[Mechanism of Biejiajian Pills against non-alcoholic steatohepatitis based on lipidomics].

This study aims to explore the potential mechanism of Biejiajian Pills in the treatment of non-alcoholic steatohepatitis(NASH) based on lipidomics. A mouse model of NASH was induced by high-fat/high cholesterol diet, and the mice of the normal group were fed with a normal diet. The therapeutic efficacy of Biejiajian Pills against NASH was evaluated through biochemical indexes in both of serum and liver, as well as the hepatic histopathology. Lipid metabolites in the liver were detected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS)-based lipidomics. Then the partial least-squares discriminant analysis, t-test and receiver operating characteristic curve analysis were performed to screen the differential lipid metabolites and the main biomarkers. The proteins and genes involved in the lipid metabolism and inflammatory response were detected by Western blot and qPCR. The results demonstrated that Biejiajian Pills notably lowered the levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), and alkaline phosphatase(ALP) in the serum and the levels of triglyceride(TG) and total cholesterol(TC) in the liver tissue. In addition, Biejiajian Pills alleviated the lipid accumulation, hepatocyte ballooning, and liver fibrosis. Lipidomics revealed that Biejiajian Pills regulated the content of 11 biomarkers including phosphatidyl choline(PC), phosphatidyl ethanolamine(PE), sphingomyelin(SM), and ceramide(Cer). The results of Western blot and qPCR demonstrated that Biejiajian Pills regulated the expression of sterol regulatory element-binding protein 1(SREBP1), peroxisome proliferator-activated receptor gamma(PPARγ) and phospho-AMP-activated protein kinase(p-AMPK), and the mRNA level of fatty acid translocase 36 gene(Cd36), Pparγ, cardiolipin synthase 1 gene(Crls1), and phospholipase Cß2 gene(Plcß2). Furthermore, Biejiajian Pills displayed inhibitory effects on phospho-p38 MAPK(p-p38 MAPK) and phospho-ERK1/2(p-ERK1/2) and the mRNA levels of interleukin-6 gene(Il-6), interleukin-1ß gene(Il-1ß) and tumor necrosis factor-α gene(Tnf-α). In conclusion, Biejiajian Pills could alleviate the lipid metabolism disorders and regulate the expression of SREBP1, PPARγ, and p-AMPK and the mRNA levels of pro-inflammatory cytokines.

The Glucose-Succinate Pathway: A Crucial Anaerobic Metabolic Pathway in the Scallop Chlamys farreri Experiencing Heat Stress.

Recently, the increase in marine temperatures has become an important global marine environmental issue. The ability of energy supply in marine animals plays a crucial role in avoiding the stress of elevated temperatures. The investigation into anaerobic metabolism, an essential mechanism for regulating energy provision under heat stress, is limited in mollusks. In this study, key enzymes of four anaerobic metabolic pathways were identified in the genome of scallop Chlamys farreri, respectively including five opine dehydrogenases (CfOpDHs), two aspartate aminotransferases (CfASTs) divided into cytoplasmic (CfAST1) and mitochondrial subtype (CfAST2), and two phosphoenolpyruvate carboxykinases (CfPEPCKs) divided into a primitive type (CfPEPCK2) and a cytoplasmic subtype (CfPEPCK1). It was surprising that lactate dehydrogenase (LDH), a key enzyme in the anaerobic metabolism of the glucose-lactate pathway in vertebrates, was absent in the genome of scallops. Phylogenetic analysis verified that CfOpDHs clustered according to the phylogenetic relationships of the organisms rather than substrate specificity. Furthermore, CfOpDHs, CfASTs, and CfPEPCKs displayed distinct expression patterns throughout the developmental process and showed a prominent expression in muscle, foot, kidney, male gonad, and ganglia tissues. Notably, CfASTs displayed the highest level of expression among these genes during the developmental process and in adult tissues. Under heat stress, the expression of CfASTs exhibited a general downregulation trend in the six tissues examined. The expression of CfOpDHs also displayed a downregulation trend in most tissues, except CfOpDH1/3 in striated muscle showing significant up-regulation at some time points. Remarkably, CfPEPCK1 was significantly upregulated in all six tested tissues at almost all time points. Therefore, we speculated that the glucose-succinate pathway, catalyzed by CfPEPCK1, serves as the primary anaerobic metabolic pathway in mollusks experiencing heat stress, with CfOpDH3 catalyzing the glucose-opine pathway in striated muscle as supplementary. Additionally, the high and stable expression level of CfASTs is crucial for the maintenance of the essential functions of aspartate aminotransferase (AST). This study provides a comprehensive and systematic analysis of the key enzymes involved in anaerobic metabolism pathways, which holds significant importance in understanding the mechanism of energy supply in mollusks.

