Non-invasive visualization of liver fibrosis with [68Ga]Ga-DOTA-FAPI-04 PET from preclinical insights to clinical translation.

PURPOSE: The reversibility of early liver fibrosis highlights the need for improved early detection and monitoring techniques. Fibroblast activation protein (FAP) is a promising theranostics target significantly upregulated during fibrosis. This preclinical and preliminary clinical study investigated a FAP-targeted probe, gallium-68-labeled FAP inhibitor 04 ([68Ga]Ga-DOTA-FAPI-04), for its capability to visualize liver fibrosis. METHODS: The preclinical study employed [68Ga]Ga-DOTA-FAPI-04 micro-positron emission tomography (PET)/computed tomography (CT) on carbon tetrachloride-induced mice model (n = 34) and olive oil-treated control group (n = 26), followed by validation of the probe's biodistribution. Hepatic uptake was correlated with fibrosis and inflammation levels, quantified through histology and serum assays. FAP and α-smooth muscle actin expression were determined by immunohistochemistry, as well as immunofluorescence. The subsequent clinical trial enrolled 26 patients with suspected or confirmed liver fibrosis to undergo [68Ga]Ga-DOTA-FAPI-04 PET/magnetic resonance imaging or PET/CT. Key endpoints included correlating [68Ga]Ga-DOTA-FAPI-04 uptake with histological inflammation grades and fibrosis stages, and evaluating its diagnostic and differential efficacy compared to established serum markers and liver stiffness measurement (LSM). RESULTS: [68Ga]Ga-DOTA-FAPI-04 mean uptake in mice livers was notably higher than in control mice, increasing from week 6 [0.70 ± 0.11 percentage injected dose per cubic centimeter (%ID/cc)], peaking at week 10 (0.97 ± 0.15%ID/cc) and slightly reducing at week 12 (0.89 ± 0.28%ID/cc). The hepatic biodistribution and FAP expression showed a consistent trend. In the patient cohort, hepatic [68Ga]Ga-DOTA-FAPI-04 uptake presented moderate correlations with inflammation grades (r = 0.517 to 0.584, all P < 0.05) and fibrosis stages (r = 0.653 to 0.698, all P < 0.01). The average SUVmax to background ratio in the liver showed superior discriminative ability, especially between stage 0 and stage 1, outperforming LSM (area under curve 0.984 vs. 0.865). CONCLUSION: [68Ga]Ga-DOTA-FAPI-04 PET shows significant potential for non-invasive visualization and dynamic monitoring of liver fibrosis in both preclinical experiment and preliminary clinical trial, especially outperforming other common clinical indicators in the early stage. TRIAL REGISTRATION: NCT04605939. Registered October 25, 2020, https://clinicaltrials.gov/study/NCT04605939.

TIR8 Protects Against Nonalcoholic Steatohepatitis by Antagonizing Lipotoxicity-induced PPARα Downregulation and Reducing the Sensitivity of Hepatocytes to LPS.

In up to one-third of nonalcoholic fatty liver disease (NAFLD) patients, simple steatosis progresses to its more severe form, nonalcoholic steatohepatitis (NASH), but the precise mechanisms underlying this transition are not fully understood. Toll/interleukin-1 receptor 8 (TIR8), a conventional innate immune regulator highly expressed in hepatic tissue, has shown potential for ameliorating various inflammation-related disorders. However, its role in NASH pathogenesis, especially its regulatory effects on lipid metabolism and inflammatory responses, is still unclear. Here, using a TIR8 knockout (TIR8KO) mouse model and mass spectrometry analyses, we found that TIR8KO mice displayed aggravated hepatic steatosis and inflammation, whereas TIR8 overexpression attenuated these adverse effects. Ectopic TIR8 expression counteracts free fatty acid (FFA)-induced PPARα inhibition and downstream signaling. A decrease in TIR8 levels in hepatocytes heightened lipopolysaccharide (LPS) sensitivity. Notably, FFA stimulation led to a direct interaction between TIR8 and proteasome subunit alpha type 4 (PSMA4), facilitating TIR8 degradation. These results revealed that TIR8 safeguards PPARα-regulated lipid metabolism and mitigates inflammation induced by external factors during NASH progression. Our study highlights TIR8 as a promising target for NASH therapy, indicating the potential of TIR8 agonists in treatment strategies.

