Progression Of Acute Liver Injury Research Articles

Neutrophil extracellular traps promote acetaminophen-induced acute liver injury in mice via AIM2.

Excessive acetaminophen (APAP) can induce neutrophil activation and hepatocyte death. Along with hepatocyte dysfunction and death, NETosis (a form of neutrophil-associated inflammation) plays a vital role in the progression of acute liver injury (ALI) induced by APAP overdose. It has been shown that activated neutrophils tend to migrate towards the site of injury and participate in inflammatory processes via formation of neutrophil extracellular traps (NETs). In this study we investigated whether NETs were involved in hepatocyte injury and contributed to APAP-induced ALI progression. ALI mouse model was established by injecting overdose (350 mg/kg) of APAP. After 24 h, blood and livers were harvested for analyses. We showed that excessive APAP induced multiple programmed cell deaths of hepatocytes including pyroptosis, apoptosis and necroptosis, accompanied by significantly increased NETs markers (MPO, citH3) in the liver tissue and serum. Preinjection of DNase1 (10 U, i.p.) for two consecutive days significantly inhibited NETs formation, reduced PANoptosis and consequently alleviated excessive APAP-induced ALI. In order to clarify the communication between hepatocytes and neutrophils, we induced NETs formation in isolated neutrophils, and treated HepaRG cells with NETs. We found that NETs treatment markedly increased the activation of GSDMD, caspase-3 and MLKL, while pre-treatment with DNase1 down-regulated the expression of these proteins. Knockdown of AIM2 (a cytosolic innate immune receptor) abolished NETs-induced PANoptosis in HepaRG cells. Furthermore, excessive APAP-associated ALI was significantly attenuated in AIM2KO mice, and PANoptosis occurred less frequently. Upon restoring AIM2 expression in AIM2KO mice using AAV9 virus, both hepatic injury and PANoptosis was aggravated. In addition, we demonstrated that excessive APAP stimulated mtROS production and mitochondrial DNA (mtDNA) leakage, and mtDNA activated the TLR9 pathway to promote NETs formation. Our results uncover a novel mechanism of NETs and PANoptosis in APAP-associated ALI, which might serve as a therapeutic target.

Preventing acute liver injury via hepatocyte-targeting nano-antioxidants.

Acute liver injury (ALI) is a severe liver disease that is characterized by sudden and massive hepatocyte necrosis and deterioration of liver functions. Oxidative stress is increasingly recognized as a key factor in the induction and progression of ALI. Scavenging excessive reactive oxygen species (ROS) with antioxidants has become a promising therapeutic option, but intrinsically hepatocyte-targeting antioxidants with excellent bioavailability and biocompatibility are yet to be developed. Herein, self-assembling nanoparticles (NPs) composed of amphiphilic polymers are introduced to encapsulate organic Selenium compound L-Se-methylselenocysteine (SeMC) and form SeMC NPs, which protect the viabilities and functions of cultured hepatocytes in drug- or chemical-induced acute hepatotoxicity models via efficient ROS removal. After further functionalization with the hepatocyte-targeting ligand glycyrrhetinic acid (GA), the resultant GA-SeMC NPs exhibit enhanced hepatocyte uptake and liver accumulation. In mouse models of ALI induced by acetaminophen (APAP) or carbon tetrachloride (CCl4 ), treatment with GA-SeMC NPs significantly decrease the levels of hepatic lipid peroxidation, tissue vacuolization and serum liver transaminases, while prominently increase that of endogenous antioxidant enzymes. Our study therefore presents a liver-targeting drug delivery strategy for the prevention and treatment of hepatic diseases.

A facile theragnostic nano-platform for the effective treatment and real-time imaging of acute liver injury.