Genetic variation and elevated liver enzymes during childhood, adolescence and early adulthood.

BACKGROUND: Genetic factors influence the risk of fatty liver disease (FLD) in adults. The aim of this study was to test if, and when, genetic risk factors known to affect FLD in adults begin to exert their deleterious effects during childhood, adolescence and early adulthood. METHODS: We included up to 4018 British children and adolescents from the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort. Three genetic variants known to associate robustly with FLD in adults (PNPLA3 rs738409, TM6SF2 rs58542926 and HSD17B13 rs72613567) were tested for association with plasma levels of alanine transaminase (ALT) and aspartate transaminase (AST) during childhood (mean age: 9.9 years), early adolescence (15.5 years), late adolescence (17.8 years), and early adulthood (24.5 years). We also tested the associations of a 17-variant score and whole-genome polygenic risk scores (PRS) derived from associations in adults with plasma ALT and AST at the same four time points. Associations with elastography-derived liver steatosis and fibrosis were tested in early adulthood. RESULTS: Genetic risk factors for FLD (individually, combined into a 3-variant score, a 17-variant score and as a genome-wide PRS), were associated with higher liver enzymes, beginning in childhood and throughout adolescence and early adulthood. The ALT-increasing effects of the genetic risk variants became larger with increasing age. The ALT-PRS was associated with liver steatosis in early adulthood. No genetic associations with fibrosis were observed. CONCLUSIONS: Genetic factors that promote FLD in adults associate with elevated liver enzymes already during childhood, and their effects get amplified with increasing age.

Effects of NR1I2 and ABCB1 Genetic Polymorphisms on Everolimus Pharmacokinetics in Japanese Renal Transplant Patients.

The purpose of this study was to evaluate the effects of NR1I2 (7635G>A and 8055C>T) and ABCB1 (1236C>T, 2677G>T/A, and 3435C>T) genetic polymorphisms on everolimus pharmacokinetics in 98 Japanese renal transplant patients. On day 15 after everolimus administration, blood samples were collected just prior to and 1, 2, 3, 4, 6, 9, and 12 h after administration. The dose-adjusted area under the blood concentration−time curve (AUC0-12) of everolimus was significantly lower in patients with the NR1I2 8055C/C genotype than in those with other genotypes (p = 0.022) and was significantly higher in male patients than female patients (p = 0.045). Significant correlations between the dose-adjusted AUC0-12 of everolimus and age (p = 0.001), aspartate transaminase (p = 0.001), and alanine transaminase (p = 0.005) were found. In multivariate analysis, aging (p = 0.008) and higher alanine transaminase levels (p = 0.032) were independently predictive of a higher dose-adjusted everolimus AUC0-12. Aging and hepatic dysfunction in patients may need to be considered when evaluating dose reductions in everolimus. In renal transplant patients, management using everolimus blood concentrations after administration may be more important than analysis of NR1I2 8055C>T polymorphism before administration.

GOT2 Silencing Promotes Reprogramming of Glutamine Metabolism and Sensitizes Hepatocellular Carcinoma to Glutaminase Inhibitors.

Hepatocellular carcinoma (HCC) is one of the primary liver malignancies with a poor prognosis. Glutamic-oxaloacetic transaminase 2 (GOT2) is a highly tissue-specific gene in the liver, but the roles GOT2 plays in the progression of HCC remain unclear. Here, we report that GOT2 is downregulated in HCC tumor tissues and that low expression of GOT2 is associated with advanced progression and poor prognosis. In HCC cells, knockdown of GOT2 promoted proliferation, migration, and invasion. In mouse models of HCC, loss of GOT2 promoted tumor growth as well as hematogenous and intrahepatic metastasis. Mechanistically, silencing of GOT2 enhanced glutaminolysis, nucleotide synthesis, and glutathione synthesis by reprogramming glutamine metabolism to support the cellular antioxidant system, which activated the PI3K/AKT/mTOR pathway to contribute to HCC progression. Furthermore, HCC with low expression of GOT2 was highly dependent on glutamine metabolism and sensitive to the glutaminase inhibitor CB-839 in vitro and in vivo. Overall, GOT2 is involved in glutamine metabolic reprogramming to promote HCC progression and may serve as a therapeutic and diagnostic target for HCC. SIGNIFICANCE: Altered glutamine metabolism induced by GOT2 loss supports HCC growth and metastasis but confers a targetable vulnerability to glutaminase inhibitors.