Group IVA Phospholipase A2 in Collagen-Producing Cells Promotes High-Fat Diet-Induced Infiltration of Inflammatory Cells into the Liver by Upregulating the Expression of MCP-1.

Nonalcoholic steatohepatitis (NASH) is characterized by hepatic inflammation and fibrosis due to excessive fat accumulation. Monocyte chemoattractant protein-1 (MCP-1) is a key chemokine that infiltrates inflammatory cells into the liver during the development of NASH. Our previous studies demonstrated that a systemic deficiency of group IVA phospholipase A2 (IVA-PLA2), an enzyme that contributes to the production of lipid inflammatory mediators, protects mice against high-fat diet-induced hepatic fibrosis and markedly suppresses the CCl4-induced expression of MCP-1 in the liver. However, it remains unclear which cell types harboring IVA-PLA2 are involved in the elevated production of MCP-1. Hence, the present study assessed the types of cells responsible for IVA-PLA2-mediated production of MCP-1 using cultured hepatic stellate cells, endothelial cells, macrophages, and hepatocytes, as well as cell-type specific IVA-PLA2 deficient mice fed a high-fat diet. A relatively specific inhibitor of IVA-PLA2 markedly suppressed the expression of MCP-1 mRNA in cultured hepatic stellate cells, but the suppression of MCP-1 expression was partial in endothelial cells and not observed in monocytes/macrophages or hepatocytes. In contrast, a deficiency of IVA-PLA2 in collagen-producing cells (hepatic stellate cells), but not in other types of cells, reduced the high-fat diet-induced expression of MCP-1 and inflammatory cell infiltration in the liver. Our results suggest that IVA-PLA2 in hepatic stellate cells is critical for hepatic inflammation in the high-fat diet-induced development of NASH. This supports a potential therapeutic approach for NASH using a IVA-PLA2 inhibitor targeting hepatic stellate cells.

Pivotal role of intestinal cholesterol and nuclear receptor LXR in metabolic liver steatohepatitis and hepatocarcinoma.

Hepatocellular carcinoma (HCC) incidence is continuously increasing worldwide, due to the rise of metabolic dysfunction-associated steatohepatitis (MASH) cases. Cholesterol is an essential driver of the metabolic dysregulations that promote HCC progression. Liver X Receptor (LXR) is a nuclear receptor best known for the regulation of lipid and cholesterol homeostasis, with a prominent function in the liver and in the intestine. Here, we aimed to explore whether modifications in intestinal lipid metabolism may contribute to the onset of HCC, particularly taking into account cholesterol metabolism and LXRs. To study the progression of MASH to HCC, we induced metabolic HCC in wild-type male mice and mice carrying an intestinal chronic activation of LXRα. Also, we analysed human hepatic transcriptome datasets. The increased consumption of fat and carbohydrates drives the intestinal activation of LXRα and accelerates the onset of the hepatic tumours. Chronic intestinal-specific activation of LXRα enhances HCC progression only in the presence of a high cholesterol intake. In HCC, despite the increased hepatic cholesterol content, LXR is not active, thus driving liver cancer development. Intriguingly, in line with these results in the mouse model, LXR transcriptome is also downregulated in human hepatocarcinoma and its expression level in liver tumours directly correlates with a decreased survival rate in patients. Overall, our findings establish the relevance of the intestine in influencing the susceptibility to MASH-HCC and point to intestinal LXRα activation as a driver of metabolic liver cancer in the presence of dietary cholesterol.

Homotherapy for heteropathy: therapeutic effect of Butein in NLRP3-driven diseases.

