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Background: Posttranslational modifications (PTM) of albumin occur in liver diseases; however, little is known about the source and function of sulfonated albumin, a significant modification of albumin occurring in nonalcoholic fatty liver disease (NAFLD). We aimed to investigate the mechanism underlying sulfonated albumin production and its role in the progression of NAFLD-related liver fibrosis., Methods: Serum samples from healthy controls and patients with NAFLD were used to measure the proportion of sulfonated albumin. Mice models with NAFLD fed with high-fat diet (HFD) and methionine choline-deficient diet (MCD) were constructed. RNA sequencing, KEGG analysis, and GSEA were used to explore the mechanism of sulfonated albumin production and its mechanism of activating hepatic stellate cells (HSCs) and promoting the progression of liver fibrosis in NAFLD., Results: Sulfonated albumin levels increased significantly in both human and mouse NAFLD serum samples. In vivo studies in mice have shown that the intraperitoneal injection of sulfonated albumin promotes inflammation, hepatic steatosis, and liver fibrosis in NAFLD. In addition, autophagy has been verified as a key mechanism in the regulation of sulfonated albumin production. We also demonstrated that reactive oxygen species (ROS) production depends on the accumulation of damaged mitochondria and affects the production of sulfonated albumin under the regulation of autophagy. Hepatocyte-derived sulfonated albumin activates HSCs through the GAL3 receptor, thereby activating the endoplasmic reticulum (ER) stress pathway and promoting profibrotic activation of HSCs., Conclusions: Our study demonstrated that sulfonated albumin activated HSCs through GAL3, thereby accelerating NAFLD-related liver fibrosis. Serum sulfonated albumin may be a potential diagnostic marker for liver fibrosis and an important target for the treatment of NAFLD-related liver fibrosis., Competing Interests: Declarations of interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Obesity due to excessive body fat accumulation remains a global problem. Patients with obesity have high cortisol levels, and its dysregulation is caused by increased 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) levels. The effects and mechanism of J2H-1702, an 11β-HSD1 inhibitor, on nonalcoholic steatohepatitis (NASH) were explored. This study compared the antiadipogenic effects of J2H-1702, elafibranor (PPARα/δ agonist), and BVT14225 (selective 11β-HSD1 inhibitor) using mouse 3T3-L1 pre-adipocytes. J2H-1702, elafibranor, and BVT14225 inhibited adipocyte differentiation and intracellular lipid accumulation in 3T3-L1 cells by downregulating phospho-extracellular signal-regulated kinase, extracellular signal-regulated kinase, phospho-c-Jun-N-terminal Kinase, c-Jun-N-terminal Kinase, phospho-P38 (P-P38), P38, CCAAT/enhancer-binding proteins alpha and β, peroxisome proliferator-activated receptor γ, and glucocorticoid receptor. Additionally, J2H-1702, elafibranor, and BVT14225 treatments effectively inhibited 11β-HSD1 activity, as revealed by cortisol concentrations, and inhibited cortisone-induced adipocyte differentiation and intracellular lipid accumulation in 3T3-L1 cells. These effects were associated with 11β-HSD1 protein inhibition. Furthermore, J2H-1702 and BVT14225 increased the expression of Akt and phosphoinositide 3-kinase involved in insulin resistance in 3T3L-1 adipocytes. In the LX-2 human hepatic stellate cell line, the relative expression of N-cadherin, 11β-HSD1, collagen1α (COLA1), α-actin of smooth muscle (α-SMA) genes in LX-2 activated with TGF-β increased significantly, and after treatment with J2H-1702, it was significantly reduced. The expression of E-cadherin is decreased in TGF-β-treated LX-2 cells and increased after treatment with J2H-1702. We tested the potential of J2H-1702 as a therapeutic agent for NASH using a high-fat diet-induced NASH model, with obeticholic acid, an FXR agonist, and elafibranor as reference drugs. All drugs significantly decreased the elevated triglyceride levels in the livers of high-fat, high-carbohydrate (HFHC-fed mice. The results may add to the benefits of targeting 11β-HSD1 inhibitors with antiadipogenic activity in developing a therapeutic agent for obesity treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Cornus officinalis (CO) has been widely used as Chinese herbal medicine and has a good clinical efficacy in liver disease. In particular, it has a significant therapeutic effect on metabolic liver disease. However, systematic pharmacological studies on its hepatoprotective effect on non-alcoholic fatty liver disease (NAFLD) are lacking., Aim of the Study: We investigated the impact of Cornus officinalis extract (COE) on two mouse models of NAFLD, screened the potential mechanisms of action by using metabolomics assays, and explored the protective effects on hepatocyte proliferation by regulating glutamate metabolism and tricarboxylic acid (TCA) cycle., Methods: The main components of COE were identified by high performance liquid chromatograph (HPLC). Male C57BL/6J mice were subjected to construct carbon tetrachloride (CCl 4 ) or methionine choline deficient (MCD) induced NAFLD mice. Liver function and lipid biochemical indicators were detected using commercial assay kits. Masson staining, Western blot, and immunohistochemistry analyses were used for assessing hepatic injury and fibrosis. LC-MS non-targeted analysis was performed using the 1290 Ultra-High Performance Liquid Chromatograph System and the 6540 Q-TOF Mass Spectrometry. Palmitic acid (PA) induced L-02 cell model was established. The mediators in glutamate metabolism and TCA cycle were assessed by assay kits., Results: In vivo experiments validated that COE significantly improved liver function in NAFLD mice, reduced lipid accumulation, and alleviated pathological damage and liver fibrosis. The non-targeted metabolomics analysis combined with Ingenuity Pathway Analysis (IPA) located glutamate metabolism and the downstream TCA cycle as potential mechanisms of COE, which was further confirmed in NAFLD model mice and PA-induced L-02 cells. Finally, we confirmed that COE could promote mitochondrial energy supply by remodeling the homeostasis of the TCA cycle, thereby enhancing hepatocyte proliferation., Conclusions: This study demonstrated that COE could significantly improve CCl 4 or MCD-induced NAFLD by promoting hepatocyte proliferation. These results highlighted the potential of COE as leads for the development of innovative treatments for NAFLD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier B.V.)

Ethnopharmacological Relevance: Nonalcoholic steatohepatitis (NASH) poses significant health risks; however, effective treatment options remain scarce. Yinchen-Gancao decoction (YG, a formula composed of Traditional Chinese Medicine Artemisia capillaris Thunb. and Glycyrrhiza uralensis Fisch.) ameliorated symptoms in NASH mouse models. However, the underlying mechanisms have not been elucidated., Aim of Study: This study aimed to assess the therapeutic efficacy of YG and explore the underlying mechanisms., Materials and Methods: YG was prepared and characterized, and then orally administered to high-fat diet (HFD) or high-fat high-sugar diet (HFHS) induced mice. Histopathological examinations and biochemical analyses were performed to evaluate the therapeutic effects. Fxr -/- mice were used to investigate the role of farnesoid X receptor (FXR) in the therapeutic effects of YG. The mechanism of action was explored by quantitative real-time PCR (RT-qPCR), western blotting, molecular docking, and cellular thermal shift assay (CETSA)., Results: YG improved liver histopathology and biochemical parameters in wild-type mice but only improved alanine aminotransferase (ALT) in Fxr -/- mice. YG upregulated FXR with Chlorogenic acid (CGA) identified as a bioactive constituent. In wild-type (WT) mice, YG downregulated de novo lipogenesis (DNL), fatty acid (FA) uptake, and upregulated FA β-oxidation. However, these effects were absent in the Fxr -/- mice. YG inhibited hepatic inflammation and endoplasmic reticulum stress (ERS) in both WT and Fxr -/- mice., Conclusion: Our study supported the use of YG as a promising therapeutic agent to attenuate NASH. Its mechanism of action involved the reduction of hepatic lipid accumulation (FXR-dependent) and the inhibition of hepatic inflammation and ERS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier B.V.)

