Ammoniagenic Action of Valproate without Signs of Hepatic Dysfunction in Rats: Possible Causes and Supporting Evidence.

(1) Background: Valproic acid (VPA) is one of the frequently prescribed antiepileptic drugs and is generally considered well tolerated. However, VPA neurologic adverse effects in the absence of liver failure are fairly common, suggesting that in the mechanism for the development of VPA-induced encephalopathy, much more is involved than merely the exposure to hyperammonemia (HA) caused by liver insufficiency to perform detoxification. Taking into account the importance of the relationship between an impaired brain energy metabolism and elevated ammonia production, and based on the ability of VPA to interfere with neuronal oxidative pathways, the current study intended to investigate a potential regional ammoniagenic effect of VPA on rats' brains by determining activities of the enzymes responsible for ammonia production and neutralization. (2) Methods: Rats received a single intraperitoneal injection of VPA (50, 100, 250, 500 mg/kg). Plasma, the neocortex, the cerebellum, and the hippocampus were collected at 30 min after injection. The levels of ammonia, urea, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured in blood plasma. The activities of glutaminase and glutamate dehydrogenase (GDH) in mitochondria and the activities of AMP deaminase (AMPD), adenosine deaminase (ADA), and glutamine synthetase (GS) in cytosolic fractions isolated from rat brain regions were measured. Ammonia, ALT, and AST values were determined in the mitochondrial and cytosolic fractions. (3) Results: Multi-dose VPA treatment did not significantly affect the plasma levels of ammonia and urea or the ALT and AST liver enzymes. Significant dose-independent increases in the accumulation of ammonia were found only in the cytosol from the cerebellum and there was a strong correlation between the ammonia level and the ADA activity in this brain structure. A significant decrease in the AMPD and AST activities was observed, while the ALT activity was unaffected. Only the highest VPA dose (500 mg/kg) was associated with significantly less activity of GS compared to the control in all studied brain structures. In the mitochondria of all studied brain structures, VPA caused a dose-independent increases in ammonia levels, a high concentration of which was strongly and positively correlated with the increased GDH and ALT activity, while glutaminase activity remained unchanged, and AST activity significantly decreased compared to the control in all studied brain structures. (4) Conclusions: This study highlights the rat brain region-specific ammoniagenic effects of VPA, which may manifest themselves in the absence of hyperammonemia. Further research should analyze how the responsiveness of the different brain regions may vary in VPA-treated animals that exhibit compromised energy metabolism, leading to increased ammoniagenesis.

Mitochondrial targets in hyperammonemia: Addressing urea cycle function to improve drug therapies.

The urea cycle (UC) is a critically important metabolic process for the disposal of nitrogen (ammonia) produced by amino acids catabolism. The impairment of this liver-specific pathway induced either by primary genetic defects or by secondary causes, namely those associated with hepatic disease or drug administration, may result in serious clinical consequences. Urea cycle disorders (UCD) and certain organic acidurias are the major groups of inherited rare diseases manifested with hyperammonemia (HA) with UC dysregulation. Importantly, several commonly prescribed drugs, including antiepileptics in monotherapy or polytherapy from carbamazepine to valproic acid or specific antineoplastic agents such as asparaginase or 5-fluorouracil may be associated with HA by mechanisms not fully elucidated. HA, disclosing an imbalance between ammoniagenesis and ammonia disposal via the UC, can evolve to encephalopathy which may lead to significant morbidity and central nervous system damage. This review will focus on biochemical mechanisms related with HA emphasizing some poorly understood perspectives behind the disruption of the UC and mitochondrial energy metabolism, namely: i) changes in acetyl-CoA or NAD+ levels in subcellular compartments; ii) post-translational modifications of key UC-related enzymes, namely acetylation, potentially affecting their catalytic activity; iii) the mitochondrial sirtuins-mediated role in ureagenesis. Moreover, the main UCD associated with HA will be summarized to highlight the relevance of investigating possible genetic mutations to account for unexpected HA during certain pharmacological therapies. The ammonia-induced effects should be avoided or overcome as part of safer therapeutic strategies to protect patients under treatment with drugs that may be potentially associated with HA.

Farnesoid X receptor agonist tropifexor detoxifies ammonia by regulating the glutamine metabolism and urea cycles in cholestatic livers.

