Acute Liver Failure Induction Research Articles

Hyperactivation of succinate dehydrogenase promotes pyroptosis of macrophage via ROS-induced GSDMD oligomerization in acute liver failure

Acute liver failure (ALF) is a life-threatening disease with high mortality. Given excessive inflammation is one of the major pathogenesis of ALF, candidates targeting inflammation could be beneficial in the condition. Now the effect of hyperactivated succinate dehydrogenase (SDH) on promoting inflammation in lipopolysaccharide (LPS)-treated macrophages has been studied. However, its role and mechanism in ALF is not well understood. Here intraperitoneal injection of D-galactosamine and LPS was conducted in male C57BL/6 J mice to induce the ALF model. Dimethyl malonate (DMM), which inhibited SDH activity, was injected intraperitoneally 30 min before ALF induction. Macrophage pyroptosis was induced by LPS plus adenosine triphosphate (ATP). Pyroptosis-related molecules and proteins including GSDMD oligomer were examined by ELISA and western blot techniques, respectively. ROS production was assessed by fluorescence staining. The study demonstrated SDH activity was increased in liver macrophages from ALF mice. Importantly, DMM administration inhibited ROS, IL-1β, and pyroptosis-associated proteins levels (NLRP3, cleaved caspase-1, GSDMD-N, and GSDMD oligomers) both in the ALF model and in macrophages stimulated with LPS plus ATP. In vitro, ROS promoted pyroptosis by facilitating GSDMD oligomerization. Additionally, when ROS levels were increased through the addition of H2O2 to the DMM group, the levels of GSDMD oligomers were reverted. In conclusion, SDH hyperactivation promotes macrophage pyroptosis by ROS-mediated GSDMD oligomerization, suggesting that targeting this pathway holds promise as a strategy for treating ALF and other inflammatory diseases.

Effects of coenzyme Q10 and N-acetylcysteine on experimental poisoning by paracetamol in Wistar rats.

Paracetamol (PAR) is a drug widely used in human and veterinary medicine as an analgesic and antipyretic, often involved in cases of intoxication. The most common clinical signs result from damage to red blood cells and hepatocytes, and this intoxication is considered a model for the induction of acute liver failure. In the present study, the hepatoprotective effects of coenzyme Q10 (CoQ10) and N-acetylcysteine (NAC) against experimental paracetamol (PAR) poisoning were analysed. Thirty-five adult Wistar rats (Rattus novergicus albinus) were randomly assigned to five groups, and thirty-one of these survived the treatments. Negative control group (CON-) received 1mL of 0.9% NaCl orally (PO). Other groups received 1.2g/kg of PAR (PO). Positive control group (CON+) received only PAR. NAC group received 800 mg/kg intraperitoneally (IP) of NAC 1h after the administration of PAR and at 12 h received 1mL of 0.9% NaCl, IP. The fourth group (CoQ10) received 1h and 12 h after intoxication, CoQ10 (10mg/kg IP). And the fifth group (NAC+CoQ10) received NAC (800mg/kg, IP) and CoQ10 (10mg/kg, IP). After 12 hours, the rats were euthanized and necropsied to collect liver and kidney tissues for histopathological evaluation and electronic microscopy. A single dose of PAR caused severe acute hepatitis. NAC couldn't reverse the liver and kidney damages. The group that received CoQ10 and NAC had moderate liver damage, while the group that received only CoQ10 had lower values of liver enzymes and mild liver and kidney damage. Animals that received treatment with CoQ10 or NAC+CoQ10 presented normal hepatocyte mitochondria and nuclei. Although CoQ10 couldn't reverse PAR organ damage, results indicate promising hepatoprotection in Wistar rats.

Preclinical Experience of the Mayo Spheroid Reservoir Bioartificial Liver (SRBAL) in Management of Acute Liver Failure

The Spheroid Reservoir Bioartificial Liver (SRBAL) is an innovative treatment option for acute liver failure (ALF). This extracorporeal support device, which provides detoxification and other liver functions using high-density culture of porcine hepatocyte spheroids, has been reported in three randomized large animal studies. A meta-analysis of these three preclinical studies was performed to establish efficacy of SRBAL treatment in terms of survival benefit and neuroprotective effect. The studies included two hepatotoxic drug models of ALF (D-galactosamine, α-amanitin/lipopolysaccharide) or a liver resection model (85% hepatectomy) in pigs or monkeys. The SRBAL treatment was started in three different settings starting at 12 h, 24 h or 48 h after induction of ALF; comparisons were made with two similar control groups in each model. SRBAL therapy was associated with significant survival and neuroprotective benefits in all three animal models of ALF. The benefits of therapy were dose dependent with the most effective configuration of SRBAL being continuous treatment of 24 h duration and dose of 200 g of porcine hepatic spheroids. Future clinical testing of SRBAL in patients with ALF appears warranted.

