{"paper_id":"321e336b-b7cb-47cb-95ce-dcb655d014ec","body_text":"Estrogen-dependent TRX2 activation reverts oxidative stress and metabolic dysfunction associated to steatotic disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Estrogen-dependent TRX2 activation reverts oxidative stress and metabolic dysfunction associated to steatotic disease Andrea Morandi, Alfredo Smiriglia, Nicla Lorito, Marina Bacci, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4259782/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jan, 2025 Read the published version in Cell Death & Disease → Version 1 posted 9 You are reading this latest preprint version Abstract Metabolic disfunction-associated steatotic liver disease (MASLD) encompasses a plethora of hepatic disorders ranging from steatosis to steatohepatitis with the worst clinical outcome represented by cirrhosis, liver failure, and hepatocellular carcinoma. According to the lower MASLD prevalence reported in pre-menopausal women compared to men, we identified a potential protective role of estrogens in counteracting the oxidative stress during disease induction and progression. We have used preclinical relevant in vitro models [i.e., immortalized cells and hepatocyte-like cells (HLC) derived from human embryonic stem cells (hESC)], exposed to sodium lactate, sodium pyruvate, and octanoic acid (LPO) to induce hepatic steatosis. This established practice of MASLD induction resulted in lipid droplet (LD) accumulation and increased mitochondrial and cytosolic reactive oxygen species (ROS) levels, paralleled by the reduction of several markers of hepatocyte function and differentiation. Here we found that estrogen replacement reduced ROS levels and LD content through the upregulation of mitochondrial thioredoxin 2 (TRX2), an antioxidant system that is under the control of the estrogen receptor alpha (hereafter referred as ER). Last, disrupting the TRX2 system using auranofin was sufficient to revert the scavenging effects exerted by estrogens, thus identifying a potential mechanism that could prevent or delay the progression of the disease. Biological sciences/Cell biology Biological sciences/Molecular biology Health sciences/Diseases/Metabolic disorders Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The metabolic dysfunction-associated steatotic liver disease (MASLD), formerly called non-alcoholic fatty liver disease (NAFLD), encompasses a wide range of pathological comorbidities and functional alterations of the liver associated with at least one cardiometabolic criteria ( 1 ). Hepatic alterations include steatosis, chronic inflammation (metabolic disfunction-associated steatohepatitis, MASH), fibrosis and cirrhosis. Furthermore, cirrhosis is a well-known risk factor for the neoplastic transformation of hepatocytes (hepatocellular carcinoma, HCC). While hepatic steatosis is defined by at least 5% of lipid/triglyceride accumulation in the liver and is potentially reversible, prolonged and severe MASH is considered a progressive disease ( 2 ). Several molecular pathways can contribute to the onset of MASLD and be responsible for the phenotypic traits displayed by hepatic cells undergoing steatosis ( 3 ), which is typically characterized by an overload and intracellular accumulation of triglycerides not related to alcohol consumption, accompanied by inflammation and oxidative stress, essential players involved in the promotion of steatohepatitis ( 4 ). Global MASLD prevalence is around 25% with an appreciably lower incidence in pre-menopausal women compared to men and post-menopausal women ( 5 ), although the female population have a greater tendency towards obesity, one of the main risk factors ( 6 ). This suggests a potential protective effect of estrogens in counteracting MASLD initiation and progression. Accumulating evidence highlights the benefits of estrogen replacement therapy in mitigating lipid accumulation and peroxidation ( 7 ), reducing lipotoxicity-induced oxidative stress in hepatic mitochondria, and ameliorating the inflammation process ( 8 – 10 ). In ovariectomized mice, it has been demonstrated that supplementation with 17β-estradiol (E2) ( 11 ), the predominant form among circulating female hormones, inhibits steatogenesis ( 12 ). However, the precise molecular mechanisms underlying the protective effects of estrogens in the onset and progression of MASLD remain poorly understood. It has been reported that estrogens primarily prevent oxidative damage via the estrogen receptor alpha (hereafter referred to as ER) which, in turn, promotes the expression and activation of a plethora of antioxidant enzymes. Importantly, nuclear factor erythroid 2-related factor 2 (NRF2) and thioredoxin 2 (TRX2) are ER-dependent genes that could regulate the cellular defense against reactive oxygen species (ROS). TRX2 directly neutralizes ROS, whereas NRF2 initiates a transcriptional regulation of scavenging mechanisms under its control ( 13 , 14 ). These actions lead to anti-steatotic effects, which alleviate the inflammation induced by MASLD ( 15 , 16 ). Here, we investigated the protective role of estrogen in counteracting ROS production through activation of the antioxidant mitochondrial TRX2 pathway, in an innovative in vitro hepatic steatosis model based on the use of immortalized and hepatocyte-like cells (HLC). Materials and methods Cell lines and general culture conditions Male and female human embryonic stem cell (hESC) lines, WA01 and WA09 respectively, were purchased from WiCell Research Institute (504 S Rosa Rd #101, Madison, WI 53719, USA) and maintained at 37°C/5%CO 2 on 5 µg/mL laminin 521 (LMN-521, STEMCELL Technologies #LN521-05) pre-coated plates with mTeSR1 PLUS serum-free medium (STEMCELL Technologies #100–0276). AML12 and HepG2 cell lines were purchased from American Type Culture Collection (ATCC): AML12 cells are hepatocytes isolated from the normal liver of a male mouse and grow in DMEM:F12 (1:1, ThermoFisher #11330032) supplemented with 10% fetal bovine serum (FBS), 10 µg/ml insulin (I), 5.5 µg/ml transferrin (T), 5 ng/ml selenium (S) (ITS, ThermoFisher #41400045), and 40 ng/ml dexamethasone (ThermoFisher #A13449); HepG2 is a human hepatoblastoma cell line of a 15-year-old male maintained in DMEM high glucose medium (ThermoFisher #11965092) supplemented with 10% FBS, 2 mM glutamax (ThermoFisher #35050061), and 1% penicillin/streptomycin (ThermoFisher #15140). Differentiation from hESC to HLC At the time of differentiation into HLC, hESC were plated as a single cell at a concentration of 400,000 cells per well onto the LMN-521 coating and 10 µM of Rho-associated kinase inhibitor Y27632 (ROCKi, Stem Cell Technologies #72302) were added to the medium. This inhibitor was used to improve the adhesion and survival of stem cells in single-cell suspensions. After 24 hours, when hESC reached 40% a confluence, we initiated the differentiation process in HLC as accurately detailed in ( 17 ). Briefly, WA01 and WA09 cells were initially differentiated in endodermal cells by culturing them in RPMI 1640 medium (Gibco #11875-093) containing B-27 Supplement, (Gibco #12587-010), 100 ng/mL Activin A (PeproTech #120-14E), and 50 ng/mL Wnt3A (Peprotech #315-20-10uG). Subsequently, the hepatoblast phenotype was obtained by differentiating endodermal cells in Knockout DMEM (Gibco, #10829), containing 20% Knockout Serum Replacement (Gibco #10828), 1% non-essential amino acids (NEAA, Gibco #11140), 0.1 mM 2-mercaptoethanol (Gibco #31350). Finally, hepatoblast cells were differentiated into HLC by culturing them, until day 17, in HepatoZYME medium (Gibco, #17705) containing 10 ng/mL hepatocyte growth factor (Peprotech, #100 − 39), 20 ng/mL oncostatin M (Peprotech, #300 − 10), and 10 µM hydrocortisone 21-hemisuccinate (Sigma Aldrich, #H4881). At day 18, HLC were used for the assays described in this manuscript. Induction of steatotic phenotype Hepatocyte steatosis was induced by administrating a mix of 10 mM sodium-L-lactate (L, Sigma-Aldrich #7022), 1 mM sodium pyruvate (P, Sigma-Aldrich P2256), and 2 mM octanoic acid (O, Sigma-Aldrich C2875) for 48 hours to HLC, AML12, and HepG2 cells. Western blotting analysis Cell lines were washed with PBS and lysed in ice using Laemmli Sample Buffer 1X (Biorad #161–0737) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich #P8340 and #P0044, respectively), and protein concentrations were measured by BCA (Sigma-Aldrich #1003290033) method. 30–35 µg of cell lysate were loaded in precast SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) gels (Biorad #456–8096) and then transferred onto nitrocellulose membrane by Trans-Blot Turbo Transfer Pack (Biorad #170–4157). The immunoblots were incubated in non-fat dry milk 2%, tween-20 0.05% in PBS at room temperature for 1 hour, and then probed with primary and appropriate secondary antibodies. The following antibodies were used: HNF4α (Cell Signaling Technology #311F12), Albumin (Santa Cruz Biotechnology #sc-271605-F10), E-cadherin (Cell Signaling Technology #3195), TRX2 (Santa Cruz Biotechnology #sc-133201-F10), ER (Abcam, # ab16660), ACTB (Cell Signaling Technology #4970), Hsp90 (Santa Cruz Biotechnology #sc-69703), and Histone H3 (Cell Signaling Technology #4499). RNA extraction and Quantitative Real-time PCR (qRT-PCR) analysis Total RNA was extracted using RNeasy plus kit (QIAGEN #74134), quantified using Nanodrop 1000 (Thermo Fisher Scientific), and 500 ng were reverse transcribed using the iScript gDNA Clear cDNA Synthesis Kit (Biorad #172–5035). qRT-PCR was performed using the CFX96 Touch Real-Time PCR Detection System (Biorad) using TaqMan Universal PCR Master Mix (Thermo Fisher Scientific #4305719). The probes used in the work are: Albumin (Hs00609411_m1), HNF4α (Hs00230853_m1), TRX2 (Hs00429399_g1), PLIN2 (Hs00605340_m1 and Mm00475794_m1), GDF15 (Hs00171132_m1), SLC34A2 (Hs00197519_m1), NRF2 (Hs00975961_g1), NQO1 (Hs01045993_g1). Data were normalized on TBP (Hs00427620_m1) and/or ACTB (Mm02619580_g1). The relative quantity was determined using ∆∆ Ct by the CFX