Thioacetamide-induced Acute Liver Research Articles

Expression of aquaporin-4 during brain edema in rats with thioacetamide-induced acute encephalopathy

To investigate the expression of aquaporin-4 (AQP4) during brain edema in rats with thioacetamide-induced acute liver failure and encephalopathy. The rat model of acute hepatic failure and encephalopathy was induced by intraperitoneal injection of thioacetamide (TAA) at a 24-hour interval for 2 consecutive days. Thirty-two SD rats were randomly divided into the model group (n = 24) and the control group (normal saline, n = 8). And then the model group was further divided into 3 subgroups by the timepoint of decapitation: 24 h (n = 8), 48 h (n = 8) and 60 h (n = 8). Then we observed their clinical symptoms and stages of HE, indices of liver function and ammonia, liver histology and brain water content. The expression of AQP4 protein in brain tissues was measured with Western blot and the expression of AQP4mRNA with RT-PCR (reverse transcription-polymerase chain reaction). Typical clinical manifestations of hepatic encephalopathy occurred in all TAA-administrated rats. The model rats showed the higher indices of ALT (alanine aminotransferase), AST (aspartate aminotransferase), TBIL (total bilirubin) and ammonia than the control rats (P < 0.05). The brain water content was significantly elevated in TAA-administrated rats compared with the control (P < 0.05). The expressions of AQP4 protein and mRNA in brain tissues significantly increased in TAA-administrated rats (P < 0.05). In addition, the expressions of AQP4 protein and mRNA were positively correlated with brain water content (r = 0.536, P < 0.01; r = 0.566, P = 0.01). The high expression of AQP4 in rats with TAA-induced acute liver failure and encephalopathy plays a significant role during brain edema. AQP4 is one of the molecular mechanisms for the occurrence of brain edema in hepatic encephalopathy.

Senescence marker protein 30 (SMP30)/regucalcin (RGN) expression decreases with aging, acute liver injuries and tumors in zebrafish

Senescence marker protein 30 (SMP30)/regucalcin (RGN) is known to be related to aging, hepatocyte proliferation and tumorigenesis. However, expression and function of non-mammalian SMP30/RGN is poorly understood. We found that zebrafish SMP30/RGN mRNA expression decreases with aging, partial hepatectomy and thioacetamide-induced acute liver injury. SMP30/RGN expression was also greatly decreased in a zebrafish liver cell line. In addition, we induced liver tumors in adult zebrafish by administering diethylnitrosamine. Decreased expression was observed in foci, hepatocellular carcinomas, cholangiocellular carcinomas and mixed tumors as compared to the surrounding area. We thus showed the importance of SMP30/RGN in liver proliferation and tumorigenesis.

Usefulness of a dual-input single-compartment model for quantitative evaluation of thioacetamide-induced acute liver injury in rats using dynamic contrast-enhanced computed tomography

We undertook this study to investigate the usefulness of a dual-input single-compartment model for evaluating thioacetamide (TAA)-induced acute liver injury in rats by use of dynamic contrast-enhanced computed tomography (DCE-CT). The rats in the TAA-treated group were injected intravenously with 140 mg/kg (n = 10) or 280 mg/kg (n = 10) of TAA, whereas those in the control group (n = 10) were injected with saline. The rate constants for the transfer of the contrast agent from the hepatic artery to the liver (K(1a)), from the portal vein to the liver (K(1p)), and from the liver to the blood (k₂), as well as liver perfusion (LP), the arterial fraction of LP (AFLP), distribution volume (DV), and mean transmit time (MTT) were estimated with use of the dual-input single-compartment model. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were also measured. K(1p), k₂, and LP decreased significantly in the TAA-treated group compared to those in the control group, whereas K(1a), AFLP, DV, and MTT increased significantly. The above parameters except for K (1a) were significantly correlated with both AST and ALT. We concluded that DCE-CT using the dual-input single-compartment model is useful for quantitative evaluation of TAA-induced acute liver injury.