Mucosal expression of defensin-5, soluble phospholipase A2 and lysozyme in the intestine in a rat model of acute liver failure and its relationship to intestinal bacterial translocation

IntroductionTo study the expression of defensin-5 (RD-5), soluble phospholipase A2 (sPLA2) and lysozyme in the intestine in a rat model of acute liver failure and its relationship with intestinal bacterial translocation (BT). Patients and methodsSprague–Dawley (SD) rats were divided into two groups. The experimental group was divided into five subgroups according to the lapsing time after the model was established, which were designated accordingly as 8h, 16h, 24h, 48h, and 72h groups. Acute liver failure (ALF) model was induced by intraperitoneal injection of 10% d-galactosamine. The homogenates of mesenteric lymph nodes (MLNs), liver and spleen from each group were cultured in agar to determine the bacterial outgrowth. The mRNA expression of RD-5, sPLA2, lysozyme and the protein expression of sPLA2, lysozyme were determined. ResultsNo bacteria grew in the organ cultures from the control group while experimental groups had positive cultures. Expression of the RD-5 and sPLA2 mRNA in the experimental groups gradually increased at early time points and peaked 16h after induction of ALF, then progressively decreased. The mRNA expression of lysozyme in the experimental group peaked at 8h after ALF induction, then progressively decreased. Similar results were obtained with Western blot and immunohistochemical staining. DiscussionThe immune barrier function of the ileal mucosa in the rat model of acute liver failure was compromised as demonstrated by the decreased expression of RD-5, sPLA2 and lysozyme in Paneth cells along with increased intestinal bacterial translocation.

Endoplasmic Reticulum Stress-Induced Upregulation of STARD1 Promotes Acetaminophen-Induced Acute Liver Failure.

Acetaminophen (APAP) overdose is a major cause of acute liver failure (ALF). Mitochondrial SH3BP5 (also called SAB) and phosphorylation of c-Jun N-terminal kinase (JNK) mediate the hepatotoxic effects of APAP. We investigated the involvement of steroidogenic acute regulatory protein (STARD1), a mitochondrial cholesterol transporter, in this process and sensitization by valproic acid (VPA), which depletes glutathione and stimulates steroidogenesis. Nonfasted C57BL/6J mice (control) and mice with liver-specific deletion of STARD1 (Stard1ΔHep), SAB (SabΔHep), or JNK1 and JNK2 (Jnk1+2ΔHep) were given VPA with or without APAP. Liver tissues were collected and analyzed by histology and immunohistochemistry and for APAP metabolism, endoplasmic reticulum (ER) stress, and mitochondrial function. Adult human hepatocytes were transplanted into Fah-/-/Rag2-/-/Il2rg-/-/NOD (FRGN) mice to create mice with humanized livers. Administration of VPA before administration of APAP increased the severity of liver damage in control mice. The combination of VPA and APAP increased expression of CYP2E1, formation of NAPQI-protein adducts, and depletion of glutathione from liver tissues of control mice, resulting in ER stress and the upregulation of STARD1. Livers from control mice given VPA and APAP accumulated cholesterol in the mitochondria and had sustained mitochondrial depletion of glutathione and mitochondrial dysfunction. Inhibition of ER stress, by administration of tauroursodeoxycholic acid to control mice, prevented upregulation of STARD1 in liver and protected the mice from hepatoxicity following administration of VPA and APAP. Administration of N-acetylcysteine to control mice prevented VPA- and APAP-induced ER stress and liver injury. Stard1ΔHep mice were resistant to induction of ALF by VPA and APAP, despite increased mitochondrial levels of glutathione and phosphorylated JNK; we made similar observations in fasted Stard1ΔHep mice given APAP alone. SabΔHepmice or Jnk1+2ΔHep mice did not develop ALF following administration of VPA and APAP. The ability of VPA to increase the severity of APAP-induced liver damage was observed in FRGN mice with humanized liver. In studies of mice, we found that upregulation of STARD1 following ER stress mediates APAP hepatoxicity via SH3BP5 and phosphorylation of JNK1 and JNK2.