Acute liver injury (ALI), induced by an imbalance of pro-inflammatory and anti-inflammatory processes, remains a major concern for disease detection and drug screening. However, current clinical blood tests for ALI diagnostics are limited by delayed estimation, invasive and non-comprehensive visualization and false results from non-specific biomarkers. Moreover, it is difficult to give timely therapy to inhibit its progression and adjust treatment regimens in time. Herein, this study developed a facile theragnostic nano-platform (BLD NP) for effective treatment and real-time imaging of acute liver injury (ALI). BLD NPs comprise peptide-caged NIR probes (CyGbF) for real-time imaging and a small molecular drug (dexamethasone sodium phosphate, Dsp) for timely treatment of ALI, in which CyGbF was conjugated and Dsp was electrostatically complexed with fluorinated polyethylene (LPOF), respectively. After systemic administration, BLD NPs passively target liver tissue and react with ALI-associated protease to in situ activate the NIR signaling moiety for non-invasive longitudinal imaging of ALI progression, while Dsp is released timely for ALI treatments, serving as a theragnostic platform and providing comprehensive estimations for ALI, comparable to standard methods including blood tests and flow cytometric analysis. Therefore, BLD NPs hold great promise for early real-time imaging, timely therapeutic treatment and prediction of the progression of ALI.

Liver infiltration of multiple immune cells during the process of acute liver injury and repair.

Immune cells, including neutrophils, natural killer (NK) cells, T cells, NKT cells and macrophages, participate in the progression of acute liver injury and hepatic recovery. To date, there has been no systematic study on the quantitative changes in these different immune cells from initial injury to subsequent recovery. To investigate the infiltration changes of various immune cells in acute liver injury models over time, and to study the relationship between the changes in leukocyte cell-derived chemotaxin 2 (LECT2) and the infiltration of several immune cells. Carbon tetrachloride- and concanavalin A-induced acute liver injury models were employed to mimic toxin-induced and autoimmune-mediated liver injury respectively. The quantitative changes in various immune cells were monitored at different time points. Serum samples were collected, and liver tissues were harvested. Ly6G, CD161, CD4, CD8 and F4/80 staining were used to indicate neutrophils, NK/NKT cells, CD4+ T cells, CD8+ T cells and macrophages, respectively. Lect2-KO mice were used to detect the function of LECT2. During the injury and repair process, different types of immune cells began to increase, reached their peaks and fell into decline at different time points. Furthermore, when the serum alanine transaminase (ALT) and aspartate transaminase (AST) indices reverted to normal levels 7 d after the injury, the infiltration of immune cells still existed even 14 d after the injury, showing an obvious lag effect. We found that the expression of LECT2 was upregulated in acute liver injury mouse models, and the liver injuries of Lect2-KO mice were less severe than those of wild-type mice. Compared with wild-type mice, Lect2-KO mice had different immune cell infiltration. The recovery time of immune cells was far behind that of serum ALT and AST during the process of liver repair. LECT2 could regulate monocyte/macrophage chemotaxis and might be used as a therapeutic target for acute liver injury.

Evaluation of the hepatoprotective effect of naringenin loaded nanoparticles against acetaminophen overdose toxicity

Acute liver injury is a common clinical disease, which easily leads to liver failure and endangers life, seriously threatening human health. Naringenin is a natural flavonoid that holds therapeutic potential against various liver injuries; however it has poor water solubility and bioavailability. In this study, we aimed to develop naringenin-loaded bovine serum albumin nanoparticles (NGNPs) and to evaluate their hepatoprotective effect and underlying mechanisms against acetaminophen overdose toxicity. In vitro data indicated that NGNPs significantly increased the drug solubility and also more effectively protected the hepatocyte cells from oxidative damage during hydrogen peroxide exposure or lipopolysaccharide (LPS) stimulation. In vivo results confirmed that NGNPs showed an enhanced accumulation in the liver tissue. In the murine model of acetaminophen-induced hepatotoxicity, NGNPs could effectively alleviate the progression of acute liver injury by reducing drug overdose-induced levels of oxidative stress, inflammation and apoptosis in hepatocytes. In conclusion, NGNPs has strong hepatoprotective effects against acetaminophen induced acute liver injury.