BACKGROUND: Aberrant inflammatory responses drive the initiation and progression of various diseases, and hyperactivation of NLRP3 inflammasome is a key pathogenetic mechanism. Pharmacological inhibitors of NLRP3 represent a potential therapy for treating these diseases but are not yet clinically available. The natural product butein has excellent anti-inflammatory activity, but its potential mechanisms remain to be investigated. In this study, we aimed to evaluate the ability of butein to block NLRP3 inflammasome activation and the ameliorative effects of butein on NLRP3-driven diseases. METHODS: Lipopolysaccharide (LPS)-primed bone-marrow-derived macrophages were pretreated with butein and various inflammasome stimuli. Intracellular potassium levels, ASC oligomerization and reactive oxygen species production were also detected to evaluate the regulatory mechanisms of butein. Moreover, mouse models of LPS-induced peritonitis, dextran sodium sulfate-induced colitis, and high-fat diet-induced non-alcoholic steatohepatitis were used to test whether butein has protective effects on these NLRP3-driven diseases. RESULTS: Butein blocks NLRP3 inflammasome activation in mouse macrophages by inhibiting ASC oligomerization, suppressing reactive oxygen species production, and upregulating the expression of the antioxidant pathway nuclear factor erythroid 2-related factor 2 (Nrf2). Importantly, in vivo experiments demonstrated that butein administration has a significant protective effect on the mouse models of LPS-induced peritonitis, dextran sodium sulfate-induced colitis, and high-fat diet-induced non-alcoholic steatohepatitis. CONCLUSION: Our study illustrates the connotation of homotherapy for heteropathy, i.e., the application of butein to broaden therapeutic approaches and treat multiple inflammatory diseases driven by NLRP3.

Risk factors for fibrosis progression in non-alcoholic steatohepatitis: Analysis of the European cohort in the real-world GAIN study / Factores de riesgo para la progresión de la fibrosis en la esteatohepatitis no alcohólica: análisis de la cohorte europea en el estudio GAIN del mundo real

Objective To better understand drivers of disease progression in non-alcoholic steatohepatitis (NASH), we assessed clinical and sociodemographic markers of fibrosis progression in adults with NASH.Patients and methodsPhysician-reported patient demographics and clinical characteristics were utilised from the real-world Global Assessment of the Impact of NASH (GAIN) study. Factors associated with likelihood of fibrosis progression since NASH diagnosis were identified using a logistic regression model.ResultsOverall, 2349 patients in Europe from the GAIN study were included; mean age was 54.6 years and 41% were women. Significant covariates included age, years since diagnosis, employment status, fibrosis stage at diagnosis, type 2 diabetes mellitus, hypertension, liver transplant and liver biopsy at diagnosis. Risk of progression was 1.16 (95% confidence interval 1.12–1.20; p<0.001) times higher for each additional year since NASH diagnosis and 5.43 (2.68–11.37; p<0.001) times higher when physicians proposed a liver transplant at diagnosis. Compared with full-time employed patients, risk of progression was 1.77 (1.19–2.60; p=0.004) times higher for unemployed patients and 3.16 (1.30–7.63; p=0.010) times higher for those unable to work due to NASH.ConclusionsDisease duration, NASH severity and presence of other metabolic comorbidities could help to assess risk of progression in patients with NASH. (AU)

Objetivo Para comprender mejor los factores que impulsan la progresión de la enfermedad en la esteatohepatitis no alcohólica (NASH), evaluamos los marcadores clínicos y sociodemográficos de la progresión de la fibrosis en adultos con NASH.Pacientes y métodosSe utilizaron las características demográficas y clínicas de los pacientes informadas por los médicos del estudio de Evaluación Global del Impacto de NASH (GAIN) del mundo real. Los factores asociados con la probabilidad de progresión de la fibrosis desde el diagnóstico de EHNA se identificaron mediante un modelo de regresión logística.ResultadosEn total, se incluyeron 2.349 pacientes en Europa del estudio GAIN; la edad media fue 54,6 años y el 41% eran mujeres. Las covariables significativas incluyeron edad, años desde el diagnóstico, situación laboral, estadio de fibrosis en el momento del diagnóstico, diabetes mellitus tipo 2, hipertensión, trasplante de hígado y biopsia de hígado en el momento del diagnóstico. El riesgo de progresión fue 1,16 (intervalo de confianza del 95% 1,12-1,20; p < 0,001) veces mayor por cada año adicional desde el diagnóstico de EHNA y 5,43 (2,68-11,37; p < 0,001) veces mayor cuando los médicos propusieron un trasplante de hígado. en el momento del diagnóstico. En comparación con los pacientes empleados a tiempo completo, el riesgo de progresión fue 1,77 (1,19-2,60; p = 0,004) veces mayor para los pacientes desempleados y 3,16 (1,30-7,63; p = 0,010) veces mayor para aquellos que no podían trabajar debido a a NASH.ConclusionesLa duración de la enfermedad, la gravedad de NASH y la presencia de otras comorbilidades metabólicas podrían ayudar a evaluar el riesgo de progresión en pacientes con NASH. (AU)

The first selective VAP-1 inhibitor in China, TT-01025-CL: safety, tolerability, pharmacokinetics, and pharmacodynamics of single- and multiple-ascending doses.

