started 10 days after MCD diet feeding; animals in the early EP therapy group were treated the same as the delayed EP group except that EP treatment started the same day when MCD diet was given; the control mice were fed with normal chow and treated with normal drinking water (n = 10 for each group). Compared to MCD group with normal drinking water, early EP treatment significantly decreased serum ALT and improved NASH histopathology; delayed EP therapy only attenuated NASH in 50% (5/10) of the animals. The beneficial effects were associated with decreased hepatic TNF-a and IL-6 mRNA expression on early 5 days, inhibited NF-kB activation, reduced liver tissue malondialdehyde levels, and decreased intestinal bacterial translocation (BT). In conclusion: EP supplemented in drinking water attenuates experimental NASH.

Serum Uric Acid Is a Mediator of the Association Between Obesity and Incident Nonalcoholic Fatty Liver Disease: A Prospective Cohort Study.

Objective: Obesity has been demonstrated to show a consistent link with the increased possibility of nonalcoholic fatty liver disease (NAFLD). Since both serum uric acid (SUA) and obesity are essential components of metabolic syndrome (MetS), it is uncertain whether the incidence of NAFLD results from serum uric acid, obesity, or other potential factors based on previous studies. Patients and methods: This study enrolled 16,839 participants with no history of alcohol consumption and no fatty liver disease in 2010. All participants completed a survey which included health and lifestyle questionnaires, and underwent physical examination, ultrasonography, and laboratory examinations of blood samples. After the four-year follow up, 5,104 (30.31%) participants were diagnosed with NAFLD. The associations between SUA, BMI or obesity, and incident NAFLD were assessed by multivariate linear regression, logistic regression analysis, and mediation analysis, respectively. Results: By adjusting demographic and serum characteristics, linear correlation coefficients between obesity and SUA were 20.26 [95% confidence interval (CI)]: 15.74, 24.77), 13.31 (95% CI: 6.63, 19.99) and 22.21 (95% CI: 16.41, 28.02) in the total population, and in the female and male groups, respectively. The odds ratios were 2.49 (95% CI: 1.61, 3.87) in the total population, 5.71 (95% CI: 2.25, 14.45) in the female group and 1.99 (95% CI: 1.15, 3.45) in the male group for the correlation between obesity and incident NAFLD. The mediation analysis showed that SUA contributed to 10.03%, 0.58%, and 12.54% of obesity-related NAFLD development in the total population, females and males, respectively. Conclusion: The findings showed mediation linkages of both obesity and SUA with the incident NAFLD. The role of SUA as a mediator constitutes clinical significance that should be recognized and considered.

Extracellular activation of arginase-1 decreases enterocyte inducible nitric oxide synthase activity during systemic inflammation.

Liver dysfunction secondary to severe inflammation is associated with the release of enzymes normally sequestered within hepatocytes. The purpose of these studies was to test the hypothesis that these enzymes are released, at least in part, to modulate potentially deleterious inflammatory processes in distant tissues like the gut. Human Caco-2(BBe) enterocyte-like cells were exposed to cytomix (IFN-gamma, TNF-alpha, and IL-1beta) in the absence or presence of human liver cytosol (LC). Nitric oxide (NO(*)) and inducible nitric oxide synthase (iNOS) protein production were measured by the Griess assay and Western analysis, respectively. Cytomix induced the expression of iNOS and release of NO(*). LC protein (400 microg/ml) added to the basal compartment but not apical compartment completely blocked the release of NO(*) but only slightly decreased the magnitude of iNOS protein induction. Ultrafiltration and ultracentrifugation studies demonstrated that microsome-associated arginase-1 activity was the iNOS-suppressing activity in LC. Liver arginase required activation by a <10-kDa factor that was present in supernatants of cytomix-stimulated cells. The selective iNOS inhibitor l-N(6)-(1-iminoethyl)-lysine.2HCl prevented production of this factor. The biotin switch assay detected increased S-nitrosylation of arginase-1 after incubation with supernatants from immunostimulated Caco-2 cells. Serum from endotoxemic mice contained significantly greater arginase activity compared with serum from control mice. Furthermore, the ratio of mucosal monomeric to dimeric iNOS increased in endotoxemic mice compared with controls. Thus reciprocal activation of arginase-1 and modulation of mucosal iNOS activity may be protective because it would be expected to decrease NO(*)-dependent intestinal barrier dysfunction on that basis.