Down‐regulation of Kir4.1 in the cerebral cortex of rats with liver failure and in cultured astrocytes treated with glutamine: Implications for astrocytic dysfunction in hepatic encephalopathy

Brain edema in acute hepatic encephalopathy (HE) is due mainly to swelling of astrocytes. Efflux of potassium is implicated in the prevention of glial swelling under hypoosmotic conditions. We investigated whether pathogenic factors of HE, glutamine (Gln) and/or ammonia, induce alterations in the expression of glial potassium channels (Kir4.1, Kir2.1) and Na(+) -K(+) -2Cl(-) cotransporter-1 (NKCC1) in rat cerebral cortex and cultured rat cortical astrocytes and whether these alterations have consequences for potassium efflux and astrocytic swelling. Thioacetamide-induced acute liver failure in rats resulted in significant decreases in the Kir4.1 mRNA and protein contents of cerebral cortex, whereas expression of Kir2.1 and NKCC1 remained unaltered. Incubation of primary cortical astrocytes for 72 hr in the presence of Gln (5 mM), but not of ammonia (5 mM or 10 mM), induced a decrease in the levels of Kir4.1 mRNA and protein. Similarly to incubation with Gln, reduction of Kir4.1 mRNA expression by RNA interference caused swelling of astrocytes as shown by confocal imaging followed by 3D computational analysis. Gln reduced the astrocytic uptake of D-[(3) H]aspartate, but, in contrast to the earlier reported effect of ammonia, this reduction was not accompanied by decreased expression of the astrocytic glutamate transporter GLT-1 mRNA. Both Gln and ammonia decreased hypoosmolarity-induced (86) Rb efflux from the cells, but the effect was more pronounced with Gln. The results indicate that down-regulation of Kir4.1 may mediate distinct aspects of Gln-induced astrocytic dysfunction in HE.

Expression of IL-1β, IL-6 and TNF-α in rats with thioacetamide-induced acute liver failure and encephalopathy: correlation with brain edema

Abstract Background: Secondary brain edema is a serious complication of hepatic encephalopathy (HE). Recently, it has been reported that proinflammatory cytokines are involved in the pathogenesis of brain edema during HE. Objectives: Observe the dynamic expressions of brain and plasma proinflammatory cytokines in encephalopathy rats, and evaluate the relationship between proinflammatory cytokines and brain edema. Methods: Acute HE rats were induced by intraperitoneal injection of thioacetamide (TAA) in 24 hours intervals for two consecutive days. Then, clinical symptom and stages of hepatic encephalopathy, motor activity counts, index of liver function, and brain water content were observed. The dynamic expressions of IL-1β, IL-6, and TNF-α in plasma and brain tissues were measured with enzyme-linked immunosorbent assay. Results: Typical clinical performances of hepatic encephalopathy were occurred in all TAA-administrated rats. The TAA rats showed lower motor activity counts and higher the index of alanine aminotransferase, aspartate aminotransferase, total bilirubin and ammonia than those in control rats. Brain water content was significantly enhanced in TAA rats compared with the control. The expressions of IL-1β, IL-6, and TNF- α in plasma and brain significantly increased in TAA rats. In addition, the expressions of cerebral proinflammatory cytokines were positively correlated with brain water content but negatively correlated with motor activity counts.Conclusion: Inflammation was involved in the pathogenesis of brain edema during TAA-induced HE.

Control of acute, chronic, and constitutive hyperammonemia by wild‐type and genetically engineered Lactobacillus plantarum in rodents†