Introduction: TT-01025-CL is an oral, irreversible small molecule that potently inhibits vascular adhesion protein-1 (VAP-1) for the treatment of inflammation associated with non-alcoholic steatohepatitis (NASH). The objectives of this study were to evaluate the safety/tolerability, pharmacokinetics, and pharmacodynamics of TT-01025-CL, a VAP-1 inhibitor, in healthy Chinese volunteers. Methods: Double-blind, placebo-controlled, dose-escalation studies were conducted in subjects randomized to receive oral once-daily TT-01025-CL (ranges: 10-300 mg [single dose]; 20-100 mg for 7 days [multiple doses]) or placebo under fasting conditions. Safety and tolerability were monitored throughout the study. Pharmacokinetic (PK) parameters were determined using non-compartment analysis. The activity of semicarbazide-sensitive amine oxidase (SSAO)-specific amine oxidase and the accumulation of methylamine in plasma were evaluated as pharmacodynamic (PD) biomarkers. Results: A total of 36 (single-dose group) and 24 (multiple-dose group) subjects were enrolled in the study. No serious adverse events (AEs) were reported, and no subject discontinued due to an AE. All treatment-emergent adverse events (TEAEs) were mild and moderate in intensity. No dose-dependent increase in the intensity or frequency of events was observed. TT-01025-CL was rapidly absorbed after administration. In the single-ascending dose (SAD) study, median Tmax ranged from 0.5 to 2 h and mean t1/2z ranged from 2.09 to 4.39 h. PK was linear in the range of 100-300 mg. The mean Emax of methylamine ranged from 19.167 to 124.970 ng/mL, with mean TEmax ranging from 13.5 to 28.0 h. The complete inhibition (>90%) of SSAO activity was observed at 0.25-0.5 h post-dose and was maintained 48-168 h post-dose. In the multiple-ascending dose (MAD) study, a steady state was reached by day 5 in the 40 mg and 100 mg dose groups. Negligible accumulation was observed after repeated dosing. PK was linear in the range of 20-100 mg. Plasma methylamine appeared to plateau at doses of 20 mg and above, with mean Emax ranging from 124.142 to 156.070 ng/mL and mean TEmax ranging from 14.2 to 22.0 h on day 7. SSAO activity in plasma was persistently inhibited throughout the treatment period. No evident change in methylamine and SSAO activity was observed in the placebo groups. Conclusion: TT-01025-CL was safe and well-tolerated at a single dose of up to 300 mg and multiple doses of up to 100 mg once daily for 7 consecutive days. Absorption and elimination occurred rapidly in healthy volunteers. Linearity in plasma exposure was observed. TT-01025-CL inhibited SSAO activity rapidly and persistently in humans. The profile of TT-01025-CL demonstrates its suitability for further clinical development.

Protective effects of dopamine against non-alcoholic steatohepatitis via inhibiting p65 pathways in vivo and in vitro.

Introduction: Currently, the role and mechanism of dopamine in non-alcoholic steatohepatitis (NASH) remains unclear. Methods: In vitro experiments utilized FFA and LPS to establish NASH cell models, while a fibrotic cell model was created using TGFß1 to investigate the impact of dopamine on cellular lipid metabolism, inflammation, and fibrosis. In vivo experiments involved the use of MCD and HFD diets to induce NASH in mouse models for observing the effects of dopamine on NASH disease progression. Results: Our study showed that dopamine significantly downregulated the expression levels of Caspase 1, IL-1ß and IL18 in the HepG2 NASH cell model. In addition, dopamine could inhibit the TGF-ß1-induced accumulation of collagen I and α-SMA in LX2 cells. In vivo experiments have shown that dopamine attenuation in mice is associated with MCD diet-induced and HFD-induced steatohepatitis. Mechanically, dopamine inhibits the p65 signaling pathway in NASH. Conclusion: In conclusion, the present study demonstrates the role of dopamine in ameliorating the symptoms of NASH and provides a direction for future research on the application of the dopaminergic system to liver disease.

Biguanides and glucagon like peptide 1 receptor agonists in the amelioration of post liver transplant weight gain; a scoping review of the mechanism of action, safety and efficacy.