Bile high-mobility group box 1 contributes to gut barrier dysfunction in experimental endotoxemia.

Lipopolysaccharide (LPS) is an important factor in sepsis. LPS given by intraperitoneal injection induces intestinal hyperpermeability and bacterial translocation in animals and stimulates hepatic Kupffer cells to release TNF-alpha into the bile. This study aims to test the hypothesis that in response to LPS stimulation, hepatic Kupffer cells and extrahepatic macrophages release a large amount of the inflammatory cytokine high-mobility group box 1 (HMGB1) into the bile and that bile containing HMGB1 contributes to gut barrier dysfunction in experimental endotoxemia. To test this, rat common bile ducts were catheterized and bile flow rate was monitored before and during the LPS administration. Eight hours after LPS challenge, anti-HMGB1 neutralizing antibody or nonimmune (sham) IgG was injected into the duodenal lumen of endotoxemic rats; normal mice were also gavaged with normal or endotoxemic rat bile (bile collected from LPS-treated rats). We found that after LPS challenge, the bile flow rate in rats was significantly decreased at the 4- to 12-h time points, TNF-alpha concentration in the bile was markedly elevated at the 3- to 4-h time points, and bile HMGB1 levels were significantly increased at the 8- to 12-h time points. Duodenal injection with anti-HMGB1 antibody reversed LPS-induced gut barrier dysfunction in rats. In addition, feeding endotoxemic rat bile to normal mice significantly increased both mucosal permeability and bacterial translocation. The increase in permeability and bacterial translocation was reversible following removal of HMGB1 from the endotoxemic rat bile. These findings document that bile HMGB1 mediates gut barrier dysfunction in experimental endotoxemia.

Ethyl pyruvate ameliorates liver injury secondary to severe acute pancreatitis.

BACKGROUND: Ethyl pyruvate (EP) is capable of significantly decreasing serum alanine aminotransferase and reducing hepatic necrosis in a murine model of severe acute pancreatitis (SAP); however, the working mechanism is still unclear. This study aims to elucidate the underlying mechanism of EP solution ameliorating SAP-induced liver injury and provide a new therapeutic agent to treat liver injury. MATERIALS AND METHODS: Acute necrotizing pancreatitis was induced in C57Bl/6 male mice by feeding the animals a choline-deficient diet supplemented with 0.5% ethionine for 24 h; then the animals were challenged with 7 hourly 50 mug/kg cerulein i.p. injections and a single i.p. injection of Escherichia coli lipopolysaccharide (4 mg/kg). Two hours after the injection of lipopolysaccharide, 40 mg/kg EP, the same volume of Ringers lactate solution (RLS), or saline solution were i.p. injected to animals of EP, RLS, and control groups every 6 h for a total 48-h period. RESULTS: When mice were treated with EP, hepatic mRNA expression of tumor necrosis factor-alpha, interleukin-6, inducible nitric oxide synthase, and cyclooxygenase-2 was significantly lower than that in pancreatitis mice treated with RLS. Compared to RLS treatment, treatment with EP significantly decreased the number of inflammatory cell infiltration and markedly inhibited hepatic nuclear factor-kappa B DNA binding; EP therapy dramatically inhibited high motility group B1 release from inflamed hepatic tissue and significantly decreased the concentration of hepatic tissue malondialdehyde, an oxidative stress parameter. EP treatment also significantly improved body circulating blood volume. CONCLUSION: EP is a potent anti-inflammatory and anti-oxidative agent to ameliorate hepatic local inflammatory response and resultantly decreases liver injury secondary to SAP.

Prolonged treatment with N-acetylcystine delays liver recovery from acetaminophen hepatotoxicity.