Hyperammonemia refers to elevated levels of ammonia in the blood, which is an important pathological feature of liver cirrhosis and hepatic failure. Preclinical studies suggest tropifexor (TXR), a novel non-bile acid agonist of Farnesoid X Receptor (FXR), has shown promising effects on reducing hepatic steatosis, inflammation, and fibrosis. This study evaluates the impact of TXR on hyperammonemia in a piglet model of cholestasis. We here observed blood ammonia significantly elevated in patients with biliary atresia (BA) and was positively correlated with liver injury. Targeted metabolomics and immunblotting showed glutamine metabolism and urea cycles were impaired in BA patients. Next, we observed that TXR potently suppresses bile duct ligation (BDL)-induced injuries in liver and brain with improving the glutamine metabolism and urea cycles. Within the liver, TXR enhances glutamine metabolism and urea cycles by up-regulation of key regulatory enzymes, including glutamine synthetase (GS), carbamoyl-phosphate synthetase 1 (CPS1), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1). In primary mice hepatocytes, TXR detoxified ammonia via increasing ureagenesis. Mechanically, TXR activating FXR to increase express enzymes that regulating ureagenesis and glutamine synthesis through a transcriptional approach. Together, these results suggest that TXR may have therapeutic implications for hyperammonemic conditions in cholestatic livers.

Protective effects of Tinospora cordifolia miers extract against hepatic and neurobehavioral deficits in thioacetamide-induced hepatic encephalopathy in rats via modulating hyperammonemia and glial cell activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora cordifolia (TC) a potential medicinal herb, has been ethnobotanically used as an eco-friendly supplement to manage various diseases, including cerebral fever. Earlier studies have shown that TC exhibits diverse beneficial effects, including hepatoprotective and neuroprotective effects. However, the effects of TC remain unexplored in animal models of encephalopathy including hepatic encephalopathy (HE). AIM OF THE STUDY: To evaluate the effects of TC stem extract against thioacetamide (TAA)-induced behavioural and molecular alterations in HE rats. METHODS AND MATERIALS: The extract was preliminarily screened through phytochemical and HR-LC/MS analysis. Animals were pre-treated with TC extract at doses 30 and 100 mg/kg, orally. Following 7 days of TC pre-treatment, HE was induced by administering TAA (300 mg/kg, i. p. thrice). Behavioural assessments were performed after 56 h of TAA first dose. The animals were then sacrificed to assess biochemical parameters in serum, liver and brain. Liver tissue was used for immunoblotting and histological studies to evaluate inflammatory and fibrotic signalling. Moreover, brain tissue was used to evaluate brain edema, activation of glial cells (GFAP, IBA-1) and NF-κB/NLRP3 downstream signalling via immunoblotting and immunohistochemical analysis in cortex and hippocampus. RESULTS: The pre-treatment with TC extract effective mitigated TAA-induced behavioural alterations, lowered serum LFT (AST, ALT, ALP, bilirubin) and oxidative stress markers in liver and brain. TC treatment significantly modulated hyperammonemia, cerebral edema and preserved the integrity of BBB proteins in HE animals. TC treatment attenuated TAA-induced histological changes, tissue inflammation (pNF-κB (p65), TNF-α, NLRP3) and fibrosis (collagen, α-SMA) in liver. In addition, immunoblotting analysis revealed TC pre-treatment inhibited fibrotic proteins such as vimentin, TGF-ß1 and pSmad2/3 in the liver. Our study further showed that TC treatment downregulated the expression of MAPK/NF-κB inflammatory signalling, as well as GFAP and IBA-1 (glial cell markers) in cortex and hippocampus of TAA-intoxicated rats. Additionally, TC-treated animals exhibited reduced expression of caspase3/9 and BAX induced by TAA. CONCLUSION: This study revealed promising insights on the protective effects of TC against HE. The findings clearly demonstrated that the significant inhibition of MAPK/NF-κB signalling and glial cell activation could be responsible for the observed beneficial effects of TC in TAA-induced HE rats.

Gut-derived ammonia contributes to alcohol-related fatty liver development via facilitating ethanol metabolism and provoking ATF4-dependent de novo lipogenesis activation.