LncRNA Gm26917 regulates inflammatory response in macrophages by enhancing Annexin A1 ubiquitination in LPS-induced acute liver injury

Long noncoding RNAs (lncRNAs) are defined as transcripts of more than 200 nucleotides that have little or no coding potential. LncRNAs function as key regulators in diverse physiological and pathological processes. However, the roles of lncRNAs in lipopolysaccharide (LPS)-induced acute liver injury (ALI) are still elusive. In this study, we report the roles of lncRNA Gm26917 induced by LPS in modulating liver inflammation. As key components of the innate immune system, macrophages play critical roles in the initiation, progression and resolution of ALI. Our studies demonstrated that Gm26917 localized in the cytoplasm of hepatic macrophages and globally regulated the expression of inflammatory genes and the differentiation of macrophages. In vivo study showed that lentivirus-mediated gene silencing of Gm26917 attenuated liver inflammation and protected mice from LPS-induced ALI. Furthermore, mechanistic study showed that the 3'-truncation of Gm26917 interacted with the N-terminus of Annexin A1, a negative regulator of the NF-κB signaling pathway. We also found that Gm26917 knockdown suppressed NF-κB activity by decreasing the ubiquitination of Annexin A1 and its interaction with NEMO. In addition, expression of Gm26917 in inflammatory macrophages was regulated by the transcription factor forkhead box M1 (FOXM1). LPS treatment dramatically increased the binding of FOXM1 to the promoter region of Gm26917 in macrophages. In summary, our findings suggest that lncRNA Gm26917 silencing protects against LPS-induced liver injury by regulating the TLR4/NF-κB signaling pathway in macrophages.

Alternatively activated macrophages promote resolution of necrosis following acute liver injury

Background & AimFollowing acetaminophen (APAP) overdose, acute liver injury (ALI) can occur in patients that present too late for N-acetylcysteine treatment, potentially leading to acute liver failure, systemic inflammation, and death. Macrophages influence the progression and resolution of ALI due to their innate immunological function and paracrine activity. Syngeneic primary bone marrow-derived macrophages (BMDMs) were tested as a cell-based therapy in a mouse model of APAP-induced ALI (APAP-ALI).MethodsSeveral phenotypically distinct BMDM populations were delivered intravenously to APAP-ALI mice when hepatic necrosis was established, and then evaluated based on their effects on injury, inflammation, immunity, and regeneration. In vivo phagocytosis assays were used to interrogate the phenotype and function of alternatively activated BMDMs (AAMs) post-injection. Finally, primary human AAMs sourced from healthy volunteers were evaluated in immunocompetent APAP-ALI mice.ResultsBMDMs rapidly localised to the liver and spleen within 4 h of administration. Injection of AAMs specifically reduced hepatocellular necrosis, HMGB1 translocation, and infiltrating neutrophils following APAP-ALI. AAM delivery also stimulated proliferation in hepatocytes and endothelium, and reduced levels of several circulating proinflammatory cytokines within 24 h. AAMs displayed a high phagocytic activity both in vitro and in injured liver tissue post-injection. Crosstalk with the host innate immune system was demonstrated by reduced infiltrating host Ly6Chi macrophages in AAM-treated mice. Importantly, therapeutic efficacy was partially recapitulated using clinical-grade primary human AAMs in immunocompetent APAP-ALI mice, underscoring the translational potential of these findings.ConclusionWe identify that AAMs have value as a cell-based therapy in an experimental model of APAP-ALI. Human AAMs warrant further evaluation as a potential cell-based therapy for APAP overdose patients with established liver injury.Lay summaryAfter an overdose of acetaminophen (paracetamol), some patients present to hospital too late for the current antidote (N-acetylcysteine) to be effective. We tested whether macrophages, an injury-responsive leukocyte that can scavenge dead/dying cells, could serve as a cell-based therapy in an experimental model of acetaminophen overdose. Injection of alternatively activated macrophages rapidly reduced liver injury and reduced several mediators of inflammation. Macrophages show promise to serve as a potential cell-based therapy for acute liver injury.