Genotypic and phenotypic spectrum of cytosolic phosphoenolpyruvate carboxykinase deficiency.

OBJECTIVES: Pathogenic biallelic variants in PCK1 coding for the cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) cause PEPCK-C deficiency, a rare disorder of gluconeogenesis presenting with hypoglycemia, lactic acidosis, and hepatopathy. To date, there has been no systematic analysis of its phenotypic, biochemical, and genetic spectrum. METHODS: All currently published individuals and a novel patient with genetically confirmed PEPCK-C deficiency were included. Clinical, biochemical, and genetic findings were analyzed. Protein and in-silico prediction score modeling was applied to analyze potential variant effects. RESULTS: Thirty-two individuals from 25 families were found, including one previously unreported patient. The typical biochemical pattern was hypoglycemia triggered by catabolic situations, elevated urinary concentrations of tricarboxylic acid cycle metabolites, mildly elevated alanine and aspartate aminotransferase and elevated lactate concentrations in serum. Plasma glutamine concentrations were elevated in some patients and may be a suitable marker for newborn screening. With adequate treatment, biochemical abnormalities usually normalized following a hypoglycemic episode. Symptom onset usually occurred in infancy with a broad range from neonatal age to adulthood. Regardless of the genotype, different phenotypes with a broad clinical spectrum were found. To date, eight genotypes with nine different PCK1 variants were identified, of which alleles with the recurrent variant c.925G > A; p.(Gly309Arg) are predominant and appear to be endemic in the Finnish population. Protein modeling suggests altered manganese- and substrate-binding as superordinate pathomechanisms. CONCLUSIONS: Environmental factors appear to be the main determinant for the phenotype in patients with biallelic variants in PCK1. Based on the biochemical pattern, PEPCK-C deficiency is a recognizable cause of childhood hypoglycemia. It is a treatable disease and early diagnosis is important to prevent metabolic derailment and morbidity. Newborn screening can identify at least a sub-cohort of affected individuals through elevated glutamine concentrations in dry blood.

Cytosolic aspartate aminotransferase moonlights as a ribosome-binding modulator of Gcn2 activity during oxidative stress.

Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors, and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of PE in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2α phosphorylation and GCN4 activation. Importantly, non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or 'moonlighting' function that modulates the ISR independent of its aspartate aminotransferase activity.

Ipragliflozin attenuates non-alcoholic steatohepatitis development in an animal model.

Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease with no decisive treatment. The sodium glucose cotransporter 2 (SGLT2) inhibitor ipragliflozin was developed as a new oral hypoglycemic drug, which can improve NASH via an insulin-independent glucose-lowering effect by inhibiting glucose reabsorption in the renal proximal tubules. However, ipragliflozin appears to modulate steatosis or inflammation via different pathways. To elucidate the new mechanism of ipragliflozin for the treatment of NASH, we evaluated its effects in a NASH mouse model (STAM mice) with beta cell depletion, and compared the expression of microRNAs (miRNAs) in STAM mice treated with or without ipragliflozin (16.7 µg/day for 5 weeks). Ipragliflozin reduced aspartate transaminase and alanine aminotransferase levels, along with reduced hepatic steatosis, hepatocyte ballooning, lobular inflammation, and liver fibrosis. In addition, ipragliflozin upregulated mitochondrial transport-related and antioxidant defensive system-related genes in the liver. Among 2555 mouse miRNA probes, miR-19b-3p was commonly differentially expressed with ipragliflozin treatment for 5 weeks in both the liver and serum but in different directions, with a decrease in the liver and increase in the serum. Therefore, ipragliflozin can improve NASH development likely through the antioxidative stress pathway and by regulating miR-19b-3p.

Influence of GSTM1, T1 genes polymorphisms on oxidative stress and liver enzymes in rural and urban pesticides-exposed workers.