Background: Non-alcoholic steatohepatitis (NASH), an advanced manifestation of non-alcoholic fatty liver disease (NAFLD), is characterized by hepatocyte injury, inflammation, and fibrosis. Saturated fatty acids (SFAs) have emerged as key contributors to hepatocyte lipotoxicity and disease progression. Toll-like receptor 4 (TLR4) acts as a sentinel for diverse ligands, including lipopolysaccharide (LPS) and endogenous molecules like palmitic acid (PA)-induced ceramide (CER) accumulation, promoting hepatocyte demise. However, the intricate mechanisms underlying TLR4's modulation of ceramide metabolism and their concerted effect on SFA-mediated hepatotoxicity remain elusive., Methods: A NASH mouse model with liver-specific TLR4 knockdown was established through palm oil feeding and AAV2/8 tail vein injection. Histological and biochemical assessments were conducted to evaluate the mice's condition and liver damage extent. Liquid chromatography-mass spectrometry (LC-MS) was employed to quantify ceramide levels in liver tissues, offering insights into NASH mechanisms., Results: The PO-fed model exhibited elevated serum ALT, AST, and liver TG levels, enhancing lipid accumulation and hepatocellular damage. TLR4 knock-down reduced liver mass and the liver-to-body weight ratio, signifying a decreased hepatic burden. Histopathological evaluations revealed substantial improvement in hepatic steatosis in TLR4-silenced PO-fed mice, with diminished lipid droplets and inflammatory infiltrates. LC-MS analysis showed a marked decrease in long-chain ceramides (C14, C16, C20) in TLR4-knockdown PO-fed mice. Furthermore, expression of MyD88, SPTLC1, SPTLC2, and inflammatory markers IL-1β, IL-6, TNF-α were significantly attenuated., Conclusion: SFAs activate the TLR4 signaling pathway via MyD88, fostering ceramide de novo synthesis, which exacerbates hepatocyte lipotoxicity and accelerates NASH progression., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) has emerged as a global health concern, placing a substantial financial strain on public health systems. Currently, no specific pharmacological treatments are recommended in existing guidelines. Abscisic acid (ABA), a natural plant hormone, is recognized for its promising potential in the healthcare field due to its diverse biological activities. Therefore, this study is aimed at exploring the protective mechanism of ABA against NAFLD. In vitro, experiments were conducted using palmitic acid (PA) to establish a fatty liver cell model, whereas in vivo, an NAFLD model was established using a continuous high-fat diet (HFD). It was found that ABA, as a natural activator of NRF2 and AMPK, reduced lipid accumulation in hepatocytes and exerted anti-inflammatory and antioxidant effects by enhancing the nuclear expression of NRF2, thereby alleviating NAFLD in mice. Furthermore, AMPK was activated by ABA through the promotion of its phosphorylation, which subsequently enhanced the p62-dependent autophagic degradation of KEAP1, leading to the release and nuclear translocation of NRF2. In conclusion, it is indicated that ABA reduces lipid accumulation, inflammation, and oxidative stress in hepatocytes via the NRF2 and AMPK pathways, potentially serving as a promising candidate for alleviating NAFLD., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD), also known as metabolic dysfunction- associated with fatty liver disease (MAFLD), is one of the most prevalent chronic liver diseases globally. NAFLD is characterized by the accumulation of liver fat unrelated to excessive alcohol consumption. Non-alcoholic steatohepatitis (NASH) is the disease progression of NAFLD and could develop into cirrhosis and hepatocellular carcinoma. In previous studies, the preclinical efficacy of alisol B and alisol B 23-acetate against NASH and metabolic syndrome was identified. However, there is a paucity of literature pertaining to the specialized structural optimization of alisol B for the treatment of NASH. In this study, a series of alisol B derivatives (1-21) were designed, synthesized and evaluated in order to improve the activity of alisol B and to obtain candidate compounds for treating NASH. The effects of the synthesized compounds on de novo lipogenesis and α-SMA gene expression were tested to explore the preliminary structure-activity relationship (SAR). Compounds 14 and 21 were selected for further in vivo investigation. The high-fat diet plus carbon tetrachloride (HFD + CCl 4 )-induced NASH mice model was employed for biological evaluation in vivo. Compounds 14 and 21 effectively improved hepatic steatosis, ballooning, inflammatory infiltration, and hepatic fibrosis in the livers of HFD + CCl 4 mice. Thus, 14 and 21 are promising lead compounds for the treatment of NASH., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Nonalcoholic fatty liver disease (NAFLD) is a common chronic disease characterized by hepatocyte steatosis, which excludes alcohol, drugs and other definite liver damage-related factors. It has been reported that OTUB1 serves a significant role in the regulation of glucose and lipid metabolism. The present study aimed to investigate the molecular mechanism underlying the effect of OTUB1 on regulating NAFLD. The NAFLD mouse model was induced via high-fat-diet, and glucose and insulin tolerance tests were then performed. In addition, the serum levels of total cholesterol (TC) and triglycerides (TG) were detected. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were assessed using the corresponding biochemical assays. Hematoxylin and eosin, and periodic acid-Schiff staining was carried out to evaluate the liver pathology in mice. The expression levels of the NAFLD-related genes and inflammatory genes were determined by reverse transcription-quantitative PCR, Western blot analysis and immunofluorescence staining. Furthermore, the regulatory association between OTUB1 and poly (adenosine diphosphate-ribose) polymerase (PARP)-1 was assessed by co-immunoprecipitation assay. The results showed that OTUB1 was significantly upregulated in both in vitro and in vivo NAFLD models. Knockout of OTUB1 significantly improved affected glucose tolerance and insulin sensitivity, decreased TG and TC content, and decreased ALT, AST and ALP levels. In addition, the results show that OTUB1 can regulate the expression of PARP1 by inhibiting the ubiquitination of PARP1, while PARP1 knockout can inhibit liver inflammation by regulating HMGB1, thereby improving NAFLD. Targeting OTUB1 could be a potential therapeutic strategy for the NAFLD., Competing Interests: Declaration of competing interest The authors report no conflict of interest., (Copyright © 2025 Elsevier Inc. All rights reserved.)

This study explored the mechanisms and effects of 1 month of peroxisome proliferator-activated receptor (PPAR)α/γ agonist aleglitazar (10 mg/kg per day) or cannabinoid receptor 2 (CB 2 R) agonist JWH015 (3 mg/kg per day), alone or combined, on visceral adipose tissue (VAT)-derived extracellular vesicle (EV) release and associated systemic/VAT inflammation, decreased VAT capillary density/fibrosis, and intestinal inflammation/hyperpermeability in nonalcoholic steatohepatitis (NASH) mice. High EV release from VAT of NASH mice was associated with severe systemic/VAT/intestinal inflammation, reduced capillary network of VAT, and intestinal hyperpermeability. Combined JWH015 with aleglitazar treatment suppressed high-fat diet-induced obesity/adiposity, inhibited VAT expansion, reduced VAT inflammation/fibrosis, normalized VAT capillary network, and attenuated intestinal mucosal injury, inflammation, and hyperpermeability in NASH + aleglitazar + JWH015 mice. The inhibition of adipose tissue (AT)-derived EV release and hypoxia-inducible factor (HIF)1α levels in AT-derived EV, normalization of CB 2 R, PPARα, PPARγ, PPARγ1, PPARγ2, tight junction proteins, vascular endothelial growth factor/CD31 expression, and down-regulation of HIF1α, monocyte chemoattractant protein-1, and transforming growth factor-β1 were observed in the VAT and intestine of the NASH + aleglitazar + jwh015 group. In vitro experiments revealed that PPARα/γ and CB 2 R activation attenuated NASH AT-derived EV-induced pathogenic changes in the J774/SVEC4-10/Caco2/3T3-L1 cell system. This study suggested that VAT-derived EVs contribute to the pathogenesis of NASH and that combined PPARα/γ and CB 2 R agonist treatment ameliorated the abovementioned abnormalities of NASH mice., Competing Interests: Disclosure Statement None declared., (Copyright © 2025 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background and Aims: NASH confers an increased liver-related and kidney morbidity. Phospholipid curcumin (Meriva) is a phospholipid formulation with ameliorated systemic curcumin absorption and delivery. We assessed the safety and efficacy of Meriva in NASH., Approach and Results: In this double-blind trial, 52 patients with biopsy-proven NASH (71% with stage ≥F2 fibrosis, 58% with stage A2-G2/A2-G3a chronic kidney disease) were randomized 1:1 to receive Meriva 2 g/d or placebo for 72 weeks. The primary endpoint was NASH resolution with no worsening of fibrosis. The secondary endpoints included a ≥1 stage liver fibrosis improvement with no NASH worsening; regression of significant (ie, stage ≥F2) fibrosis and CKD; and improvement in renal, glucose, lipid, and inflammatory parameters. We also explored the treatment effect on hepatic activation of NF-kB, a key proinflammatory transcription factor and a major target of curcumin. Fifty-one patients (26 on Meriva and 25 on placebo) completed the trial. Sixteen (62%) patients on Meriva versus 3 (12%) patients on placebo had NASH resolution (RR = 5.33 [95% CI = 1.76-12.13]; p = 0.003). Thirteen (50%) patients on Meriva versus 2 (8%) patients on placebo had ≥1 stage fibrosis improvement (RR = 6.50 [1.63-21.20]; p = 0.008). Eleven (42%) patients on Meriva versus 0 (0%) on placebo had regression of significant liver fibrosis (RR = 18.01 [1.43-36.07]; p = 0.02). Hepatic NF-kB inhibition predicted NASH resolution (AUC = 0.90, 95% CI = 0.84-0.95) and fibrosis improvement (AUC = 0.89, 95% CI = 0.82-0.96). Thirteen (50%) patients on Meriva versus 0 (0%) on placebo had chronic kidney disease regression (RR = 10.71 [1.94-17.99)]; p = 0.004). Compared with placebo, Meriva improved eGFR (difference in adjusted eGFR change: +3.59 [2.96-4.11] mL/min/1.73 m 2 /y, p = 0.009), fasting glucose(-17 mg/dL; 95% CI = -22, -12), HbA1c (-0.62%; 95% CI = -0.87%, -0.37%), LDL-C (-39 mg/dL; 95% CI = -45, -33), triglycerides (-36 mg/dL, 95% CI = -46, -26), HDL-C (+10 mg/dL; 95% CI = +8, +11), and inflammatory markers. Adverse events were rare, mild, and evenly distributed., Conclusions: In patients with NASH, Meriva administration for 72 weeks was safe, well-tolerated, and improved liver histology, possibly through NF-kB inhibition, kidney disease, and metabolic profile., (Copyright © 2024 American Association for the Study of Liver Diseases.)

Nonalcoholic fatty liver disease (NAFLD) poses a great global challenge to public health, yet it holds promise for amelioration through plant-derived polysaccharide. Ginkgo biloba seeds have long been used as medicine and food, which has potential benefits for various chronic diseases. However, the protective role of Ginkgo biloba seed polysaccharide against NAFLD remains unclear. In this study, we isolated and purified polysaccharide (GBSP-2) from Ginkgo biloba seeds. GBSP-2 is composed of α-d-glucopyranose residues, which are interconnected with α-d-glucopyranose units linked by (1→4) bonds, (1→4,6) bonds and (1→3,4) bonds, the ratio distribution is 15:1:1. By studying a mouse model, we investigated the effect of GBSP-2 (100 or 200 mg/kg) on high-fat-diet-induced NAFLD. We demonstrated that GBSP-2 significantly alleviated NAFLD, as evidenced by reduced hepatic steatosis, decreased inflammation, improved oxidative stress and ameliorative glucolipid metabolic disorders. Furthermore, GBSP-2 mitigated gut microbiota disturbance of NAFLD mice and markedly increased the abundance of Akkermansia, Romboutsia, Lactobacillus and Bacteroides. Mechanistically, GBSP-2 could activate AMPK/ACC signaling pathway to inhibit lipid synthesis by generating 3,4-dihydroxyphenylpropionic acid (DHPPA). Overall, these findings suggest that GBSP-2 plays a multi-channel and multi-target role in improving NAFLD through the gut-liver axis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Non‑alcoholic steatohepatitis (NASH), the more progressive form of non‑alcoholic fatty liver disease, has become a major cause of cirrhosis and liver cancer. The aim of the present study was to investigate the anti‑NASH effect of the nonabsorbable antibiotic rifaximin and its specific molecular mechanisms. A methionine‑choline deficient (MCD) diet was used to induce NASH formation in mice. The mice with NASH were treated with rifaximin to observe its effects on liver fat deposition, hepatocyte inflammation and liver fibrosis. Furthermore, the intestinal microbiota of mice with NASH was analysed by 16S rRNA sequencing and terminal ileal bile acid levels were assessed using liquid chromatography‑electrospray ionization‑tandem mass spectrometry analysis. Furthermore, the correlation between the intestinal microflora and bile acid levels in the terminal ileum was investigated, and the effects of rifaximin on the intestinal Helicobacter ‑deoxycholic acid (DCA)‑farnesoid X receptor (Fxr)‑hepatocyte nuclear factor 1α (Hnf1α) signalling pathway were examined. Moreover, analyses of mice after intestinal decontamination with broad‑spectrum antibiotics and of hepatocyte‑specific Hnf1α knockout (Hnf1α H‑KO ) mice were used to elucidate the molecular mechanisms by which rifaximin improves NASH. Notably, treatment with rifaximin markedly ameliorated liver steatosis, hepatocyte inflammation and liver fibrosis in mice with MCD diet‑induced NASH. Rifaximin modulated the gut microbiota, especially Helicobacter hepaticus , in mice with NASH. In addition, rifaximin inhibited the intestinal Helicobacter‑DCA‑Fxr‑Hnf1α signalling pathway in mice with NASH. By contrast, rifaximin did not exert an anti‑NASH effect on decontamination‑treated mice or Hnf1α H‑KO mice. Taken together, these results indicated that rifaximin can ameliorate NASH in mice by modulating the Helicobacter ‑DCA‑Fxr‑Hnf1α signalling pathway, providing a theoretical basis for the clinical treatment of patients with NASH with rifaximin.