Weight gain post-liver transplant can lead to adverse patient outcomes in the post-transplant period. Pharmacotherapy and other measures can be utilised to reduce the burden and occurrence of weight gain in this population. We explored the mechanism of action, safety, and efficacy of these medications, specifically GLP-1 receptor agonists and metformin, focusing on liver transplant patients. This scoping review was conducted in line with the scoping review structure as outlined by the PRISMA guidelines. Metformin and GLP-1 receptor agonists have been observed to be safe and effective in liver transplant patients. Experimental models have found liver-centric weight loss mechanisms in this drug cohort. There is a paucity of evidence about the use of antihyperglycemics in a post-transplant population for weight loss purposes. However, some small studies have shown strong safety and efficacy data. The evidence in relation to using these medications in patients with metabolic syndrome for weight loss warrants further study in a transplant population.

Significance of serum ferritin and De Ritis ratio in non-alcoholic fatty liver disease as diagnostic markers.

Objectives: To ascertain the significance of serum ferritin and De Ritis ratio as diagnostic markers in patients of nonalcoholic fatty liver disease with and without type 2 diabetes mellitus. METHODS: The comparative cross-sectional study was conducted from February to October 2022 at the Radiology Department of Combined Military Hospital, Rawalpindi, Pakistan, and comprised individuals aged 30-65 who were divided into 3 groups. Healthy controls formed group I, non-alcoholic fatty liver disease patients without type 2 diabetes mellitus formed group II and non-alcoholic fatty liver disease patients with type 2 diabetes mellitus were in group III. Blood 5ml was withdrawn and assessed for alkaline phosphatase, aspartate transaminase, alanine transaminase and ferritin. De Ritis ratio was calculated and subjected to intergroup comparison. Data was analysed using SPSS 22. RESULTS: Of the 210 subjects, 110(52.4%) were males and 100(47.6%) were females, with 70(33.3%) in each of the three groups. Group I had 38(54.3%) females and 32(45.7%) males with mean age 37.50±4.513. In group II, there were 27(38.6%) females and 43(61.4%) males with mean age 45.86±9.646, while in group III there were 35(50%) females and 35(50%) males with mean age 54.01±9.243 years. Serum ferritin levels were significantly increased in patient groups II and III compared to control group I (p<0.05). De Ritis ratio was markedly raised in groups II and III compared to group I (p<0.05). Ferritin was significantly correlated to age, weight, height, fasting blood glucose, haemoglobin, alkaline phosphatase, aspartate aminotransferase, alanine transaminase and bilirubin (p<0.05). De Ritis ratio had a significant correlation with body mass index and fasting blood glucose (p<0.05). CONCLUSIONS: Serum ferritin and De Ritis ratio were found to be useful diagnostic indicators for non-alcoholic fatty liver disease, highlighting their importance in improving disease screening.

Single-Cell Data Analysis Reveals Critical Hepatic Cells Subpopulations in the Progression of Non-alcoholic Fatty Liver Disease to Non-Alcoholic Steatohepatitis.

AIMS: The aim of this study was to reveal the hepatic cell landscape and function in the progression of NAFLD to NASH. BACKGROUND: Non-alcoholic steatohepatitis (NASH) is the progressive form and turning point of nonalcoholic fatty liver disease (NAFLD), which severely causes irreversible cirrhosis as well as hepatocellular carcinoma. The mechanism underlying the progression of NAFLD to NASH has not been revealed. Unraveling the mechanism of action of NAFLD-NASH is an important goal in improving the survival of patients with liver disease. OBJECTIVE: The aim of this study is to discover heterogeneous hepatic cells during the progression of NAFLD to NASH. METHODS: Single-nucleus RNA-seq (snRNA-seq) data containing NASH in NAFLD samples were obtained from the Gene Expression Omnibus (GEO) database. Cell types in liver tissues from NASH and NAFLD were identified after dimensionality reduction analysis, cluster analysis, and cell annotation. The cell pathways in which differences existed were identified by analyzing metabolic pathways in Hepatic cells. We also identified cell subpopulations in Hepatic cells. The developmental trajectories of Hepatic cells were characterized by pseudotime trajectory analysis. Single-cell regulatory network inference and clustering analysis identified key transcription factors and gene regulatory networks in Hepatic cells. Moreover, cell communication analysis determined the potential interactions between Hepatic cells and immune cells, and heapatic stellate cells. RESULTS: Seven cell types were identified in NAFLD and NASH. The proportion of Hepatic cells was lower in NASH and showed greater energy metabolism and glucose metabolism activity. Hepatic cells exhibited heterogeneity, showing two cell subpopulations, Hepatic cells 1 and Hepatic cells 2. Dysregulation of lipid metabolism in Hepatic Cell 2 resulted in lipid accumulation in the liver, which might be involved in the progression of NAFLD. Four key transcription factors, BHLHE40, NFEL2L, RUNX1, and INF4A, were primarily found in Hepatic cells 2. The transcription factors within the hepatic cells 2 subpopulation mainly regulated genes related to lipid metabolism, energy metabolism, and inflammatory response. The cell communication analysis showed that hepatocyte interactions with immune cells were associated with inflammatory responses, while interactions with hepatic astrocytes were associated with liver injury and hepatocyte fibrosis. CONCLUSION: The hepatic cells 2 might promote the progression of NAFLD to NASH by regulating metabolic activity, which might contribute to liver injury through inflammation.