Several studies discussed the relationship between the toxicity of organophosphates (OPs) and carbamates pesticides and oxidative stress which affects human health. This study aimed to evaluate the effects of pesticides on the induction of oxidative stress and hepatotoxicity. It was also focused on glutathione-S-transferase gene polymorphism in the modulation of these effects. In addition, the role of the educational level of exposed workers was studied. Acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), liver enzymes, malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), and glutathione S transferase (GST) were estimated at 100 pesticide-exposed workers (50 urban researchers (UE) and 50 rural sprayers (RE)), and 100 matched controls (50 urban controls (UC)and 50 rural controls (RC)). AChE and BuChE were decreased in RE and UE compared to RC. Aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activity were elevated in UE and UC compared to the RE and RC. Alanine aminotransferase (ALT) was elevated in UE compared to RE. MDA in RE and UE showed elevation compared to RC. There was a significant reduction in the levels of GSH, GST, and GPx in UE compared to RE and RC. The most sensitive pesticide-induced hepatotoxicity group were exposed workers with the GSTT1 genotype. Within these workers, ALT and ALP were significantly correlated with MDA and inversely correlated with AChE and BuChE, while AST was inversely correlated with AChE and BuChE only in UE. Conclusion: GST gene polymorphisms appeared to have a significant role in workers' susceptibility to hepatotoxic effects due to occupational exposure to pesticides; GSTT1 was the most sensitive genotype.

High protein diet-induced metabolic changes are transcriptionally regulated via KLF15-dependent and independent pathways.

High protein diet (HPD) is an affordable and positive approach in prevention and treatment of many diseases. It is believed that transcriptional regulation is responsible for adaptation after HPD feeding and Kruppel-like factor 15 (KLF15), a zinc finger transcription factor that has been proved to perform transcriptional regulation over amino acid, lipid and glucose metabolism, is known to be involved at least in part in this HPD response. To gain more insight into molecular mechanisms by which HPD controls expressions of genes involved in amino acid metabolism in the liver, we performed RNA-seq analysis of mice fed HPD for a short period (3 days). Compared to a low protein diet, HPD feeding significantly increased hepatic expressions of enzymes involved in the breakdown of all the 20 amino acids. Moreover, using KLF15 knockout mice and in vivo Ad-luc analytical system, we were able to identify Cth (cystathionine gamma-lyase) as a new target gene of KLF15 transcription as well as Ast (aspartate aminotransferase) as an example of KLF15-independent gene despite its remarkable responsiveness to HPD. These findings provide us with a clue to elucidate the entire transcriptional regulatory mechanisms of amino acid metabolic pathways.

The FibroScan-aspartate aminotransferase score can stratify the disease severity in a Japanese cohort with fatty liver diseases.

This study aimed to prove that the FibroScan-aspartate aminotransferase (FAST) scores can be used to stratify disease severity in a Japanese cohort with fatty liver diseases [metabolic dysfunction-associated fatty liver disease (MAFLD) and nonalcoholic fatty liver disease (NAFLD)]. All the participants (n = 2254) underwent liver stiffness measurements and controlled attenuation parameter assessments. We compared the clinical characteristics of the patients with MAFLD and NAFLD using the FAST scores and explored the independent determinants of FAST scores ≥ 0.35, which indicated possible progressive disease. Overall, MAFLD was diagnosed in 789 patients (35.0%), while NAFLD was diagnosed in 618 (27.4%). The proportion of patients that had a condition that suggested progressive liver disease was higher in those with MAFLD than in those with NAFLD [68 (8.6%) vs 48 (7.7%)]. The area under the receiver-operating characteristic curve of the FAST score for diagnosing advanced fibrosis was 0.969 in MAFLD and 0.965 in NAFLD. Multivariate analyses determined that diabetes mellitus, alanine aminotransferase (ALT) levels, fatty liver index, and Fibrosis-4 index independently predict FAST scores ≥ 0.35 in patients with MAFLD. ALT levels had the strongest correlation with the FAST scores (p = 0.7817). The FAST score could stratify the disease severity in the Japanese cohort with fatty liver diseases.

A triterpenoid-enriched extract of bitter melon leaves alleviates hepatic fibrosis by inhibiting inflammatory responses in carbon tetrachloride-treated mice.

