Haoshu Fang

Oxidation of HMGB1 Causes Attenuation of Its Pro-Inflammatory Activity and Occurs during Liver Ischemia and Reperfusion

High mobility group box 1 (HMGB1) is a nuclear transcription factor. Once HMGB1 is released by damaged cells or activated immune cells, it acts as danger molecule and triggers the inflammatory signaling cascade. Currently, evidence is accumulating that posttranslational modifications such as oxidation may modulate the pro-inflammatory potential of danger signals. We hypothesized that oxidation of HMGB1 may reduce its pro-inflammatory potential and could take place during prolonged ischemia and upon reperfusion. Liver grafts were cold preserved for 24 h and flushed with saline in hourly intervals to collect the effluent. Liver grafts, cold-preserved for 6 h, were transplanted into syngeneic recipients to obtain serum and liver samples 24 h after initiation of reperfusion. Addition of the effluent to a macrophage culture induced the synthesis of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6. The stimulatory activity of graft effluent was reduced after depletion of HMGB1 via immunoprecipitation. Oxidation of the effluent HMGB1 using H2O2 attenuated its stimulatory activity as well. Liver transplantation of cold preserved grafts caused HMGB1 translocation and release as determined by immunohistochemistry and ELISA-assay, respectively. Using Western blot with non-reducing conditions revealed the presence of oxidized HMGB1 in liver samples obtained after 12 h and in effluent samples after 16 h of cold preservation as well as in liver and serum samples obtained 24 h after reperfusion. These observations confirm that post-translational oxidation of HMGB1 attenuates its pro-inflammatory activity. Oxidation of HMGB1 as induced during prolonged ischemia and by reoxygenation during reperfusion in vivo might also attenuate its pro-inflammatory activity. Our findings also call for future studies to investigate the mechanism of the inhibitory effect of oxidized HMGB1 on the pro-inflammatory potential.

Ischemic preconditioning protects against liver ischemia/reperfusion injury via heme oxygenase-1-mediated autophagy

Objectives:Ischemic preconditioning exerts a protective effect in hepatic ischemia/reperfusion injury. The exact mechanism of ischemic preconditioning action remains largely unknown. Recent studies suggest that autophagy plays an important role in protecting against ischemia/reperfusion injury. However, the role of autophagy in ischemic preconditioning–afforded protection and its regulatory mechanisms in liver ischemia/reperfusion injury remain poorly understood. This study was designed to determine whether ischemic preconditioning could protect against liver ischemia/reperfusion injury via heme oxygenase-1-mediated autophagy. Design:Laboratory investigation. Setting:University animal research laboratory. Subjects:Male inbred Lewis rats and C57BL/6 mice. Interventions:Ischemic preconditioning was produced by 10 minutes of ischemia followed by 10 minutes of reperfusion prior to 60 minutes of ischemia. In a rat model of hepatic ischemia/reperfusion injury, rats were pretreated with wortmannin or rapamycin to evaluate the contribution of autophagy to the protective effects of ischemic preconditioning. Heme oxygenase-1 was inhibited with tin protoporphyrin IX. In a mouse model of hepatic ischemia/reperfusion injury, autophagy or heme oxygenase-1 was inhibited with vacuolar protein sorting 34 small interfering RNA or heme oxygenase-1 small interfering RNA, respectively. Measurements and Main Results:Ischemic preconditioning ameliorated liver ischemia/reperfusion injury, as indicated by lower serum aminotransferase levels, lower hepatic inflammatory cytokines, and less severe ischemia/reperfusion-associated histopathologic changes. Ischemic preconditioning treatment induced autophagy activation, as indicated by an increase of LC3-II, degradation of p62, and accumulation of autophagic vacuoles in response to ischemia/reperfusion injury. When ischemic preconditioning–induced autophagy was inhibited with wortmannin in rats or vacuolar protein sorting 34–specific small interfering RNA in mice, liver ischemia/reperfusion injury was worsened, whereas rapamycin treatment increased autophagy and mimicked the protective effects of ischemic preconditioning. Furthermore, ischemic preconditioning increased heme oxygenase-1 expression. The inhibition of heme oxygenase-1 with tin protoporphyrin IX in rats or heme oxygenase-1-specific small interfering RNA in mice decreased ischemic preconditioning–induced autophagy and diminished the protective effects of ischemic preconditioning against ischemia/reperfusion injury. Conclusions:Ischemic preconditioning protects against liver ischemia/reperfusion injury, at least in part, via heme oxygenase-1-mediated autophagy.