Maestro software (BioRad). Confocal image acquisition and analysis Cells were plated at a concentration of 10,000 cells per well in a Nunc™Lab-Tek™Chamber Slide System coverglass (Thermo Fisher Scientific #155383). Once steatosis has been induced, cells were stained at 37°C for (i) 15 minutes with BODIPY 493/503 (Thermo Fisher Scientific #D3922) to reveal LD content, (ii) 30 minutes with MitoSOX (Thermo Fisher Scientific #M36008) and CellRox (Thermo Fisher Scientific #C10444) probes to reveal mitochondrial and cytoplasmatic ROS respectively. When the incubation with the probes was completed, cells were fixed using a 4% formaldehyde solution for 10 minutes. Subsequently, the formaldehyde was removed, and three washes were performed with PBS. To stain the nuclei, cells were then incubated for 10 minutes with the DAPI dye (excitation, ex: 358 nm; emission, em: 461 nm) diluted 1:1000 in PBS and subsequently washed an additional 3 times in PBS. Finally, images were acquired using a TCS SP8 microscope (Leica Microsystems) with LAS-AF image acquisition software. The quantification of intracellular lipids was performed using CellProfiler software from three representative 63x images from 3 independent experiments. Flow cytometry analysis AML12 cells (7x10 4 cells/well) were seeded into 12-well plates. After LPO administration, cells were stained at 37°C for 15 minutes with BODIPY 493/503 to reveal intracellular lipid content. Live cells resuspended in PBS with 0.1% FBS were subjected to flow cytometry analysis using a FACS Canto II (BD Biosciences). ROS analysis Cell lines (7x10 4 cells/well) were seeded into 12-well plates. Once steatosis was induced, the cells were incubated with the CellRox probe in the dark for 30 minutes. Then, cells were lysed with RIPA buffer and fluorescence was measured on a Synergy H1 Hybrid Multi-Mode Reader (Biotek) plate reader (ex: 485 nm; em:520 nm). In silico analysis for TRX2 expression Transcriptomic data are available at the National Center for Biotechnology Information GEO repository (accession number GSE163211). The GSE163211 dataset was analyzed with GEO2R and R-program. The data used represent the expression values of TRX2. The statistical test performed is the Wilcox test. Statistical significance was defined as p < 0.05. Statistical analysis All experiments were conducted at least 3 times independently. Statistical analysis of the data was performed using GraphPad Prism Software 10. Unless stated otherwise, comparisons between the 2 groups were made using the two-tailed, unpaired Student’s t-test. Comparisons between multiple groups were made using one-way or two-way analysis of variance (ANOVA). Bonferroni and Dunnett's post-testing analysis with a confidence interval of 95% was used for individual comparisons as reported in figure legends. Statistical significance was defined as: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 ; when differences were not statistically significant, or the comparison was not biologically relevant, no indication was reported in the figures. Results Phenotypic and functional characterization of HLC derived from hESC Male WA01 and female WA09 hESC were differentiated into HLC using a previously published 18-day protocol ( 17 ). The differentiation of hESC into HLC was confirmed by the classic acquisition of the cobblestone-like hepatic morphology (Fig. 1 a), in parallel to the expression of established hepatic markers, such as increased mRNA albumin expression and hepatocyte nuclear factor-4-alpha (HNF4α), whose mRNA and protein expression levels were enhanced in HLC when compared to hESC cells (Fig. 1 b-c). Moreover, we also observed a higher protein level of a crucial molecule responsible for the assembly and functionality of adherent junctions (i.e., E-Cadherin, E-Cad, Fig. 1 c), a characteristic feature of differentiated hepatocytes. Thus, using sex-matched hESC differentiated into HLC we reproduced a relevant hepatocyte model, useful for studying the onset and progression of MASLD and the subsequent impact of estrogen administration. Lactate, pyruvate, and octanoic acid-induced steatosis in HLC and immortalized cells To extensively mimic an in vitro model of steatosis, in addition to WA01 and WA09 hESC-derived HLC, we used two immortalized liver cell lines that have been widely employed to study MASLD initiation and progression: AML12, hepatocytes isolated from a normal murine liver ( 18 ), and HepG2, a hepatoblastoma cell line, the most commonly employed experimental model for in vitro liver cancer research ( 19 ). The four cell models were treated with a cocktail of compounds composed of sodium L-lactate (L), sodium pyruvate (P), and octanoic acid (O), hereafter referred to as LPO, for 48 hours to induce steatosis (Fig. 2 a) ( 20 ). Following LPO treatment, both hESC-derived HLC models significantly reduced the mRNA (Fig. 2 b) and/or protein (Fig. 2 c) expression levels of Albumin, HNF4α and E-Cad, a sign of hepatocyte dysfunction. Steatosis is characterized by the intracellular accumulation of lipids within the cytoplasm of the hepatocyte, in the form of lipid droplets (LD), that are dynamic and multifunctional organelles involved in energy metabolism, signaling, and inflammatory mediator production ( 21 ). To determine whether LPO could also induce LD accumulation, we performed confocal and FACS analyses using the fluorescent neutral lipid dye BODIPY 493/503 which is retained in LD. Confocal analysis and subsequent quantification showed that LPO-treated WA01 and WA09 cells are significantly enriched in LD content (Fig. 2 d). Similarly, confocal and/or FACS analyses confirmed that LPO treatment significantly increased LD accumulation also in AML12 and HepG2 cell lines (Fig. 2 e). In parallel, all the LPO-exposed cell models displayed significantly higher levels of PLIN2 (Fig. 2 f), a member of the perilipin protein family which we demonstrated to be associated with LD accumulation ( 22 ). LPO treatment increased oxidative stress in HLC and immortalized cells In addition to LD accumulation, oxidative stress is another major determinant of MASLD onset and progression ( 23 ). We therefore monitored mitochondrial and cytoplasmatic ROS levels, using respectively MitoSOX and CellRox fluorescent probes, which revealed increased ROS levels in LPO-treated HLC when compared to untreated (i.e., healthy) HLC (Fig. 3 a). Similarly, AML12 and HepG2 cell lines upon LPO administration showed enhanced CellRox levels, detected either by confocal (Fig. 3 b) or fluorometric (Fig. 3 c) analyses. These data are in line with a previously published functional metabolic analysis showing that LPO administration decreases mitochondrial oxygen consumption of HLC cells, hence indicating a potential dysfunction of the electron transport chain induced by LPO ( 24 ). Taken together, these data show that the steatotic hepatic model based on LPO-mediated induction in human and murine cells is a robust platform for studying the molecular determinants of MASLD disease. Estrogen reduced LD accumulation and oxidative stress in LPO-treated hepatocytes Since estrogens could have a protective role in MASLD onset and progression ( 25 ), we evaluated the effects exerted by 17β-estradiol (E2) administration in LPO-treated cells. First, we confirmed the expression of ER in both untreated and LPO-induced HLC (Fig. 4 a). In line with the hypothesis, we observed that 48-hour E2 treatment led to the transcriptional activation of established E2-dependent liver-specific genes ( 26 ), such as growth differentiation factor 15 (GDF15) and solute carrier family 34 member 2 (SLC34A2), in LPO-treated HLC (Fig. 4 b). Due to the inherent limitation of hESC-derived HLC cells being hardly transfectable, we employed AML12 and HepG2 cells that enable easier genetic manipulation and analysis. Indeed, endogenous ER activity was monitored by luciferase reporter assay in LPO-treated AML12 and HepG2 cells upon E2 administration (Fig. 4 c), sustaining the hypothesis that the E2-induced transcriptional program is mediated by ER. Indeed, the transcriptional activation induced by E2 in the LPO models had important functional implications. In particular, 48-hour E2 treatment abrogated the LD accumulation induced by LPO (Fig. 4 d) and concomitantly reduced the ROS levels in both WA01 and WA09-derived HLC (Fig. 4 e ). Moreover, the higher levels of ROS induced by LPO administration in AML12 and HepG2 cells are mitigated following E2 treatment, as detected using the CellRox probe (Fig. 4 f,g). Interestingly, male and female-derived HLC have a comparable response to E2 administration, suggesting that the “gender-relevant phenotype” is independent of the genetic background of the HLC. Importantly, these results confirm the potential protective role of E2 in counteracting MASLD in our models. TRX2 inhibition reverted estrogen-mediated ROS buffering To investigate the mechanism responsible for the protective role exerted by E2, we explored the potential involvement of antioxidant systems that have been reported to be controlled by E2. Contrary to expectations, NRF2, a well-established ER-dependent gene ( 27 , 28 ), exhibited no response to E2 treatment, neither in its expression levels nor in its subsequent activation, as assessed through the expression of the downstream target gene NQO1 in both HLC ( Supplementary Fig. 1 ). Therefore, we investigated whether thioredoxin 2 (TRX2), which is part of the mitochondrial thioredoxin system and is known to be controlled by E2 ( 14 ), could be responsible for the E2-mediated ROS buffering observed in LPO-treated models. In line with this hypothesis, treatment with E2 determined an increase in TRX2 mRNA and protein levels in LPO-exposed WA01 and WA09 HLC (Fig. 5 a). To explore the contribution of the TRX2 system to the protective effects exerted by E2, we employed auranofin, a pharmacological agent approved for the treatment of rheumatoid arthritis, known to disrupt the TRX2 antioxidant system ( 29 ). The treatment of cellular models exposed to LPO with