Non-alcoholic fatty liver disease (NAFLD) has evolved to become a major cause of hepatocellular carcinoma (HCC). Visualization of NAFLD-HCC deterioration process imaging is essential to understand the underlying pathophysiological processes. However, currently relevant probes with short emission wavelengths, univariate and the inability to achieve theranostics functionality have encountered obstacles in further evaluating the NAFLD-HCC process. Here, we present a carboxylesterase (CE)-activated NIR fluorescent probe (BODJ) which has lipid droplets (LDs)-targeting ability and emits at a wavelength of 858 nm with a fluorescence quantum yield of 19.06%. CE-activated BODJ was used as a visual tool to successfully visualize both NAFLD deterioration processes and HCC in situ based on changes in the average number of LDs and the associated fluorescence intensity fluctuations. Imaging results showed that changes associated with CE and LDs in the modelled cells varied during the transition from nonalcoholic fatty liver to nonalcoholic steatohepatitis and later progression to HCC, highlighting the close association between bivariate and disease. We also demonstrate that BODJ has photodynamic (PDT) and photothermal therapy (PTT) capabilities, allowing image-guided dual phototherapy to damage HCC in situ. This NIR probe, which takes advantage of bivariate to track the deterioration process that illuminates NAFLD-HCC and has dual phototherapy capabilities, provides new ideas for the design of probes related to the diagnosis and treatment of hepatic metabolic diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: Hepatic steatosis, the early stage of nonalcoholic fatty liver disease (NAFLD), currently lacks targeted pharmacological treatments. G protein-coupled receptors (GPCRs) in hepatocytes differentially regulate lipid metabolism depending on their coupling profile of G protein subtypes. Unlike G s , G i , and G q signaling, the role of G 12 signaling in hepatic steatosis remains elusive. The objective of this study was to investigate the effect of G 12 signaling on hepatic steatosis and obesity and its mechanisms., Methods: We generated mice expressing a G 12 -coupled designer GPCR in a liver-specific manner. We performed phenotypic analysis in the mice under the condition of fasting (acute hepatic steatosis model) or high-fat diet feeding (chronic hepatic steatosis model)., Results: In acute and chronic hepatic steatosis models, chemogenetic activation of hepatic G 12 signaling suppressed the progression of hepatic steatosis. The treatment led to an increased triglyceride secretion with little effect on mitochondrial respiratory activity, fatty acid oxidation, de novo lipogenesis, and fatty acid uptake. Furthermore, in a high-fat-diet-induced obesity model, activation of the G 12 -coupled designer GPCR exerted anti-obesity effects with increased whole-body energy expenditure and fat oxidation. Anti-FGF21 antibody treatment showed that the anti-obesity effects of the hepatic G 12 D activation relied in part on the hepatokine FGF21., Conclusions: Our findings indicate that the activation of G 12 signaling in the liver has the potential to prevent hepatic steatosis and obesity. This discovery provides a strong rationale for the development of drugs targeting G 12 -coupled GPCRs expressed in the liver., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Nonalcoholic fatty liver disease (NAFLD), a major cause of chronic liver disorders, has become a serious public health issue. Although the Smad4 signaling pathway has been implicated in the progression of NAFLD, the specific role of Smad4 in hepatocytes in NAFLD pathogenesis remains unclear. Hepatocyte-specific knockout Smad4 mice (Alb Smad4-/- ) were first constructed using the Cre-Loxp recombinant system to establish a high-fat diet induced NAFLD model. The role of Smad4 in the occurrence and development of NAFLD was determined by monitoring the body weight of mice, detecting triglycerides and free fatty acids in serum and liver tissue homogenates, staining the tissue sections to observe the accumulation of liver fat, and RT-qPCR detecting the expression of genes related to lipogenesis, fatty acid intake, and fatty acid β oxidation. The molecular mechanism of Smad4 in hepatocytes affecting NAFLD was therefore investigated through combining in vitro and in vivo experiments. Smad4 deficiency in hepatocytes mitigated NAFLD progression and decreased inflammatory cell infiltration. Moreover, Smad4 deficiency inhibited CXCL1 secretion by suppressing the activation of the ASK1/P38/JNK signaling pathway. Furthermore, targeting CXCL1 using CXCR2 inhibitors diminished hepatocyte lipogenesis and inhibited the polarization of M1-type macrophages. Collectively, these results suggested that Smad4 plays a vital role in exacerbating NAFLD and may be a promising candidate for anti-NAFLD therapy., Competing Interests: Competing interests: The authors declare no competing interests. Ethics approval: All animal studies were performed after approval by the Institutional Laboratory Animal Care and Use Committee of the Institute of Biophysics, Chinese Academy of Sciences., (© 2025. The Author(s).)

Non-alcoholic fatty liver disease (NAFLD) is a disease with various levels varying from fatty liver steatosis to acute steatosis which is non-alcoholic steatohepatitis (NASH), which can develop into hepatic failure, as well as in some conditions it can develop into hepatocellular carcinoma (HCC). In the NAFLD and NASH context, aberrant microRNA (miRNA) expression has a thorough contribution to the incidence and development of these liver disorders by influencing key biological actions, involving lipid metabolism, inflammation, and fibrosis. Dysregulated miRNAs can disrupt the balance between lipid accumulation and clearance, exacerbate inflammatory responses, and promote fibrogenesis, thus advancing the severeness of the disorder from simple steatosis to more complex NASH. In the current review, the latest development concerned with the activity of complex regulatory networks of miRNA in the incidence as well as the evolution of NAFLD is to be discussed, also conferring about the miRNAs' role in the onset, pathogenesis as well as diagnosis of NAFLD and NASH discussing miRNAs' role as diagnostic biomarkers and their therapeutic effects on NAFLD/NASH., Competing Interests: Declarations. Ethics approval and consent to participate: Not Applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests. Conflict of interest: The author declares they have no conflict of interest. Human and animal ethics: Not applicable., (© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Neuregulin (NRG) family is involved in energy metabolism, among which NRG1 is a neuregulin proved to play a protective role in MAFLD cells. But the presice echanism has not been fully illustrated. This study aimed to investigate the role of NRG1 via the ERK/SIRT1 signaling in the pathogenesis of MAFLD., Methods: C57BL/6 mice were fed with high-fat diet for 8 weeks, and then injected with NRG1 (0.3 mg/kg/d) and PD98059 (0.3 mg/kg/d) via tail vein for 5 weeks. HepG2 cells induced by oleic acid and palmitic acid were treated with 20ng/mL NRG1 and 10µmol/L PD98059. The changes of histopathological, biochemical indexes, inflammatory factors, lipid metabolism, apoptosis and autophagy parameters were measured., Results: The expressions of NRG1 in MAFLD cell and animal models were significantly lower than that in the control group. After the intervention of ERK inhibitor PD98059, the expression of NRG1 decreased significantly in vivo, but no significant change was observed in vitro. Moreover, NRG1 ameliorated hepatic steatosis, enhanced cell viability, reduced cell apoptosis, and attenuated liver injury both in vitro and in vivo. After NRG1 intervention, the expressions of ERBB2, ERBB3, p-ERK1/2, SIRT1 and p-FOXO1 as well as the LC3II/I ratio in MAFLD cells and liver tissues of MAFLD mice were significantly increased, while the expression of SREBP1c was decreased. The aforementioned therapeutic effect of NRG1 was lost after the intervention of PD98059., Conclusion: NRG1 might play a protective role in the pathogenesis of MAFLD by activating the downstream ERK1/2 through ErbB2-ErbB3, which promotes the expression of SIRT1 and autophagy markers. This study might indicate a new therapeutic strategy for MAFLD., Competing Interests: Declarations. Ethics approval and consent to participate: The authors declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. All experiments were performed with the approval of the Animal Care and Use Committee of Hangzhou Medical College (Permit number: 20180035). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Non-alcoholic fatty liver disease (NAFLD) is the main cause of chronic liver disease worldwide, affecting one-fourth of the world's population. With more than half of the world's population, the Asia-Pacific region contributed 62.6 % of liver-related fatal incidents in 2015. Currently, liver imaging techniques such as computed tomography (CT), nuclear magnetic resonance (NMR) spectroscopy, and ultrasound are non-invasive imaging methods to diagnose the disease. A liver biopsy is the gold standard test for establishing the definite diagnosis of non-alcoholic steatohepatitis (NASH). However, there are still significant problems with sample variability and the procedure's invasiveness. Numerous studies have indicated various non-invasive biomarkers for both fibrosis and steatosis to counter the invasiveness of diagnostic procedures. Metabolomics could be a promising method for detecting early liver diseases, investigating pathophysiology, and developing drugs. Metabolomics, when utilized with other omics technologies, can result in a deeper understanding of biological systems. Metabolomics has emerged as a prominent research topic, offering extensive opportunities to investigate biomarkers for liver diseases that are both sensitive and specific. In this review, we have described the recent studies involving the use of a metabolomics approach in the diagnosis of liver diseases, which would be beneficial for the early detection and treatment of liver diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Non-alcoholic steatohepatitis (NASH) is the most common cause of chronic liver diseases with its pathophysiological mechanism poorly understood. In this work, serological, histological, molecular biological, biochemical, and immunological methods were applied to explore the pathological significance and action of zinc finger protein 281 (ZFP281 in mouse, ZNF281 in human) and targeted strategies. We reported that ZFP281/ZNF281 abundance in hepatocytes was positively correlated with the progression of NASH. Hepatocyte-specific knockdown of Zfp281 prevented mice from NASH diet-induced liver injury, steatosis, inflammation, and fibrosis. Consistently, the metabolic syndromes in NASH mice, characterized by obesity, hyperglycemia, insulin resistance, and hyperlipidemia, were also relieved by hepatocyte-specific Zfp281 deficiency. Mechanistically, incremental ZNF281 led to the upregulation of proinflammatory signaling, receptor-interacting protein kinase 1 (RIPK1)/RIPK3/mixed lineage kinase domain like pseudokinase (MLKL) axis in hepatocytes bearing free fatty acid stress. Activated MLKL translocated to the mitochondrial membrane, disrupting mitochondrial fatty acid β-oxidation and facilitating lipid accumulation in hepatocytes exposed to free fatty acid stimulation; also, MLKL in activated form orientated to the plasma membrane, triggering the lytic death mode in hepatocytes and launching hepatocellular proinflammatory responses. Moreover, we screened a ZFP281 inhibitor, pterostilbene, and demonstrated that pterostilbene, by inhibiting ZFP281 elevation in NASH livers, reduced hepatocyte injury, steatosis, inflammatory responses and fibrogenesis. In conclusion, this work proposes that induction of ZFP281/ZNF281-RIPK1/RIPK3/MLKL signaling disrupts fatty acid metabolism, promoting lipid accumulation, and triggers proinflammatory cell death, accelerating hepatic necroinflammation. Our work identifies ZFP281/ZNF281 as a promising target as well as pterostilbene as a potential strategy for NASH managing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The effectiveness of established biomarkers for non-alcoholic fatty liver disease (NAFLD) within the updated framework of steatotic liver disease (SLD) remains uncertain. This cohort study examines the association of four metabolic biomarkers-retinol-binding protein 4 (RBP-4), fibroblast growth factor 21 (FGF-21), adiponectin, and osteocalcin-with SLD and its subtypes: metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction with alcohol-related liver disease (MetALD)/alcohol-related liver disease (ALD). Among 3,504 Chinese participants aged 55-70, 938 (26.8%) have developed SLD over 5 years, including 871 with MASLD and 67 with MetALD/ALD. The findings indicate that models incorporating RBP-4, FGF-21, adiponectin, and osteocalcin improve predictive accuracy for SLD beyond conventional models. Notably, adiponectin emerges as the most versatile marker, while elevated baseline levels of FGF-21 or RBP-4 indicate specific needs for metabolic or alcohol-related interventions, respectively, supporting tailored precision medicine strategies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Aldehyde dehydrogenase 2 (Aldh2) Glu504Lys mutation, common in East Asians, is linked to various alcohol-related pathologies, notably fatty liver disease. Recent findings suggest that high ethanol-producing Klebsiella pneumoniae(HiAlc Kpn) exacerbates liver injury in non-alcoholic fatty liver disease (NAFLD). Our study investigated the combined effects of Aldh2 deficiency and HiAlc Kpn on NAFLD liver injury, transcriptome analyses to unearth potential mechanisms and therapeutic targets. In our controlled experiment with Aldh2-deficient mice, we induced fatty liver via alcohol and HiAlc Kpn gavage, followed by comprehensive analyses to detect gene expression and epigenetic changes. The results showed that Aldh2-deficient mice were particularly vulnerable to ethanol and HiAlc Kpn, with notable gene expression changes in key metabolic and liver injury pathways. Our analysis identified crucial differentially expressed genes (DEGs) and pathways, highlighting the significant roles of genes like Cyp8b1, Cyp7a1, and Ugt2b1 in liver metabolism and suggesting them as therapeutic targets. The study underscores the impact of Aldh2 deficiency and HiAlc Kpn on NAFLD progression, revealing potential therapeutic strategies. Despite these insights, further research is needed to clarify the systemic effects on aldehyde metabolism and the full implications of Aldh2 deficiency and HiAlc Kpn in liver injury., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: The animal study was reviewed and approved by the animal study was reviewed and approved by the Experimental Animal Ethics Subcommittee of the Capital Institute of Pediatrics, a licensed individual with license number DWLL2021009, and our manuscript confirming the study is reported by ARRIVE guidelines ( https://arriveguidelines.org )., (© 2025. The Author(s).)

