Among these mechanisms, ferroptosis has gained increasing attention as a distinct, non-apoptotic form of regulated cell death driven by iron-dependent lipid peroxidation and the accumulation of lipid-derived reactive oxygen species. In the progression of MASLD/MASH, multiple pathophysiological pathways establish a hepatic microenvironment that predisposes hepatocytes to ferroptosis, which is in turn accompanied by profound alterations in the intracellular microenvironment [4]. Specifically, these environmental changes arises from disruption of hepatic iron homeostasis and Acyl-CoA synthetase long-chain family member 4 (ACSL4)-mediated lipid remodeling increase peroxidationprone substrates, while dysfunction of the cystine-glutathione-glutathione peroxidase 4 (GPx4) axis compromises the cellular capacity to detoxify lipid hydroperoxides [5-7]. As a consequence, progressive lipid peroxidation reduces membrane structural fluidity and promotes the accumulation of oxidized lipid species within cellular and organelle membranes. These changes are associated with perturbations in fundamental physicochemical parameters, including viscosity, polarity, and pH, which critically regulate diffusion-dependent reactions, redox signaling, and organelle function [8]. Among these parameters, viscosity plays a particularly pivotal role by modulating intracellular reaction kinetics, thereby influencing signal transduction, metabolite transport, and biomolecular interactions. Emerging evidence suggests that dysregulated intracellular represents a characteristic feature of ferroptosis-associated pathological states in the context of MASLD/MASH progression.

These insights highlight the potential value of viscosity-responsive fluorescent probes as tools for monitoring ferroptosis-associated perturbations within the intracellular microenvironment. While existing probes have enabled the detection of lipid peroxidation within specific organelles, single-organelle-targeted approaches provide only limited insight into the interorganelle crosstalk that underlies ferroptotic signaling. Given that ferroptosis arises from tightly interconnected metabolic and redox processes spanning multiple organelles, there is a critical need for highly sensitive fluorescent probes capable of capturing its spatiotemporal dynamics across interconnected organelle networks. Such tools would enable sensitive and early detection of ferroptosis in chronic liver diseases, with substantial implications for early diagnosis and prevention of progression to irreversible liver fibrosis.

In this context, a recent study by Pham et al. [8] reported the development of a highly sensitive near-infrared (NIR) fluorescent probe designed to monitor lipid peroxidation–mediated viscosity changes during ferroptosis. The probe was constructed using a donor-π bridge-acceptor framework, incorporating 1H-indene-1,3(2H)-dione or its malononitrile-substituted derivatives as acceptor units and dimethylamino-or methoxy-phenyl groups as donor moieties. Extension of π-conjugation through the insertion of an additional double bond between the thiophene and phenyl rings induced a bathochromic shift in absorption. In this study, the authors developed TTM-4 as a highly sensitive NIR fluorescent probe for the detection of ferroptosis. TTM- 4 exhibited particularly favorable optical properties, including an absorption maximum near 570 nm and broad NIR emission spanning 670–840 nm in various solvents. Notably, the emission intensity of TTM-4 increased by approximately 508-fold in high-viscosity environments, and the probe displayed the longest emission wavelength, strongest fluorescence intensity, and extended fluorescence lifetime in glycerol. Whereas BODIPY 581/591 C11 primarily reflects neutral lipid oxidation, TTM-4 detects lipid peroxidation-associated microenvironmental changes within lipid droplets, requires lower probe concentrations, and exhibits minimal cytotoxicity, making it more sensitive and better suited for in vivo imaging applications. Organelle colocalization studies further demonstrated that TTM-4 fluorescence was predominantly localized to lipid droplets and the endoplasmic reticulum, with minimal signal detected in mitochondria, lysosomes, or the Golgi apparatus. This selective labeling suggests that TTM-4 preferentially detects oxidized lipid droplets and endoplasmic reticulum (ER)-associated lipid peroxidation microenvironments, supporting its utility for capturing early ferroptotic events. Consistently, TTM-4 fluorescence was markedly enhanced in hepatocytes exposed to oxidative stress, whereas lipid peroxidation inhibitors significantly attenuated the signal.

Furthermore, this study systematically investigated ferroptosis-associated lipid peroxidation at the cellular, animal, and human liver tissue levels using the NIR fluorescent probe TTM-4. In vitro analyses in HepG2, AML12, and mouse primary hepatocytes revealed that pharmacological inhibition of lysosomal acid lipase markedly reduced TTM-4 signaling in the ER surrounding cytosolic lipid droplets and decreased oxidized nuclear lipid droplets, while cytoplasmic lipid droplet oxidation remained unchanged. These findings indicate that the formation of nuclear lipid droplet during ferroptosis was not a passive consequence of lipid accumulation but rather depends on lipid droplet degradation and ER-mediated lipid trafficking. To extend these observations to a metabolic disease context, HepG2 cells were treated with palmitic acid to induce MASLD-relevant lipotoxic stress. This treatment led to a pronounced increase in oxidized lipid droplets and nuclear lipid droplets, accompanied by enhanced TTM-4 fluorescence. Consistently, treatment of AML12 cells with the ferroptosis inducer (RAS-selective lethal 3) significantly elevated TTM-4 signals, which were effectively suppressed by the iron chelator deferoxamine, thereby confirming the ferroptosis specificity and high sensitivity of TTM-4. The applicability of TTM-4 was further validated in vivo using a choline-deficient, amino acid-defined, high-fat diet mouse model. Notably, TTM-4 identified ferroptotic signals prior to detection by conventional probes, highlighting its enhanced sensitivity in liver tissue and its capacity to capture the spatial redistribution of oxidized lipids during the progression of ferroptosis, particularly at chronic stages. Consistent with in vitro and in vivo results, TTM-4 signals in human MASLD liver samples were also predominantly localized to α-smooth muscle actin-positive fibrotic regions, suggesting a spatial association between ferroptotic lipid peroxidation and fibrotic remodeling. Finally, integrative analysis of MASLD patient cohorts and public transcriptomic datasets revealed a progressive increase in cell death-inducing DFFA-like effector C (CIDEC) expression with advancing disease severity, which showed a strong positive correlation with both steatosis and fibrosis severity. CIDEC is known to promote lipid droplet fusion and enlargement, providing a biological basis for its association with the spatial redistribution of lipid peroxidation. Notably, CIDEC expression spatially co-localized with TTM-4 signals in liver tissue and cultured hepatocytes, supporting a potential role for CIDEC in the formation of oxidized lipid droplets and the amplification of ferroptotic stress during MASLD progression. Collectively, these findings support a model in which CIDEC upregulation facilitates oxidized lipid droplet formation, thereby exacerbating ferroptotic stress and contributing to disease progression.

In summary, this study advances a revised framework for MASLD/MASH progression in which ferroptosis is positioned as a self-reinforcing and microenvironment-altering pathogenic mechanism, rather than a passive consequence of lipotoxic injury. Through the integration of iron dysregulation, lipid remodeling, and impaired antioxidant capacity with viscosity-mediated intracellular remodeling, ferroptosis emerges as a dynamic phenotypic process operating across interconnected organelle networks. The viscosity-responsive NIR fluorescent probe TTM-4 enables early and spatially resolved detection of ferroptotic lipid peroxidation, while the identification of CIDEC is associated with lipid droplet remodeling to amplified ferroptotic stress. Collectively, these findings position ferroptosis-driven microenvironmental remodeling as a central pathogenic axis and establish TTM-4 as a promising translational tool for chronic liver diseases.

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