The antagonistic effect of tamoxifen against d-galactosamine/lipopolysaccharide-induced acute liver failure is associated with reactivation of hepatic nuclear factor-κB

Context: Tamoxifen (TAM) ameliorates D-galactosamine/lipopolysaccharide (Gal/LPS)-induced acute liver failure (ALF) through its antioxidative effect; thus, this study was designed to determine whether the effectiveness of TAM is related to nuclear factor-κB (NF-κB) reactivation.Materials and methods: Experimental mice were injected with TAM once daily for 3 consecutive days intraperitoneally (i.p). Twelve hours after pretreatment, Gal/LPS was given to mice (i.p) for ALF induction. In the positive control group, N-acetylcysteine (NAC) was administered immediately after ALF establishment. Except for survival observation, other animals were sacrificed 7 h after Gal/LPS treatment. Survival and hepatic failure were evaluated. For the oxidation assessment, the reduced/oxidized glutathione (GSH/GSSG) ratio and hepatic superoxide dismutase (SOD) activity were analyzed using both colorimetry and Western blotting. Lastly, hepatic NF-κB activation was measured through Western blot analysis of p65 and IκBα.Results: The results indicated that pretreatment with TAM dramatically attenuated Gal/LPS-induced ALF, as demonstrated by improved survival (70%), decreased transaminase levels, and reversed histopathological manifestation. In addition, the hepatic GSH/GSSG ratio and SOD activity were decreased in the ALF model. However, to some degree, TAM and NAC effectively prevented this undesirable phenomenon in contrast to the ALF model. Western blotting revealed that compared with mice in the ALF model group, mice treated with TAM or NAC showed reactivation of hepatic NF-κB.Conclusions: Taking the results together with those of other studies, we conclude that TAM may attenuate Gal/LPS-induced ALF by antagonizing oxidative stress through NF-κB reactivation.

Mesenchymal stem cells increase heme oxygenase 1-activated autophagy in treatment of acute liver failure

In recent years, transplantation of mesenchymal stem cells (MSCs) has attracted much attention as a potential cell-based therapy for acute liver failure (ALF). As an inducible enzyme, heme oxygenase 1 (HO-1) has been reported to have cytoprotective, anti-apoptotic and immunoregulatory effects. Autophagy, a conserved catabolic process in cells, may be an important pathway for MSCs to treat ALF. In this study, we aimed to explore whether MSCs treat ALF by regulating autophagy and whether HO-1 was involved in the same pathway. Bone marrow-derived MSCs were isolated from Sprague-Dawley rats and cultured according to an established protocol. Co-culture systems of MSCs and hepatocytes were used to assess autophagy in the treatment of ALF. Meanwhile, MSCs were transplanted into rats with d-galactosamine (Gal)-induced ALF. Autophagy inhibitor (3-methyladenine, 3-MA), HO-1 inhibitor (zinc protoporphyrin, ZnPP) and PI3K specific inhibitor (LY294002) were employed in the study. Blood samples and liver tissues were collected before euthanasia. Survival rate, liver function, inflammatory factors, histology, Ki67 and TUNEL staining were determined. MSCs transplantation alleviated ALF both in vivo and in vitro. Autophagy and autophagy-related proteins were significantly up-regulated during MSCs treatment. 3-MA attenuated the therapeutic effect of MSCs. Administration of LY294002 before ALF induction inhibited hepatocyte autophagy. During the MSCs treatment, the HO-1 expression was increased, while inhibiting HO-1 attenuated the therapeutic effect of MSCs as well as hepatocyte autophagy. These findings suggested MSCs could alleviate ALF by increasing the HO-1 expression, which played an important role in activating autophagy through PI3K/AKT signaling pathway.

Effect of tumor necrosis factor-\u03b1 on the expression of the ammonia transporter Rhcg in the brain in mice with acute liver failure

BackgroundAmmonia and tumor necrosis factor-alpha (TNF-α) play important roles in the mechanisms of hepatic encephalopathy (HE). Rhesus glycoprotein C (Rhcg) is important for ammonia transport especially in the kidney. The aim of the present study was to investigate the role of Rhcg in the brain in acute liver failure (ALF) and the effect of TNF-α on Rhcg expression.MethodsALF mouse models were generated by treatment with d-galactosamine (D-GalN) and lipopolysaccharide (LPS), or D-GalN and TNF-α. ALF induction was blocked by pretreatment with anti-TNF-α IgG. The levels of serum TNF-α were determined by ELISA. Blood ammonia and brain ammonia concentrations were detected using an ammonia assay kit. The expression and distribution of Rhcg in the brain tissues of ALF mice were examined by western blotting, real-time PCR, immunohistochemical, and immunofluorescence analyses.ResultsSerum TNF-α levels were increased in the LPS/D-GalN group. Blood and brain ammonia were increased in the LPS/D-GalN- and TNF-α/D-GalN-induced ALF groups. Rhcg mRNA and protein levels were elevated in both ALF groups, consistent with the increase in blood and brain ammonia. Rhcg was mainly expressed in vascular endothelial cells and astrocytes. Pretreatment with anti-TNF-α IgG antibody downregulated Rhcg in brain tissues in the LPS/D-GalN group, prevented the occurrence of ALF, and reduced blood and brain ammonia levels in the LPS/D-GalN group.ConclusionTNF-α promoted the transport of ammonia from the blood to brain tissues and exacerbated the toxic effects of ammonia by upregulating Rhcg.