Hyperammonemia is a common complication of acute and chronic liver diseases. Often accompanied with side effects, therapeutic interventions such as antibiotics or lactulose are generally targeted to decrease the intestinal production and absorption of ammonia. In this study, we aimed to modulate hyperammonemia in three rodent models by administration of wild-type Lactobacillus plantarum, a genetically engineered ammonia hyperconsuming strain, and a strain deficient for the ammonia transporter. Wild-type and metabolically engineered L. plantarum strains were administered in ornithine transcarbamoylase-deficient Sparse-fur mice, a model of constitutive hyperammonemia, in a carbon tetrachloride rat model of chronic liver insufficiency and in a thioacetamide-induced acute liver failure mice model. Constitutive hyperammonemia in Sparse-fur mice and hyperammonemia in a rat model of chronic hepatic insufficiency were efficiently decreased by Lactobacillus administration. In a murine thioacetamide-induced model of acute liver failure, administration of probiotics significantly increased survival and decreased blood and fecal ammonia. The ammonia hyperconsuming strain exhibited a beneficial effect at a lower dose than its wild-type counterpart. Improved survival in the acute liver failure mice model was associated with lower blood ammonia levels but also with a decrease of astrocyte swelling in the brain cortex. Modulation of ammonia was abolished after administration of the strain deficient in the ammonium transporter. Intestinal pH was clearly lowered for all strains and no changes in gut flora were observed. Hyperammonemia in constitutive model or after acute or chronic induced liver failure can be controlled by the administration of L. plantarum with a significant effect on survival. The mechanism involved in this ammonia decrease implicates direct ammonia consumption in the gut.

Fulminant Hepatic Failure in Rats Induces Oxidative Stress Differentially in Cerebral Cortex, Cerebellum and Pons Medulla

Hepatic Encephalopathy (HE) is one of the most common complications of acute liver diseases and is known to have profound influence on the brain. Most of the studies, available from the literature are pertaining to whole brain homogenates or mitochondria. Since brain is highly heterogeneous with functions localized in specific areas, the present study was aimed to assess the oxidative stress in different regions of brain-cerebral cortex, cerebellum and pons medulla during acute HE. Acute liver failure was induced in 3-month old adult male Wistar rats by intraperitoneal injection of thioacetamide (300 mg/kg body weight for two days), a well known hepatotoxin. Oxidative stress conditions were assessed by free radical production, lipid peroxidation, nitric oxide levels, GSH/GSSG ratio and antioxidant enzyme machinery in three distinct structures of rat braincerebral cortex, cerebellum and pons medulla. Results of the present study indicate a significant increase in malondialdehyde (MDA) levels, reactive oxygen species (ROS), total nitric oxide levels [(NO) estimated by measuring (nitrites + nitrates)] and a decrease in GSH/GSSG ratio in all the regions of brain. There was also a marked decrease in the activity of the antioxidant enzymes-glutathione peroxidase, glutathione reductase and catalase while the super oxide dismutase activity (SOD) increased. However, the present study also revealed that pons medulla and cerebral cortex were more susceptible to oxidative stress than cerebellum. The increased vulnerability to oxidative stress in pons medulla could be due to the increased NO levels and increased activity of SOD and decreased glutathione peroxidase and glutathione reductase activities. In summary, the present study revealed that oxidative stress prevails in different cerebral regions analyzed during thioacetamide-induced acute liver failure with more pronounced effects on pons medulla and cerebral cortex.

Role of Hepatic Nitric Oxide Synthases in Rats with Thioacetamide-induced Acute Liver Failure and Encephalopathy

Hepatic encephalopathy is neuropsychiatric derangement secondary to hepatic decompensation or portal-systemic shunting. Nitric oxide (NO) synthase inhibition aggravates encephalopathy and increases mortality in rats with thioacetamide (TAA)-induced acute liver failure, suggesting a protective role of NO. This study investigated the roles of endothelium-derived constitutive NO synthase (eNOS) and inducible NOS (iNOS) in the liver of rats with fulminant hepatic failure and encephalopathy. Male Sprague-Dawley rats (300-350 g) were randomized to receive TAA 350 mg/kg/day, by intraperitoneal injection or normal saline for 3 days. Severity of encephalopathy was assessed with the Opto-Varimex animal activity meter. Plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, and bilirubin were measured. Hepatic iNOS and eNOS RNA and protein expressions were assessed by reverse transcription-polymerase chain reaction and Western blot analyses, respectively. The TAA group showed lower motor activity counts than the normal saline group. Hepatic eNOS, but not iNOS, mRNA and protein expressions were enhanced in the TAA group. In addition, hepatic eNOS mRNA expression was negatively correlated with total movement but positively correlated with ALT and AST. Protein expression of hepatic eNOS was positively correlated with ALT, AST and bilirubin. Upregulation of hepatic eNOS was observed in rats with TAA-induced fulminant hepatic failure and encephalopathy, which might play a regulatory role.