INTRODUCTION: Acetaminophen (APAP) toxicity is the most common cause of acute liver failure in the US and Europe. Massive hepatocyte necrosis is the predominant feature of APAP-induced acute liver injury (ALI). Liver regeneration is a vital process for survival after a toxic insult, it occurs at a relative late time point after the injurious phase. Currently, N-acetylcysteine (NAC), a glutathione precursor, is the antidote for acetaminophen overdose. However, NAC is effective only for patients who present within hours of an acute overdose, and is less effective for late-presenting patients. It is possible that in delayed patients, previously reduced endogenous glutathione (GSH) level has restored and prolonged treatment with NAC might be toxic and impair liver regeneration. Therefore, we hypothesize that prolonged treatment with NAC impairs liver regeneration in ALI induced by APAP. METHODS: ALI was induced in C57BL/6 male mice by a single dose of APAP (350 mg/kg) by intraperitoneal injection. After two hours of APAP challenge, the mice were given 100 mg/kg NAC dissolved in 0.6 mL saline, or saline treatment every 12 hours for a total of 72 hours. RESULTS: Seventy-two hours after APAP challenge, compared with saline treatment, NAC treatment significantly increased serum transaminases (alanine transaminase/aspartate aminotransferase), induced evident hepatocyte vacuolation in the periportal area and delayed liver regeneration seen in histopathology. This detrimental effect was associated with reduced hepatic nuclear factor (NF)-kappaB DNA binding and decreased expression of cell cycle protein cyclin D1, two important factors in liver regeneration. CONCLUSIONS: Prolonged treatment with NAC impairs liver regeneration in ALI induced by APAP.

DAMPs and sterile inflammation in drug hepatotoxicity.

Drug hepatotoxicity is the leading cause of acute liver failure (ALF) in the developed countries. The early diagnosis and treatment are still problematic, and one important reason is the lack of reliable mechanistic biomarkers and therapeutic targets; therefore, searching for new biomarkers and therapeutic targets is urgent. Drug hepatotoxicity induces severe liver cells damage and death. Dead and damaged cells release endogenous damage-associated molecular patterns (DAMPs). Increased circulating levels of DAMPs (HMGB1, histones and DNA) can reflect the severity of drug hepatotoxicity. Elevated plasma HMGB1 concentrations can serve as early and sensitive mechanistic biomarker for clinical acetaminophen hepatotoxicity. DAMPS significantly contribute to liver injury and inhibiting the release of DAMPs ameliorates experimental hepatotoxicity. In addition, HMGB1 mediates 80% of gut bacterial translocation (BT) during acetaminophen toxicity. Gut BT triggers systemic inflammation, leading to multiple organ injury and mortality. Moreover, DAMPs can trigger and extend sterile inflammation, which contributes to early phase liver injury but improves liver regeneration at the late phase of acetaminophen overdose, because anti-inflammatory treatment reduces liver injury at early phase but impairs liver regeneration at late phase of acetaminophen toxicity, whereas pro-inflammatory therapy improves late phase liver regeneration. DAMPs are promising mechanistic biomarkers and could also be the potential therapeutic targets for drug hepatotoxicity. DAMPs-triggered sterile inflammation contributes to liver injury at early phase but improves liver regeneration at later phase of acetaminophen hepatotoxicity; therefore, anti-inflammatory therapy would be beneficial at early phase but should be avoided at the late phase of acetaminophen overdose.

Ischemia/reperfusion injury in porcine intestine - Viability assessment.