Dendritic cells with increased expression of suppressor of cytokine signaling 1(SOCS1) gene ameliorate lipopolysaccharide/d-galactosamine-induced acute liver failure

Acute liver failure is a devastating clinical syndrome with extremely terrible inflammation reaction, which is still lack of effective treatment in clinic. Suppressor of Cytokine Signaling 1 protein is inducible intracellular negative regulator of Janus kinases (JAK)/signal transducers and activators of transcription (STAT) pathway that plays essential role in inhibiting excessive intracellular signaling cascade and preventing autoimmune reaction. In this paper, we want to explore whether dendritic cells (DCs) with overexpression of SOCS1 have a therapeutic effect on experimental acute liver failure. Bone marrow derived dendritic cells were transfected with lentivirus encoding SOCS1 and negative control lentivirus, thereafter collected for costimulatory molecules analysis, allogeneic Mixed Lymphocyte Reaction and Western blot test of JAK/STAT pathway. C57BL/6 mice were randomly separated into normal control and treatment groups which respectively received tail vein injection of modified DCs, negative control DCs and normal saline 12 h earlier than acute liver failure induction. Our results indicated that DCs with overexpression of SOCS1 exhibited like regulatory DCs (DCregs) with low level of costimulatory molecules and poor allostimulatory ability in vitro, which was supposed to correlate with block of JAK2/STAT1 signaling. In vivo tests, we found that infusion of modified DCs increased survival rate of acute liver failure mice and alleviate liver injury via inhibition of TLR4/HMGB1 pathway. We concluded that DCs transduced with SOCS1 gene exhibit as DCregs through negative regulation of JAK2/STAT1 pathway and ameliorated lipopolysaccharide/d-galactosamine induced acute liver failure via inhibition of TLR4 pathway.

Tamoxifen Prevents D-galactosamine/Lipopolysaccharide-Induced Murine Acute Hepatic Failure through Inhibition of Oxidative Stress and Mmd-2 Upregulation

ABSTRACTOxidative stress is considered one of the major mechanisms underlying lipopolysaccharide (LPS)-induced acute liver failure (ALF). Tamoxifen has been reported to ameliorate LPS-induced ALF via the induction of monocyte to macrophage differentiation-associated 2 (Mmd-2). Whether antioxidant effects are involved remains unknown. Mice were given tamoxifen (TAM) once a day for 3 days. Twelve hours later, d-galactosamine (GaIN) and LPS were injected intraperitoneally to induce ALF. N-Acetylcysteine (NAC) was administered immediately after ALF induction as a positive control. The results showed that serum transaminases increased and hepatic antioxidants decreased significantly in the model group. ALF was alleviated markedly by TAM or NAC treatment. This demonstrated that ALF may be associated with excessive oxidative stress caused by decreased expression of antioxidant enzymes. Both TAM and NAC increased the levels and activity of these antioxidant enzymes significantly (p < 0.05). Hepatic Mmd-2 expression was downregulated in the control group while remaining stable or exhibiting elevated levels in the TAM or NAC groups. The results indicate that TAM may protect mice from GaIN/LPS-induced ALF through increased activity of antioxidant enzymes and upregulation of Mmd-2 expression.

Novel spheroid reservoir bioartificial liver improves survival of nonhuman primates in a toxin-induced model of acute liver failure.