Liver fibrosis is a progression of chronic liver disease characterized by excess deposition of fibrillary collagen. The aim of this study was to investigate the protective effect of a triterpenoid-enriched extract (TEE) from bitter melon leaves against carbon tetrachloride (CCl4)-induced hepatic fibrosis in mice. Male ICR mice received TEE (100 or 150 mg kg-1) by daily oral gavage for one week before starting CCl4 administration and throughout the entire experimental period. After intraperitoneal injection of CCl4 for nine weeks, serum and liver tissues of the mice were collected for biochemical, histopathological and molecular analyses. Our results showed that TEE supplementation reduced CCl4-induced serum aspartate aminotransferase and alanine aminotransferase activities. Histopathological examinations revealed that CCl4 administration results in hepatic fibrosis, while TEE supplementation significantly suppressed hepatic necroinflammation and collagen deposition. In addition, TEE supplementation decreased α-smooth muscle actin (α-SMA)-positive staining and protein levels of α-SMA and transforming growth factor-ß1. TEE-supplemented mice had lower mRNA expression levels of interleukin-6, tumor necrosis factor-α, and toll-like receptor 4. Moreover, TEE (150 mg kg-1) supplementation significantly reduced intrahepatic inflammatory Ly6C+ monocyte infiltration. We demonstrated that TEE could ameliorate hepatic fibrosis by regulating inflammatory cytokine secretion and α-SMA expression in the liver to reduce collagen accumulation.

Circular RNA circ_0003028 contributes to tumorigenesis by regulating GOT2 via miR-1298-5p in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a common malignant tumor, with high morbidity and mortality. Circular RNA (circRNA) circ_0003028 was reported to be upregulated in NSCLC. This study is designed to explore the role and mechanism of circ_0003028 on NSCLC progression. In this work, circ_0003028, microRNA-1298-5p (miR-1298-5p), and glutamic oxaloacetic transaminase 2 (GOT2) level were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The localization of circ_0003028 was analyzed by subcellular fractionation assay. Cell proliferation, colony number, cell cycle progression, apoptosis, migration, invasion, and angiogenesis were measured by Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, transwell, and tube formation assays. Protein levels of Beclin1, light chain 3 (LC3)-II/LC3-I, GOT2, proliferating cell nuclear antigen (PCNA) were examined by western blot assay. The binding relationship between miR-1298-5p and circ_0003028 or GOT2 was predicted by circular RNA Interactome or starbase and then verified by dual-luciferase reporter, RNA Immunoprecipitation (RIP), and RNA pull-down assays. The biological role of circ_0003028 on NSCLC tumor growth was examined by the xenograft tumor model in vivo. We reported that circ_0003028 and GOT2 were upregulated, and miR-1298-5p was decreased in NSCLC tissues and cells. Moreover, circ_0003028 knockdown curbed cell proliferative ability, migration, invasion, angiogenesis, and facilitate apoptosis and autophagy in NSCLC cells in vitro. Mechanical analysis discovered that circ_0003028 regulated GOT2 expression by sponging miR-1298-5p. Circ_0003028 silencing hindered the cell growth of NSCLC in vivo. Taken together, circ_0003028 knockdown could suppress NSCLC progression partly by regulating the miR-1298-5p/GOT2 axis, providing an underlying therapeutic target for NSCLC.

Inborn disorders of the malate aspartate shuttle.

Over the last few years, various inborn disorders have been reported in the malate aspartate shuttle (MAS). The MAS consists of four metabolic enzymes and two transporters, one of them having two isoforms that are expressed in different tissues. Together they form a biochemical pathway that shuttles electrons from the cytosol into mitochondria, as the inner mitochondrial membrane is impermeable to the electron carrier NADH. By shuttling NADH across the mitochondrial membrane in the form of a reduced metabolite (malate), the MAS plays an important role in mitochondrial respiration. In addition, the MAS maintains the cytosolic NAD+ /NADH redox balance, by using redox reactions for the transfer of electrons. This explains why the MAS is also important in sustaining cytosolic redox-dependent metabolic pathways, such as glycolysis and serine biosynthesis. The current review provides insights into the clinical and biochemical characteristics of MAS deficiencies. To date, five out of seven potential MAS deficiencies have been reported. Most of them present with a clinical phenotype of infantile epileptic encephalopathy. Although not specific, biochemical characteristics include high lactate, high glycerol 3-phosphate, a disturbed redox balance, TCA abnormalities, high ammonia, and low serine, which may be helpful in reaching a diagnosis in patients with an infantile epileptic encephalopathy. Current implications for treatment include a ketogenic diet, as well as serine and vitamin B6 supplementation.

Optimum protein requirement of juvenile orange-spotted grouper (Epinephelus coioides).