Von Willebrand factor delays liver repair after acetaminophen-induced acute liver injury in mice

Acetaminophen (APAP)-induced acute liver failure is associated with substantial alterations in the hemostatic system. In mice, platelets accumulate in the liver after APAP overdose and appear to promote liver injury. Interestingly, patients with acute liver injury have highly elevated levels of the platelet-adhesive protein von Willebrand factor (VWF), but a mechanistic connection between VWF and progression of liver injury has not been established. We tested the hypothesis that VWF contributes directly to experimental APAP-induced acute liver injury. Wild-type mice and VWF-deficient (Vwf-/-) mice were given a hepatotoxic dose of APAP (300 mg/kg, i.p.) or vehicle (saline). VWF plasma levels were measured by ELISA, and liver necrosis or hepatocyte proliferation was measured by immunohistochemistry. Platelet and VWF deposition were measured by immunofluorescence. In wild-type mice, VWF plasma levels, high molecular weight (HMW) VWF multimers, and VWF activity decreased 24 h after APAP challenge. These changes coupled to robust hepatic VWF and platelet deposition, although VWF deficiency had minimal effect on peak hepatic platelet accumulation or liver injury. VWF plasma levels were elevated 48 h after APAP challenge, but with relative reductions in HMW multimers and VWF activity. Whereas hepatic platelet aggregates persisted in livers of APAP-challenged wild-type mice, platelets were nearly absent in Vwf-/- mice 48 h after APAP challenge. The absence of platelet aggregates was linked to dramatically accelerated repair of the injured liver. Complementing observations in Vwf-/- mice, blocking VWF or the platelet integrin αIIbβ3 during development of injury significantly reduced hepatic platelet aggregation and accelerated liver repair in APAP-challenged wild-type mice. These studies are the first to suggest a mechanistic link between VWF, hepatic platelet accumulation, and liver repair. Targeting VWF might provide a novel therapeutic approach to improve repair of the APAP-injured liver. Patients with acute liver injury due to acetaminophen overdose have highly elevated levels of the platelet-adhesive protein von Willebrand factor. It is not known whether von Willebrand factor plays a direct role in the progression of acute liver injury. We discovered that von Willebrand factor delays repair of the acetaminophen-injured liver in mice and that targeting von Willebrand factor, even in mice with established liver injury, accelerates liver repair.

From the Editor’s desk....: July 2018

From the Editor’s desk....: July 2018

The Role of Lipocalin‐2 (LCN2) and High Mobility Box Group 1 (HMGB1) in Acetaminophen‐induced Acute Liver Failure