This study aims to evaluate the effectiveness and safety of the spheroid reservoir bioartificial liver (SRBAL) with porcine hepatocyte organoids in a preclinical nonhuman primate model of acute liver failure (ALF).Methods: Thirty healthy rhesus monkeys were infused with α-amanitin and lipopolysaccharide and randomized into five groups (ALF alone control group; sham no-cell SRBAL treatment group; groups A, B and C with SRBAL treatment started at 12 h, 24 h and 36 h after induction of ALF, respectively). Animals were continuously treated with the SRBAL device for 6 h and followed for up to 336 h.Results: Survival of ALF monkeys improved with hepatocyte SRBAL treatment compared to control groups. Blood ammonia and total bilirubin were lower, and albumin levels were higher in all hepatocyte SRBAL treatment groups. No evidence of porcine endogenous retrovirus was identified in monkey liver or blood after SRBAL treatment. Titers of monkey antibody (IgG, IgM) did not rise after SRBAL treatment. In survival cases, the proportion of necrotic and apoptotic hepatocytes was lower in SRBAL-treated groups, with earlier liver regeneration leading to recovery. Cytokines TNF-α, IL-6, IL-12, IL-1β, IL-8, IFN-γ and IL-2 were ameliorated by the SRBAL treatment, while levels of M-CSF; HGF, EGF and VEGF; IL-1RA and MIF rose on priming, proliferation and the late phase of liver regeneration.Conclusions: The benefit of SRBAL therapy included preventive effects and therapeutic effects. SRBAL improved survival rate and prolonged median survival time in a nonhuman primate model of drug-induced ALF, and these benefits declined with a delay in the initiation of therapy. Improved survival and recovery of ALF monkeys was associated with a reduction in blood ammonia levels, inhibition of the pro-inflammatory response of ALF, and provided a microenvironment more suitable for regeneration of the injured liver.

Piperidylmethyloxychalcone improves immune-mediated acute liver failure via inhibiting TAK1 activity

Mice deficient in the toll-like receptor (TLR) or the myeloid differentiation factor 88 (MyD88) are resistant to acute liver failure (ALF) with sudden death of hepatocytes. Chalcone derivatives from medicinal plants protect from hepatic damages including ALF, but their mechanisms remain to be clarified. Here, we focused on molecular basis of piperidylmethyloxychalcone (PMOC) in the treatment of TLR/MyD88-associated ALF. C57BL/6J mice were sensitized with D-galactosamine (GalN) and challenged with Escherichia coli lipopolysaccharide (LPS, TLR4 agonist) or oligodeoxynucleotide containing unmethylated CpG motif (CpG ODN, TLR9 agonist) for induction of ALF. Post treatment with PMOC sequentially ameliorated hepatic inflammation, apoptosis of hepatocytes, severe liver injury and shock-mediated death in ALF-induced mice. As a mechanism, PMOC inhibited the catalytic activity of TGF-β-activated kinase 1 (TAK1) in a competitive manner with respect to ATP, displaced fluorescent ATP probe from the complex with TAK1, and docked at the ATP-binding active site on the crystal structure of TAK1. Moreover, PMOC inhibited TAK1 auto-phosphorylation, which is an axis in the activating pathways of nuclear factor-κB (NF-κB) or activating protein 1 (AP1), in the liver with ALF in vivo or in primary liver cells stimulated with TLR agonists in vitro. PMOC consequently suppressed TAK1-inducible NF-κB or AP1 activity in the inflammatory injury, an early pathogenesis leading to ALF. The results suggested that PMOC could contribute to the treatment of TLR/MyD88-associated ALF with the ATP-binding site of TAK1 as a potential therapeutic target.

C5a/C5aR pathway is essential for up-regulating SphK1 expression through p38-MAPK activation in acute liver failure.

AIMTo investigate the role of the complement 5a (C5a)/C5a receptor (C5aR) pathway in the pathogenesis of acute liver failure (ALF) in a mouse model.METHODSBALB/c mice were randomly assigned to different groups, and intraperitoneal injections of lipopolysaccharide (LPS)/D-galactosamine (D-GalN) (600 mg/kg and 10 μg/kg) were used to induce ALF. The Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase (ALT) levels, at different time points within a 1-wk period, were detected with a biochemistry analyzer. Pathological examination of liver tissue was performed 36 h after ALF induction. Serum complement 5 (C5), C5a, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, high-mobility group protein B1 (HMGB1) and sphingosine-1-phosphate levels were detected by enzyme-linked immunosorbant assay. Hepatic morphological changes at 36 h after ALF induction were assessed by hematoxylin and eosin staining. Expression of C5aR, sphingosine kinase 1 (SphK1), p38-MAPK and p-p38-MAPK in liver tissue, peripheral blood mononuclear cells (PBMCs) and peritoneal exudative macrophages (PEMs) of mice or RAW 264.7 cells was analyzed by western blotting. C5aR mRNA levels were detected by quantitative real-time PCR.RESULTSActivation of C5 and up-regulation of C5aR were observed in liver tissue and PBMCs of mice with ALF. Blockade of C5aR with a C5aR antagonist (C5aRa C5aRa) significantly reduced the levels of serum ALT, inflammatory cytokines (TNF-α, IL-1β and IL-6) and HMGB1, as well as the liver tissue damage, but increased the survival rates (P < 0.01 for all). Blockade of C5aR decreased SphK1 expression in both liver tissue and PBMCs significantly at 0.5 h after ALF induction. C5aRa pretreatment significantly down-regulated the phosphorylation of p38-MAPK in liver tissues of ALF mice and C5a stimulated PEMs or RAW 264.7 cells. Moreover, inhibition of p38-MAPK activity with SB203580 reduced SphK1 protein production significantly in PEMs after C5a stimulation.CONCLUSIONThe C5a/C5aR pathway is essential for up-regulating SphK1 expression through p38 MAPK activation in ALF in mice, which provides a potential immunotherapeutic strategy for ALF in patients.