Endocrine-disrupting chemicals (EDCs), including phthalates, have been implicated in the development of non-alcoholic fatty liver disease (NAFLD) and hepatic fibrosis. This study investigates the age-dependent effects of butyl benzyl phthalate (BBP) exposure on lipid metabolism in the livers of young and aged mice. Young (2-month-old) and aged (20-month-old) male C57BL/6 mice were exposed to BBP through drinking water at a dose of 169 μg/kg/day for 6 and 4 months, respectively. Young mice exposed to BBP showed fatty liver, with downregulation of key fatty acid oxidation genes (CPT1A, CPT1B, CPT2, and Acox1) and elevated pro-inflammatory cytokines (TNF-α and IL-6). In contrast, aged mice exhibited hepatic fibrosis, with increased collagen deposition and upregulation of genes related to fibrosis (Acta2, MMP2, TGF-ß1, and Col1a2), cirrhosis (CXCR4, SOX9, DCN, and MFAP4), and cancer (Bcl2, CDKN2a, c-Myc, and Fn1). Overall, these findings emphasize the importance of age when evaluating the risks of EDC exposure, such as BBP. Future research should focus on understanding the molecular mechanisms behind these age-related differences and explore Grem1 and SOCS3 as potential therapeutic targets for treating EDC-induced and age-related liver diseases.

Non-alcoholic fatty liver disease (NAFLD) is a liver disease without approved treatment. Oxymatrine (OMT) has protective effects in various liver diseases. We aimed to investigate the roles and mechanisms of OMT in NAFLD. NAFLD models were established using high-fat and high-sucrose diet-fed rats and oleic acid (OA)-stimulated hepatocytes, respectively. Then, OMT was used to treat the NAFLD models, with metformin as a positive control. Liver damage, lipid accumulation and hepatic lipid profile of NAFLD rats were assessed. Peroxisome proliferator activated receptor alpha (PPARα), sirtuin 1 (Sirt1)/adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway- and fatty acid oxidation (acyl-CoA oxidase 1 and carnitine palmitoyltransferase 1A)-associated proteins were measured both in vivo and in vitro. Furthermore, hepatocytes were transfected with si-Sirt1 and oe-PPARα to verify the mechanisms of OMT in NAFLD. NAFLD rats supplemented with OMT displayed reduced liver damage and lipid accumulation. After OMT intervention, the liver lipid profile of NAFLD rats was changed greatly, most of the top differentially expressed lipid metabolites were triglyceride, moreover, diacylglycerol content was decreased in NAFLD rats. OMT activated the Sirt1/AMPK pathway and PPARα, and upregulated acyl-CoA oxidase 1 and carnitine palmitoyltransferase 1A expressions in NAFLD models. In vitro, OMT enhanced viability, and improved lipid accumulation in OA-stimulated hepatocytes. However, the protective functions of OMT in OA-exposed hepatocytes were offset by Sirt1 knockdown, while PPARα overexpression further counteracted the effects of Sirt1 knockdown. OMT could relieve NAFLD by promoting Sirt1/AMPK pathway- and PPARα-mediated hepatic fatty acid oxidation, indicating that OMT is a potential approach for NAFLD treatment., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The diagnostic criteria for Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD) and Metabolic Associated Steatotic Liver Disease (MASLD) aim to refine the classification of fatty liver diseases previously grouped under Non-Alcoholic Fatty Liver Disease (NAFLD). This study evaluates the applicability of the MAFLD and MASLD frameworks in NAFLD patients, exploring their clinical utility in identifying high-risk patients. A total of 369 NAFLD patients were assessed using MAFLD and MASLD diagnostic criteria. Baseline characteristics, metabolic profiles, hepatic fibrosis, and cardiovascular risks were compared across the groups. Among NAFLD patients, 97.55% (n = 359) met MASLD criteria, and 97.01% (n = 357) fulfilled MAFLD criteria. Both frameworks MAFLD and MASLD captured overlapping populations, with MASLD encompassing slightly more cases. No significant differences were observed in metabolic risk factors, fibrosis indices (APRI, FIB-4, NAFLD fibrosis score), or cardiovascular risk (10-year ASCVD score). A small subset of lean NAFLD patients (10 cases) with distinct profiles remained uncategorized by either framework. Pure NAFLD cases (n = 10) were with mild insulin resistance (HOMA-IR: 3.07 ± 0.33) and slightly elevated LDL (102.5 ± 42.87 mg/dL), while fibrosis indices indicated low fibrosis risk. Steatosis indices supported the diagnosis of early-stage NAFLD with preserved liver function. These patients do not meet the criteria for inclusion in the MAFLD or MASLD frameworks, highlighting a gap in the current diagnostic systems. MAFLD and MASLD criteria align closely with NAFLD in capturing patients with metabolic risk with MASLD-enhanced inclusivity. Further refinement is required to address heterogeneity, particularly in lean NAFLD patients. Hypertension prevalence was comparable (17.4% in NAFLD, 18.2% in MAFLD, 17.8% in MASLD; p = 0.960), as was diabetes mellitus (36.7%, 37.8%, and 37.6%, respectively; p = 0.945). Body mass index was also similar across groups, with medians of 33.25, 33.6, and 33.4 kg/m 2 (p = 0.731). Non-invasive markers of hepatic fibrosis, including APRI, FIB-4, and NAFLD fibrosis scores, did not differ significantly, with median FIB-4 scores around 1.05 (p = 0.953). Similarly, were the results of hepatic steatosis index and ASCVD score., Competing Interests: Declarations. Conflict of Interest: The authors declare no competing interests. Ethics approval: “This study was conducted per the principles of the Declaration of Helsinki. This study was approved by the Ethics Committee of the National Liver Institute, Menoufia University as unique research and not a part of an approved student thesis. Informed consent was obtained from all patients or their legal representatives before enrolment in the study. Consent to participate and publish.: Not applicable., (© 2025. The Author(s).)

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of liver conditions, ranging from hepatic steatosis to steatohepatitis, fibrosis, and severe outcomes such as cirrhosis or cancer. The progression from hepatic steatosis to fibrosis involves significant extracellular matrix (ECM) remodeling, characterized by increased collagen deposition and cross-linking of ECM proteins, causing increased tissue stiffness and altered MMP expression patterns. Dysregulated MMP expression and extracellular acidosis are key contributors to NAFLD progression. Unlike other MMPs, which may be relevant only at specific disease stages, MMP-9 serves as a universal marker, allowing for monitoring of its expression in relation to disease states and ECM parameters. Understanding dysregulated MMP-9 expression across different NAFLD stages can provide crucial insights into disease progression and serve as both a diagnostic and a prognostic biomarker, identifying potential therapeutic targets. This study introduces a three-dimensional (3D) collagen/alginate-based liver disease model designed to investigate how matrix collagen content, elasticity, and diseased cell conditions influence MMP expression and pH levels in situ using nanoprobes. The platform offered an understanding of the relationships between these factors and their role in NAFLD progression, offering valuable insights into disease progression and potential resolution. To examine how various physicochemical and biological factors, particularly MMP expression and collagen deposition, drive NAFLD progression, three 3D NAFLD models were developed, simulating healthy (HL), steatotic (SL), and fibrotic (FL) liver matrices. Additionally, the role of collagenase treatment in the FL matrix in enhancing MMP expression and potentially mitigating fibrosis was also explored. By employing dual-sensitive fluorescent nanoprobes to monitor real-time in situ changes in MMP-9 expression and pH levels, this platform offers a novel approach to understanding the in vitro roles of matrix stiffness, collagen deposition, and diseased cell conditions in NAFLD pathogenesis.