AIM: To investigate viability assessment of segmental small bowel ischemia/reperfusion in a porcine model. METHODS: In 15 pigs, five or six 30-cm segments of jejunum were simultaneously made ischemic by clamping the mesenteric arteries and veins for 1 to 16 h. Reperfusion was initiated after different intervals of ischemia (1-8 h) and subsequently monitored for 5-15 h. The intestinal segments were regularly photographed and assessed visually and by palpation. Intraluminal lactate and glycerol concentrations were measured by microdialysis, and samples were collected for light microscopy and transmission electron microscopy. The histological changes were described and graded. RESULTS: Using light microscopy, the jejunum was considered as viable until 6 h of ischemia, while with transmission electron microscopy the ischemic muscularis propria was considered viable until 5 h of ischemia. However, following ≥ 1 h of reperfusion, only segments that had been ischemic for ≤ 3 h appeared viable, suggesting a possible upper limit for viability in the porcine mesenteric occlusion model. Although intraluminal microdialysis allowed us to closely monitor the onset and duration of ischemia and the onset of reperfusion, we were unable to find sufficient level of association between tissue viability and metabolic markers to conclude that microdialysis is clinically relevant for viability assessment. Evaluation of color and motility appears to be poor indicators of intestinal viability. CONCLUSION: Three hours of total ischemia of the small bowel followed by reperfusion appears to be the upper limit for viability in this porcine mesenteric ischemia model.

HMGB1 and Histones Play a Significant Role in Inducing Systemic Inflammation and Multiple Organ Dysfunctions in Severe Acute Pancreatitis.

Severe acute pancreatitis (SAP) starts as a local inflammation of pancreatic tissue that induces the development of multiple extrapancreatic organs dysfunction; however, the underlying mechanisms are still not clear. Ischemia-reperfusion, circulating inflammatory cytokines, and possible bile cytokines significantly contribute to gut mucosal injury and intestinal bacterial translocation (BT) during SAP. Circulating HMGB1 level is significantly increased in SAP patients and HMGB1 is an important factor that mediates (at least partly) gut BT during SAP. Gut BT plays a critical role in triggering/inducing systemic inflammation/sepsis in critical illness, and profound systemic inflammatory response syndrome (SIRS) can lead to multiple organ dysfunction syndrome (MODS) during SAP, and systemic inflammation with multiorgan dysfunction is the cause of death in experimental SAP. Therefore, HMGB1 is an important factor that links gut BT and systemic inflammation. Furthermore, HMGB1 significantly contributes to multiple organ injuries. The SAP patients also have significantly increased circulating histones and cell-free DNAs levels, which can reflect the disease severity and contribute to multiple organ injuries in SAP. Hepatic Kupffer cells (KCs) are the predominant source of circulating inflammatory cytokines in SAP, and new evidence indicates that hepatocyte is another important source of circulating HMGB1 in SAP; therefore, treating the liver injury is important in SAP.

Toll-like receptor-4 signaling mediates hepatic injury and systemic inflammation in hemorrhagic shock.

BACKGROUND: Hemorrhagic shock and resuscitation (HS/R) activates inflammatory pathways leading to organ injury after trauma. Toll-like receptors (TLRs), such as TLR4, are required for activation of proinflammatory cellular signaling pathways in response to microbial products, but can also recognize endogenous molecules released from damaged tissues. Using mouse strains deficient in TLR4 protein or signaling, we hypothesized that TLR4 would be important for development of systemic inflammation and hepatic injury after HS/R. We sought to determine the role of lipolysaccharide through use of CD14-/- mice. STUDY DESIGN: TLR4-mutant (C[3H]/HeJ), TLR4-deficient (TLR4-/-), CD14-/-, TLR2-/- mice and wild-type (WT) controls were subjected to HS/R or sham procedure (Sham). At 6.5 hours, mice were euthanized for determination of serum interleukin (IL)-6, IL-10, and alanine aminotransferase concentrations. Hepatic nuclear factor-kappaB DNA-binding (electrophoretic mobility shift assay) and tumor necrosis factor, IL-10, and inducible nitric oxide synthase mRNA expression (semiquantitative reverse transcriptase-polymerase chain reaction) were determined. RESULTS: Relative to sham, TLR4-competent (C[3H]/HeOuJ) mice exhibited a significant increase in serum alanine aminotransferase, IL-6, and IL-10 after HS/R (p < 0.05). TLR4-mutant (C[3H]/HeJ) mice were protected from HS/R-induced hepatocellular injury and had lower circulating IL-6 and IL-10 levels than WT (p < 0.05). Similarly, TLR4-/- mice had lower circulating IL-6 and IL-10 levels than WT after HS/R (p < 0.05). Hepatic nuclear factor-kappaB activation and tumor necrosis factor, IL-10, and inducible nitric oxide synthase mRNA expression were lower in TLR4-mutant compared with TLR4-competent mice after HS/R. In contrast, serum ALT concentrations were comparable between CD14-/- and TLR2-/- mice and their WT counterparts after HS/R. CONCLUSIONS: These results suggest that TLR4, but not TLR2, signaling is required for initiation of the systemic inflammatory response and development of hepatocellular injury after HS/R. Lack of involvement of CD14 argues for a lipolysaccharide-independent role for TLR4 in this process.