Sphingosine kinase 1 dependent protein kinase C-δ activation plays an important role in acute liver failure in mice.

To investigate the role of protein kinase C (PKC)-δ activation in the pathogenesis of acute liver failure (ALF) in a well-characterized mouse model of D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced ALF. BALB/c mice were randomly assigned to five groups, and ALF was induced in mice by intraperitoneal injection of D-GaIN (600 mg/kg) and LPS (10 μg/kg). Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at different time points within one week were determined using a multiparameteric analyzer. Serum levels of high-mobility group box 1 (HMGB1), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 as well as nuclear factor (NF)-κB activity were determined by enzyme-linked immunosorbent assay. Hepatic morphological changes at 36 h after ALF induction were assessed by hematoxylin and eosin staining. Expression of PKC-δ in liver tissue and peripheral blood mononuclear cells (PBMCs) was analyzed by Western blot. The expression and activation of PKC-δ were up-regulated in liver tissue and PBMCs of mice with D-GalN/LPS-induced ALF. Inhibition of PKC-δ activation with rottlerin significantly increased the survival rates and decreased serum ALT/AST levels at 6, 12 and 24 h compared with the control group (P < 0.001). Rottlerin treatment also significantly decreased serum levels of HMGB1 at 6, 12, and 24 h, TNF-α, IL-6 and IL-1 β at 12 h compared with the control group (P < 0.01). The inflammatory cell infiltration and necrosis in liver tissue were also decreased in the rottlerin treatment group. Furthermore, sphingosine kinase 1 (SphK1) dependent PKC-δ activation played an important role in promoting NF-κB activation and inflammatory cytokine production in ALF. SphK1 dependent PKC-δ activation plays an important role in promoting NF-κB activation and inflammatory response in ALF, and inhibition of PKC-δ activation might be a potential therapeutic strategy for this disease.

Expression of intestinal defensin-5, soluble phospholipase A2 and lysozyme and the relation to bacterial translocation in rat models of acute liver failure

To study the intestinal expression of defensin-5 (RD-5), soluble phospholipase A2 (sPLA2) and lysozyme in acute liver failure (ALF) using rat models, and to determine the relation of these expressions to intestinal bacterial translocation. Forty-eight healthy male Sprague-Dawley rats were divided into a control group (n=8) and a model group (n=40; intraperitoneal injection of 10% D-galactosamine). The model group was further divided into five subgroups according to the time lapse after model establishment (8, 16, 24, 48, and 72 hours). At the end of the experiments, homogenates of mesenteric lymph nodes, liver and spleen were cultured in agar for bacterial outgrowth.Hematoxylin-eosin stained sections of liver and terminal ileum were examined under an optical microscope to assess pathological changes. mRNA expression of RD-5, sPLA2 and lysozyme in the terminal ileum was determined by reverse transcription-polymerase reaction (RT-PCR), and protein expression of sPLA2 and lysozyme from the same anatomic location was determined by western blotting and immunohistochemistry. Means between groups were compared with one-way analysis of variance. ALF was successfully induced in the D-galactosamine injected rats. No bacteria grew in the organ cultures from the control group, while 8.3%, 37.5% and 58.3% of the rats in the 24-, 48-and 72-hour model groups showed positive cultures. Despite this, the structure of the terminal ileum from the rats in the 72-hour model group was nearly intact, without obvious necrosis of mucosal epithelial cells. Expression of RD-5 and sPLA2 mRNA in the model groups gradually increased at early time points and peaked at 16 hours after induction of ALF (1.291+/-0.153 and 1.131+/-0.128), which was significantly higher than that detected in the control group (0.725+/-0.116 and 0.722+/-0.112, t=69.25, 95.71, all P<0.01). After that, the expression of RD-5 and sPLA2 mRNA progressively decreased, and by 72 hours after the induction of ALF, the expression (0.415+/-0.104 and 0.425+/-0.076) was significantly lower than that of the control group (t=31.55 and 44.98, all P<0.01). Lysozyme mRNA expression in the model group peaked at 8 hours after ALF induction (1.211+/-0.107), which was higher than that of the control group at this time point (0.853+/-0.093), and by 72 hours after ALF induction it declined to 0.704+/-0.103, which was significantly lower than that of the control group (t=9.224; all P=0.009). In addition, at 72 hours after ALF induction the protein expression of both lysozyme and sPLA2 was significantly lower in the model group (0.327+/-0.086 and 0.382+/-0.057) than in the control group (0.583+/-0.121 and 0.650+/-0.093, t=12.28 and 15.83, P=0.004 and 0.001). Similar results were obtained with immunohistochemical staining. The function of the ileal mucosal immune barrier in the rat model of acute liver failure decreased, along with decreases in expression of RD-5, sPLA2 and lysozyme in the Paneth cells.At the same time, the rate of organ bacterial translocation increased without obvious injury to the intestinal mucosa structure.