Reactive oxygen species exacerbate nonalcoholic steatohepatitis (NASH) by oxidizing macromolecules; yet how they promote NASH remains poorly understood. Here, we show that peroxidase activity of global hepatic peroxiredoxin (PRDX) is significantly decreased in NASH, and palmitic acid (PA) binds to PRDX1 and inhibits its peroxidase activity. Using three genetic models, we demonstrate that hepatic PRDX1 protects against NASH in male mice. Mechanistically, PRDX1 suppresses STAT signaling and protects mitochondrial function by scavenging hydrogen peroxide, and mitigating the oxidation of protein tyrosine phosphatases and lipid peroxidation. We further identify rosmarinic acid (RA) as a potent agonist of PRDX1. As revealed by the complex crystal structure, RA binds to PRDX1 and stabilizes its peroxidatic cysteine. RA alleviates NASH through specifically activating PRDX1's peroxidase activity. Thus, beyond revealing the molecular mechanism underlying PA promoting oxidative stress and NASH, our study suggests that boosting PRDX1's peroxidase activity is a promising intervention for treating NASH., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Clinical studies have suggested that tirzepatide may also possess hepatoprotective effects; however, the molecular mechanisms underlying this association remain unclear. In our study, we performed biochemical analyses of serum and histopathological examinations of liver tissue in mice. To preliminarily explore the molecular mechanisms of tirzepatide on metabolic dysfunction-associated fatty liver disease (MAFLD), liquid chromatography-mass spectrometry (LC-MS) was employed for comprehensive metabolomic, lipidomic, and proteomic analyses in MAFLD mice fed a high-fat diet (HFD). The results demonstrated that tirzepatide significantly reduced serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), as well as hepatic triglycerides (TG) and total cholesterol (TC), indicating its efficacy in treating MAFLD. Further findings revealed that tirzepatide reduced fatty acid uptake by downregulating Cd36 and Fabp2/4, as well as enhance the mitochondrial-lysosomal function by upregulating Lamp1/2. In addition, tirzepatide promoted cholesterol efflux and reduced cholesterol reabsorption by upregulating the expression of Hnf4a, Abcg5, and Abcg8. These results suggest that tirzepatide exerts its therapeutic effects on MAFLD by reducing fatty acid uptake, promoting cholesterol excretion, and enhancing mitochondrial-lysosomal function, providing a theoretical basis for a comprehensive understanding of tirzepatide., Competing Interests: Declarations. Ethics approval and consent to participate: All animal experiments were approved by the Institutional Animal Care and Use Committee of Guangzhou Jennio Biotechnology Co., Ltd. (Approval No. JENNIO-IACUC-2023-A066). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Testosterone deficiency is linked to an increased prevalence of non-alcoholic fatty liver disease (NAFLD), although the mechanisms underlying this association are not fully understood. Ferroptosis, a regulated cell death pathway driven by iron-dependent lipid peroxidation, has been suggested to play a role in NAFLD pathogenesis. Since testosterone deficiency is associated with lipid disorders and iron deposition, we hepothesize that ferroptosis may be involved in the pathogenesis of diet-induced NAFLD exacerbated by testosterone deficiency., Methods: Apolipoprotein E (APOE -/- ) mice were subjected to sham surgery or bilateral castration and subsequently fed a high-fat diet for 16 weeks. Liver gene expression was analyzed using RNA sequencing. Additional assessments included blood analysis, histological staining, measurement of iron and antioxidant enzyme levels, quantitative real-time PCR, Western blotting, and electron microscopy. The effects of testosterone on ferroptosis induced by free fatty acids (FFAs) and Erastin were further investigated in HepG2 cells in vitro., Results: Testosterone deficiency resulted in increased hepatic lipid accumulation and macrovesicular steatosis in high-fat diet-fed APOE -/- mice, accompanied by hepatic inflammation, fibrosis, and elevated liver enzyme levels. Transcriptomic analysis revealed that testosterone deficiency affects ferroptosis and circadian rhythm-related signaling pathways. Castrated APOE -/- mice exhibited significantly higher hepatic iron deposition, lipid peroxidation, and expression of key ferroptosis-related proteins, along with decreased Brain and muscle ARNT-like gene 1 (BMAL1) protein expression. In vitro, testosterone treatment reduced lipid and iron accumulation and lipid peroxidation in HepG2 cells subjected to FFAs and Erastin. Moreover, BMAL1 knockdown negated the protective effects of testosterone against ferroptosis in hepatocytes., Conclusion: Our study demonstrated that testosterone deficiency exacerbates NAFLD induced by a high-fat diet by promoting hepatocyte ferroptosis through modulation of the circadian protein BMAL1., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Non-alcoholic fatty liver disease (NAFLD) is a multisystem metabolic disorder, marked by abnormal lipid accumulation and intricate inter-organ interactions, which contribute to systemic metabolic imbalances. NAFLD may progress through several stages, including simple steatosis (NAFL), non-alcoholic steatohepatitis (NASH), cirrhosis, and potentially liver cancer. This disease is closely associated with metabolic disorders driven by overnutrition, with key pathological processes including lipid dysregulation, impaired lipid autophagy, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and local inflammation. While hepatic lipid metabolism in NAFLD is well-documented, further research into inter-organ communication mechanisms is crucial for a deeper understanding of NAFLD progression. This review delves into intrahepatic networks and tissue-specific signaling mediators involved in NAFLD pathogenesis, emphasizing their impact on distal organs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2025 Fan, Zhang, Wang, Wang, Li and Bai.)

Hepatocellular carcinoma (HCC) is a heterogeneous tumor associated with several risk factors, with non-alcoholic fatty liver disease (NAFLD) emerging as an important cause of liver tumorigenesis. Due to the obesity epidemics, the occurrence of NAFLD has significantly increased with nearly 30% prevalence worldwide. HCC often arises in the background of chronic liver disease (CLD), such as nonalcoholic steatohepatitis (NASH) and cirrhosis. Gut microbiome (GM) alterations have been linked to NAFLD progression and HCC development, with several investigations reporting a crucial role for the gut-liver axis and microbial metabolites in promoting CLD. Moreover, the GM affects liver homeostasis, energy status, and the immune microenvironment, influencing the response to immunotherapy with interesting therapeutic implications. In this review, we summarize the main changes in the GM and derived metabolites (e.g., short-chain fatty acids and bile acids) occurring in HCC patients and influencing NAFLD progression, emphasizing their potential as early diagnostic biomarkers and prognostic tools. We discuss the weight loss effects of diet-based interventions and healthy lifestyles for the treatment of NAFLD patients, highlighting their impact on the restoration of the intestinal barrier and GM structure. We also describe encouraging preclinical findings on the modulation of GM to improve liver functions in CLD, boost the antitumor immune response (e.g., probiotic supplementations or anti-hypercholesterolemic drug treatment), and ultimately delay NAFLD progression to HCC. The development of safe and effective strategies that target the gut-liver axis holds promise for liver cancer prevention and treatment, especially if personalized options will be considered.

Purpose: The prevalence of obesity, along with that of its associated health conditions, including cardiovascular diseases, diabetes mellitus, and liver diseases, such as non-alcoholic fatty liver disease (NAFLD), is increasing annually. Bariatric surgery is indicated for the treatment of obesity if conservative treatment fails. While various liver fibrosis scores have been proposed for assessing liver function, they are typically used prior to bariatric surgery. This study aimed to determine whether fibrosis scores calculated from non-invasive parameters are effective in monitoring liver function after bariatric surgery., Methods: This study analyzed data from 151 patients who underwent sleeve gastrectomy (SG) and were followed up at 3, 6, 9, 12, 24, and 36 months postoperatively. From the routinely collected parameters, liver fibrosis scores (APRI, Fib-4, BARD, Forns index [FORNS], Lok score [LOK], and NAFLD scores) were calculated retrospectively and compared to diabetes status % excess weight loss (%EWL) and % total weight loss (%TWL) over a 3-year follow-up period., Results: After SG, APRI, FORNS, and NAFLD scores showed significant improvements, whereas Fib-4, BARD, and LOK scores did not improve. Similarly, body mass index, %EWL, %TWL, and diabetes status also improved significantly. Throughout the 3-year follow-up period, only the APRI and NAFLD scores showed significant improvement., Conclusion: Only APRI and NAFLD scores changed significantly after SG. Thus, these two scores may be used to reflect and monitor liver function in patients who have undergone SG., Competing Interests: Declarations. Ethical approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent: For this type of study, formal consent is not required. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Nutrition and its associated inflammation have been acknowledged as vital factors in the etiopathogenesis of non-alcoholic fatty liver disease (NAFLD) and liver fibrosis. The nutritional risk index (NRI) has been widely recognized as a valid indicator of nutritional status in several diseases, including osteoporosis and cardiovascular disease. However, the role of NRI in NAFLD and liver fibrosis remains unclear., Methods: Participants were selected from the National Health and Nutrition Examination Survey data for the 2017-2018 cycle. Association between NRI and both NAFLD and liver fibrosis was evaluated using multiple logistic regression and restricted cubic spline (RCS) analysis. Mediation analysis was employed to assess the influence of inflammation on the association between NRI and both NAFLD and liver fibrosis., Results: Compared to their respective control groups, individuals with NAFLD and liver fibrosis exhibited higher NRI levels. Multiple logistic regression analyses indicated that NRI was positively associated with the odds of NAFLD and liver fibrosis across both continuous scales and quantile groups, with adjustments for relevant covariables. The RCS model demonstrated a dose-response effect between NRI and the odds of NAFLD, but not with liver fibrosis. Receiver operating characteristic (ROC) analysis revealed the area under the ROC curves of 0.798 and 0.775 for NAFLD and liver fibrosis, respectively. Mediation analysis showed that inflammation accounted for 3.139% of the effect of NRI on the odds of NAFLD, suggesting inflammation might partially mediate the impact of NRI on NAFLD., Conclusions: Our findings indicate that NRI may serve as a potential associated marker for these liver diseases, underscoring the importance of nutritional status in their etiopathogenesis., Competing Interests: Declarations. Ethics approval and consent to participate: The NHANES protocol (Protocol number: 2018-01) was examined and approved by the Research Ethics Review Board of the National Center for Health Statistics and adhered to the provisions of the Declaration of Helsinki. Since the NHANES data released by the NCHS undergo de-identification and anonymization during analysis, conducting secondary analyses on this dataset does not necessitate additional ethical approval or informed consent. The NHANES website provides access to the approval granted by the NCHS Research Ethics Review Board ( https://www.cdc.gov/nchs/nhanes/irba98.htm ). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests. Informed consent: Each patient/participant had completed a written statement of informed consent before taking part., (© 2025. The Author(s).)