auranofin reversed the protective, antioxidant effects induced by E2. This reversal was evidenced by the increase of both mitochondrial and cytoplasmic ROS levels in both immortalized and HLC models (Fig. 5 b,c). However, it is important to acknowledge that the administration of auranofin into LPO-exposed HLC still induced oxidative stress, indicating that auranofin may exert a pro-oxidant effect. This effect could occur either through direct interaction with the TRX2 system or by affecting other redox regulatory mechanisms. TRX2 expression profile in a cohort of female MASLD patients Finally, to investigate the clinical relevance of our findings, we evaluated whether TRX2 expression correlates with different stages of MASLD progression and could be associated with its emergence. Subudhi et al. conducted a comprehensive analysis of approximately 800 genes in liver tissue samples obtained from 318 donors. These samples were classified into four distinct subsets based on liver histology: normal liver histology (Normal), steatosis only (Steatosis), metabolic dysfunction-associated steatohepatitis without fibrosis (early MASH), and MASH with fibrosis stages 1–4 (late MASH) ( 30 ). For our analysis, we specifically extracted the TRX2 expression data from the 243 female liver samples. Within this cohort of female donors, we categorized the liver specimens into pre- and post-menopausal groups based on age, with 50 years old serving as the threshold for distinguishing between the two conditions. Although TRX2 expression levels were comparable or even higher in post-menopausal healthy donors when compared to pre-menopausal (normal, mean premenopausal women = 831.4502 and mean post-menopausal women = 878.5661), it was interesting to observe higher TRX2 expression levels in pre-menopausal liver specimens in the three stages of MASLD progression when compared to the post-menopausal counterpart (Steatosis: mean premenopausal = 860.9288 and mean post-menopausal = 834.9340; Early MASH: mean premenopausal = 789.2069 and mean postmenopausal = 783.4695; Late MASH: mean premenopausal = 768.2529 and mean post-menopausal = 702.7858, Fig. 6 a-c). Notably, the decline in TRX2 expression among post-menopausal patients was more pronounced compared to pre-menopausal individuals as MASLD advanced (Fig. 6 d). This observation underscores the potential clinical significance of monitoring TRX2 expression in tracking MASLD progression. Given the pivotal role of ROS formation and handling in the progression of the pathology, it is plausible that the diminished presence of estrogen and consequent reduction in estrogen-dependent TRX2-mediated ROS scavenging contribute to the heightened decline observed in the post-menopausal female cohort. Discussion MASLD is a global health problem, with its prevalence on the rise, that is characterized by the association of liver steatosis to additional comorbidities including obesity, diabetes, and cardiovascular and renal diseases ( 1 ). The incidence of MASLD displays a notable disparity between the sexes, with men exhibiting a higher prevalence compared to premenopausal women ( 31 ). This observation underscores the pivotal role of sex hormones, particularly estrogens, in MASLD pathogenesis. Indeed, the decline in estrogen levels in post-menopausal women contributes to an increased susceptibility to MASLD, highlighting estrogens’ significance in the context of liver health ( 32 ). Estrogens are known to exert protective effects against hepatic lipid accumulation through various mechanisms, including the modulation of lipid metabolism, insulin sensitivity, and inflammation ( 33 ). The current study aimed to expand further this area of investigation, increasing the mechanistic insights involved in the estrogens’ mediated protective role. Since both genomic and environmental factors contribute to the onset and progression of MASLD, it is difficult to study the molecular mechanisms underlying the disease and, consequently, to find effective therapeutic approaches. A further aspect complicating the study of MASLD is the lack of in vitro and in vivo experimental models capable of recapitulating the complexity of the liver tissue. Currently, in vivo murine models serve as the primary research tool for MASLD because they have demonstrated a higher likelihood of extrapolating findings from studies on the disease's pathophysiology in humans and a more accurate assessment of possible therapeutic targets ( 34 ). On the other hand, utilizing in vitro models allows a more precise manipulation and observation of specific pathways and better control over experimental variables, essential for elucidating the underlying mechanisms involved in liver diseases. In this study, we complemented immortalized liver cell lines with HLC obtained from hESC that were subjected to LPO administration. LPO generated hepatic steatosis and increased ROS levels in all the cell models employed. Such an approach allowed the establishment of a robust platform for studying mechanistic determinants involved in MASLD onset and progression, including the role of estrogens. Besides its role in reproduction and sexual development, estrogens can influence the developing of several complex pathologies, including metabolic and liver disease ( 18 ). Indeed, the prevalence of metabolic syndrome rises with menopause, most likely as a secondary effect of the metabolic reprogramming from central fat redistribution induced by reduced levels of estrogens ( 26 ), since it is known that estrogens regulate the activity of a series of enzymes involved in de novo synthesis and oxidation of fatty acids ( 35 ). Indeed, also in experimental conditions, estrogens reduce the susceptibility to steatosis development in liver cells of female mice fed a high-fat diet (HFD) subjected to ovariectomy ( 20 ). Furthermore, independent of genetic background, ER deletion in Western-type diet-fed mice of both sexes (female and male) resulted in impaired high-density lipoprotein (HDL) internalization by isolated hepatocytes. Interestingly, this effect was observed despite elevated serum cholesterol and HDL levels specifically in female mice ( 36 ). While dysfunction in lipid metabolism should be considered the basis of MASLD onset, its progression requires multiple additional hits, including oxidative stress ( 37 ). It has been reported that estrogens can reduce oxidative stress and cell damage in cardiovascular diseases ( 38 ), where the incidence, like MASLD, is much higher in men and post-menopausal women than in fertile women. Specifically, it has been reported that E2 reduces ROS production and protects against oxidative stress by increasing the activation of the NRF2-mediated antioxidant response ( 39 ). This led us to hypothesize that such a mechanism could be also implicated in cyto-protection during MASLD. We, therefore, verified if E2 administration could lower ROS levels in our in vitro experimental models, preventing or postponing the disease's progression. While we noted that E2 played a protective role in reducing the enhanced levels of ROS and lipid accumulation associated with LPO-induced MASLD independently from genetic background (Fig. 7 ), we did not observe a clear involvement of NRF2 activation ( Supplementary Figure S1 ). Nonetheless, we identified TRX2 as implicated in the protective mechanism dependent on E2 in our experimental setups. TRX2, localized within mitochondria, serves as a crucial antioxidant defense system, scavenging ROS and maintaining mitochondrial redox balance (Fig. 7 ). Dysregulation of TRX2 could exacerbate mitochondrial dysfunction, perpetuating oxidative stress and contributing to MASLD progression. Indeed, strategies aimed at enhancing TRX2 activity or expression hold the potential to mitigate oxidative stress and subsequent liver injury in MASLD ( 40 ). An aspect not investigated in our study pertained to the potential interaction between E2, ER, and TRX2, particularly behind the E2-dependent transcriptional regulation of TRX2. The interplay between TRX2 and estrogens can occur at multiple levels: as shown in our study, TRX2 appears to be an E2-dependent gene. However, estrogens can influence ROS level regulating mitochondrial biogenesis and subsequent function. Since TRX2 is primarily located in the mitochondria, E2 effects on mitochondrial function may also impact TRX2 activity. These effects can be reverted by the administration of the TRX2 inhibitor, namely auranofin, which resulted in an impairment of the antioxidant effects of estrogens in our MASLD-models. Indeed, auranofin showed to induce oxidative damage and modifications of cellular redox status, resulting in the overproduction of ROS and apoptosis of gastric cancer cells ( 41 ). Nonetheless, treatment with auranofin decreased palmitic acid-induced steatosis in HepG2 and lipogenesis and fibrosis in western diet-induced MASLD model mice ( 42 ). Therefore, estrogens, through their antioxidant properties ( 43 ), can modulate redox signaling pathways hence, shaping the overall antioxidant cellular response: TRX2 may play a role and exert compensative antioxidant function and therefore, the role we have described in our in vitro study has to be related to the MASLD pathogenesis. Further studies are needed to address the specific mechanisms underlying TRX2 and ER interaction and potential therapeutic implications, including also the importance of stromal and immune cells in this crosstalk, which will certainly participate in the progression of MASLD disease. In conclusion, understanding the gender-specific disparities, the influence of estrogens, the significance of redox balance, and the role of the TRX2 system in MASLD onset and progression provides valuable insights into the pathophysiology of this complex disorder. Targeted therapeutic interventions aimed at restoring hormonal balance, preserving redox homeostasis, and enhancing mitochondrial function hold promise in mitigating MASLD burden and improving clinical outcomes. Collaborative efforts integrating basic