Acute Liver Failure Induced by Thioacetamide: Selection of Optimal Dosage in Wistar and Lewis Rats

Acute liver failure (ALF) is a clinical condition with very high mortality rate. Its pathophysiological background is still poorly understood, which necessitates a search for optimal experimental ALF models with features resembling those of the human disorder. Taking into consideration reproducibility of induction of ALF, adequate animal size, cost of animals, the required time gap between insult and death of animals ("therapeutic window"), potential risk to investigator and other aspects, administration of thioacetamide (TAA) in rats is currently most recommended. However, the fundamental details of this ALF model have not yet been evaluated. This prompted us to investigate, first, the course of ALF as induced by intraperitoneal TAA at doses increasing from 175 to 700 mg/kg BW per day. The animals' survival rate, plasma alanine and aspartate aminotransferase activities, and bilirubin and ammonia levels were determined over the follow-up period. Second, we examined whether Wistar and Lewis rats exhibit any differences in the course of ALF induced by different TAA doses. We found that the optimal dose for ALF induction in rats is 350 mg.kg(-1) i.p., given as a single injection. Wistar rats proved more susceptible to the development of TAA-induced ALF compared with Lewis rats. Collectively, our present findings provide a sound methodological background for experimental studies aimed at evaluation of pathophysiology and development of new approaches in the therapy of ALF.

Sodium butyrate protects against toxin-induced acute liver failure in rats

Acute liver failure (ALF) is a serious clinical syndrome with high mortality. Sodium butyrate has been shown to alleviate organ injury in a wide variety of preclinical models of critical diseases. The aim of this study was to investigate the protective effect of sodium butyrate on ALF in rats. All rats were randomly divided into control, model and sodium butyrate treatment groups. Except the control group, the rats were induced ALF animal model by subcutaneous injection of human serum albumin+ D-galactosamine+lipopolysaccharide. After induction of ALF, the rats in the treatment group received sodium butyrate (500 mg/kg) at 12-hour or 24-hour time point. Fourty-eight hours after ALF induction, the animals were sacrificed and samples were harvested. Serum endotoxin, high mobility group box-1 (HMGB1), liver function parameters, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were measured. The expression of HMGB1 and nuclear factor-kappa B (NF-kappaB) p65 protein in liver tissue was detected by Western blotting. The histological changes of liver and intestine were examined. The survival duration was also observed. Serum endotoxin, alanine aminotransferase, HMGB1, TNF-alpha and IFN-gamma were significantly increased and the liver histology showed more severe histopathological injury in the model group compared with the control group (P<0.05). Compared to the model group, sodium butyrate treatment significantly improved the histopathological changes in the liver and intestine, reduced serum endotoxin and inflammatory cytokines, suppressed HMGB1 and NF-kappaB p65 proteins in liver tissue, and prolonged the survival duration regardless of treatment at 12 hours or 24 hours after induction of ALF (P<0.05). Sodium butyrate protected the liver from toxin-induced ALF in rats. The mechanisms may be due to direct hepatoprotection and decreased intestinal permeability.