During Thioacetamide-Induced Acute Liver Failure, the Proliferative Response of Hepatocytes to Thyroid Hormone Is Maintained, Indicating a Potential Therapeutic Approach to Toxin-Induced Liver Disease

In toxic liver injury, proliferation of preexisting hepatocytes helps restore liver mass and function. While loss of liver mass per se stimulates hepatocyte proliferation, exogenous mitogens have a potential role in enhancing liver regeneration. The aim of this study was to characterize the effects of the mitogen, tri-iodothyonine, on the regenerative capacity of hepatocytes during thioacetamide-induced liver failure. Rats received (two) thioacetamide injections and, 12 hr later, either tri-iodothyonine or vehicle-only control. Liver cell proliferation was assessed and comparison made with other control groups receiving tri-iodothyonine or vehicle only. In rats with thioacetamide-induced hepatitis the proportion of hepatocytes in S-phase was greater in the tri-iodothyonine group (27+/-3.5%) compared to the vehicle-only group (20+/-2.5%; P < 0.05), with, notably, a greater number of midzonal (BrdU) positive hepatocytes in the tri-iodothyonine group. We conclude that the ability of hepatocytes in the midzonal areas of rat liver to proliferate in response to tri-iodothyonine is maintained during severe acute toxic injury.

α‐Melanocyte‐stimulating hormone gene therapy reverses carbon tetrachloride induced liver fibrosis in mice

Hepatic fibrosis represents a process of healing and scarring in response to chronic liver injury. Effective therapies are lacking. We have previously demonstrated that alpha-melanocyte-stimulating hormone (alpha-MSH) gene therapy protects against thioacetamide-induced acute liver failure in mice. Recent reports showed that collagen metabolism is a novel target of alpha-MSH. Therefore, the aim of this study is to investigate whether alpha-MSH gene therapy possesses anti-hepatic fibrogenic effect in mice. Liver fibrosis was induced in mice by administering carbon tetrachloride (CCl4) continuously for 10 weeks. Alpha-MSH expression plasmid was delivered via electroporation after liver fibrosis had been established. Histopathology, reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and gelatin zymography were used to investigate its possible mechanisms of action. Alpha-MSH gene therapy reversed established liver fibrosis in CCl4-treated mice. RT-PCR revealed that alpha-MSH gene therapy attenuated the liver TGF-beta1, collagen alpha1, and cell adhesion molecule mRNA upregulation. Following gene transfer, both the activation of alpha-smooth muscle actin (alpha-SMA) and cyclooxygenase-2 (COX-2) was significantly attenuated. Further, alpha-MSH significantly increased matrix metalloproteinase (MMP) activity with tissue inhibitors of matrix metalloproteinase (TIMP) inactivation. We have demonstrated that alpha-MSH gene therapy reversed established liver fibrosis in mice. It also prevented the upregulated fibrogenic and proinflammatory gene response after CCl4 administration. Its collagenolytic effect may be attributed to MMP and TIMP modulation. In summary, alpha-MSH gene therapy may be an effective therapeutic modality against liver fibrosis with potential clinical use.

Single injection of naked plasmid encoding α-melanocyte-stimulating hormone protects against thioacetamide-induced acute liver failure in mice

Oxidative stress has been implicated in the propagation of acute liver injury. The aim of our study was to investigate whether gene transfer of α-melanocyte-stimulating hormone (α-MSH), a potent anti-inflammatory peptide, could prevent fulminant hepatic failure in mice. Acute liver damage was induced by intraperitoneal administration of thioacetamide. Hydrodynamics-based gene transfection with α-MSH expression plasmid via rapid tail vein injection was initiated 1 day prior to intoxication. The mortality in the α-MSH-treated mice was significantly lower compared to the vehicle group 3 days after injury. Liver histology significantly improved and TUNEL-positive hepatocytes decreased in the treated mice. The degradation of IκBα, endogenous inhibitor of nuclear factor κB, and upregulation of inducible nitric oxide synthase and tumor necrosis factor-α mRNA levels were prevented in the α-MSH-treated group, indicating decreased oxidative stress and inflammation. These results suggest α-MSH gene therapy might protect against acute hepatic necroinflammatory damage with further potential applications.