research and clinical translation are imperative in advancing our understanding and management of MASLD in both sexes. Declarations Acknowledgment We thank Giulia Mariottini that, during their BSc internship, discussed this study and helped with cell culturing and Alice Guida and Tommaso Mello that helped with AML12 cell lines. The work was funded by by Ministero dell’Istruzione dell’Università e della Ricerca – Progetto di Ricerca di Rilevante Interesse Nazionale (PRIN) 2022RCFZZ3 to A.M., by Associazione Italiana Ricerca sul Cancro (AIRC) and Fondazione Cassa di Risparmio di Firenze (grant Multiuser 19515 and grant IG 22941 to A.M.). M.B. was supported by Fondazione Pezcoller/SIC Prof.ssa De Gasperi Ronc. N.L. was supported by an AIRC fellowship. We thank Fondazione Annastaccatolisa ODV for supporting A.Su. fellowship. The data presented in the current study were in part generated using the equipment of the Facility di Medicina Molecolare, funded by “Ministero dell’Istruzione dell’Università e della Ricerca – Bando Dipartimenti di Eccellenza 2018-2022”. The illustration was created with BioRender.com. Author contributions The authors contributed to this work in different capacities, described as follows. A.Sm. performed cell-based and biochemistry experiments, data acquisition, analysis, and interpretation and wrote the original draft of the manuscript; N.L., M.B., A.Su., G.C., F.B., M.A.K, and A.P performed cell-based and biochemistry experiments and participated in the interpretation of the data;, performed the data analysis and interpretation and revised the manuscript; A.M. conceived, designed, and supervised the study, was responsible for the financial support, analyzed and interpreted the data, wrote the original draft of the manuscript. All the authors reviewed the prepared manuscript. Conflict of interest All the other authors declare no competing interests. References Rinella ME, Lazarus JV, Ratziu V, Francque SM, Sanyal AJ, Kanwal F, et al. A multisociety Delphi consensus statement on new fatty liver disease nomenclature. Hepatology. 2023;78(6):1966–86. Day CP, Saksena S. Non-alcoholic steatohepatitis: definitions and pathogenesis. 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Ishii T, Warabi E. Mechanism of Rapid Nuclear Factor-E2-Related Factor 2 (Nrf2) Activation via Membrane-Associated Estrogen Receptors: Roles of NADPH Oxidase 1, Neutral Sphingomyelinase 2 and Epidermal Growth Factor Receptor (EGFR). Antioxidants (Basel). 2019;8(3). Zhu C, Wang S, Wang B, Du F, Hu C, Li H, et al. 17β-Estradiol up-regulates Nrf2 via PI3K/AKT and estrogen receptor signaling pathways to suppress light-induced degeneration in rat retina. Neuroscience. 2015;304:328–39. May HC, Yu JJ, Guentzel MN, Chambers JP, Cap AP, Arulanandam BP. Repurposing Auranofin, Ebselen, and PX-12 as Antimicrobial Agents Targeting the Thioredoxin System. Front Microbiol. 2018;9:336. Subudhi S, Drescher HK, Dichtel LE, Bartsch LM, Chung RT, Hutter MM, et al. Distinct Hepatic Gene-Expression Patterns of NAFLD in Patients With Obesity. Hepatol Commun. 2022;6(1):77–89. Chen XY, Wang C, Huang YZ, Zhang LL. Nonalcoholic fatty liver disease shows significant sex dimorphism. World J Clin Cases. 2022;10(5):1457–72. Henschen A, Hökfelt T, Elde R, Fahrenkrug J, Frey P, Terenius L, et al. Expression of eight neuropeptides in intraocular spinal cord grafts: organotypical and disturbed patterns as evidenced by immunohistochemistry. Neuroscience. 1988;26(1):193–213. Palmisano BT, Zhu L, Stafford JM. Role of Estrogens in the Regulation of Liver Lipid Metabolism. Adv Exp Med Biol. 2017;1043:227–56. Ramos MJ, Bandiera L, Menolascina F, Fallowfield JA. models for non-alcoholic fatty liver disease: Emerging platforms and their applications. iScience. 2022;25(1):103549. Motomura W, Inoue M, Ohtake T, Takahashi N, Nagamine M, Tanno S, et al. Up-regulation of ADRP in fatty liver in human and liver steatosis in mice fed with high fat diet. Biochem Biophys Res Commun. 2006;340(4):1111–8. Zhu L, Shi J, Luu TN, Neuman JC, Trefts E, Yu S, et al. Hepatocyte estrogen receptor alpha mediates estrogen action to promote reverse cholesterol transport during Western-type diet feeding. Mol Metab. 2018;8:106–16. Arroyave-Ospina JC, Wu Z, Geng Y, Moshage H. Role of Oxidative Stress in the Pathogenesis of Non-Alcoholic Fatty Liver Disease: Implications for Prevention and Therapy. Antioxidants (Basel). 2021;10(2). Xiang D, Liu Y, Zhou S, Zhou E, Wang Y. Protective Effects of Estrogen on Cardiovascular Disease Mediated by Oxidative Stress. Oxid Med Cell Longev. 2021;2021:5523516. Rooney J, Oshida K, Vasani N, Vallanat B, Ryan N, Chorley BN, et al. Activation of Nrf2 in the liver is associated with stress resistance mediated by suppression of the growth hormone-regulated STAT5b transcription factor. PLoS One. 2018;13(8):e0200004. Zhu M, Dagah OMA, Silaa BB, Lu J. Thioredoxin/Glutaredoxin Systems and Gut Microbiota in NAFLD: Interplay, Mechanism, and Therapeutical Potential. Antioxidants (Basel). 2023;12(9). 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Additional Declarations (Not answered) Supplementary Files Supplementaryfigure1.png Supplementary figure 1 Cite Share Download PDF Status: Published Journal Publication published 31 Jan, 2025 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 17 May, 2024 Review # 2 received at journal 14 May, 2024 Review # 1 received at journal 26 Apr, 2024 Reviewer # 2 agreed at journal 26 Apr, 2024 Reviewer # 1 agreed at journal 23 Apr, 2024 Reviewers invited by journal 23 Apr, 2024 Submission checks completed at journal 15 Apr, 2024 First submitted to journal 12 Apr, 2024 Editor assigned by journal 12 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4259782\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":294401548,\"identity\":\"133a37e5-47f2-413f-8581-2d48907b5c57\",\"order_by\":0,\"name\":\"Andrea 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22:10:19\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4259782/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4259782/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1038/s41419-025-07331-7\",\"type\":\"published\",\"date\":\"2025-01-31T05:00:00+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":55431802,\"identity\":\"67f3b66d-651e-45d8-ae8c-77f7872afbc4\",\"added_by\":\"auto\",\"created_at\":\"2024-04-27 13:34:36\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":686814,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003ePhenotypic and functional characterization of HLC derived from hESC.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Representative bright field images of the differentiation phases that lead WA01 and WA09 hESC to differentiate into HLC. Magnification: 40X (\\u003cstrong\\u003eb\\u003c/strong\\u003e) HLC were subjected to qRT-PCR analysis. Relative expression is shown using the hESC as a comparator (set as 1). Data represent means ± SEM. *p \\u0026lt; 0.05, **p \\u0026lt; 0.01; ***p \\u0026lt; 0.001. Each dot represents a biological replicate, n=3. (\\u003cstrong\\u003ec\\u003c/strong\\u003e) Total protein lysates from WA01 and WA09 hESC and derived HLC were subjected to western blot analysis with the antibodies indicated. The Histone H3 is used as a protein loading control normalizer.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4259782/v1/23c6b06b88cb1aecc852b27c.png\"},{\"id\":55431803,\"identity\":\"d74101ad-fb28-4e59-b6d1-fe3a2eef1857\",\"added_by\":\"auto\",\"created_at\":\"2024-04-27 13:34:36\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":623436,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eLactate, pyruvate, and octanoic acid-induced steatosis in HLC and immortalized cells\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Schematic representation of steatosis induction in hepatic cell models: treatment with 10 mM sodium L-lactate (L), 1 mM sodium pyruvate (P), and 2 mM octanoic acid (O) (LPO) for 48 hours. (\\u003cstrong\\u003eb-c\\u003c/strong\\u003e) Untreated and LPO-exposed WA01 and WA09 HLC were subjected to qRT-PCR (\\u003cstrong\\u003eb\\u003c/strong\\u003e) and western blotting (\\u003cstrong\\u003ec\\u003c/strong\\u003e) analyses using the assays described in the figure. The Histone H3 is used as a protein loading control normalizer. Data represent means ± SEM. Student’s t-test *p \\u0026lt; 0.05, ***p \\u0026lt; 0.001, ****p \\u0026lt; 0.0001. Each dot represents a biological replicate, n=3. (\\u003cstrong\\u003ed,e\\u003c/strong\\u003e) Untreated and LPO-exposed WA01, WA09 HLC (\\u003cstrong\\u003ed\\u003c/strong\\u003e), AML12, and HepG2 cells (\\u003cstrong\\u003ee\\u003c/strong\\u003e) were subjected to confocal and/or FACS analyses as indicated in the figure. Representative confocal images of BODIPY\\u003csup\\u003e493/503\\u003c/sup\\u003e stained cells are shown (orange/yellow: LD; blue: DAPI, nuclei. Scale bar, 10 µm). Quantification of BODIPY\\u003csup\\u003e493/503 \\u003c/sup\\u003espots/cell was reported. The mean fluorescence intensity (MFI) of the populations positive for BODIPY\\u003csup\\u003e493/503\\u003c/sup\\u003e recovered by FACS analysis was reported. Data represent means ± SEM.\\u0026nbsp; Student’s t test **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001, ****p \\u0026lt; 0.0001. Each dot represents a biological replicate, n≥3. (\\u003cstrong\\u003ef\\u003c/strong\\u003e) Untreated and LPO-exposed WA01, WA09, AML12, and HepG2 cells were subjected to qRT-PCR analysis. Data represent means ± SEM. Student’s t test *p \\u0026lt; 0.05, **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001, ****p \\u0026lt; 0.0001. Each dot represents a biological replicate, n=3.