BACKGROUND & AIMS: Dysbiosis contributes to alcohol-associated liver disease (ALD); however, the precise mechanisms remain elusive. Given the critical role of the gut microbiota in ammonia production, we herein aim to investigate whether and how gut-derived ammonia contributes to ALD. METHODS: Blood samples were collected from human subjects with/without alcohol drinking. Mice were exposed to the Lieber-DeCarli isocaloric control or ethanol-containing diets with and without rifaximin (a nonabsorbable antibiotic clinically used for lowering gut ammonia production) supplementation for five weeks. Both in vitro (NH4Cl exposure of AML12 hepatocytes) and in vivo (urease administration for 5 days in mice) hyperammonemia models were employed. RNA sequencing and fecal amplicon sequencing were performed. Ammonia and triglyceride concentrations were measured. The gene and protein expression of enzymes involved in multiple pathways were measured. RESULTS: Chronic alcohol consumption causes hyperammonemia in both mice and human subjects. In healthy livers and hepatocytes, ammonia exposure upregulates the expression of urea cycle genes, elevates hepatic de novo lipogenesis (DNL), and increases fat accumulation. Intriguingly, ammonia promotes ethanol catabolism and acetyl-CoA formation, which, together with ammonia, synergistically facilitates intracellular fat accumulation in hepatocytes. Mechanistic investigations uncovered that ATF4 activation, as a result of ER stress induction and general control nonderepressible 2 activation, plays a central role in ammonia-provoked DNL elevation. Rifaximin ameliorates ALD pathologies in mice, concomitant with blunted hepatic ER stress induction, ATF4 activation, and DNL activation. CONCLUSIONS: An overproduction of ammonia by gut microbiota, synergistically interacting with ethanol, is a significant contributor to ALD pathologies.

L-Isoleucine reverses hyperammonemia-induced myotube mitochondrial dysfunction and post-mitotic senescence.

Perturbations in the metabolism of ammonia, a cytotoxic endogenous metabolite, occur in a number of chronic diseases, with consequent hyperammonemia. Increased skeletal muscle ammonia uptake causes metabolic, molecular, and phenotype alterations including cataplerosis of (loss of tricarboxylic acid cycle (TCA) cycle intermediate) α-ketoglutarate (αKG), mitochondrial oxidative dysfunction, and senescence-associated molecular phenotype (SAMP). L-Isoleucine (Ile) is an essential, branched-chain amino acid (BCAA) that simultaneously provides acetyl-CoA as an oxidative substrate and succinyl-CoA for anaplerosis (providing TCA cycle intermediates). Our multiomics analyses in myotubes and skeletal muscle from hyperammonemic mice and human patients with cirrhosis showed perturbations in BCAA transporters and catabolism. We, therefore, determined if Ile reverses hyperammonemia-induced impaired mitochondrial oxidative function and SAMP. Studies were performed in differentiated murine C2C12 myotubes that were early passage, late passage (senescent), or those depleted of LAT1/SLC7A5 and human induced pluripotent stem cell-derived myotubes (hiPSCM). Ile reverses hyperammonemia-induced reduction in the maximum respiratory capacity, complex I, II, and III functions in early passage murine myotubes and hiPSCM. Consistently, low ATP content and impaired global protein synthesis (high energy requiring cellular process) during hyperammonemia are reversed by Ile in murine myotubes and hiPSCM. Lower abundance of critical regulators of protein synthesis in mTORC1 signaling, and increased phosphorylation of eukaryotic initiation factor 2α are also reversed by Ile. Genetic depletion studies showed that Ile responses are independent of the amino acid transporter LAT1/SLC7A5. Our studies show that Ile reverses the hyperammonemia-induced impaired mitochondrial oxidative function, cataplerosis, and SAMP in a LAT1/SLC7A5 transporter-independent manner.

miRNA-ome plasma analysis unveils changes in blood-brain barrier integrity associated with acute liver failure in rats.