Obesity is related to liver fibrosis, a condition marked by the collection of scar tissue in the liver due to the development of a profibrotic environment, which includes increased hepatocellular death and elevated reactive oxygen species production. The aim of study is to evaluate the effect of bariatric surgery on the association between liver fibrosis indices and obesity. This is a retrospective cohort, evaluating 1205 individuals diagnosed with type 2 diabetes (T2D) and living with obesity, who experienced bariatric surgery. These patients living with T2D and obesity were monitored after bariatric surgery for two years. The trajectory of biochemical markers and liver fibrosis indices were evaluated at five visits. These liver indices were Fibrosis-4 (FIB-4) index, aspartate aminotransferase (AST) to platelet ratio index, and non-alcoholic fatty liver disease (NAFLD) fibrosis score. FIB-4 index demonstrated notable trends based on its values. It showed an initial increase observed at the three-months visit, followed by a decline up to one year with a slight increase at the last follow-up (P-trend < 0.001). It should be mentioned that, mean FIB-4 in patients with FIB-4 ≥ 1.3 (pre-operation) did not exceed the value of 2.00, which is lower than the cut-off value of high risk for liver cirrhosis (FIB-4 ≥ 2.67). In addition, the NAFLD fibrosis score (NFS) demonstrated a substantial decline from - 0.32 ± 1.32 pre-operation to -0.86 ± 1.15 at the two-year mark (P-trend < 0.001). Finally, the AST to platelet ratio index (APRI) decreased from 0.27 ± 0.20 pre-operation to 0.23 ± 0.12 at the 12-month follow-up. Bariatric surgery significantly improves NFS and cause alterations in APRI and Fib-4 index levels without increasing the risk of liver cirrhosis development among patients with T2D and obesity., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: The ethical committee of the Tehran University of Medical Sciences (TUMS) approved the study with the registered number of IR.TUMS.IKHC.REC.1401.162. Informed consent for participation and publication was obtained from all individual participants included in the study. Artificial Intelligence (AI) statement: Generative AI and AI-assisted technologies were only used during the writing process to improve the readability and language of the manuscript., (© 2025. The Author(s).)

Circular RNAs (circRNAs) play an important role in regulating inflammation and oxidative stress during the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD); however, the underlying mechanism is unclear. This study aimed to determine the role of mmu&#95;circ&#95;0009303 in MASLD. We used a bioinformatics approach to identify potential targets and established an in vitro model of MASLD. Oil red O staining, cell transfection and dual-luciferase reporter assay were used to determine the role of mmu&#95;circ&#95;0009303. The results indicated that the mmu&#95;circ&#95;0009303 expression was significantly increased in the MASLD model both in vitro and in vivo and was associated with oxidative stress levels and inflammation. Moreover, bioinformatics analyses revealed that miRNA-182-5p and Foxo3 are targets of mmu&#95;circ&#95;0009303 and miRNA-182-5p, respectively. In the in vitro MASLD model, mmu&#95;circ&#95;0009303 promoted fat deposition in NCTC1469 cells, which was induced by free fatty acid (FFA) through the regulation of miRNA-182-5p/Foxo3. The expression of miRNA-182-5p and Forkhead box O3 (Foxo3) was associated with mmu&#95;circ&#95;0009303 expression in the liver of mice with MASLD, which was induced by a high-fat diet. Furthermore, mmu&#95;circ&#95;0009303 may be involved in regulating the expression of lipid metabolism-related regulatory proteins, such as CPT1A, SLC27A4, ACBD3, SREBP1, FAS, PPARα, and PPARγ. Taken together, mmu&#95;circ&#95;0009303 promotes oxidative stress, inflammation, and excessive fat accumulation in NCTC1469 cells induced by FFA through the regulation of miRNA-182-5p/Foxo3 and lipid metabolism-related regulatory proteins. These findings provide a potential target for the treatment of MASLD.

Excessive hepatic lipid accumulation and inflammatory injury are significant pathological manifestations of nonalcoholic fatty liver disease (NAFLD). Our previous research discovered that diosgenin, a natural steroidal saponin derived from Chinese herbs, can reduce hepatic lipid accumulation and steatosis; however, the exact mechanism remains unclear. This study aimed to investigate the protective mechanisms of diosgenin against NAFLD. We utilized network pharmacology and molecular docking approaches to identify the pathways through which diosgenin improves NAFLD. In high-fat diet (HFD)-fed rats, we measured biochemical markers in the serum and liver. Liver histopathology was assessed using HE and oil-red O staining. In free fatty acids (FFAs)-induced HepG2 cells, we employed the cell transfection overexpression method to verify the regulatory relationship of the identified pathways. The mechanisms in vitro and in vivo were examined using quantitative polymerase chain reaction and Western blot analyses. Bioinformatics analysis indicated that the mTOR-FASN/HIF-1α/RELA/VEGFA pathway may be the target pathway for diosgenin in alleviating NAFLD. Diosgenin inhibited hepatic lipid accumulation and pro-inflammatory cytokines in HFD-fed rats, and reduced intracellular lipid accumulation as well as TG, TC, IL-1β, and TNF-α levels in FFAs-induced HepG2 cells. Mechanistically, diosgenin downregulated the expression of p-mTOR, FASN, HIF-1α, RELA, and VEGFA, which are associated with lipid synthesis and inflammation. Overexpression of mTOR abolished the beneficial effects of diosgenin on lipid reduction and inflammation, as well as its inhibitory effects on the expression of FASN, HIF-1α, RELA, and VEGFA. In conclusion, diosgenin alleviates NAFLD through mTOR-mediated inhibition of lipid accumulation and inflammation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Fengxia Zhang reports financial support was provided by National Natural Science Foundation of China. Fengxia Zhang reports financial support was provided by Natural Science Foundation of Shandong Province. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Protein N-glycosylation is vital in the human liver and influences functions such as lipid metabolism, apoptosis, and inflammation. However, site-specific N-glycosylation patterns and variations in liver biopsy samples between healthy individuals and those with nonalcoholic fatty liver disease (NAFLD) remain incompletely characterized, primarily due to the limitations of current clinical glycoproteomic methods, including a large demand for clinical samples, low efficiency of tissue protein extraction, and a low recovery rate of intact N-glycopeptides (IGPs). To address this issue, we developed GlycoPCT, a quantitative glycoproteomic method based on pressure cycling technology. It enables efficient recovery of IGPs and accurate analysis of trace liver biopsy samples. Our research revealed a total of 4,459 unique IGPs and 361 glycans from 758 glycoproteins. High-mannose type, complex type, fucosylation type, and sialylation type N-glycans were significantly upregulated in the NAFLD group ( p < 0.001, t test). Notably, we also identified 182 upregulated IGPs from 67 proteins ( p < 0.05, FC > 1.50) and 108 downregulated IGPs from 44 proteins ( p < 0.05, FC < 0.67) in the NAFLD group. Furthermore, we highlighted an essential acute phase glycoprotein, alpha-1-acid glycoprotein 1 (A1TA), which is synthesized in the liver and plays a significant role in NAFLD progression. These novel glyco-signatures provide crucial clues for the diagnosis and pathogenesis of NAFLD.

Background: Nonalcoholic fatty liver disease (NAFLD) is the extremely usual reason of chronic liver disease, extending from simple hepatic steatosis (HS), nonalcoholic steatohepatitis (NASH) to advanced hepatic fibrosis and cirrhosis. Though orlistat is a Food and Drug Administration (FDA) approved drug for long-duration management of obesity, few cases of severe hepatic insult were declared. Melatonin is an efficient antioxidant; it also regulates metabolic processes that lead to fat accumulation and obesity., Aim of the Work: The current research aimed to compare the impact of orlistat, melatonin, and their combination on the structural changes of the hepatic tissue of adult male albino rats supplied with high fat diet (HFD)., Material and Methods: Thirty adult male albino rats divided into five groups. Liver specimens were divided into two parts. One-half was processed to obtain paraffin blocks, and the other half was processed to obtain semithin sections. Morphometric study and statistical analysis were done., Results: Hepatic tissue from the HFD group showed steatosis, ballooning, and inflammation and all these parameters were moderately improved - except for inflammation which worsened with therapy. Combined orlistat and melatonin-treated groups showed marked improvement of all parameters as well as marked improvement in the hepatic fibrosis.Orlistat/Melatonin combination therapy is both safe and effective in comparison to orlistat and melatonin monotherapy.