The purpose of subject was to explore the optimum protein requirement of juvenile grouper (Epinephelus coioides). In the test, 450 juveniles with an average weight (10.02 ± 0.22) g were randomly divided into six groups with triplicate, and were fed with 350, 400, 450, 500, 550 and 600 g/kg iso-lipid test diet twice 1 day for 8 weeks, respectively. The results showed that: (1) With the increase of protein level, the body weight gain rate and specific growth rate first increased and then reduced, while the feed coefficient rate first decreased and then increased, while the protein efficiency significantly decreased (P < 0.05). (2) With the increase of protein level, the condition factor, hepaticsomatic index and visceralsomatic index significantly reduced (P < 0.05). (3) With the increase of protein level, the crude protein content of whole fish and muscle gradually increased, while the crude lipid content gradually decreased. (4) High-protein diet (550-600 g/kg) significantly increased the plasma total protein content and decreased the triglyceride content of orange-spotted grouper (P < 0.05). (5) Compared with the 350 g/kg group, 500, 550, 600 g/kg groups significantly increased the activities of glutamic-pyruvic transaminase and glutamic oxaloacetic transaminase in liver (P < 0.05). (6) With the increase of protein level, the protease activity of intestine first increased and then decreased, and reached the maximum at the protein level of 500 g/kg, while lipase and amylase decreased significantly (P < 0.05). (7) The activities of acid phosphatase, superoxide dismutase and lysozyme in liver increased first and then decreased with the increase of protein level, and reached the maximum in the 400 g/kg protein group. According to the analysis specific growth rate, the optimum protein level of juvenile orange-spotted grouper is 521.84 g/kg.

Gensenoside Rg1 protects against lipopolysaccharide- and d-galactose-induced acute liver failure via suppressing HMGB1-mediated TLR4-NF-κB pathway.

AIM: Acute liver failure (ALF) is a life-threatening acute liver injury (ALI) with high mortality. Gensenoside Rg1 (G-Rg1) effects on Lipopolysaccharide- (LPS-) and d-galactose-(D-gal-) induced ALI, but its effects on ALF remained unclear. This paper aimed to validate its possible efficacy on ALF prevention. METHODS: For in vivo studies, histological examination was performed using hematoxylin-eosin (H&E) staining, and alanine aminotransferase (ALT), aspartate aminotransminase (AST), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) contents were measured. Levels of inflammatory cytokines tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) were quantified via enzyme-linked immunosorbent assay (ELISA). Human bronchial epithelial cell line BEAS-2B was used for ALF model in vitro and its viability was measured by MTT assay. Expressions of high mobility group box 1 (HMGB1) and toll-like receptor 4-Nuclear Factor-κB (TLR4-NF-κB) pathway-related proteins were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. RESULTS: G-Rg1 relieved LPS- and D-gal-induced hepatic injury, and reduced ALT, AST and MDA levels but upregulated SOD and GSH levels, with downregulation on TNF-α and IL-6 levels. Expressions of HMGB1, TLR4 and NF-κB pathway-related proteins were also down-regulated after G-Rg1 treatment both in vivo and in vitro, while BEAS-2B cell viability was increased. However, overexpressed HMGB1 reversed the effects of G-Rg1 treatment in vitro. CONCLUSION: G-Rg1 had a protective effect against LPS- and D-gal-induced ALF both in vitro and in vivo, which might be related to inhibited HMGB1-mediated TLR4-NF-κB Pathway. These discoveries suggested that G-Rg1 could be a potential agent for prevention against ALF.

Genome-wide association study of serum liver enzymes implicates diverse metabolic and liver pathology.

Serum liver enzyme concentrations are the most frequently-used laboratory markers of liver disease, a major cause of mortality. We conduct a meta-analysis of genome-wide association studies of liver enzymes from UK BioBank and BioBank Japan. We identified 160 previously-unreported independent alanine aminotransferase, 190 aspartate aminotransferase, and 199 alkaline phosphatase genome-wide significant associations, with some affecting multiple different enzymes. Associated variants implicate genes that demonstrate diverse liver cell type expression and promote a range of metabolic and liver diseases. These findings provide insight into the pathophysiology of liver and other metabolic diseases that are associated with serum liver enzyme concentrations.

Effects of Bardoxolone Methyl on Hepatic Enzymes in Patients with Type 2 Diabetes Mellitus and Stage 4 CKD.