BACKGROUND: Hepatic encephalopathy (HE) symptoms associated with liver insufficiency are linked to the neurotoxic effects of ammonia and other toxic metabolites reaching the brain via the blood-brain barrier (BBB), further aggravated by the inflammatory response. Cumulative evidence documents that the non-coding single-stranded RNAs, micro RNAs (miRs) control the BBB functioning. However, miRs' involvement in BBB breakdown in HE is still underexplored. Here, we hypothesized that in rats with acute liver failure (ALF) or rats subjected to hyperammonemia, altered circulating miRs affect BBB composing proteins. METHODS: Transmission electron microscopy was employed to delineate structural alterations of the BBB in rats with ALF (thioacetamide (TAA) intraperitoneal (ip.) administration) or hyperammonemia (ammonium acetate (OA) ip. administration). The BBB permeability was determined with Evans blue dye and sodium fluorescein assay. Plasma MiRs were profiled by Next Generation Sequencing (NGS), followed by in silico analysis. Selected miRs, verified by qRT-PCR, were examined in cultured rat brain endothelial cells. Targeted protein alterations were elucidated with immunofluorescence, western blotting, and, after selected miR mimics transfection, through an in vitro resistance measurement. RESULTS: Changes in BBB structure and increased permeability were observed in the prefrontal cortex of TAA rats but not in the brains of OA rats. The NGS results revealed divergently changed miRNA-ome in the plasma of both rat models. The in silico analysis led to the selection of miR-122-5p and miR-183-5p with their target genes occludin and integrin ß1, respectively, as potential contributors to BBB alterations. Both proteins were reduced in isolated brain vessels and cortical homogenates in TAA rats. We documented in cultured primary brain endothelial cells that ammonia alone and, in combination with TNFα increases the relative expression of NGS-selected miRs with a less pronounced effect of TNFα when added alone. The in vitro study also confirmed miR-122-5p-dependent decrease in occludin and miR-183-5p-related reduction in integrin ß1 expression. CONCLUSION: This work identified, to our knowledge for the first time, potential functional links between alterations in miRs residing in brain endothelium and BBB dysfunction in ALF.

Enhanced Activation of the S1PR2-IL-1ß-Src-BDNF-TrkB Pathway Mediates Neuroinflammation in the Hippocampus and Cognitive Impairment in Hyperammonemic Rats.

Hyperammonemia contributes to hepatic encephalopathy. In hyperammonemic rats, cognitive function is impaired by altered glutamatergic neurotransmission induced by neuroinflammation. The underlying mechanisms remain unclear. Enhanced sphingosine-1-phosphate receptor 2 (S1PR2) activation in the cerebellum of hyperammonemic rats contributes to neuroinflammation. in In hyperammonemic rats, we assessed if blocking S1PR2 reduced hippocampal neuroinflammation and reversed cognitive impairment and if the signaling pathways were involved. S1PR2 was blocked with intracerebral JTE-013, and cognitive function was evaluated. The signaling pathways inducing neuroinflammation and altered glutamate receptors were analyzed in hippocampal slices. JTE-013 improved cognitive function in the hyperammonemic rats, and hyperammonemia increased S1P. This increased IL-1ß, which enhanced Src activity, increased CCL2, activated microglia and increased the membrane expression of the NMDA receptor subunit GLUN2B. This increased p38-MAPK activity, which altered the membrane expression of AMPA receptor subunits and increased BDNF, which activated the TrkB → PI3K → Akt → CREB pathway, inducing sustained neuroinflammation. This report unveils key pathways involved in the induction and maintenance of neuroinflammation in the hippocampus of hyperammonemic rats and supports S1PR2 as a therapeutic target for cognitive impairment.

Limited role for hyperammonemia in the progression of diet-induced nonalcoholic steatohepatitis.

OBJECTIVES: To determine whether hyperammonemia has a direct impact on steatohepatitis in mice fed with a high-fat diet (HFD). METHODS: Male C57BL/6 mice were divided into two groups receiving either chow diet or HFD. After 12-week NASH modeling, hyperammonemia was induced by intragastric administration of ammonium chloride solution (NH4 Cl) or liver-specific carbamoyl phosphate synthetase 1 (Cps1) knockdown. In vitro experiments were performed in HepG2 cells induced by free fatty acid (FFA) and NH4 Cl. RESULTS: NH4 Cl administration led to increased levels of plasma and hepatic ammonia in NASH mice. NH4 Cl-induced hyperammonemia did not influence liver histological changes in mice fed with HFD; however, elevated plasma cholesterol level, and an increasing trend of liver lipid content were observed. No significant effect of hyperammonemia on hepatic inflammation and fibrosis in NASH mice was found. In vitro cell experiments showed that NH4 Cl treatment failed to increase the lipid droplet content and the expressions of de novo lipogenesis genes in HepG2 cells induced by FFA. The knockdown of Cps1 in HFD-fed mice resulted in elevated plasma ammonia levels but did not cause histological change in the liver. CONCLUSIONS: Our study revealed a limited role of ammonia in aggravating the progression of NASH. Further studies are needed to clarify the role and mechanism of ammonia in NASH development.

The interaction of ammonia and manganese in abnormal metabolism of minimal hepatic encephalopathy: A comparison metabolomics study.