Acetaminophen (APAP) overdose is the most common cause of Acute Liver Failure (ALF). Following APAP overdose, toxicity is initiated by the conversion of APAP to its reactive metabolic N‐acetyl‐p‐benzoquinone imine (NAPQI) by CYP2E1/CYP3A4. NAPQI binds covalently to mitochondrial proteins forming 3‐(cysteine‐S‐yl)‐acetaminophen (APAP‐Cys). This generates initial hepatotoxicity in the form of centrilobular necrosis and sterile inflammation. Lipocalin‐2 (LCN2), is an acute‐phase innate immune protein, upregulated during tissue injury in various organs including the liver. Recently we have demonstrated that LCN2 is pro‐inflammatory and elevated during APAP overdose in mouse model. Interestingly, LCN2 knockout (LCN2 KO) mice exhibit protection from APAP overdose compared to its wild type (WT) littermates. During APAP overdose there is necrosis of hepatocytes, these hepatocytes release Damage Associated Molecular Patterns (DAMPS) that further exacerbate the inflammatory process. One such DAMP is High Mobility Group Box 1 (HMGB1). However, the role of LCN2 and HMGB1 in APAP overdose has not yet been investigated.Present study investigates the molecular mechanism of LCN2 during the progression of acute liver injury in APAP overdose model. We hypothesize that during APAP induced ALF, LCN2 induces the expression and translocation of HMGB1 which in turn mediates inflammation and progression of injury. An in vitro model of HepG2 cells as well as an in vivo model of WT and LCN2 KO mice will be utilized to test our hypothesis. HepG2 cells will be treated with recombinant LCN2 to investigate whether exogenous LCN2 can cause induction and translocation of HMGB1. Translocation of HMGB1 will be assessed by immunofluorescence staining. Serum and tissues samples of WT and LCN2 KO mice subjected to APAP overdose will be assessed for expression of HMGB1. APAP‐cys formation will be measured in WT and LCN2 KO using the High‐Performance Liquid Chromatography method to ensure comparable bioactivation based injury between the WT and LCN2 KO mice. Lastly, histopathology will be performed on liver samples from both groups to assess liver pathology. It is anticipated that LCN2 mediates progression of liver injury in APAP overdose via HMGB1.Support or Funding InformationPhiladelphia College of Osteopathic Medicine‐Division of ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

IL-33 signalling in liver immune cells enhances drug-induced liver injury and inflammation.

The aim of this study was to investigate the contribution of IL-33/ST2 axis in the onset and progression of acute liver injury using a mice model of drug-induced liver injury (DILI). DILI was induced by overdose administration of acetaminophen (APAP) by oral gavage in wild-type BALB/c, ST2-deficient mice and in different bone marrow chimeras. Neutrophils were depleted by anti-Ly6G and macrophages with clodronate liposomes (CLL). Blood and liver were collected for biochemical, immunologic and genetic analyses. Mice were imaged by confocal intravital microscopy and liver non-parenchymal cells and hepatocytes were isolated for flow cytometry, genetic and immunofluorescence studies. Acetaminophen overdose caused a massive necrosis and accumulation of immune cells within the liver, concomitantly with IL-33 and chemokine release. Liver non-parenchymal cells were the major sensors for IL-33, and amongst them, neutrophils were the major players in amplification of the inflammatory response triggered by IL-33/ST2 signalling pathway. Blockage of IL-33/ST2 axis reduces APAP-mediated organ injury by dampening liver chemokine release and activation of resident and infiltrating liver non-parenchymal cells.

Hypercoagulability progresses to hypocoagulability during evolution of acetaminophen-induced acute liver injury in pigs

Increases in prothrombin time (PT) and international normalised ratio (INR) characterise acute liver injury (ALI) and failure (ALF), yet a wide heterogeneity in clotting abnormalities exists. This study defines evolution of coagulopathy in 10 pigs with acetaminophen (APAP)-induced ALI compared to 3 Controls. APAP administration began at 0 h and continued to ‘ALF’, defined as INR >3. In APAP pigs, INR was 1.05 ± 0.02 at 0 h, 2.15 ± 0.43 at 16 h and > 3 at 18 ± 1 h. At 12 h thromboelastography (TEG) demonstrated increased clot formation rate, associated with portal vein platelet aggregates and reductions in protein C, protein S, antithrombin and A Disintegrin and Metalloprotease with Thrombospondin type 1 repeats–13 (ADAMTS-13) to 60%, 24%, 47% and 32% normal respectively. At 18 ± 1 h, INR > 3 was associated with: hypocoagulable TEG profile with heparin-like effect; falls in thrombin generation, Factor V and Factor VIII to 52%, 19% and 17% normal respectively; further decline in anticoagulants; thrombocytopenia; neutrophilia and endotoxemia. Multivariate analysis, found that ADAMTS-13 was an independent predictor of a hypercoagulable TEG profile and platelet count, endotoxin, Protein C and fibrinogen were independent predictors of a hypocoagulable TEG profile. INR remained normal in Controls. Dynamic changes in coagulation occur with progression of ALI: a pro-thrombotic state progresses to hypocoagulability.