Disparate outcomes in Hispanic patients with metabolic dysfunction-associated steatotic liver disease/steatohepatitis and type 2 diabetes: Large cohort study.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are a growing health burden across a significant portion of the global patient population. However, these conditions seem to have disparate rates and outcomes between different ethnic populations. The combination of MASLD/MASH and type 2 diabetes increases the risk of hepatocellular carcinoma (HCC), and Hispanic patients experience the greatest burden, particularly those in South Texas. AIM: To compare outcomes between Hispanic and non-Hispanic patients in the United States, while further focusing on the Hispanic population within Southeast Texas to determine whether the documented disparity in outcomes is a function of geographical circumstance or if there is a more widespread reason that all clinicians must account for in prognostic consideration. METHODS: This cohort analysis was conducted with data obtained from TriNetX, LLC ("TriNetX"), a global federated health research network that provides access to deidentified medical records from healthcare organizations worldwide. Two cohort networks were used: University of Texas Medical Branch (UTMB) hospital and the United States national database collective to determine whether disparities were related to geographic regions, like Southeast Texas. RESULTS: This study findings revealed Hispanics/Latinos have a statistically significant higher occurrence of HCC, type 2 diabetes mellitus, and liver fibrosis/cirrhosis in both the United States and the UTMB Hispanic/Latino groups. All-cause mortality in Hispanics/Latinos was lower within the United States group and not statistically elevated in the UTMB cohort. CONCLUSION: This would appear to support that Hispanic patients in Southeast Texas are not uniquely affected compared to the national Hispanic population.

Resmetirom's approval: Highlighting the need for comprehensive approaches in NASH therapeutics.

The recent FDA approval of Rezdiffra (resmetirom), an oral partial agonist of the thyroid hormone receptor-beta (THR-beta), for the treatment of noncirrhotic non-alcoholic steatohepatitis (NASH) with moderate to advanced fibrosis, has challenged conventional approaches to NASH drug development. Despite extensive efforts targeting typical pathways involved in NASH progression, such as lipogenesis, oxidative stress, and inflammation, these approaches have yet to yield any approved therapies. The success of resmetirom highlights the potential advantages of targeting THR-beta, which exerts pleiotropic effects on multiple pathways involved in NASH pathogenesis, including lipid metabolism, glucose homeostasis, and inflammation. In the phase 3 MAESTRONASH trial, resmetirom significantly improved NASH resolution, fibrosis, and LDL cholesterol levels compared to placebo, with a favorable safety profile. The tissue-specific action of resmetirom may also contribute to its efficacy and safety. The approval of resmetirom has opened new avenues for NASH drug development, emphasizing the importance of exploring novel mechanisms of action, developing targeted therapies, and embracing a more comprehensive approach to treatment. As the global burden of NASH continues to grow, the lessons learned from the success of resmetirom should inform future drug development strategies, offering hope to the millions of patients affected by this disease worldwide.

Blood Level of Nitric Oxide Metabolites and Expression of Genes Regulating NO Synthesis in Early Forms of Non-Alcoholic Fatty Liver Disease.