This study was to investigate the effects of ammonia and manganese in the metabolism of minimal hepatic encephalopathy (MHE). A total of 32 Sprague-Dawley rats were divided into four subgroups: chronic hyperammonemia (CHA), chronic hypermanganese (CHM), MHE and control group (CON). 1H-NMR-based metabolomics was used to detect the metabolic changes. Sparse projection to latent structures discriminant analysis was used for identifying and comparing the key metabolites. Significant elevated blood ammonia were shown in the CHA, CHM, and MHE rats. Significant elevated brain manganese (Mn) were shown in the CHM, and MHE rats, but not in the CHA rats. The concentrations of γ-amino butyric acid (GABA), lactate, alanine, glutamate, glutamine, threonine, and phosphocholine were significantly increased, and that of myo-inositol, taurine, leucine, isoleucine, arginine, and citrulline were significantly decreased in the MHE rats. Of all these 13 key metabolites, 10 of them were affected by ammonia (including lactate, alanine, glutamate, glutamine, myo-inositol, taurine, leucine, isoleucine, arginine, and citrulline) and 5 of them were affected by manganese (including GABA, lactate, myo-inositol, taurine, and leucine). Enrichment analysis indicated that abnormal metabolism of glutamine and TCA circle in MHE might be affected by the ammonia, and abnormal metabolism of GABA might be affected by the Mn, and abnormal metabolism of glycolysis and branched chain amino acids metabolism might be affected by both ammonia and Mn. Both ammonia and Mn play roles in the abnormal metabolism of MHE. Chronic hypermanganese could lead to elevated blood ammonia. However, chronic hyperammonemia could not lead to brain Mn deposition.

Hyperammonemia induced gut microbiota dysbiosis and motor coordination disturbances in mice: new insight into gut­brain axis involvement in hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neuropsychiatric hepatic­induced syndrome in which several factors are involved in promoting brain perturbations, with ammonia being the primary factor. Motor impairment, incoordination, and gut dysbiosis are some of the well­known symptoms of HE. Nevertheless, the link between the direct effect of hyperammonemia and associated gut dysbiosis in the pathogenesis of HE is not well established. Thus, this work aimed to assess motor function in hyperammonemia and gut dysbiosis in mice. Twenty­eight Swiss mice were distributed into three groups: two­week and four­week hyperammonemia groups were fed with an ammonia­rich diet (20% w/w), and the control group was pair­fed with a standard diet. Motor performance in the three groups was measured through a battery of motor tests, namely the rotarod, parallel bars, beam walk, and static bars. Microbial analysis was then carried out on the intestine of the studied mice. The result showed motor impairments in both hyperammonemia groups. Qualitative and quantitative microbiological analysis revealed decreased bacterial load, diversity, and ratios of both aerobic and facultative anaerobic bacteria, following two and four weeks of ammonia supplementation. Moreover, the Shannon diversity index revealed a time­dependent cutback of gut bacterial diversity in a treatment­time­dependent manner, with the presence of only Enterobacteriaceae, Streptococcaceae, and Enterococcaceaeat at four weeks. The data showed that ammonia­induced motor coordination deficits may develop through direct and indirect pathways acting on the gut­brain axis.

The pathogenesis of gut microbiota in hepatic encephalopathy by the gut-liver-brain axis.

Hepatic encephalopathy (HE) is a neurological disease occurring in patients with hepatic insufficiency and/or portal-systemic blood shunting based on cirrhosis. The pathogenesis is not completely clear till now, but it is believed that hyperammonemia is the core of HE. Hyperammonemia caused by increased sources of ammonia and decreased metabolism further causes mental problems through the gut-liver-brain axis. The vagal pathway also plays a bidirectional role in the axis. Intestinal microorganisms play an important role in the pathogenesis of HE through the gut-liver-brain axis. With the progression of cirrhosis to HE, intestinal microbial composition changes gradually. It shows the decrease of potential beneficial taxa and the overgrowth of potential pathogenic taxa. Changes in gut microbiota may lead to a variety of effects, such as reduced production of short-chain fatty acids (SCFAs), reduced production of bile acids, increased intestinal barrier permeability, and bacterial translocation. The treatment aim of HE is to decrease intestinal ammonia production and intestinal absorption of ammonia. Prebiotics, probiotics, antibiotics, and fecal microbiota transplantation (FMT) can be used to manipulate the gut microbiome to improve hyperammonemia and endotoxemia. Especially the application of FMT, it has become a new treated approach to target microbial composition and function. Therefore, restoring intestinal microbial homeostasis can improve the cognitive impairment of HE, which is a potential treatment method.