Augmenter of liver regeneration protects against carbon tetrachloride-induced liver injury by promoting autophagy in mice.

BackgroundAugmenter of liver regeneration (ALR) exerts strong hepatoprotective properties in various animal models of liver injury, but its protective mechanisms have not yet been explored. Autophagy is a recently recognized rudimentary cellular response to inflammation and injury. The aim of this study was to test the hypothesis that ALR may protect against acute liver injury through the autophagic pathway.MethodsThe level and role of ALR in liver injury were studied in a mouse model of acute liver injury induced by carbon tetrachloride (CCl4). The effect of ALR on autophagy was analyzed in vitro and in vivo. After autophagy was inhibited by 3-methyladenine (3-MA), apoptosis and proliferation were detected in the mouse model with acute liver injury. The ALR and autophagic levels were measured in patients with liver cirrhosis (LC) and acute liver failure (ALF), respectively.ResultsDuring the progression of acute liver injury, the ALR levels increased slightly in early stage and significantly decreased in late stage in mice Treatment with an ALR plasmid via tail vein injection protected mice against acute liver injury. The protective effect of ALR relied on the induction of autophagy, which was supported by the following evidence: (1) ALR overexpression directly induced autophagy flux in vitro and in vivo; and (2) ALR treatment suppressed apoptosis and promoted proliferation in mice exposed to CCl4, but the inhibition of autophagy reversed these effects. More importantly, the ALR levels decreased in patients with LC and ALF compared with normal controls.ConclusionWe demonstrated that ALR ameliorated liver injury via an autophagic mechanism, which indicates a potential therapeutic application for liver injury.

Acute liver failure in dogs and cats.

To define acute liver failure (ALF), review the human and veterinary literature, and discuss the etiologies and current concepts in diagnostic and treatment options for ALF in veterinary and human medicine. In veterinary medicine ALF is most commonly caused by hepatotoxin exposure, infectious agents, inflammatory diseases, trauma, and hypoxic injury. A patient may be deemed to be in ALF when there is a progression of acute liver injury with no known previous hepatic disease, the development of hepatic encephalopathy of any grade that occurs within 8 weeks after the onset of hyperbilirubinemia (defined as plasma bilirubin >50 μM/L [>2.9 mg/dL]), and the presence of a coagulopathy. Diagnostic testing to more specifically characterize liver dysfunction or pathology is usually required. Supportive care to aid the failing liver and compensate for the lost functions of the liver remains the cornerstone of care of patients with ALF. Advanced therapeutic options such as extracorporeal liver assist devices and transplantation are currently available in human medicine. The prognosis for ALF depends upon the etiology, the degree of liver damage, and the response to therapy. In veterinary medicine, the prognosis is generally poor.

Administration of phospholipase D1 inhibitor attenuates aminoacetophenone-induced acute liver injury (CAM5P.245)

Abstract A phospholipase D1 (PLD1)-selective inhibitor, N-[2-[4-(5-chloro-2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)-1-piperidinyl]-1-methylethyl]-2-naphthalenecarboxamide, strongly blocked aminoacetophenone-induced acute liver injury. Administration of 500 mg/kg aminoacetophenone strongly increased alanine transaminase levels in serum, which was markedly decreased by PLD1 inhibitor administration in a dose-dependent manner, showing almost complete inhibition by 4 mg/kg of the inhibitor. During the pathological progress of acute liver injury, GSH levels are decreased, which is recovered by the administration of PLD1 inhibitor. JNK mediates aminoacetophenone-induced oxidative stress and liver injury. PLD1 inhibitor administration strongly blocked JNK phosphorylation in aminoacetophenone-induced acute liver injury model. Several inflammatory cytokines including TNF-α, IFN-γ, CCL2, and IL-1β were strongly increased from liver in aminoacetophenone-induced acute liver injury, which were also markedly decreased by PLD1 inhibitor administration. Taken together, the results indicate that the PLD1 inhibitor strongly show therapeutic effects reverse the progression of acute liver injury, and PLD1 can be regarded as an important target molecule to develop therapeutic agents against acute liver injury.