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4259782/v1/8e47140bd9ba95ab6f08e792.png\"},{\"id\":55431810,\"identity\":\"48950e21-e936-4467-8fc5-66797907409a\",\"added_by\":\"auto\",\"created_at\":\"2024-04-27 13:34:37\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":477690,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eLPO treatment increased oxidative stress in HLC and immortalized cells\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Untreated and LPO-exposed WA01 and WA09 cells were subjected to confocal analysis. Representative pictures of MitoSOX and CellRox stained cells are shown (Red: MitoSOX; Green: CellRox; blue: DAPI, nuclei. Scale bar, 10 µm), n≥3. (\\u003cstrong\\u003eb-c\\u003c/strong\\u003e) AML12 and HepG2 cells were treated with LPO for 48 hours and subjected to confocal and fluorescent analyses. (\\u003cstrong\\u003eb\\u003c/strong\\u003e) Representative pictures of CellRox-stained cells are shown (Green: CellRox; blue: DAPI, nuclei. Scale bar, 10 µm). Quantification of CellRox was reported. (\\u003cstrong\\u003ec\\u003c/strong\\u003e) ROS levels were measured using the fluorescent probe CellRox in AML12 and HepG2 cells treated with LPO (48 hours). Untreated cells were used as comparator. Data represent means ± SEM. Student’s t-test *p \\u0026lt; 0.05, **p \\u0026lt; 0.01; ***p \\u0026lt; 0.001; ****p \\u0026lt; 0.0001. Each dot represents a biological replicate, n≥3.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4259782/v1/058eb4fa1ff2a4cafd1fe1ea.png\"},{\"id\":55432171,\"identity\":\"9d5857e6-a89a-4131-b327-6763e72b1361\",\"added_by\":\"auto\",\"created_at\":\"2024-04-27 13:42:36\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":588899,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eEstrogen reduced LD accumulation and oxidative stress in LPO-treated hepatocytes\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Untreated and LPO-exposed WA01 and WA09 HLC were subjected to western blot analysis with the antibodies indicated. ER+ MCF7 breast cancer cell line is used as a positive control (CTR+) for estrogen receptor (ER) expression. The Histone H3 is used as protein loading control normalizer. (\\u003cstrong\\u003eb\\u003c/strong\\u003e) LPO-treated WA01 and WA09 HLC were cultured with or without 1 nM of 17β-estradiol (E2) for 48 hours and subjected to qRT-PCR using the assays described in the figure. Data represent means ± SEM. Student’s t-test *p \\u0026lt; 0.05, ***p \\u0026lt; 0.001. Each dot represents a biological replicate, n≥3. (\\u003cstrong\\u003ec\\u003c/strong\\u003e) LPO-treated AML12 and HepG2 were cultured with or without 50 nM of E2 for 48 hours and subjected to ERE-luciferase reporter assay. Data represent means ± SEM. Student’s t-test *p \\u0026lt; 0.05, **p \\u0026lt; 0.01. Each dot represents a biological replicate, n=3. (\\u003cstrong\\u003ed\\u003c/strong\\u003e) LPO-treated WA01 and WA09 HLC were cultured with or without 1 nM of E2 for 48 hours and subjected to confocal analysis. Representative confocal images of BODIPY\\u003csup\\u003e493/503\\u003c/sup\\u003e stained cells are shown (orange/yellow: LDs; blue: DAPI, nuclei. Scale bar, 10 µm). Quantification of BODIPY\\u003csup\\u003e493/503\\u003c/sup\\u003e spots/cell was reported. *p \\u0026lt; 0.05, **p \\u0026lt; 0.01. n≥3 (\\u003cstrong\\u003ee\\u003c/strong\\u003e) LPO-treated WA01 and WA09 HLC were cultured with or without 1 nM of E2 for 48 hours and subjected to confocal analysis. Representative confocal images of MitoSOX stained cells are shown (red: MitoSOX; blue: DAPI, nuclei. Scale bar, 10 µm). (\\u003cstrong\\u003ef\\u003c/strong\\u003e) LPO-treated AML12 cells were cultured with or without 50 nM of E2 for 48 hours and subjected to confocal analysis. Representative images of CellRox-stained cells are shown (Green: CellRox; blue: DAPI, nuclei. Scale bar, 10 µm). Quantification of CellRox intensity is reported. ****p \\u0026lt; 0.0001. n≥3 (\\u003cstrong\\u003eg\\u003c/strong\\u003e) Intracellular ROS levels were measured by CellRox staining in LPO-treated HepG2 cells cultured with or without 50 nM of E2 for 48 hours. Data represent means ± SEMs. Student’s t-test **p \\u0026lt; 0.01. Each dot represents a biological replicate, n≥3.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4259782/v1/522f7fd6bea74df9daf70c14.png\"},{\"id\":55431809,\"identity\":\"23c53a98-c99a-4f3d-8d71-33515f3722c1\",\"added_by\":\"auto\",\"created_at\":\"2024-04-27 13:34:37\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":323005,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eTRX2 inhibition reverted estrogen-mediated ROS buffering\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) LPO-treated WA01 and WA09 HLC were cultured with or without 1nM E2 for 48 hours and subjected to qRT-PCR and western blotting analysis using the assays described in the figure. The Hsp90 is used as a protein loading control normalizer. Data represent means ± SEMs. Student’s t-test ****p \\u0026lt; 0.0001. Each dot represents a biological replicate, n=6. (\\u003cstrong\\u003eb\\u003c/strong\\u003e) Intracellular ROS levels were measured with a CellRox probe in LPO-treated AML12 and HepG2 cells cultured with 50 nM of E2 and 250 nM of auranofin (AU) for 48 hours. Data represent means ± SEMs. Student’s t-test. **p \\u0026lt; 0.01. Each dot represents a biological replicate, n=3. (\\u003cstrong\\u003ec\\u003c/strong\\u003e) LPO-treated WA01 and WA09 cells were cultured with or without 1nM of E2 and 500 nM of auranofin (AU) for 48 hours and subjected to confocal analysis. Representative images of MitoSOX stained cells are shown (Red: MitoSOX; blue: DAPI, nuclei. Scale bar, 10 µm).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4259782/v1/1fe269d02c1a2006b9a9d864.png\"},{\"id\":55431806,\"identity\":\"29d0d69d-43df-4862-a49c-1a7c385b0605\",\"added_by\":\"auto\",\"created_at\":\"2024-04-27 13:34:37\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":117291,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eTRX2 expression profile in a cohort of female MASLD patients\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eGene expression of TRX2 was analyzed with GEO2R and \\u003cem\\u003eR\\u003c/em\\u003e-program in a cohort of normal or MASLD-bearing patients (GSE163211) divided by age. TRX2 expression levels (\\u003cstrong\\u003ea\\u003c/strong\\u003e) in the whole female population, (\\u003cstrong\\u003eb\\u003c/strong\\u003e) in the postmenopausal (Age≥50), and (\\u003cstrong\\u003ec\\u003c/strong\\u003e) in premenopausal women (Age\\u0026lt;50). (\\u003cstrong\\u003ed\\u003c/strong\\u003e) Differences in TRX2 expression between postmenopausal and premenopausal women compared to normal tissue (Normal-Steatosis, Normal-early MASH, and Normal-late MASH stage). Wilcox test, *p \\u0026lt; 0.05, **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001, ****p \\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4259782/v1/b7d4c8d0b25e3e3372167ac7.png\"},{\"id\":55431808,\"identity\":\"a704cbc1-aeae-43c3-a9c4-05fd0b8d1d6f\",\"added_by\":\"auto\",\"created_at\":\"2024-04-27 13:34:37\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":239490,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eGraphical abstract of the study.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(\\u003cstrong\\u003e1\\u003c/strong\\u003e) Human embryonic stem cells (hESC) were differentiated into hepatocyte-like cells (HLC) using an 18-day differentiation protocol; (\\u003cstrong\\u003e2\\u003c/strong\\u003e) HLC and hepatic immortalized cell lines (AML12, HepG2) were then treated with sodium L lactate (L), sodium pyruvate (P) and octanoic acid (O), a protocol of induction called LPO. LPO administration induces distinct steatotic traits, such as the accumulation of lipid droplets and an increase in reactive oxygen species (ROS) levels. (\\u003cstrong\\u003e3\\u003c/strong\\u003e) Treatment with 17-β estradiol (E2) results in a reduction of the distinctive traits of MASLD through the activation of the mitochondrial antioxidant mechanism of thioredoxin 2 (TRX2), which is inhibited by the administration of auranofin.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4259782/v1/ada03b7f4ba0b84c8a44995a.png\"},{\"id\":75217912,\"identity\":\"b59db77b-7a0f-498b-8440-3c230712bee2\",\"added_by\":\"auto\",\"created_at\":\"2025-02-01 08:06:07\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":5086307,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4259782/v1/cf77a10f-d091-417d-8029-9ceba41417e4.pdf\"},{\"id\":55431805,\"identity\":\"aaaf1b99-d429-4ee4-ae4c-1d476072d9f1\",\"added_by\":\"auto\",\"created_at\":\"2024-04-27 13:34:37\",\"extension\":\"png\",\"order_by\":6,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":63090,\"visible\":true,\"origin\":\"\",\"legend\":\"Supplementary figure 1\",\"description\":\"\",\"filename\":\"Supplementaryfigure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4259782/v1/df690a6ab4ae26c4740404c9.png\"}],\"financialInterests\":\"(Not answered)\",\"formattedTitle\":\"Estrogen-dependent TRX2 activation reverts oxidative stress and metabolic dysfunction associated to steatotic disease\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eThe metabolic dysfunction-associated steatotic liver disease (MASLD), formerly called non-alcoholic fatty liver disease (NAFLD), encompasses a wide range of pathological comorbidities and functional alterations of the liver associated with at least one cardiometabolic criteria (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e). Hepatic alterations include steatosis, chronic inflammation (metabolic disfunction-associated steatohepatitis, MASH), fibrosis and cirrhosis. Furthermore, cirrhosis is a well-known risk factor for the neoplastic transformation of hepatocytes (hepatocellular carcinoma, HCC). While hepatic steatosis is defined by at least 5% of lipid/triglyceride accumulation in the liver and is potentially reversible, prolonged and severe MASH is considered a progressive disease (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e). Several molecular pathways can contribute to the onset of MASLD and be responsible for the phenotypic traits displayed by hepatic cells undergoing steatosis (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e), which is typically characterized by an overload and intracellular accumulation of triglycerides not related to alcohol consumption, accompanied by inflammation and oxidative stress, essential players involved in the promotion of steatohepatitis (\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eGlobal MASLD prevalence is around 25% with an appreciably lower incidence in pre-menopausal women compared to men and post-menopausal women (\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e), although the female population have a greater tendency towards obesity, one of the main risk factors (\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e). This suggests a potential protective effect of estrogens in counteracting MASLD initiation and progression. Accumulating evidence highlights the benefits of estrogen replacement therapy in mitigating lipid accumulation and peroxidation (\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e), reducing lipotoxicity-induced oxidative stress in hepatic mitochondria, and ameliorating the inflammation process (\\u003cspan additionalcitationids=\\\"CR9\\\" citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e). In ovariectomized mice, it has been demonstrated that supplementation with 17β-estradiol (E2) (\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e), the predominant form among circulating female hormones, inhibits steatogenesis (\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e). However, the precise molecular mechanisms underlying the protective effects of estrogens in the onset and progression of MASLD remain poorly understood.