Although significant progress has been made in developing preclinical models for metabolic dysfunction-associated steatotic liver disease (MASLD), few have encapsulated the essential biological and clinical outcome elements reflective of the human condition. We conducted a comprehensive literature review of English-language original research articles published from 1990 to 2023, sourced from PubMed, Embase, and Web of Science, aiming to collate studies that provided a comparative analysis of physiological, metabolic, and hepatic histological characteristics between MASLD models and control groups. The establishment of a robust metabolic dysfunction-associated steatotic liver rodent model hinges on various factors, including animal species and strains, sex, induction agents and methodologies, and the duration of induction. Through this review, we aim to guide researchers in selecting suitable induction methods and animal species for constructing preclinical models aligned with their specific research objectives and laboratory conditions. Future studies should strive to develop simple, reliable, and reproducible models, considering the model's sensitivity to factors such as light-dark cycles, housing conditions, and environmental temperature. Additionally, the potential of diverse in vitro models, including 3D models and liver organ technology, warrants further exploration as valuable tools for unraveling the cellular mechanisms underlying fatty liver disease., (© 2024 The Author(s). Journal of Gastroenterology and Hepatology published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Rationale and Objectives: To investigate the diagnostic performance of water-specific T1 mapping for staging liver fibrosis in a non-alcoholic fatty liver disease (NAFLD) rabbit model, in comparison to Modified Look-Locker Inversion recovery (MOLLI) T1 mapping., Materials and Methods: 60 rabbits were randomly divided into the control group (12 rabbits) and NAFLD model groups (eight rabbits per subgroup) corresponding to different durations of high-fat high cholesterol diet feeding. All rabbits underwent MRI examination including MOLLI T1 mapping and 3D multi-echo variable flip angle (VFAME- GRE) sequences were acquired before and 20 min after the administration of Gd- EOB-DTPA. Histological assessments were performed to evaluate steatosis, inflammation, ballooning, and fibrosis. Statistical analysis included the intraclass correlation coefficient, analysis of variance, spearman correlation, multiple linear regression, and receiver operating characteristic curve., Results: A moderate correlation was observed between conventional native T1 and MRI-PDFF (r = -0.513, P < 0.001), as well as between conventional native T1 and liver steatosis grades (r = -0.319, P = 0.016). However, no significant correlation was found between the native wT1 and PDFF (r = 0.137, P = 0.314), or between the native wT1 and steatosis grades (r = 0.106, P = 0.435). In the multiple regression analysis, liver fibrosis, and hepatocellular ballooning were identified as independent factors influencing native wT1 in this study (R2 =0.545, P < 0.05), while steatosis was independently associated with conventional native T1 (R2 =0.321, P < 0.05). The AUC values for native T1, native wT1, HBP T1, and HBP wT1 were 0.549(0.410-0.682), 0.811(0.684-0.903), 0.775(0.644-0.876), and 0.752(0.619-0.858) for F1 or higher, 0.581(0.441-0.711), 0.828(0.704-0.916), 0.832(0.708-0.919), and 0.854(0.734-0.934) for F2 or higher, respectively., Conclusion: The native wT1 may provide a more reliable assessment of early liver fibrosis in the context of NAFLD compared to conventional native T1., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Fubi Hu reports financial support was provided by the Health Commission of Sichuan Province. Chao Zou reports financial support was provided by National Natural Science Foundation of China. Fubi Hu reports financial support was provided by Sichuan Medical Association. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.)

Background: Association of genetic factors with non-invasive tests (NITs) for MASLD has not been well established., Methods: Clinical and laboratory data, liver biopsy and/or liver stiffness measurement (LSM) by transient elastography were collected from MASLD patients seen in tertiary care hepatology practices. Minor allele frequency for genomic loci rs641738 (MBOAT7), rs58542926 (TM6SF2), rs738409 (PNPLA3), rs62305723 (HSD1713B) were evaluated for association with high ELF (≥11.3), high FIB-4 (≥3.25), high LSM (≥10 kPa), histologic fibrosis (stage 3/4 vs. stages 0-2)., Results: Among 2289 MASLD patients with available polymorphism and liver fibrosis/NIT data [52 ± 13 years, 46 % male, BMI 36.6 ± 9.9, 35 % type 2 diabetes (T2D)], 53 % had high-risk allele (C > G) at rs738409 (PNPLA3), 70 % high-risk allele (C > T) at rs641738 (MBOAT7), 18 % high-risk minor allele (C > T) at rs58542926 (TM6SF2), 11 % low-risk minor allele (G > A) at rs62305723 (HSD17b13). Only PNPLA3-rs738409 (47 % CC, 40 % CG, 13 % GG) was significantly associated with higher NIT scores and histologic fibrosis: high ELF 2.8 % CC vs. 8.1 % CG/GG; high FIB-4 4.7 % CC vs. 11.6 % CG/GG; high LSM 10 % vs. 19 %; advanced histologic fibrosis 34 % CC vs. 60 % CG/GG (all p < 0.01). Similar associations of PNPLA3-rs738409 with NITs were observed in a subgroup of MASLD patients with T2D (n = 799; all p < 0.05). The PNPLA3-rs738409 CG/GG genotype, older age and T2D were independently associated with high ELF [OR (95 % CI) = 3.25 (2.03-5.20)], FIB-4 [OR = 2.75 (1.90-3.98)], LSM [OR = 2.71 (1.60-4.59)] scores and advanced histologic fibrosis [OR = 2.56 (1.81-3.62)]., Conclusions: The polymorphism rs738409 in the PNPLA3 gene, T2D, and older age were independent predictors of high-risk MASLD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) is becoming a global public health burden, yet effective therapeutic strategies are notably lacking. NAFLD development may be mediated by mitochondrial dysfunction, according to new research. Producing mitochondrial regulators from plant-based substances to treat mitochondrial dysfunction is an appealing approach to treating NAFLD. Hesperetin (HES) is a flavonoid that is found naturally and is a member of the flavanone family. This study aims to clarify the mechanism of HES in preventing NAFLD which is caused by a high-fat diet (HFD). Serum and liver biochemical parameters, liver histology, lipid profiles, and mitochondrial function were evaluated in HFD-induced NAFLD Sprague-Dawley (SD) rats. HES treatment significantly reduced body weight gain, liver weight, and the liver index, while also improving hepatic steatosis, lipid metabolism disorders, and mitochondrial dysfunction in rats with NAFLD. The mechanism was investigated and confirmed using western blot and real-time quantitative polymerase chain reaction (RT-qPCR). We showed that in the liver of NAFLD rats, HES decreased the expression of dynamic-related protein 1 (Drp1), phosphorylated Drp1 at serine-616 (Drp1-pS616) and induced phosphorylated Drp1 at serine-637 (Drp1-pS637), PTEN-induced kinase 1 (PINK1), and E3 Ubiquitin-Protein Ligase Parkin (Parkin) via an AMP-activated protein kinase alpha (AMPKα)-dependent mechanism. Moreover, HES increased the expression of the mitochondrial fusion proteins mitofusin-2 (Mfn2) and optic atrophy 1 (Opa1) while suppressing the expression of fission protein 1 (Fis1). In this work, we identify a unique mechanism by which HES prevents NAFLD from developing. HES may be an attractive potential therapeutic agent to cure NAFLD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background/introduction: To evaluate the performance of pre-trained deep learning schemes (DLS) in hepatic steatosis (HS) grading of Non-Alcoholic Fatty Liver Disease (NAFLD) patients, using as input B-mode US images containing right kidney (RK) cortex and liver parenchyma (LP) areas indicated by an expert radiologist., Methods: A total of 112 consecutively enrolled, biopsy-validated NAFLD patients underwent a regular abdominal B-mode US examination. For each patient, a radiologist obtained a B-mode US image containing RK cortex and LP and marked a point between the RK and LP, around which a window was automatically cropped. The cropped image dataset was augmented using up-sampling, and the augmented and non-augmented datasets were sorted by HS grade. Each dataset was split into training (70%) and testing (30%), and fed separately as input to InceptionV3, MobileNetV2, ResNet50, DenseNet201, and NASNetMobile pre-trained DLS. A receiver operating characteristic (ROC) analysis of hepatorenal index (HRI) measurements by the radiologist from the same cropped images was used for comparison with the performance of the DLS., Results: With the test data, the DLS reached 89.15 %-93.75 % accuracy when comparing HS grades S0-S1 vs. S2-S3 and 79.69 %-91.21 % accuracy for S0 vs. S1 vs. S2 vs. S3 with augmentation, and 80.45-82.73 % accuracy when comparing S0-S1 vs. S2-S3 and 59.54 %-63.64 % accuracy for S0 vs. S1 vs. S2 vs. S3 without augmentation. The performance of radiologists' HRI measurement after ROC analysis was 82 %, 91.56 %, and 96.19 % for thresholds of S ≥ S1, S ≥ S2, and S = S3, respectively., Conclusion: All networks achieved high performance in HS assessment. DenseNet201 with the use of augmented data seems to be the most efficient supplementary tool for NAFLD diagnosis and grading., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Associazione Italiana di Fisica Medica e Sanitaria. Published by Elsevier Ltd. All rights reserved.)

Metabolic dysfunction-associated steatohepatitis (MASH) is a major cause of liver-related morbidity and mortality, yet treatment options are limited. Manual scoring of liver biopsies, currently the gold standard for clinical trial enrollment and endpoint assessment, suffers from high reader variability. This study represents the most comprehensive multisite analytical and clinical validation of an artificial intelligence (AI)-based pathology system, AI-based measurement of metabolic dysfunction-associated steatohepatitis (AIM-MASH), to assist pathologists in MASH trial histology scoring. AIM-MASH demonstrated high repeatability and reproducibility compared to manual scoring. AIM-MASH-assisted reads by expert MASH pathologists were superior to unassisted reads in accurately assessing inflammation, ballooning, MAS ≥ 4 with ≥1 in each score category and MASH resolution, while maintaining non-inferiority in steatosis and fibrosis assessment. These findings suggest that AIM-MASH could mitigate reader variability, providing a more reliable assessment of therapeutics in MASH clinical trials., Competing Interests: Competing interests: H.P., H.H., A.S.-M., R.E., N.P., A.H.B. and N.P.A. are full-time, salaried employees of PathAI. K.E.W. was a full-time employee of PathAI during all phases of the study and is now a paid consultant of PathAI. S.A.H. is a paid consultant for Akero Therapeutics, Aligos Therapeutics, Altimmune, Boehringer Ingelheim, Bluejay Therapeutics, Echosens North America, Galecto, Gilead Sciences, GlaxoSmithKline, Hepion Pharmaceuticals, Hepta Bio, HistoIndex, Kriya Therapeutics, Madrigal Pharmaceuticals, Medpace, MGGM Therapeutics, NeuroBo Pharmaceuticals, Northsea Therapeutics, Novo Nordisk, Pfizer, Sagimet Biosciences, Terns and Viking Therapeutics and a shareholder of Akero, Cirius Therapeutics, Galectin Therapeutics, HistoIndex and Northsea Therapeutics. S.S.M., M.C.V., L.C.M., S.P.M.C., S.H.M., C.E.T. and M.C.M. were PathAI employees at the time of study conduct. J.G. and M.R. are paid contractors of PathAI. R.P.M. and G.M.S. are full-time, salaried employees of OrsoBio. C.C. is a full-time, salaried employee of Inipharm. S.D.P. is a full-time salaried employee of Gilead Sciences. A.-S.S. is a full-time, salaried employee of Novo Nordisk. A.M. was a paid consultant for Bristol Myers Squibb. V.B. is a full-time, salaried employee of Bristol Myers Squibb. A.J.S. has stock options in Genfit, Akarna, Tiziana, Indalo, Durect Inversago and Galmed; is a consultant to AstraZeneca, Nimbus, Takeda, Janssen, Gilead, Terns, Merck, Boehringer Ingelheim, Bristol Myers Squibb, Lilly, Novartis, Novo Nordisk, Pfizer and Genfit; and has been an unpaid consultant to Intercept, Echosens, Immuron, Galectin and Affimune Prosciento. His institution has received grant support from Gilead, Bristol Myers Squibb, Intercept, Merck, AstraZeneca and Novartis. He receives royalties from Elsevier and UptoDate. Q.M.A. is a coordinator of the EU IMI-2 LITMUS consortium, which is funded by the EU Horizon 2020 program and the EFPIA. This multistakeholder consortium includes industry partners. He has research grant funding from AstraZeneca, Boehringer Ingelheim and Intercept. He is a consultant on behalf of Newcastle University to Alimentiv, Akero, AstraZeneca, 89bio, Boehringer Ingelheim, Bristol Myers Squibb, Galmed, Genfit, Genentech, Gilead, GlaxoSmithKline, HistoIndex, Intercept, Inventiva, QVIA, Janssen, Madrigal, Merck, NGM Bio, Novartis, Novo Nordisk, PathAI, Pfizer, PharmaNest, Prosciento, Roche and Terns. He is a speaker for Novo Nordisk, Madrigal and Springer Healthcare and receives royalties from Elsevier. R.L. is a consultant to Aardvark Therapeutics, Altimmune, Alnylam–Regeneron, Amgen, Arrowhead Pharmaceuticals, AstraZeneca, Bluejay Therapeutics, Bristol Myers Squibb, Eli Lilly, Galmed, Gilead, Inipharma, Intercept, Inventiva, Ionis, Janssen, Madrigal, NGM Biopharmaceuticals, Novartis, Novo Nordisk, Merck, Pfizer, Sagimet, Theratechnologies, 89bio, Terns Pharmaceuticals and Viking Therapeutics. He is a cofounder of LipoNexus. V.R. is a paid consultant for Novo Nordisk, Northsea Madrigal, Enyo, Poxel, Bristol Myers Squibb, Intercept, NGM Bio and Sagimet., (© 2024. The Author(s).)