Mechanistic biomarkers in acute liver injury: Are we there yet?

Although the outcome of acute liver failure (ALF) has improved due to developments in the general intensive care techniques, mortality rates without transplantation in patients who fulfil poor prognostic criteria are still in excess of 80%. Besides N-acetylcysteine, there are no specific treatment options for ALF that occurs on the background of acetaminophen (APAP) toxicity. Also, strategies to limit progression of acute liver injury in patients who are progressing to ALF remain an unmet need. Further difficulties in the management of patients with ALF are the lack of biomarkers that may indicate progression of liver failure early as decision making regarding listing for transplantation in patients with ALF is challenging.

Circulating microRNAs: promising candidates serving as novel biomarkers of acute hepatitis

Acute liver failure as life threatening condition comprises a difficult diagnostic situation to evaluate potential outcomes and therapeutic options. Thus, prognostic indicators are urgently needed for evaluation of progression of liver injury, clinical outcome, prognosis, and for therapeutic response. Recently, circulating microRNA, in particular miR-122, was described as a potential biomarker of acute liver injury after intoxication of mice. Circulating microRNA (miRNA) molecules are very stable and RNase-resistant due to protein aggregation and vesicle enclosure. Since miRNA species are known to be associated with chronic liver damage or with liver cancer, circulating miRNA patterns are suggested to serve also as reporters for progression of acute liver failure. miRNA profiling analyses using PCR arrays or next generation sequencing, may achieve identification of miRNA species that are linked to the rapid progression of acute liver injury, to the outcome of liver failure, or to the therapeutic response. Therefore, circulating miRNAs are promising, non-invasive biomarkers of future diagnostic approaches. However, normalisation of circulating miRNA levels is essential and further standardisation of miRNA quantification assays is needed.

Contrast‐enhanced ultrasonography using Sonazoid to evaluate changes in hepatic hemodynamics in acute liver injury

Disturbances in hepatic microcirculation are believed to be involved in the mechanisms regulating the progression of acute liver injury (ALI). Evaluation of hepatic hemodynamics in patients with acute liver injury might be helpful in understanding the extent of the intrahepatic microcirculatory disturbances. Therefore, we investigated whether contrast-enhanced ultrasonography (CEUS) is useful to evaluate the changes in hepatic hemodynamics in patients with ALI. CEUS was performed in 21 patients with ALI and coagulopathy. Participants were injected with 0.0075 mL Sonazoid/kg body weight, and time-intensity curves were simultaneously recorded for the hepatic and portal veins. The data were compared with those of 10 healthy volunteers. The arrival time of Sonazoid in the hepatic vein was similar to that in the portal vein in the patients, whereas the arrival time in the hepatic vein was delayed relative to that in the portal vain in the controls (interval between the hepatic and portal vein arrival times, control vs patients 6.74 ± 3.07 s vs 1.13 ± 1.07 s, P < 0.001). Repeated examination revealed that the interval between the hepatic and portal vein arrival times was extended by improvements in hepatic function. The early arrival of Sonazoid in the hepatic vein in the patients is likely to reflect the formation of intrahepatic shunts as a result of hepatic microcirculatory disturbances. CEUS using Sonazoid is a useful method to estimate the changes in hepatic hemodynamics in patients with ALI.