The levels of NO metabolites in the plasma and mRNA of the NOS3, ATG9B, and NOS2 genes in peripheral blood leukocytes of healthy people and patients with early forms of non-alcoholic fatty liver disease (steatosis and weak activity non-alcoholic steatohepatitis) were studied. In patients with steatohepatitis, the concentration of NO metabolites in the blood and the level of mRNA of the NOS2 gene were higher than in patients with steatosis and healthy people. These differences can be of diagnostic value for distinguishing between steatosis and weak activity steatohepatitis in non-alcoholic fatty liver disease. A correlation between the levels of NO metabolites and the expression of the NOS2 gene in weak activity steatohepatitis was established, which indicates activation of NO synthesis in non-alcoholic steatohepatitis due to the expression of the inducible NO synthase gene. The level of the NOS2 gene mRNA in peripheral blood leukocytes of patients with weak activity steatohepatitis correlated with the level of TNFα and IL-6 cytokines. An increase in the level of NO in the blood in weak activity steatohepatitis correlated with the level of MDA, an indicator of oxidative stress.

WFUMB Guideline/Guidance on Liver Multiparametric Ultrasound: Part 1. Update to 2018 Guidelines on Liver Ultrasound Elastography.

The World Federation for Ultrasound in Medicine and Biology (WFUMB) endorsed the development of this document on multiparametric ultrasound. Part 1 is an update to the WFUMB Liver Elastography Guidelines Update released in 2018 and provides new evidence on the role of ultrasound elastography in chronic liver disease. The recommendations in this update were made and graded using the Oxford classification, including level of evidence (LoE), grade of recommendation (GoR) and proportion of agreement (Oxford Centre for Evidence-Based Medicine [OCEBM] 2009). The guidelines are clinically oriented, and the role of shear wave elastography in both fibrosis staging and prognostication in different etiologies of liver disease is discussed, highlighting advantages and limitations. A comprehensive section is devoted to the assessment of portal hypertension, with specific recommendations for the interpretation of liver and spleen stiffness measurements in this setting.

Exploring shared molecular signatures and regulatory mechanisms in nonalcoholic steatohepatitis and inflammatory bowel disease using integrative bioinformatics analysis.

The co-existence of inflammatory bowel disease (IBD) and non-alcoholic steatohepatitis (NASH) has raised interest in identifying shared molecular mechanisms and potential therapeutic targets. However, the relationship between these two diseases remains unclear and effective medical treatments are still lacking. Through the bioinformatics analysis in this study, 116 shared differentially expressed genes (SDEGs) were identified between IBD and NASH datasets. GO and KEGG pathway analyses revealed significant involvement of SDEGs in apoptotic processes, cell death, defense response, cytokine and chemokine activity, and signaling pathways. Furthermore, weighted gene co-expression network analysis (WGCNA) identified five shared signature genes associated specifically with IBD and NASH, they were CXCL9, GIMAP2, ADAMTS5, GRAP, and PRF1. These five genes represented potential diagnostic biomarkers for distinguishing patients with diseases from healthy individuals by using two classifier algorithms and were positively related to autophagy, ferroptosis, angiogenesis, and immune checkpoint factors in the two diseases. Additionally, single-cell analysis of IBD and NASH samples highlighted the expression of regulatory genes in various immune cell subtypes, emphasizing their significance in disease pathogenesis. Our work elucidated the shared signature genes and regulatory mechanisms of IBD and NASH, which could provide new potential therapies for patients with IBD and NASH.

Does Autophagy have a Role in the Pathogenesis of Pediatric Hepatic Steatosis?

Hepatic steatosis has become the most common cause of chronic liver disease among children worldwide.  Lipophagy has been considered as a pathway affecting steatosis development and progression. OBJECTIVE: this study aimed to evaluate the immunohistochemical expression of Beclin1 and LC3A in pediatric hepatic tissues with steatosis and to correlate their expression with clinicopathological parameters. METHODS: this study included 81 Egyptian pediatric patients with hepatic steatosis and 21 pediatric cases without hepatic steatosis. All specimens were stained by Beclin1 and LC3A antibodies. According to final diagnosis obtained from Pediatric Hepatology department, patients were divided into two groups: chronic liver disease (CLD) group that included 45 cases and inborn error of metabolism (IEM) group that included 36 cases. RESULTS: higher beclin1 expression was significantly correlated with higher stages of fibrosis and distorted liver architecture in CLD group, (P=0.043) for both. The control group showed higher positivity, percentage, as well as the median values of the H score of LC3A expression than did the CLD group or the IEM group (P=0.055, 0.001, and 0.008, respectively). Higher positivity of LC3A was significantly associated with higher stages of fibrosis and distorted liver architecture in the studied IEM group (P=0.021) for both. CONCLUSIONS: Varying intensity grades of LC3A and Beclin 1 immunohistochemical expression demonstrate the variation of autophagy at different phases of pediatric hepatic steatosis and varied disease etiology.