Limb expression 1-like protein (LIX1L) is an essential player in liver disorders, but its function in metabolic dysfunction-associated steatohepatitis (MASH) and associated hepatocellular carcinoma (HCC) progression remains obscure. Here, we identify LIX1L as a key integrative regulator linking lipid metabolism and inflammation, adipose tissue and hepatic microenvironment, which promotes MASH progression. LIX1L significantly upregulates in MASH patients, mouse models, and palmitic acid-stimulated hepatocytes. Lix1l deletion inhibits hepatic lipid accumulation, inflammation, and fibrosis as well as adipocyte differentiation by downregulating CD36, alleviating MASH and associated HCC progression in mice. Mechanistically, metabolic stress promotes PARP1-mediated poly-ADP-ribosylation of LIX1L to increase stability and RNA binding ability of LIX1L. Subsequently, LIX1L binds to AU-rich element in the 3'UTR and CDS of CD36 mRNA, thus mitigating CD36 mRNA decay. Furthermore, LIX1L deficiency-mediated downregulation of CD36 reprograms the tumor-prone liver microenvironment with increased cytotoxic T lymphocytes and reduced immunosuppressive cell proportions. These data indicate a systematic function of LIX1L in the pathogenesis of MASH and underscore targeting PARP1/LIX1L/CD36 axis as a feasible strategy for treatment of MASH and associated HCC., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mitochondrial dynamics plays a crucial role in the occurrence and development of non-alcoholic fatty liver diseases (NAFLD). SENP1, a SUMO-specific protease, catalyzes protein de-SUMOylation and involves in various physiological and pathological processes. However, the exact role of SENP1 in NAFLD remains unclear. Therefore, we investigated the regulatory role of SENP1 in mitochondrial dynamics during the progression of NAFLD. In the study, the NAFLD in vivo model induced by high fat diet (HFD) and in vitro model induced by free fatty acids (FFA) were established to investigate the role and underlying mechanism of SENP1 through detecting mitochondrial morphology and dynamics. Our results showed that the down-regulation of SENP1 expression and the mitochondrial dynamics dysregulation occurred in the NAFLD, evidenced as mitochondrial fragmentation, up-regulation of p-Drp1 ser616 and down-regulation of MFN2, OPA1. However, over-expression of SENP1 significantly alleviated the NAFLD, rectified the mitochondrial dynamics disorder, reduced Cyt-c release and ROS levels induced by FFA or HFD; moreover, the over-expression of SENP1 also reduced the SUMOylation levels of Drp1 and prevented the Drp1 translocation to mitochondria. Our findings suggest that the possible mechanisms of SENP1 were through rectifying the mitochondrial dynamics disorder, reducing Cyt-c release and ROS-mediated oxidative stress. The findings would provide a novel target for the prevention and treatment of NALFD., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent chronic liver disease associated with type 2 diabetes, which doubles the risk of developing this condition. Various flavonoid compounds have a positive effect on lipid metabolism, inflammation, and insulin resistance and can contribute to slowing down the progression of MASLD. In the current study, we investigated the biological effects of Licochalcone D (Lico D), a flavonoid, in a metabolic disease model. Oil Red O staining, quantitative real-time polymerase chain reaction, and western blotting were performed. For in vivo experiments, we used high-fat diet (60 %) plus low-dose streptozotocin (30 mg/kg; 5 days; intraperitoneal)-induced metabolic disease mouse model and evaluated lipid metabolism. We observed that Lico D reduced lipid accumulation and increased lipid metabolism both in vitro and in vivo. Additionally, we identified that the AMP-activated protein kinase and Sirtuin 1 pathways were activated in the mouse liver and hepatic steatosis cell model. In silico analysis revealed that Lico D passes the "Lipinski Rule of Five," which indicates drug-likeness properties. In conclusion, our findings highlight the role of Lico D in improving metabolic dysfunction-associated steatotic liver disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background and Aims: Blood-based biomarkers have been proposed as an alternative to liver biopsy for noninvasive liver disease assessment in chronic liver disease. Our aims for this systematic review were to evaluate the diagnostic utility of selected blood-based tests either alone, or in combination, for identifying significant fibrosis (F2-4), advanced fibrosis (F3-4), and cirrhosis (F4), as compared to biopsy in chronic liver disease., Approach and Results: We included a comprehensive search of databases including Ovid MEDLINE(R), EMBASE, Cochrane Database, and Scopus through to April 2022. Two independent reviewers selected 286 studies with 103,162 patients. The most frequently identified studies included the simple aspartate aminotransferase-to-platelet ratio index and fibrosis (FIB)-4 markers (with low-to-moderate risk of bias) in HBV and HCV, HIV-HCV/HBV coinfection, and NAFLD. Positive (LR+) and negative (LR-) likelihood ratios across direct and indirect biomarker tests for HCV and HBV for F2-4, F3-4, or F4 were 1.66-6.25 and 0.23-0.80, 1.89-5.24 and 0.12-0.64, and 1.32-7.15 and 0.15-0.86, respectively; LR+ and LR- for NAFLD F2-4, F3-4, and F4 were 2.65-3.37 and 0.37-0.39, 2.25-6.76 and 0.07-0.87, and 3.90 and 0.15, respectively. Overall, the proportional odds ratio indicated FIB-4 <1.45 was better than aspartate aminotransferase-to-platelet ratio index <0.5 for F2-4. FIB-4 >3.25 was also better than aspartate aminotransferase-to-platelet ratio index >1.5 for F3-4 and F4. There was limited data for combined tests., Conclusions: Blood-based biomarkers are associated with small-to-moderate change in pretest probability for diagnosing F2-4, F3-4, and F4 in viral hepatitis, HIV-HCV coinfection, and NAFLD, with limited comparative or combination studies for other chronic liver diseases., (Copyright © 2024 American Association for the Study of Liver Diseases.)

The incidence of nonalcoholic fatty liver disease (NAFLD) has been steadily increasing in Western society in recent years and has been recognized as a risk factor for the development of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying the progression from NAFLD to HCC are still unclear, despite the use of suitable mouse models. To identify the transcriptional and lipid profiles of livers from mice with NAFLD-HCC, we induced both NAFLD and NAFLD-HCC pathologies in C57BL/6J mice and performed RNA-sequencing (RNA-seq) and targeted lipidomic analysis. Our RNA-seq analysis revealed that the transcriptional signature of NAFLD in mice is characterized by changes in inflammatory response and fatty acid metabolism. Moreover, the signature of NAFLD-HCC is characterized by processes typically observed in cancer, such as epithelial to mesenchymal transition, angiogenesis and inflammatory responses. Furthermore, we found that the diet used in this study inhibited cholesterol synthesis in both models. The analysis of lipid composition also showed a significant impact of the provided diet. Therefore, our study supports the idea that a Western diet (WD) affects metabolic processes and hepatic lipid composition. Additionally, the combination of a WD with the administration of a carcinogen drives the progression from NAFLD to HCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Nonalcoholic fatty liver disease (NAFLD) comprises multiple heterogeneous pathophysiological conditions commonly evaluated by suboptimal liver biopsies. This study aimed to elucidate the role of 13 diverse histological liver scores in assessing NAFLD disease activity using an in silico pharmacometric model-based approach. We further sought to investigate various noninvasive patient characteristics for their ability to reflect all 13 histological scores and the NAFLD activity score (NAS)., Methods: A histological liver score model was built upon 13 biopsy-based pathological features (binary and categorical scores) from the extensive NASH-CRN (Nonalcoholic Steatohepatitis-Clinical Research Network) observational NAFLD Database study (n = 914 adults) using the concept of item response theory. The impact of 69 noninvasive biomarkers potentially reflecting NAFLD activity was quantitatively described across the entire spectrum of all 13 histological scores., Results: The model suggested that four different disease facets underlie the cardinal NAFLD features (steatosis, inflammation, hepatocellular ballooning (= NAS); fibrosis; highest correlations: corr ballooning-fibrosis  = 0.69/corr inflammation-ballooning  = 0.62/corr steatosis-inflammation  = 0.60). The 13 histological liver scores were best described by contrasting noninvasive biomarkers: Age and platelets best reflected the fibrosis score, while alanine and aspartate aminotransferase best described the NAS, with diverging contributions of the three individual NAS components to the results of the overall NAS., Conclusions: An in silico histological liver score model allowed to simultaneously quantitatively analyze 13 features beyond NAS and fibrosis, characterizing different disease facets underlying NAFLD and revealing the contrasting ability of 69 noninvasive biomarkers to reflect the diverse histological (sub-)scores., Competing Interests: Declarations. Conflicts of Interest: The authors declare that they have no conflicts of interest that are relevant to the content of this article., (© 2024. The Author(s).)