\\u003c/p\\u003e \\u003cp\\u003eIt has been reported that estrogens primarily prevent oxidative damage via the estrogen receptor alpha (hereafter referred to as ER) which, in turn, promotes the expression and activation of a plethora of antioxidant enzymes. Importantly, nuclear factor erythroid 2-related factor 2 (NRF2) and thioredoxin 2 (TRX2) are ER-dependent genes that could regulate the cellular defense against reactive oxygen species (ROS). TRX2 directly neutralizes ROS, whereas NRF2 initiates a transcriptional regulation of scavenging mechanisms under its control (\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e). These actions lead to anti-steatotic effects, which alleviate the inflammation induced by MASLD (\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e). Here, we investigated the protective role of estrogen in counteracting ROS production through activation of the antioxidant mitochondrial TRX2 pathway, in an innovative \\u003cem\\u003ein vitro\\u003c/em\\u003e hepatic steatosis model based on the use of immortalized and hepatocyte-like cells (HLC).\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell lines and general culture conditions\\u003c/h2\\u003e \\u003cp\\u003eMale and female human embryonic stem cell (hESC) lines, WA01 and WA09 respectively, were purchased from WiCell Research Institute (504 S Rosa Rd #101, Madison, WI 53719, USA) and maintained at 37\\u0026deg;C/5%CO\\u003csub\\u003e2\\u003c/sub\\u003e on 5 \\u0026micro;g/mL laminin 521 (LMN-521, STEMCELL Technologies #LN521-05) pre-coated plates with mTeSR1 PLUS serum-free medium (STEMCELL Technologies #100\\u0026ndash;0276).\\u003c/p\\u003e \\u003cp\\u003eAML12 and HepG2 cell lines were purchased from American Type Culture Collection (ATCC): AML12 cells are hepatocytes isolated from the normal liver of a male mouse and grow in DMEM:F12 (1:1, ThermoFisher #11330032) supplemented with 10% fetal bovine serum (FBS), 10 \\u0026micro;g/ml insulin (I), 5.5 \\u0026micro;g/ml transferrin (T), 5 ng/ml selenium (S) (ITS, ThermoFisher #41400045), and 40 ng/ml dexamethasone (ThermoFisher #A13449); HepG2 is a human hepatoblastoma cell line of a 15-year-old male maintained in DMEM high glucose medium (ThermoFisher #11965092) supplemented with 10% FBS, 2 mM glutamax (ThermoFisher #35050061), and 1% penicillin/streptomycin (ThermoFisher #15140).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDifferentiation from hESC to HLC\\u003c/h2\\u003e \\u003cp\\u003eAt the time of differentiation into HLC, hESC were plated as a single cell at a concentration of 400,000 cells per well onto the LMN-521 coating and 10 \\u0026micro;M of Rho-associated kinase inhibitor Y27632 (ROCKi, Stem Cell Technologies #72302) were added to the medium. This inhibitor was used to improve the adhesion and survival of stem cells in single-cell suspensions. After 24 hours, when hESC reached 40% a confluence, we initiated the differentiation process in HLC as accurately detailed in (\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e). Briefly, WA01 and WA09 cells were initially differentiated in endodermal cells by culturing them in RPMI 1640 medium (Gibco #11875-093) containing B-27 Supplement, (Gibco #12587-010), 100 ng/mL Activin A (PeproTech #120-14E), and 50 ng/mL Wnt3A (Peprotech #315-20-10uG). Subsequently, the hepatoblast phenotype was obtained by differentiating endodermal cells in Knockout DMEM (Gibco, #10829), containing 20% Knockout Serum Replacement (Gibco #10828), 1% non-essential amino acids (NEAA, Gibco #11140), 0.1 mM 2-mercaptoethanol (Gibco #31350). Finally, hepatoblast cells were differentiated into HLC by culturing them, until day 17, in HepatoZYME medium (Gibco, #17705) containing 10 ng/mL hepatocyte growth factor (Peprotech, #100\\u0026thinsp;\\u0026minus;\\u0026thinsp;39), 20 ng/mL oncostatin M (Peprotech, #300\\u0026thinsp;\\u0026minus;\\u0026thinsp;10), and 10 \\u0026micro;M hydrocortisone 21-hemisuccinate (Sigma Aldrich, #H4881). At day 18, HLC were used for the assays described in this manuscript.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eInduction of steatotic phenotype\\u003c/h2\\u003e \\u003cp\\u003eHepatocyte steatosis was induced by administrating a mix of 10 mM sodium-L-lactate (L, Sigma-Aldrich #7022), 1 mM sodium pyruvate (P, Sigma-Aldrich P2256), and 2 mM octanoic acid (O, Sigma-Aldrich C2875) for 48 hours to HLC, AML12, and HepG2 cells.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWestern blotting analysis\\u003c/h2\\u003e \\u003cp\\u003eCell lines were washed with PBS and lysed in ice using Laemmli Sample Buffer 1X (Biorad #161\\u0026ndash;0737) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich #P8340 and #P0044, respectively), and protein concentrations were measured by BCA (Sigma-Aldrich #1003290033) method. 30\\u0026ndash;35 \\u0026micro;g of cell lysate were loaded in precast SDS-PAGE (sodium dodecyl sulfate\\u0026ndash;polyacrylamide gel electrophoresis) gels (Biorad #456\\u0026ndash;8096) and then transferred onto nitrocellulose membrane by Trans-Blot Turbo Transfer Pack (Biorad #170\\u0026ndash;4157). The immunoblots were incubated in non-fat dry milk 2%, tween-20 0.05% in PBS at room temperature for 1 hour, and then probed with primary and appropriate secondary antibodies. The following antibodies were used: HNF4α (Cell Signaling Technology #311F12), Albumin (Santa Cruz Biotechnology #sc-271605-F10), E-cadherin (Cell Signaling Technology #3195), TRX2 (Santa Cruz Biotechnology #sc-133201-F10), ER (Abcam, # ab16660), ACTB (Cell Signaling Technology #4970), Hsp90 (Santa Cruz Biotechnology #sc-69703), and Histone H3 (Cell Signaling Technology #4499).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eRNA extraction and Quantitative Real-time PCR (qRT-PCR) analysis\\u003c/h2\\u003e \\u003cp\\u003eTotal RNA was extracted using RNeasy plus kit (QIAGEN #74134), quantified using Nanodrop 1000 (Thermo Fisher Scientific), and 500 ng were reverse transcribed using the iScript gDNA Clear cDNA Synthesis Kit (Biorad #172\\u0026ndash;5035). qRT-PCR was performed using the CFX96 Touch Real-Time PCR Detection System (Biorad) using TaqMan Universal PCR Master Mix (Thermo Fisher Scientific #4305719). The probes used in the work are: Albumin (Hs00609411_m1), HNF4α (Hs00230853_m1), TRX2 (Hs00429399_g1), PLIN2 (Hs00605340_m1 and Mm00475794_m1), GDF15 (Hs00171132_m1), SLC34A2 (Hs00197519_m1), NRF2 (Hs00975961_g1), NQO1 (Hs01045993_g1). Data were normalized on TBP (Hs00427620_m1) and/or ACTB (Mm02619580_g1). The relative quantity was determined using \\u003csup\\u003e∆∆\\u003c/sup\\u003eCt by the CFX Maestro software (BioRad).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eConfocal image acquisition and analysis\\u003c/h2\\u003e \\u003cp\\u003eCells were plated at a concentration of 10,000 cells per well in a Nunc\\u0026trade;Lab-Tek\\u0026trade;Chamber Slide System coverglass (Thermo Fisher Scientific #155383). Once steatosis has been induced, cells were stained at 37\\u0026deg;C for (i) 15 minutes with BODIPY\\u003csup\\u003e493/503\\u003c/sup\\u003e (Thermo Fisher Scientific #D3922) to reveal LD content, (ii) 30 minutes with MitoSOX (Thermo Fisher Scientific #M36008) and CellRox (Thermo Fisher Scientific #C10444) probes to reveal mitochondrial and cytoplasmatic ROS respectively. When the incubation with the probes was completed, cells were fixed using a 4% formaldehyde solution for 10 minutes. Subsequently, the formaldehyde was removed, and three washes were performed with PBS. To stain the nuclei, cells were then incubated for 10 minutes with the DAPI dye (excitation, ex: 358 nm; emission, em: 461 nm) diluted 1:1000 in PBS and subsequently washed an additional 3 times in PBS. Finally, images were acquired using a TCS SP8 microscope (Leica Microsystems) with LAS-AF image acquisition software. The quantification of intracellular lipids was performed using CellProfiler software from three representative 63x images from 3 independent experiments.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eFlow cytometry analysis\\u003c/h2\\u003e \\u003cp\\u003eAML12 cells (7x10\\u003csup\\u003e4\\u003c/sup\\u003e cells/well) were seeded into 12-well plates. After LPO administration, cells were stained at 37\\u0026deg;C for 15 minutes with BODIPY\\u003csup\\u003e493/503\\u003c/sup\\u003e to reveal intracellular lipid content. Live cells resuspended in PBS with 0.1% FBS were subjected to flow cytometry analysis using a FACS Canto II (BD Biosciences).