Inhibition of cyclooxygenase-2 aggravates secretory phospholipase A 2-mediated progression of acute liver injury

Our previous study [Bhave, V. S., Donthamsetty, S., Latendresse, J. R., Muskhelishvili, L., and Mehendale, H. M. 2008-this issue. Secretory phospholipase A 2 mediates progression of acute liver injury in the absence of sufficient COX-2. Toxicol Appl Pharmacol] showed that in the absence of sufficient induction and co-presence of cyclooxygenase-2 (COX-2), secretory phospholipase A 2 (sPLA 2) appearing in the intercellular spaces for cleanup of post-necrotic debris seems to contribute to the progression of toxicant-initiated liver injury, possibly by hydrolysis of membrane phospholipids of hepatocytes in the perinecrotic areas. To further test our hypothesis on the protective role of COX-2, male Fisher-344 rats were administered a selective COX-2 inhibitor, NS-398, and then challenged with a moderately toxic dose of CCl 4. This led to a 5-fold increase in the susceptibility of the COX-2 inhibited rats to CCl 4 hepatotoxicity and mortality. The CCl 4 bioactivating enzyme CYP2E1 protein, CYP2E1 enzyme activity, and the 14CCl 4-derived radiolabel covalently bound to the liver proteins were unaffected by the COX-2 inhibitor suggesting that the increased hepatotoxic sensitivity of the COX-2 inhibited rats was not due to higher bioactivation of CCl 4. Further investigation showed that this increased mortality was due to higher plasma and hepatic sPLA 2 activities, inhibited PGE 2 production, and progression of liver injury as compared to the non-intervened rats . In conclusion, inhibition of COX-2 mitigates the tissue protective mechanisms associated with COX-2 induction, which promotes sPLA 2-mediated progression of liver injury in an acute liver toxicity model. Because increased sPLA 2 activity in the intercellular space is associated with increased progression of injury, and induced COX-2 is associated with hepatoprotection, ratios of hepatic COX-2 and sPLA 2 activities may turn out to be a useful tool in predicting the extent of hepatotoxicities.

Secretory phospholipase A 2 mediates progression of acute liver injury in the absence of sufficient cyclooxygenase-2

Previous studies have shown that injury initiated by toxicants progresses even after most of the toxicant is eliminated from the body. One mechanism of progression of injury is the extracellular appearance of hydrolytic enzymes following leakage or upon cell lyses. Under normal conditions, after exposure to low to moderate doses of toxicants, secretory phospholipase A 2 (sPLA 2) and other hydrolytic enzymes are known to appear in the extracellular spaces in order to cleanup the post-necrotic debris in tissues. We tested the hypothesis that sPLA 2 contributes to progression of toxicant-initiated liver injury because of hydrolysis of membrane phospholipids of hepatocytes in the perinecrotic areas in the absence of sufficient cyclooxygenase-2 (COX-2). Male Sprague–Dawley rats were administered either a moderately hepatotoxic dose (MD, 2 ml CCl 4/kg, ip) or a highly hepatotoxic dose (HD, 3 ml CCl 4/kg, ip) of CCl 4. After MD, liver sPLA 2 and COX-2 were co-localized in the necrotic and perinecrotic areas and their activities in plasma and liver increased before decreasing in tandem with liver injury (ALT and histopathology) leading to 100% survival. In contrast, after the HD, high extracellular and hepatic sPLA 2 activities were accompanied by minimal COX-2 activity and localization in the liver throughout the time course. This led to progression of liver injury and 70% mortality. These data suggested a destructive role of sPLA 2 in the absence of sufficient COX-2. Time- and dose-dependent destruction of hepatocytes by sPLA 2 in isolated hepatocyte incubations confirmed the destructive ability of sPLA 2 when present extracellularly, suggesting its ability to spread injury in vivo. These findings suggest that sPLA 2, secreted for cleanup of necrotic debris upon initiation of hepatic necrosis, requires the co-presence of sufficiently induced COX-2 activity to prevent the run-away destructive action of sPLA 2 in the absence of the tissue protective mechanisms afforded by COX-2 induction.