Dysregulated cellular redox status during hyperammonemia causes mitochondrial dysfunction and senescence by inhibiting sirtuin-mediated deacetylation.

Perturbed metabolism of ammonia, an endogenous cytotoxin, causes mitochondrial dysfunction, reduced NAD+ /NADH (redox) ratio, and postmitotic senescence. Sirtuins are NAD+ -dependent deacetylases that delay senescence. In multiomics analyses, NAD metabolism and sirtuin pathways are enriched during hyperammonemia. Consistently, NAD+ -dependent Sirtuin3 (Sirt3) expression and deacetylase activity were decreased, and protein acetylation was increased in human and murine skeletal muscle/myotubes. Global acetylomics and subcellular fractions from myotubes showed hyperammonemia-induced hyperacetylation of cellular signaling and mitochondrial proteins. We dissected the mechanisms and consequences of hyperammonemia-induced NAD metabolism by complementary genetic and chemical approaches. Hyperammonemia inhibited electron transport chain components, specifically complex I that oxidizes NADH to NAD+ , that resulted in lower redox ratio. Ammonia also caused mitochondrial oxidative dysfunction, lower mitochondrial NAD+ -sensor Sirt3, protein hyperacetylation, and postmitotic senescence. Mitochondrial-targeted Lactobacillus brevis NADH oxidase (MitoLbNOX), but not NAD+ precursor nicotinamide riboside, reversed ammonia-induced oxidative dysfunction, electron transport chain supercomplex disassembly, lower ATP and NAD+ content, protein hyperacetylation, Sirt3 dysfunction and postmitotic senescence in myotubes. Even though Sirt3 overexpression reversed ammonia-induced hyperacetylation, lower redox status or mitochondrial oxidative dysfunction were not reversed. These data show that acetylation is a consequence of, but is not the mechanism of, lower redox status or oxidative dysfunction during hyperammonemia. Targeting NADH oxidation is a potential approach to reverse and potentially prevent ammonia-induced postmitotic senescence in skeletal muscle. Since dysregulated ammonia metabolism occurs with aging, and NAD+ biosynthesis is reduced in sarcopenia, our studies provide a biochemical basis for cellular senescence and have relevance in multiple tissues.

Hyperammonemia induces microglial NLRP3 inflammasome activation via mitochondrial oxidative stress in hepatic encephalopathy.

BACKGROUND: The role of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in the pathogenesis of hepatic encephalopathy (HE) is unclear. Mitochondrial reactive oxygen species (mtROS) is a signal for NLRP3 inflammasome activation. Therefore, we aimed to determine whether mtROS-dependent NLRP3 inflammasome activation is involved in HE, using in vivo and in vitro models. METHODS: Bile duct ligation (BDL) in C57/BL6 mice was used as an in vivo HE model. NLRP3 activation was assessed in the hippocampus. Immunofluorescence staining was performed to determine the cellular source of NLRP3 in the hippocampal tissue. For the in vitro experiment, BV-2 microglial cells were primed with lipopolysaccharide (LPS), followed by ammonia treatment. NLRP3 activation and mitochondrial dysfunction were measured. Mito-TEMPO was used to suppress mtROS production. RESULTS: BDL mice showed cognitive impairment with hyperammonemia. Both the priming and activation steps of NLRP3 inflammasome activation were processed in the hippocampus of BDL mice. Moreover, intracellular ROS levels increased in the hippocampus, and NLRP3 was mainly expressed in the microglia of the hippocampus. In LPS-primed BV-2 cells, ammonia treatment induced NLRP3 inflammasome activation and pyroptosis, with elevation of mtROS and altered mitochondrial membrane potential. Pretreatment with Mito-TEMPO suppressed mtROS production and the subsequent NLRP3 inflammasome activation and pyroptosis under LPS and ammonia treatment in BV-2 cells. CONCLUSIONS: Hyperammonemia in HE may be involved in mtROS overproduction and subsequent NLRP3 inflammasome activation. Further studies using NLRP3-specific inhibitor or NLRP3 knockout mice are needed to elucidate the important role of NLRP3 inflammasome in HE development.

Extracellular vesicles from hyperammonemic rats induce neuroinflammation in hippocampus and impair cognition in control rats.