\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eROS analysis\\u003c/h2\\u003e \\u003cp\\u003eCell lines (7x10\\u003csup\\u003e4\\u003c/sup\\u003e cells/well) were seeded into 12-well plates. Once steatosis was induced, the cells were incubated with the CellRox probe in the dark for 30 minutes. Then, cells were lysed with RIPA buffer and fluorescence was measured on a Synergy H1 Hybrid Multi-Mode Reader (Biotek) plate reader (ex: 485 nm; em:520 nm).\\u003c/p\\u003e \\u003cp\\u003e \\u003cspan type=\\\"ItalicUnderline\\\" class=\\\"ItalicUnderline\\\" name=\\\"Emphasis\\\"\\u003eIn silico\\u003c/span\\u003e \\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003eanalysis for TRX2 expression\\u003c/span\\u003e\\u003c/p\\u003e \\u003cp\\u003eTranscriptomic data are available at the National Center for Biotechnology Information GEO repository (accession number GSE163211). The GSE163211 dataset was analyzed with GEO2R and R-program. The data used represent the expression values of TRX2. The statistical test performed is the Wilcox test. Statistical significance was defined as p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eAll experiments were conducted at least 3 times independently. Statistical analysis of the data was performed using GraphPad Prism Software 10. Unless stated otherwise, comparisons between the 2 groups were made using the two-tailed, unpaired Student\\u0026rsquo;s t-test. Comparisons between multiple groups were made using one-way or two-way analysis of variance (ANOVA). Bonferroni and Dunnett's post-testing analysis with a confidence interval of 95% was used for individual comparisons as reported in figure legends. Statistical significance was defined as: \\u003cem\\u003e*p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05; **p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01; ***p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001; ****p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001\\u003c/em\\u003e; when differences were not statistically significant, or the comparison was not biologically relevant, no indication was reported in the figures.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePhenotypic and functional characterization of HLC derived from hESC\\u003c/h2\\u003e \\u003cp\\u003eMale WA01 and female WA09 hESC were differentiated into HLC using a previously published 18-day protocol (\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e). The differentiation of hESC into HLC was confirmed by the classic acquisition of the cobblestone-like hepatic morphology (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea), in parallel to the expression of established hepatic markers, such as increased mRNA albumin expression and hepatocyte nuclear factor-4-alpha (HNF4α), whose mRNA and protein expression levels were enhanced in HLC when compared to hESC cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb-c). Moreover, we also observed a higher protein level of a crucial molecule responsible for the assembly and functionality of adherent junctions (i.e., E-Cadherin, E-Cad, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec), a characteristic feature of differentiated hepatocytes. Thus, using sex-matched hESC differentiated into HLC we reproduced a relevant hepatocyte model, useful for studying the onset and progression of MASLD and the subsequent impact of estrogen administration.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eLactate, pyruvate, and octanoic acid-induced steatosis in HLC and immortalized cells\\u003c/h2\\u003e \\u003cp\\u003eTo extensively mimic an \\u003cem\\u003ein vitro\\u003c/em\\u003e model of steatosis, in addition to WA01 and WA09 hESC-derived HLC, we used two immortalized liver cell lines that have been widely employed to study MASLD initiation and progression: AML12, hepatocytes isolated from a normal murine liver (\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e), and HepG2, a hepatoblastoma cell line, the most commonly employed experimental model for \\u003cem\\u003ein vitro\\u003c/em\\u003e liver cancer research (\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe four cell models were treated with a cocktail of compounds composed of sodium L-lactate (L), sodium pyruvate (P), and octanoic acid (O), hereafter referred to as LPO, for 48 hours to induce steatosis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea) (\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e). Following LPO treatment, both hESC-derived HLC models significantly reduced the mRNA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb) and/or protein (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec) expression levels of Albumin, HNF4α and E-Cad, a sign of hepatocyte dysfunction.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eSteatosis is characterized by the intracellular accumulation of lipids within the cytoplasm of the hepatocyte, in the form of lipid droplets (LD), that are dynamic and multifunctional organelles involved in energy metabolism, signaling, and inflammatory mediator production (\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e). To determine whether LPO could also induce LD accumulation, we performed confocal and FACS analyses using the fluorescent neutral lipid dye BODIPY\\u003csup\\u003e493/503\\u003c/sup\\u003e which is retained in LD. Confocal analysis and subsequent quantification showed that LPO-treated WA01 and WA09 cells are significantly enriched in LD content (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ed). Similarly, confocal and/or FACS analyses confirmed that LPO treatment significantly increased LD accumulation also in AML12 and HepG2 cell lines (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ee). In parallel, all the LPO-exposed cell models displayed significantly higher levels of PLIN2 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ef), a member of the perilipin protein family which we demonstrated to be associated with LD accumulation (\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eLPO treatment increased oxidative stress in HLC and immortalized cells\\u003c/h2\\u003e \\u003cp\\u003eIn addition to LD accumulation, oxidative stress is another major determinant of MASLD onset and progression (\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e). We therefore monitored mitochondrial and cytoplasmatic ROS levels, using respectively MitoSOX and CellRox fluorescent probes, which revealed increased ROS levels in LPO-treated HLC when compared to untreated (i.e., healthy) HLC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea). Similarly, AML12 and HepG2 cell lines upon LPO administration showed enhanced CellRox levels, detected either by confocal (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb) or fluorometric (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ec) analyses. These data are in line with a previously published functional metabolic analysis showing that LPO administration decreases mitochondrial oxygen consumption of HLC cells, hence indicating a potential dysfunction of the electron transport chain induced by LPO (\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eTaken together, these data show that the steatotic hepatic model based on LPO-mediated induction in human and murine cells is a robust platform for studying the molecular determinants of MASLD disease.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEstrogen reduced LD accumulation and oxidative stress in LPO-treated hepatocytes\\u003c/h2\\u003e \\u003cp\\u003eSince estrogens could have a protective role in MASLD onset and progression (\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e), we evaluated the effects exerted by 17β-estradiol (E2) administration in LPO-treated cells. First, we confirmed the expression of ER in both untreated and LPO-induced HLC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea). In line with the hypothesis, we observed that 48-hour E2 treatment led to the transcriptional activation of established E2-dependent liver-specific genes (\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e), such as growth differentiation factor 15 (GDF15) and solute carrier family 34 member 2 (SLC34A2), in LPO-treated HLC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb). Due to the inherent limitation of hESC-derived HLC cells being hardly transfectable, we employed AML12 and HepG2 cells that enable easier genetic manipulation and analysis. Indeed, endogenous ER activity was monitored by luciferase reporter assay in LPO-treated AML12 and HepG2 cells upon E2 administration (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec), sustaining the hypothesis that the E2-induced transcriptional program is mediated by ER. Indeed, the transcriptional activation induced by E2 in the LPO models had important functional implications. In particular, 48-hour E2 treatment abrogated the LD accumulation induced by LPO (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed) and concomitantly reduced the ROS levels in both WA01 and WA09-derived HLC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee\\u003cb\\u003e).\\u003c/b\\u003e Moreover, the higher levels of ROS induced by LPO administration in AML12 and HepG2 cells are mitigated following E2 treatment, as detected using the CellRox probe (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ef,g). Interestingly, male and female-derived HLC have a comparable response to E2 administration, suggesting that the \\u0026ldquo;gender-relevant phenotype\\u0026rdquo; is independent of the genetic background of the HLC. Importantly, these results confirm the potential protective role of E2 in counteracting MASLD in our models.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTRX2 inhibition reverted estrogen-mediated ROS buffering\\u003c/h2\\u003e \\u003cp\\u003eTo investigate the mechanism responsible for the protective role exerted by E2, we explored the potential involvement of antioxidant systems that have been reported to be controlled by E2. Contrary to expectations, NRF2, a well-established ER-dependent gene (\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e), exhibited no response to E2 treatment, neither in its expression levels nor in its subsequent activation, as assessed through the expression of the downstream target gene NQO1 in both HLC (\\u003cb\\u003eSupplementary Fig.