Ethyl pyruvate is a novel anti-inflammatory agent to treat multiple inflammatory organ injuries.

Ethyl pyruvate (EP) is a simple derivative of pyruvic acid, which is an important endogenous metabolite that can scavenge reactive oxygen species (ROS). Treatment with EP is able to ameliorate systemic inflammation and multiple organ dysfunctions in multiple animal models, such as acute pancreatitis, alcoholic liver injury, acute respiratory distress syndrome (ARDS), acute viral myocarditis, acute kidney injury and sepsis. Recent studies have demonstrated that prolonged treatment with EP can ameliorate experimental ulcerative colitis and slow multiple tumor growth. It has become evident that EP has pharmacological anti-inflammatory effect to inhibit multiple early inflammatory cytokines and the late inflammatory cytokine HMGB1 release, and the anti-tumor activity is likely associated with its anti-inflammatory effect. EP has been tested in human volunteers and in a clinical trial of patients undergoing cardiac surgery in USA and shown to be safe at clinical relevant doses, even though EP fails to improve outcome of the heart surgery, EP is still a promising agent to treat patients with multiple inflammatory organ injuries and the other clinical trials are on the way. This review focuses on how EP is able to ameliorate multiple organ injuries and summarize recently published EP investigations. Graphical AbstractThe targets of the anti-inflammatory agent EP.

The ethyl pyruvate analogues, diethyl oxaloproprionate, 2-acetamidoacrylate, and methyl-2-acetamidoacrylate, exhibit anti-inflammatory properties in vivo and/or in vitro.

Ethyl pyruvate (EP) is a simple aliphatic ester derived from the endogenous metabolite, pyruvic acid. EP has been shown to decrease the expression of various pro-inflammatory mediators, including nitric oxide (NO*), tumor necrosis factor (TNF), cyclooxygenase-2, and interleukin (IL)-6, in a variety of in vitro and in vivo model systems. In an effort to better understand the chemical features that might explain the anti-inflammatory properties of EP, we screened 15 commercially available compounds for cytoprotective or anti-inflammatory effects using two in vitro assay systems: TNF and NO* production by lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage-like cells and changes in the permeability of Caco-2 human enterocyte-like monolayers stimulated with a cocktail of pro-inflammatory cytokines called cytomix (1000U/ml IFN-gamma plus 10ng/ml TNF-alpha plus 1ng/ml IL-1beta). Two compounds, namely diethyl oxaloproprionate (DEOP) and 2-acetamidoacrylate (2AA), demonstrated consistent anti-inflammatory or cytoprotective pharmacological properties in this screening process. Treatment of mice with either of these compounds ameliorated LPS-induced ileal mucosal hyperpermeability to the fluorescent probe, fluorescein isothiocyanate-labeled dextran (average molecular mass 4kDa), and bacterial translocation to mesenteric lymph nodes. Treatment with either of these compounds also improved survival in mice challenged with a lethal dose of LPS. Finally, in a study that compared 2AA to its methyl ester, we showed that methyl-2-acetamidoacrylate is at least 100-fold more potent than the parent carboxylate as an inhibitor of LPS-induced NO* production by RAW 264.7 cells. Collectively, these data are consistent with the view that anti-inflammatory activity is demonstrable for a number of compounds that either incorporate an olefinic linkage conjugated to a carbonyl moiety or are capable of undergoing tautomeric rearrangement to form such a structure. Moreover, our findings suggest that esters with these general characteristics, perhaps because of their greater lipophilicity or electrophilicity, are more potent anti-inflammatory agents than are the parent carboxylates.