Protective effect of MDL28170 against thioacetamide-induced acute liver failure in mice.

Liver injury is known to often progress even after the hepatotoxicant is dissipated. The hydrolytic enzyme calpain, which is released from dying hepatocytes, destroys the surrounding cells and results in progression of injury. Therefore, control of calpain activation may be a suitable therapeutic intervention in cases of fulminant hepatic failure. This study evaluated the effects of a potent cell-permeable calpain inhibitor, MDL28170, and its mechanisms of action on thioacetamide (TAA)-induced hepatotoxicity in mice. We found that MDL28170 significantly decreased mortality and change in serum transaminase after TAA administration. The necroinflammatory response in the liver was also suppressed. Furthermore, a significant suppression of hepatocyte apoptosis could be found by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. The upregulation of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha), both of which are known to mediate the propagation of inflammation, was abolished. MDL2810 also effectively blocked hepatic stellate cell activation, which is assumed to be the early step in liver fibrosis. These results demonstrated that MDL28170 attenuated TAA-induced acute liver failure by inhibiting hepatocyte apoptosis, abrogating iNOS and TNF-alpha mRNA upregulation and blocking hepatic stellate cell activation.

803 Inhibited NF-ϰB transcriptional regulation may explain failed tissue repair in thioacetamide-induced acute liver failure in type I diabetic rats

803 Inhibited NF-ϰB transcriptional regulation may explain failed tissue repair in thioacetamide-induced acute liver failure in type I diabetic rats

Role of tryptophan in the elevated serotonin-turnover in hepatic encephalopathy.

The increase of the brain levels of 5-hydroxyindoleacetic acid (5-HIAA) in hepatic encephalopathy (HE) suggests an increased turnover of serotonin (5-HT). To study the role of tryptophan on the increased brain 5-HT metabolism in HE, we attempted to monitor brain levels of tryptophan in rats with thioacetamide-induced acute liver failure by intravenous infusion of branched-chain amino acids (BCAA). The effect of this treatment on 5-HT synthesis and metabolism was investigated in five brain areas. BCAA-infusions (1 and 2 gm/kg/24 h) increased the ratio BCAA/aromatic amino acids in plasma two- and fourfold, respectively, and lowered both plasma and brain levels of tryptophan. At the higher BCAA-dose all parameters suggesting an altered brain 5-HT metabolism (increased brain levels of 5-HT and 5-HIAA, increased 5-HIAA/5-HT ratio) were almost completely normalized. These results provide further evidence for the role of tryptophan in the elevation of brain 5-HT metabolism and for a potential role of BCAA in the treatment of HE.

The glial glutamate transporter in hyperammonemia and hepatic encephalopathy: Relation to energy metabolism and glutamatergic neurotransmission

Abnormalities in glutamate metabolism and glutamatergic neurotransmission appear to play a major role in the pathogenesis of hyperammonemia and hepatic encephalopathy. Astrocytes may be involved in these derangements as ammonia has been shown to impair the ability of these cells to take up glutamate. This study presents a northern blot analysis of the GLT-1 glutamate transporter in hyperammonemic rats, and in rats with thioacetamide-induced acute liver failure. Our findings demonstrate a downregulation of GLT-1 mRNA in both conditions. This article examines the potential impact of deficits in glutamate uptake on energy metabolism and glutamatergic neurotransmission in the context of abnormalities in glial-neuronal interactions. We propose that an ammonia-induced abnormality in astroglial glutamate uptake constitutes a critical aspect in the pathogenesis of hepatic encephalopathy and other hyperammonemic conditions.