Mycophenolate Mofetil and Sirolimus in Hepatocyte Transplantation in an Experimental Model of Toxic Acute Liver Failure

ABSTRACTIntroduction: Hepatocyte transplantation is proposed as a solution for liver failure. The allotransplantation of hepatocytes has been studied extensively, however, graft rejection remains a major problem. The aim of the present study was to evaluate the immunosuppressive activity of mycophenolate mofetil (MMF), sirolimus, and their combination in an experimental model of hepatocyte allotransplantation in rats with acute liver failure. Materials and methods: Five male Wistar rats were used as hepatocyte donors and 60 male Lewis rats as recipients. The recipients were divided into five groups of 12 animals each. Group 1: no treatment. Group 2: cyclosporine. Group 3: sirolimus. Group 4: MMF. Group 5: MMF and sirolimus. All surviving animals were preserved for 15 days. For the induction of acute liver failure the recipients were injected with N-dimethyl-nitrosamine 24 hr before transplantation. The isolated hepatocytes were transplanted intrasplenically. Results: Analysis of the results showed a statistically significant prolongation of animal survival for groups 3, 4, and 5. More animals in group 5 survived than those in groups 3 and 4, although the difference was not statistically significant. Transplant hepatocyte survival was significantly better in groups 3, 4, and 5. Hepatocytes transplanted in the spleen of animals of group 5 showed better survival compared with those of groups 3 and 4. Conclusion: Use of MMF and sirolimus, as monotherapy or in combination, is both effective and safe as immunosuppressive treatment in hepatocyte transplantation, as was proven in this experimental protocol.

Aquaporin-4 deletion in mice reduces encephalopathy and brain edema in experimental acute liver failure

Brain edema and associated astrocyte swelling leading to increased intracranial pressure are hallmarks of acute liver failure (ALF). Elevated blood and brain levels of ammonia have been implicated in the development of brain edema in ALF. Cultured astrocytes treated with ammonia have been shown to undergo cell swelling and such swelling was associated with an increase in the plasma membrane expression of aquaporin-4 (AQP4) protein. Further, silencing the AQP4 gene in cultured astrocytes was shown to prevent the ammonia-induced cell swelling. Here, we examined the evolution of brain edema in AQP4-null mice and their wild type counterparts (WT-mice) in different models of ALF induced by thioacetamide (TAA) or acetaminophen (APAP). Induction of ALF with TAA or APAP significantly increased brain water content in WT mice (by 1.6%±0.3 and 2.3±0.4%, respectively). AQP4 protein was significantly increased in brain plasma membranes of WT mice with ALF induced by either TAA or APAP. In contrast to WT-mice, brain water content did not increase in AQP4-null mice. Additionally, AQP4-null mice treated with either TAA or APAP showed a remarkably lesser degree of neurological deficits as compared to WT mice; the latter displayed an inability to maintain proper gait, and demonstrated a markedly reduced exploratory behavior, with the mice remaining in one corner of the cage with its head tilted downwards. These results support a central role of AQP4 in the brain edema associated with ALF.

Cerebral microdialysis reflects the neuroprotective effect of fractionated plasma separation and adsorption in acute liver failure better and earlier than intracranial pressure: a controlled study in pigs

BackgroundCerebral edema is a well-recognized and potentially fatal complication of acute liver failure (ALF). The effectiveness of treatments that address intracranial hypertension is generally assessed by measuring intracranial pressure (ICP). The aim of this study was to determine the role of cerebral microdialysis in monitoring the efficacy of fractionated plasma separation and adsorption (FPSA) treatment for ALF. We hypothesized that in ALF cerebral microdialysis reflects the benefits of FPSA treatment on cerebral edema before ICP.MethodsA surgical resection model of ALF was used in 21 pigs. We measured plasma ammonia concentration, brain concentrations of glucose, lactate, pyruvate, glutamate and glutamine, and ICP. Animals were randomized into three groups: in one group eight animals received 6 hours of FPSA treatment 2 hours after induction of ALF; in another group 10 animals received supportive treatment for ALF only; and in the final group three underwent sham surgery.ResultsThe ICP was significantly higher in the ALF group than in the FPSA group 9 hours after surgery. The lactate/pyruvate (L/P) ratio was significantly lower in the FPSA group than the ALF group 5 hours after surgery, before any significant difference in ICP was detected. Indeed, significant changes in the L/P ratio could be observed within 1 hour of treatment. Glutamine levels were significantly lower in the FPSA group than the ALF group between 6 hours and 10 hours after surgery.ConclusionsBrain lactate/pyruvate ratio and concentration of glutamine measured by cerebral microdialysis reflected the beneficial effects of FPSA treatment on cerebral metabolism more precisely and rapidly than ICP in pigs with fulminant ALF. The role of glutamine as a marker of the efficacy of FPSA treatment for ALF appears promising, but needs further evaluation.