Patients with liver cirrhosis show hyperammonemia and peripheral inflammation and may show hepatic encephalopathy with cognitive impairment, reproduced by rats with chronic hyperammonemia. Peripheral inflammation induces neuroinflammation in hippocampus of hyperammonemic rats, altering neurotransmission and leading to cognitive impairment. Extracellular vesicles (EVs) may transmit pathological effects from the periphery to the brain. We hypothesized that EVs from peripheral blood would contribute to cognitive alterations in hyperammonemic rats. The aims were to assess whether EVs from plasma of hyperammonemic rats (HA-EVs) induce cognitive impairment and to identify the underlying mechanisms. Injection of HA-EVs impaired learning and memory, induced microglia and astrocytes activation and increased TNFα and IL-1ß. Ex vivo incubation of hippocampal slices from control rats with HA-EVs reproduced these alterations. HA-EVs increased membrane expression of TNFR1, reduced membrane expression of TGFßR2 and Smad7 and IκBα levels and increased IκBα phosphorylation. This led to increased activation of NF-κB and IL-1ß production, altering membrane expression of NR2B, GluA1 and GluA2 subunits, which would be responsible for cognitive impairment. All these effects of HA-EVs were prevented by blocking TNFα, indicating that they were mediated by enhanced activation of TNFR1 by TNFα. We show that these mechanisms are very different from those leading to motor incoordination, which is due to altered GABAergic neurotransmission in cerebellum. This demonstrates that peripheral EVs play a key role in the transmission of peripheral alterations to the brain in hyperammonemia and hepatic encephalopathy, inducing neuroinflammation and altering neurotransmission in hippocampus, which in turn is responsible for the cognitive deficits.

Cellular Pathogenesis of Hepatic Encephalopathy: An Update.

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome derived from metabolic disorders due to various liver failures. Clinically, HE is characterized by hyperammonemia, EEG abnormalities, and different degrees of disturbance in sensory, motor, and cognitive functions. The molecular mechanism of HE has not been fully elucidated, although it is generally accepted that HE occurs under the influence of miscellaneous factors, especially the synergistic effect of toxin accumulation and severe metabolism disturbance. This review summarizes the recently discovered cellular mechanisms involved in the pathogenesis of HE. Among the existing hypotheses, ammonia poisoning and the subsequent oxidative/nitrosative stress remain the mainstream theories, and reducing blood ammonia is thus the main strategy for the treatment of HE. Other pathological mechanisms mainly include manganese toxicity, autophagy inhibition, mitochondrial damage, inflammation, and senescence, proposing new avenues for future therapeutic interventions.

Sustained Hyperammonemia Activates NF-κB in Purkinje Neurons Through Activation of the TrkB-PI3K-AKT Pathway by Microglia-Derived BDNF in a Rat Model of Minimal Hepatic Encephalopathy.

Chronic hyperammonemia is a main contributor to the cognitive and motor impairment in patients with hepatic encephalopathy. Sustained hyperammonemia induces the TNFα expression in Purkinje neurons, mediated by NF-κB activation. The aims were the following: (1) to assess if enhanced TrkB activation by BDNF is responsible for enhanced NF-κB activation in Purkinje neurons in hyperammonemic rats, (2) to assess if this is associated with increased content of NF-κB modulated proteins such as TNFα, HMGB1, or glutaminase I, (3) to assess if these changes are due to enhanced activation of the TNFR1-S1PR2-CCR2-BDNF-TrkB pathway, (4) to analyze if increased activation of NF-κB is mediated by the PI3K-AKT pathway. It is shown that, in the cerebellum of hyperammonemic rats, increased BDNF levels enhance TrkB activation in Purkinje neurons leading to activation of PI3K, which enhances phosphorylation of AKT and of IκB, leading to increased nuclear translocation of NF-κB which enhances TNFα, HMGB1, and glutaminase I content. To assess if the changes are due to enhanced activation of the TNFR1-S1PR2-CCR2 pathway, we blocked TNFR1 with R7050, S1PR2 with JTE-013, and CCR2 with RS504393. These changes are reversed by blocking TrkB, PI3K, or the TNFR1-SP1PR2-CCL2-CCR2-BDNF-TrkB pathway at any step. In hyperammonemic rats, increased levels of BDNF enhance TrkB activation in Purkinje neurons, leading to activation of the PI3K-AKT-IκB-NF-κB pathway which increased the content of glutaminase I, HMGB1, and TNFα. Enhanced activation of this TrkB-PI3K-AKT-NF-κB pathway would contribute to impairing the function of Purkinje neurons and motor function in hyperammonemic rats and likely in cirrhotic patients with minimal or clinical hepatic encephalopathy.