[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.

Muscle Quality as a Potential Diagnostic Marker of Advanced Liver Fibrosis in Patients with Non-alcoholic Fatty Liver Disease.

Background: Muscle-liver crosstalk plays an important role in the development and progression of non-alcoholic fatty liver disease (NAFLD). The measurement of muscle echo-intensity during ultrasonography is a real-time, non-invasive method of assessing muscle quality. In this retrospective study, we investigated the significance of poor muscle quality (namely, a greater mass of non-contractile tissue, including intramuscular fat) as a risk factor for advanced liver fibrosis and considered whether it may represent a useful tool for the diagnosis of advanced liver fibrosis. Methods: We analyzed data from 307 patients with NAFLD (143 men and 164 women) who visited the University of Tsukuba Hospital between 2017 and 2022. The patients were stratified into the following tertiles of muscle quality according to their muscle echo-intensity on ultrasonography: modest (84.1 A.U.), intermediate (97.4 A.U.), and poor (113.6 A.U.). We then investigated the relationships between muscle quality and risk factors for advanced liver fibrosis and calculated appropriate cutoff values. Results: Patients with poor muscle quality showed a significant, 7.6-fold greater risk of liver fibrosis compared to those with modest muscle quality. Receiver operating characteristic curve analysis showed that muscle quality assessment was as accurate as the Fibrosis-4 index and NAFLD fibrosis score in screening for liver fibrosis and superior to the assessment of muscle quantity and strength, respectively. Importantly, a muscle echo-intensity of ≥92.4 A.U. may represent a useful marker of advanced liver fibrosis. Conclusion: Muscle quality may represent a useful means of identifying advanced liver fibrosis, and its assessment may become a useful screening tool in daily practice.

Morroniside delays the progression of non-alcoholic steatohepatitis by promoting AMPK-mediated lipophagy.

BACKGROUND: Non-alcoholic steatohepatitis (NASH), the inflammatory subtype in the progression of non-alcoholic fatty liver disease, is becoming a serious burden threatening human health, but no approved medication is available to date. Mononoside is a natural active substance derived from Cornus officinalis and has been confirmed to have great potential in regulating lipid metabolism in our previous studies. However, its effect and mechanism to inhibit the progression of NASH remains unclear. PURPOSE: Our work aimed to explore the action of mononoside in delaying the progression of NASH and its regulatory mechanisms from the perspective of regulating lipophagy. METHODS AND RESULTS: Male C57BL/6 mice were fed with a high-fat and high-fructose diet for 16 weeks to establish a NASH mouse model. After 8 weeks of high-fat and high-fructose feeding, these mice were administrated with different doses of morroniside. H&E staining, ORO staining, Masson staining, RNA-seq, immunoblotting, and immunofluorescence were performed to determine the effects and molecular mechanisms of morroniside in delaying the progression of NASH. In this study, we found that morroniside is effective in attenuating hepatic lipid metabolism disorders and inflammatory response activation, thereby limiting the progression from simple fatty liver to NASH in high-fat and high-fructose diet-fed mice. Mechanistically, we identified AMPK signaling as the key molecular pathway for the positive efficacy of morroniside by transcriptome sequencing. Our results revealed that morroniside maintained hepatic lipid metabolism homeostasis and inhibited NLRP3 inflammasome activation by promoting AMPKα phosphorylation-mediated lipophagy and fatty acid oxidation. Consistent results were observed in palmitic acid-treated cell models. Of particular note, silencing AMPKα both in vivo and in vitro reversed morroniside-induced lipophagy flux enhancement and NLRP3 inflammasome inhibition, emphasizing the critical role of AMPKα activation in the effect of morroniside in inhibiting NASH progression. CONCLUSION: In summary, the present study provides strong evidence for the first time that morroniside inhibits NASH progression by promoting AMPK-dependent lipophagy and inhibiting NLRP3 inflammasome activation, suggesting that morroniside is expected to be a potential molecular entity for the development of therapeutic drugs for NASH.