Induction of peripheral-type benzodiazepine receptors in mouse brain following thioacetamide-induced acute liver failure

To investigate the possible role of peripheral-type benzodiazepine receptors (PBR) in hepatic encephalopathy, we examined expression of PBR in mouse brain following thioacetamide (TAA)-induced acute liver failure. Treatment of mice with TAA resulted in an increase in the number of the binding sites of the PBR ligand [ 3H] Ro5-4864 to brain homogenates, with no significant change in affinity of the ligand. The order of potency of different ligands to compete against [ 3H] Ro5-4864 binding in the brain of TAA-treated mice was Ro5-4864 > PK11195 > diazepam > protoporphyrin IX, findings similar to those in the control. Northern blot analysis revealed an increase in PBR/isoquinoline binding protein (PBR/IBP) mRNA in mouse brain following TAA treatment, in a time- and dose-dependent manner. These results indicate that the increased number of PBR in the brains of TAA-treated mice relates to the induction of PBR/IBP expression and suggest that the induction of PBR in brain may contribute to pathogenesis of hepatic encephalopathy.

P-147 Induction of peripheral-type benzodiazepine receptors in mouse brain following thioacetamide-induced acute liver failure

P-147 Induction of peripheral-type benzodiazepine receptors in mouse brain following thioacetamide-induced acute liver failure

Decreased brain concentrations of choline and acetylcholine in mice with thioacetamide-induced acute liver failure

To investigate the alteration of the cholinergic neurotransmitter system in hepatic encephalopathy (HE), we measured the concentrations of choline (Ch) and acetylcholine (ACh) in the brain of mice with thioacetamide (TAA)-induced acute liver failure using a high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The concentrations of Ch and ACh in TAA-treated mice decreased in most regional brain areas as compared to control mice. In particular, Ch significantly decreased in the cortex ( P < 0.05), hippocampus ( P < 0.01), hypothalamus ( P < 0.05), midbrain ( P < 0.01) and medulla oblongata ( P < 0.01). Similarly, ACh significantly decreased in the striatum ( P < 0.01), hippocampus ( P < 0.01), midbrain ( P < 0.01) and medulla oblongata ( P < 0.01). Our findings suggest that the suppression of the cholinergic neurotransmitter system may play a role in the pathogenesis of HE in acute liver failure.

Decreased brain concentrations of choline and acetylcholine in mice with thioacetamide-induced acute liver failure

Decreased brain concentrations of choline and acetylcholine in mice with thioacetamide-induced acute liver failure

Hepatic encephalopathy in rats with thioacetamide-induced acute liver failure is not mediated by endogenous benzodiazepines

Background: To distinguish whether the improvement of hepatic encephalopathy by benzodiazepine receptor antagonists is mediated by their antagonistic or their inverse agonistic properties, the neurobehavioral effects of a variety of benzodiazepine receptor ligands in rats with thioacetamide-induced acute liver failure were tested. Methods: The neural inhibitory effect of the benzodiazepine agonist flunitrazepam and its reversibility by the “pure” antagonist Ro 14-7437 were examined in thioacetamide-treated rats and controls. The effects of Ro 14-7437, of the partial inverse agonist Ro 15-4513, and the inverse agonist DMCM in rats with hepatic encephalopathy grade II/III were tested. Encephalopathic rats were pretreated with Ro 147437 or vehicle and then injected with Ro 15-4513. Results: Thioacetamide-treated rats were more sensitive to flunitrazepam than controls. In both groups, its effect was completely antagonized with Ro 14-7437. Encephalopathy was significantly improved by Ro 154513, although Ro 14-7437 and vehicle had no effect. DMCM worsened the condition of encephalopathic rats but had no effect in controls. Pretreatment with Ro 14-7437 abolished the beneficial effects of Ro 154513. Conclusions: In rats with thioacetamide-induced liver failure, endogenous benzodiazepines do not precipitate hepatic encephalopathy. The amelioration of hepatic encephalopathy is mediated by benzodiazepine receptor ligands with both antagonistic and inverse agonistic properties.