Early release of macrophage migration inhibitory factor after liver ischemia and reperfusion injury in rats.

Macrophage migration inhibitory factor (MIF) is an important mediator of ischemia/reperfusion (I/R) injury in heart, brain and intestine. We previously demonstrated that MIF was released during warm/cold ischemia in vitro. However, the role of MIF in liver I/R injury remains unclear. We aimed to test the hypothesis that MIF acts as an early proinflammatory cytokine and could mediate the inflammatory injury in liver I/R. Rats (n=6 per group) were subjected to 90 min warm ischemia followed by 0.5h, 6h and 24h reperfusion, respectively to liver transplantation (LTx) after 6h of cold ischemia followed by 24h of reperfusion. The expression of MIF, its receptor (cluster of differentiation 74 (CD74)) and the downstream inflammatory cytokines (tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)) were analyzed. Peritoneal macrophages were cultured for 6h alone or in the presence of effluent from cold-preserved livers or effluent depleted of MIF. Warm I/R increased hepatic MIF-mRNA and protein expression. MIF-protein was released into peripheral circulation in vivo with a maximum at 0.5h after reperfusion. Induction of MIF-expression was associated with the expression of proinflammatory cytokines and its receptor in both models. MIF released by isolated cold preserved livers, induced TNF-α and IL-1β production by cultured peritoneal macrophages. Intrahepatic upregulation of MIF, release into systemic circulation and the associated upregulation of the proinflammatory mediators suggest a role of MIF in mediating the inflammatory response to I/R injury. Blocking experiments will help to elucidate its role as potential molecular target for preventing hepatic I/R injury.

HMGB1 translocation and expression is caused by warm ischemia reperfusion injury, but not by partial hepatectomy in rats

Mechanical injury or ischemia/reperfusion (I/R) injury induces high mobility of group box 1 (HMGB1) translocation and release. However, the surgical procedure itself can initiate pathophysiologic processes causing damage to the respective organ. A liver resection, as an example, leads to portal hyperperfusion injury of the remnant liver. Therefore, we aimed to elucidate the impact of different hepatic surgical injury models on cellular localization and expression of HMGB1. Focal warm I/R injury was induced by clamping the vascular blood supply to the median and left lateral liver lobes for 90 min followed by 0.5 h, 6 h and 24 h reperfusion, as reported previously. Liver injury by PH was induced by subjecting rats to 30%, 70% or 90% partial hepatectomy (PH) followed by a 24 h observation period. Additional 12 rats were subjected to 90% PH and sacrificed at 1 h and 6 h to investigate the expression and release pattern of HMGB1. Elevation of serum liver enzymes indicating hepatic injury peaked at 6 h and recovered thereafter in models, warm I/R injury and PH. Liver injury was confirmed by liver histology. HMGB1 was translocated from the nucleus to the cytoplasm in livers subjected to warm I/R; but not in livers subjected to PH. Both protein and mRNA expression of HMGB1 were significantly up-regulated in livers subjected to warm I/R. In contrast, neither 30% PH, 70% PH nor 90% PH caused an elevation of hepatic HMGB1 mRNA and protein expression. High serum levels of HMGB1 (30 ng/ml) were measured at 0.5 h reperfusion period after warm I/R, much lower levels thereafter (<5 ng/ml). Similar low serum levels were measured at all time points after 90% PH. Subsequently expression levels of TNF-a should be changed to tumor necrosis factor-alpha (TNF-α) reached a peak (26-fold elevation) at 6 h and decreased down to 5-fold at 24 h after warm I/R. TNF-α expression levels after PH never exceeded a 5-fold elevation. In conclusion, HMGB1 translocation and expression depends on the type of liver injury as it is induced by ischemia, but not by liver resection/hyperperfusion. These results suggest that HMGB1 may be used as molecular marker to visualize ischemic damage. Mechanic injury in hepatic surgery is associated with focal warm ischemia, and thereby HMGB1 translocation reflects surgical quality in experimental PH. Expression of hepatic TNF-α follows the kinetic pattern of HMGB1, pointing to a muss less pronounced inflammatory response after successful PH compared to warm I/R injury.