Extracellular vesicles from mesenchymal stem cells reduce neuroinflammation in hippocampus and restore cognitive function in hyperammonemic rats.

Chronic hyperammonemia, a main contributor to hepatic encephalopathy (HE), leads to neuroinflammation which alters neurotransmission leading to cognitive impairment. There are no specific treatments for the neurological alterations in HE. Extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) reduce neuroinflammation in some pathological conditions. The aims were to assess if treatment of hyperammonemic rats with EVs from MSCs restores cognitive function and analyze the underlying mechanisms. EVs injected in vivo reach the hippocampus and restore performance of hyperammonemic rats in object location, object recognition, short-term memory in the Y-maze and reference memory in the radial maze. Hyperammonemic rats show reduced TGFß levels and membrane expression of TGFß receptors in hippocampus. This leads to microglia activation and reduced Smad7-IkB pathway, which induces NF-κB nuclear translocation in neurons, increasing IL-1ß which alters AMPA and NMDA receptors membrane expression, leading to cognitive impairment. These effects are reversed by TGFß in the EVs from MSCs, which activates TGFß receptors, reducing microglia activation and NF-κB nuclear translocation in neurons by normalizing the Smad7-IkB pathway. This normalizes IL-1ß, AMPA and NMDA receptors membrane expression and, therefore, cognitive function. EVs from MSCs may be useful to improve cognitive function in patients with hyperammonemia and minimal HE.

The role and control of arginine levels in arginase 1 deficiency.

Arginase 1 Deficiency (ARG1-D) is a rare urea cycle disorder that results in persistent hyperargininemia and a distinct, progressive neurologic phenotype involving developmental delay, intellectual disability, and spasticity, predominantly affecting the lower limbs and leading to mobility impairment. Unlike the typical presentation of other urea cycle disorders, individuals with ARG1-D usually appear healthy at birth and hyperammonemia is comparatively less severe and less common. Clinical manifestations typically begin to develop in early childhood in association with high plasma arginine levels, with hyperargininemia (and not hyperammonemia) considered to be the primary driver of disease sequelae. Nearly five decades of clinical experience with ARG1-D and empirical studies in genetically manipulated models have generated a large body of evidence that, when considered in aggregate, implicates arginine directly in disease pathophysiology. Severe dietary protein restriction to minimize arginine intake and diversion of ammonia from the urea cycle are the mainstay of care. Although this approach does reduce plasma arginine and improve patients' cognitive and motor/mobility manifestations, it is inadequate to achieve and maintain sufficiently low arginine levels and prevent progression in the long term. This review presents a comprehensive discussion of the clinical and scientific literature, the effects and limitations of the current standard of care, and the authors' perspectives regarding the past, current, and future management of ARG1-D.

The effects of Ibuprofen and 1, 8- cineol on anxiety and spatial memory in hyperammonemic rats.

In hepatic encephalopathy, hyperammonemia (HA) causes cognitive impairment and anxiety by causing neuroinflammation. Ibuprofen and 1,8- cineol have anti-inflammatory and antioxidant properties, respectively. The aim of this study was to evaluate the effects of ibuprofen alone and in combination with 1,8- cineol on anxiety and oxidative stress in a HA rat animal model. For this purpose, 36 rats were divided into six groups (n = 6) including the HA (received intraperitoneally (IP) ammonium acetate 2.5 mg/kg for four week), ibuprofen (induced HA rats that received 15 mg/kg, IP), cineol (induced HA rats that received 5 and 10 mg/kg, IP), Ib + cineol (induced HA rats that received 15 and 10 mg/kg, respectively, IP), and the control groups (received normal saline, IP). Except the HA group, all other groups received the aforementioned treatment for two weeks.. The Morris water maze and elevated plus maze were used to assess cognitive function and anxiety in the animals, respectively. Superoxide dismutase (SOD) activity was measured to evaluate oxidative stress. The mRNA expression levels of interleukin (IL)-6 and IL-1ß was assessed by real-time PCR in the animal's brain. The results showed a significant improvement in spatial memory and anxiety of the Ib group compared to the HA group (P < 0.01), but no significant change was observed in SOD activity (P > 0.05). There was a significant improvement in spatial memory and anxiety as well as a significant increase in SOD activity in the Ib + cineol group (P < 0.01) compared to the HA group. These results indicate that the Ib + cineol, not only improve cognitive function and reduce anxiety, also reduce oxidative stress, therefore, the simultaneous use of these two compounds may be useful in improving HA-induced cognitive disorders and anxiety.