In a multinational placebo-controlled phase III clinical trial in 2,185 patients with type 2 diabetes mellitus and stage 4 chronic kidney disease, treatment with the Nrf2 activator bardoxolone methyl increased estimated glomerular filtration rate, a measure of kidney function, but also resulted in increases in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transferase. These increases in liver enzyme level(s) were maximal after 4 weeks of treatment and reversible, trending back toward baseline through week 48. Total bilirubin concentrations did not increase, and no cases met Hy's Law criteria, although two subjects had ALT concentrations that exceeded 10 × the upper limit of the population reference range leading to discontinuation of treatment. Animal and cell culture experiments suggested that the increases in ALT and AST induced by bardoxolone methyl may be related to its pharmacological activity. Bardoxolone methyl significantly induced the mRNA expression of ALT and AST isoforms in cultured cells. Expression of ALT and AST isoforms in liver and kidney also positively correlated with Nrf2 status in mice. Overall, these data suggest that the increases in ALT and AST observed clinically were, at least in part, related to the pharmacological induction of aminotransferases via Nrf2 activation, rather than to any intrinsic form of hepatotoxicity.

Toll-like receptor 7 mRNA is reduced in hepatitis C-based liver cirrhosis and hepatocellular carcinoma, out-performs alpha-fetoprotein levels, and with age and serum aspartate aminotransferase is a new diagnostic index.

BACKGROUND: Hepatitis B and C viruses are leading causes of liver cirrhosis and hepatocellular carcinoma (HCC). Toll-like receptor 7 (TLR-7) has been implicated in the pathogenesis of HCC linked to hepatitis B. We hypothesised a role of leukocyte TLR-7 mRNA in hepatitis C related liver cirrhosis and HCC, using alpha-fetoprotein (AFP) and liver function tests as comparators. METHODS: We recruited 102 patients with HCV-related HCC, 97 with HCV-related liver cirrhosis and 60 healthy controls. Quantification of TLR-7 mRNA was performed using real-time PCR, AFP and routine LFTs by standard techniques. RESULTS: TLR-7 mRNA levels were significantly lower in HCC patients compared to cirrhotic patients and lower again in healthy controls (p < 0.001 for trend). In multivariate analysis, age, aspartate transaminase (AST), AFP, and TLR-7 mRNA were significant predictors of HCC. The ROCC/AUC for age, AST and TLR-7 mRNA were all between 0.64 and 0.78 (all P < 0.01), but for AFP was 0.57 (95% CI 0.48-0.65, P = 0.09). We derived an index score using age, AST and TLR-7 mRNA for the diagnosis of HCC. The ROCC/AUC for the index was superior to all three root indices in the prediction of HCC. The index linked significantly with the Tokyo and Vienna liver cancer staging systems, but not with those of the CLIP and Okuda systems, in distinguishing HCC from liver cirrhosis. CONCLUSION: The combination of TLR-7 mRNA levels with age and AST improves the performance of TLR-7 in HCC diagnosis, out-performs alpha-fetoprotein and predicts early HCC.

A Mouse Model of Acute Liver Injury by Warm, Partial Ischemia-Reperfusion for Testing the Efficacy of Virus-Derived Therapeutics.

Ischemia-reperfusion injury (IRI) drives early and long-term damage to organs as well as compounding damage from acute transplant rejection and surgical trauma. IRI initiates an aggressive and prolonged inflammation leading to tissue injury, organ failure, and death. However, there are few effective therapeutic interventions for IRI. The destructive inflammatory cell activity in IRI is part of an aberrant innate immune response that triggers multiple pathways. Hence, immune-modulating treatments to control pathways triggered by IRI hold great therapeutic potential. Viruses, especially large DNA viruses, have evolved highly effective immune-modulating proteins for the purpose of immune evasion and to protect the virus from the host immune defenses. A number of these immune-modulating proteins have proven therapeutically effective in preclinical models, many with function targeting pathways known to be involved in IRI. The use of virus-derived immune-modulating proteins thus represents a promising source for new treatments to target ischemia-reperfusion injury. Laboratory small animal models of IRI are well established and are able to reproduce many aspects of ischemia-reperfusion injury seen in humans. This chapter will discuss the methods used to perform the IRI procedure in mice, as well as clinically relevant diagnostic tests to evaluate liver injury and approaches for assessing histological damage while testing novel immune modulating protein treatments.