GSK-3β Inhibition Attenuates CLP-Induced Liver Injury by Reducing Inflammation and Hepatic Cell Apoptosis

Liver dysfunction has been known to occur frequently in cases of sepsis. Excessive inflammation and apoptosis are pathological features of acute liver failure. Recent studies suggest that activation of glycogen synthase kinase- (GSK-) 3β is involved in inflammation and apoptosis. We aimed to investigate the protective effects of GSK-3β inhibition on polymicrobial sepsis-induced liver injury and to explore the possible mechanisms. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP), and SB216763 was used to inhibit GSK-3β in C57BL/6 mice. GSK-3β was activated following CLP. Administration of SB216763 decreased mortality, ameliorated liver injury, and reduced hepatic apoptosis. The inhibition of GSK-3β also reduced leukocyte infiltration and hepatic inflammatory cytokine expression and release. Moreover, GSK-3β inhibition suppressed the transcriptional activity of nuclear factor-kappa B (NF-κB) but enhanced the transcriptional activity of cAMP response element binding protein (CREB) in the liver. In in vitro studies, GSK-3β inhibition reduced inflammatory cytokine production via modulation of NF-κB and CREB signaling pathways in lipopolysaccharide-stimulated macrophages. In conclusion, these findings suggest that GSK-3β blockade protects against CLP-induced liver via inhibition of inflammation by modulating NF-κB and CREB activity and suppression of hepatic apoptosis.

Granulocyte Colony Stimulating Factor Induces Lipopolysaccharide (LPS) Sensitization via Upregulation of LPS Binding Protein in Rat

Liver is the main organ for lipopolysaccharide (LPS) clearance. Sensitization to LPS is associated with the upregulation of LPS-binding protein (LBP) in animal models. Therefore, we hypothesized that LBP could induce LPS sensitization through enhancing hepatic uptake of LPS. In this study, we examined the role of LBP in pathogenesis of LPS induced systemic inflammatory response syndrome (SIRS). LBP expression was upregulated after granulocyte colony stimulating (G-CSF) pretreatment. The effect of LBP was further confirmed by blockade of LBP using LBP blocking peptide – LBPK95A. After G-CSF pretreatment, upregulation of LBP was observed in bone marrow cells and liver. The G-CSF induced LBP upregulation caused LPS hypersensitization in rats as indicated by higher mortality and severer liver damage. Of note, LBP blockade increased the survival rate and attenuated the liver injury. The LBP induced LPS hypersensitization was associated with increased hepatic uptake of LPS and augmented hepatic expression of LPS receptors, such as toll-like receptor (TLR)-4. Furthermore, LBP mediated early neutrophil infiltration, which led to increased monocyte recruitment in liver after LPS administration. In conclusion, G-CSF induced LBP expression could serve as a new model for investigation of LPS sensitization. We demonstrated the crucial role of LBP upregulation in pathogenesis of LPS induced SIRS.

Chronic Lithium Treatment Protects Against Liver Ischemia/Reperfusion Injury in Rats

Lithium has long been widely used in the treatment of bipolar mood disorders. Recent studies have demonstrated that lithium is able to decrease ischemia/reperfusion (I/R) injury in the brain, kidneys, and heart. Because lithium may act on a number of stress and survival pathways, it is of great interest to explore this compound also in the setting of liver I/R injury. In this study, we aimed to evaluate the effects of lithium in a model of liver I/R injury in rats. Chronic treatment with lithium (2 mmol/kg for 3 days before ischemia) decreased I/R injury, whereas acute treatment with a single dose of lithium (2 mmol/kg 1 hour before ischemia) did not confer any protection in a partial hepatic I/R model. Furthermore, rats subjected to chronic lithium treatment had a significantly better survival rate (60%) than saline‐treated rats (27%) in a total hepatic I/R survival model. Chronic lithium treatment protected against liver I/R injury, as indicated by lower serum aminotransferase levels, fewer I/R‐associated histopathological changes, lower hepatic inflammatory cytokine levels, less neutrophil infiltration, and lower hepatic high‐mobility group box expression and serum levels. The mechanism of action of lithium appears to involve its ability to inhibit glycogen synthase kinase 3β activation, modulate mitogen‐activated protein kinase activation, inhibit hepatic apoptosis, and induce autophagy. On the basis of these data, we conclude that lithium treatment may be a simple and applicable preconditioning intervention for protecting against liver I/R injury. Liver Transpl 19:762–772, 2013..

