Non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) are the leading causes of liver disease worldwide. To identify disease-specific pathomechanisms, we analyzed the lipidome, metabolome and immune cell recruitment in livers in both diseases. Mice harboring ASH or NASH had comparable disease severities regarding mortality rate, neurological behavior, expression of fibrosis marker and albumin levels. Lipid droplet size was higher in NASH than ASH and qualitative differences in the lipidome were mainly based on incorporation of diet-specific fatty acids into triglycerides, phosphatidylcholines and lysophosphatidylcholines. Metabolomic analysis showed downregulated nucleoside levels in both models. Here, the corresponding uremic metabolites were only upregulated in NASH suggesting stronger cellular senescence, which was supported by lower antioxidant levels in NASH as compared to ASH. While altered urea cycle metabolites suggest increased nitric oxide synthesis in both models, in ASH, this depended on increased L-homoarginine levels indicating a cardiovascular response mechanism. Interestingly, only in NASH were the levels of tryptophan and its anti-inflammatory metabolite kynurenine upregulated. Fittingly, high-content immunohistochemistry showed a decreased macrophage recruitment and an increased polarization towards M2-like macrophages in NASH. In conclusion, with comparable disease severity in both models, higher lipid storage, oxidative stress and tryptophan/kynurenine levels were seen in NASH, leading to distinct immune responses.

This study demonstrates the effectiveness of propidium iodide as a reliable marker for detecting dead or dying cells in frozen liver tissue sections. By comparing propidium iodide staining with the widely used Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, both methods showed consistent results in disease models such as alcohol-induced fibrosis and Western diet-induced fatty liver. Additionally, propidium iodide was successfully co-stained with other fluorescent markers, like phalloidin (for actin filaments) and antibodies targeting collagen, enabling detailed spatial analysis of dying cells within tissue. This multiplex approach allows for a deeper understanding of tissue organization and cell death localization, particularly in complex conditions like liver fibrosis. Moreover, our results suggest that propidium iodide staining can be applied beyond current models, offering a more accessible and cost-effective alternative to traditional methods, like TUNEL. Furthermore, its integration with other markers enables simultaneous analysis of immune responses and tissue damage, making it a powerful tool for future studies on liver disease and other inflammatory conditions. This technique has the potential to advance research into disease mechanisms and improve the evaluation of novel therapeutic strategies targeting tissue regeneration and inflammation control.

17-β-Hydroxysteroid dehydrogenase 13 (HSD17B13) is a lipid droplet protein primarily expressed in the liver hepatocytes. HSD17B13 is associated with the clinical outcome of chronic liver diseases and is therefore a target for the development of drugs. Here, we demonstrate the promising therapeutic effect of BI-3231 as a potent inhibitor of HSD17B13 based on its ability to inhibit triglyceride accumulation in lipid droplets (LDs), restore lipid metabolism and homeostasis, and increase mitochondrial activity in vitro.

Background: The Rho-kinase ROCK II plays a major role in the activation of hepatic stellate cells (HSC), which are the key profibrotic and contractile cells contributing to the development of chronic liver disease. Inhibition of ROCK II ultimately blocks the phosphorylation of the myosin light chain (MLC) and thus inhibits stress fibre assembly and cell contraction. We investigated the effects of the ROCK inhibitors Y-33075 as well as Y-27632 in murine and human hepatic stellate cells.Methods: Primary isolated HSC from FVB/NJ mice and the immortalized human HSC line TWNT-4 were culture-activated and incubated with Y-27632 and Y-33075 (10nM to 10μM) for 24h. Protein expression levels were analyzed by Western Blots and transcriptional levels of pro-fibrotic markers and proliferative markers were evaluated using real-time qPCR. Migration was investigated by wound-healing assay. Proliferation was assessed by BrdU assay. Contraction of HSC was measured using 3D collagen matrices after incubation with Y-27632 or Y-33075 in different doses.Results: Both Rho-kinase inhibitors, Y-27632 and Y-33075, reduced contraction, fibrogenesis and proliferation in activated primary mouse HSC (FVB/NJ) and human HSC line (TWNT-4) significantly. Y-33075 demonstrated a 10-times increased potency compared to Y-27632. Surprisingly, both inhibitors mediated a substantial and unexpected increase in migration of HSC in FVB/NJ.Conclusion: ROCK inhibition by the tested compounds decreased contraction but increased migration. Y-33075 proved more potent than Y27632 in the inhibition of contraction of HSCs and should be further evaluated in chronic liver disease.

Phospholipids are essential for RNA virus replication, yet their role in modulating conformational dynamics of membrane-associated viral proteins remains poorly understood. For NS5A, a key replication factor of hepatitis C virus, previous crystallographic models fail to capture the lipid-driven conformational mechanics we uncover here. Using structural informatics and biochemical probing of pharmacophore-guided mutants in defined lipid environments, we evaluated competing NS5A domain 1 dimerization models. Our data reveal an alternative membrane-specific fold stabilized by polyproline hinges and phospholipids (PIPs) such as phosphatidylinositol-4-phosphate, a host lipid enriched at replication membranes. PIP binding promotes a conformational switch that drives dimerization, linking lipid sensing to membrane remodeling and host factor recruitment. This reciprocal mechanism—where a lipid allosterically modulates a viral protein that reshapes membranes—is blocked by the antiviral pibrentasvir. These findings define a lipid-driven structural switch that governs NS5A pleiotropy and highlight dynamic lipid-protein interfaces as targets for antiviral intervention.

Having identified LC3-interacting region (LIR) motifs 1/2 of A20 binding inhibitor of NF-κB-1 (ABIN-1) as mitophagy regulators in vitro, this study generated CRISPR-engineered ABIN1-ΔLIR1/2 mice lacking both LIR motifs to elucidate its in vivo significance. These mice exhibit enhanced hepatic lipid droplet accumulation, dysregulated mitochondrial metabolism, and impaired mitophagy, through modulation of the AMPK-TFEB axis. Liver biopsies from patients with metabolic dysfunction-associated steatotic liver disease corroborate these findings, suggesting therapeutic implications for liver diseases.