Balancing Innate Immunity and Inflammatory State via Modulation of Neutrophil Function: A Novel Strategy to Fight Sepsis

Sepsis and SIRS (systemic inflammatory response syndrome) belong to a severe disease complex characterized by infection and/or a whole-body inflammatory state. There is a growing body of evidence that neutrophils are actively involved in sepsis and are responsible for both release of cytokines and phagocytosis of pathogens. The neutrophil level is mainly regulated by G-CSF, a cytokine and drug, which is widely used in the septic patient with neutropenia. This review will briefly summarize the role of neutrophils and the therapeutic effect of G-CSF in sepsis. We further suggest that targeting neutrophil function to modulate the balance between innate immunity and inflammatory injury could be a worthwhile therapeutic strategy for sepsis.

Lipopolysaccharide-binding Protein (lbp) Blockade Augments the Protective Effect of Granulocyte Colony-stimulating Factor (g-csf) in a Rat Sepsis Model

ABSTRACT The effect of granulocyte colony-stimulating factor (G-CSF) on sepsis is discussed controversially in clinical studies. We previously demonstrated that G-CSF treatment induced lipopolysaccharide (LPS) sensitization via up-regulation of LPS-binding protein (LBP). We hypothesized that the futile effect of G-CSF-treatment in sepsis might be due to its ability to up-regulate LBP. Therefore, blockade of LBP may attenuate the G-CSF–induced LPS sensitization and protect animals from polymicrobial sepsis. Endogenous LBP levels were up-regulated by pretreatment with G-CSF, and the LBP protein was blocked by administration of a specific blocking peptide—LBPK95A. Polymicrobial sepsis was induced by intraperitoneal injection of feces slurry. Rats were monitored every 3 up to 72 h to observe the survival rate. Tissue injury, bacterial infiltration, local inflammatory response, and neutrophil infiltration at 0, 2, and 12 h after the septic insult were analyzed. The survival benefit of G-CSF pretreatment was improved when combined with LBPK95A treatment (control vs. G-CSF vs. combi: 36% vs. 56% vs. 93%; P < 0.05). Combined treatment of G-CSF and LBPK95A was associated with the minimal tissue damage. Treatment with LBPK95A significantly inhibited the neutrophil infiltration without interfering with the bacterial clearance. The G-CSF–induced inflammatory sensitization effect was inhibited by LBPK95A, indicated by the decrease of cytokines expression, and the activation of nuclear factor kappa B and signal transducer and activator of transcription 3 signaling pathway. In conclusion, these results suggested that the effect of prophylactic augmentation of the host’s response via G-CSF pretreatment was further enhanced by inhibition of the up-regulation of LBP.

The fibrin-derived peptide bβ15-42 attenuates liver damage in a rat model of liver ischemia/reperfusion injury.

ABSTRACT The inflammatory response after liver ischemia/reperfusion (I/R) contributes to increased risk of liver failure after liver surgery. Strategies aimed to preventing inflammation could be beneficial in reducing liver I/R injury. Recent studies have demonstrated that peptide B&bgr;15-42 is able to decrease the injury of I/R in heart and kidney by inhibition of leukocyte migration and preserving endothelial barrier function. Prompted by these results, we hypothesized that B&bgr;15-42 could also possess anti-inflammatory abilities to protect from or reduce hepatic I/R injury. Therefore, in this study, we aimed to evaluate the effects of B&bgr;15-42 in a model of liver I/R injury in rats. Rats were treated with B&bgr;15-42 at initiation of reperfusion and 2 h thereafter. Rats were killed at 0.5, 6, 24, and 48 h after reperfusion. Hepatic mRNA levels of fibrinogen-&agr; (Fg&agr;), Fg&bgr;, Fg&ggr; were significantly increased after I/R. Treatment with Fg-derived B&bgr;15-42 ameliorated liver I/R injury, as indicated by lower serum aminotransferase levels and fewer I/R-associated histopathologic changes. B&bgr;15-42 treatment decreased leukocyte infiltration and expression of hepatic inflammatory cytokines. Moreover, B&bgr;15-42 significantly reduced high-mobility group box 1 release and altered mitogen-activated protein kinase activation. In conclusion, B&bgr;15-42 treatment protected against liver warm I/R injury. The mechanism of protective action of B&bgr;15-42 seemed to involve its ability to reduce hepatic inflammatory response through preventing high-mobility group box 1 release and altering mitogen-activated protein kinase activation.