\\u0026nbsp;1\\u003c/b\\u003e). Therefore, we investigated whether thioredoxin 2 (TRX2), which is part of the mitochondrial thioredoxin system and is known to be controlled by E2 (\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e), could be responsible for the E2-mediated ROS buffering observed in LPO-treated models. In line with this hypothesis, treatment with E2 determined an increase in TRX2 mRNA and protein levels in LPO-exposed WA01 and WA09 HLC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea). To explore the contribution of the TRX2 system to the protective effects exerted by E2, we employed auranofin, a pharmacological agent approved for the treatment of rheumatoid arthritis, known to disrupt the TRX2 antioxidant system (\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e). The treatment of cellular models exposed to LPO with auranofin reversed the protective, antioxidant effects induced by E2. This reversal was evidenced by the increase of both mitochondrial and cytoplasmic ROS levels in both immortalized and HLC models (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb,c). However, it is important to acknowledge that the administration of auranofin into LPO-exposed HLC still induced oxidative stress, indicating that auranofin may exert a pro-oxidant effect. This effect could occur either through direct interaction with the TRX2 system or by affecting other redox regulatory mechanisms.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTRX2 expression profile in a cohort of female MASLD patients\\u003c/h2\\u003e \\u003cp\\u003eFinally, to investigate the clinical relevance of our findings, we evaluated whether TRX2 expression correlates with different stages of MASLD progression and could be associated with its emergence. Subudhi \\u003cem\\u003eet al.\\u003c/em\\u003e conducted a comprehensive analysis of approximately 800 genes in liver tissue samples obtained from 318 donors. These samples were classified into four distinct subsets based on liver histology: normal liver histology (Normal), steatosis only (Steatosis), metabolic dysfunction-associated steatohepatitis without fibrosis (early MASH), and MASH with fibrosis stages 1\\u0026ndash;4 (late MASH) (\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e). For our analysis, we specifically extracted the TRX2 expression data from the 243 female liver samples. Within this cohort of female donors, we categorized the liver specimens into pre- and post-menopausal groups based on age, with 50 years old serving as the threshold for distinguishing between the two conditions. Although TRX2 expression levels were comparable or even higher in post-menopausal healthy donors when compared to pre-menopausal (normal, mean premenopausal women\\u0026thinsp;=\\u0026thinsp;831.4502 and mean post-menopausal women\\u0026thinsp;=\\u0026thinsp;878.5661), it was interesting to observe higher TRX2 expression levels in pre-menopausal liver specimens in the three stages of MASLD progression when compared to the post-menopausal counterpart (Steatosis: mean premenopausal\\u0026thinsp;=\\u0026thinsp;860.9288 and mean post-menopausal\\u0026thinsp;=\\u0026thinsp;834.9340; Early MASH: mean premenopausal\\u0026thinsp;=\\u0026thinsp;789.2069 and mean postmenopausal\\u0026thinsp;=\\u0026thinsp;783.4695; Late MASH: mean premenopausal\\u0026thinsp;=\\u0026thinsp;768.2529 and mean post-menopausal\\u0026thinsp;=\\u0026thinsp;702.7858, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea-c). Notably, the decline in TRX2 expression among post-menopausal patients was more pronounced compared to pre-menopausal individuals as MASLD advanced (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ed). This observation underscores the potential clinical significance of monitoring TRX2 expression in tracking MASLD progression. Given the pivotal role of ROS formation and handling in the progression of the pathology, it is plausible that the diminished presence of estrogen and consequent reduction in estrogen-dependent TRX2-mediated ROS scavenging contribute to the heightened decline observed in the post-menopausal female cohort.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eMASLD is a global health problem, with its prevalence on the rise, that is characterized by the association of liver steatosis to additional comorbidities including obesity, diabetes, and cardiovascular and renal diseases (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe incidence of MASLD displays a notable disparity between the sexes, with men exhibiting a higher prevalence compared to premenopausal women (\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e). This observation underscores the pivotal role of sex hormones, particularly estrogens, in MASLD pathogenesis. Indeed, the decline in estrogen levels in post-menopausal women contributes to an increased susceptibility to MASLD, highlighting estrogens\\u0026rsquo; significance in the context of liver health (\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e). Estrogens are known to exert protective effects against hepatic lipid accumulation through various mechanisms, including the modulation of lipid metabolism, insulin sensitivity, and inflammation (\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e). The current study aimed to expand further this area of investigation, increasing the mechanistic insights involved in the estrogens\\u0026rsquo; mediated protective role.\\u003c/p\\u003e \\u003cp\\u003eSince both genomic and environmental factors contribute to the onset and progression of MASLD, it is difficult to study the molecular mechanisms underlying the disease and, consequently, to find effective therapeutic approaches. A further aspect complicating the study of MASLD is the lack of \\u003cem\\u003ein vitro\\u003c/em\\u003e and \\u003cem\\u003ein vivo\\u003c/em\\u003e experimental models capable of recapitulating the complexity of the liver tissue. Currently, \\u003cem\\u003ein vivo\\u003c/em\\u003e murine models serve as the primary research tool for MASLD because they have demonstrated a higher likelihood of extrapolating findings from studies on the disease's pathophysiology in humans and a more accurate assessment of possible therapeutic targets (\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e). On the other hand, utilizing \\u003cem\\u003ein vitro\\u003c/em\\u003e models allows a more precise manipulation and observation of specific pathways and better control over experimental variables, essential for elucidating the underlying mechanisms involved in liver diseases.\\u003c/p\\u003e \\u003cp\\u003eIn this study, we complemented immortalized liver cell lines with HLC obtained from hESC that were subjected to LPO administration. LPO generated hepatic steatosis and increased ROS levels in all the cell models employed. Such an approach allowed the establishment of a robust platform for studying mechanistic determinants involved in MASLD onset and progression, including the role of estrogens.\\u003c/p\\u003e \\u003cp\\u003eBesides its role in reproduction and sexual development, estrogens can influence the developing of several complex pathologies, including metabolic and liver disease (\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e). Indeed, the prevalence of metabolic syndrome rises with menopause, most likely as a secondary effect of the metabolic reprogramming from central fat redistribution induced by reduced levels of estrogens (\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e), since it is known that estrogens regulate the activity of a series of enzymes involved in \\u003cem\\u003ede novo\\u003c/em\\u003e synthesis and oxidation of fatty acids (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e). Indeed, also in experimental conditions, estrogens reduce the susceptibility to steatosis development in liver cells of female mice fed a high-fat diet (HFD) subjected to ovariectomy (\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e). Furthermore, independent of genetic background, ER deletion in Western-type diet-fed mice of both sexes (female and male) resulted in impaired high-density lipoprotein (HDL) internalization by isolated hepatocytes. Interestingly, this effect was observed despite elevated serum cholesterol and HDL levels specifically in female mice (\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eWhile dysfunction in lipid metabolism should be considered the basis of MASLD onset, its progression requires multiple additional hits, including oxidative stress (\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e). It has been reported that estrogens can reduce oxidative stress and cell damage in cardiovascular diseases (\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e), where the incidence, like MASLD, is much higher in men and post-menopausal women than in fertile women. Specifically, it has been reported that E2 reduces ROS production and protects against oxidative stress by increasing the activation of the NRF2-mediated antioxidant response (\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e). This led us to hypothesize that such a mechanism could be also implicated in cyto-protection during MASLD. We, therefore, verified if E2 administration could lower ROS levels in our \\u003cem\\u003ein vitro\\u003c/em\\u003e experimental models, preventing or postponing the disease's progression. While we noted that E2 played a protective role in reducing the enhanced levels of ROS and lipid accumulation associated with LPO-induced MASLD independently from genetic background (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e), we did not observe a clear involvement of NRF2 activation (\\u003cb\\u003eSupplementary Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e\\u003c/b\\u003e). Nonetheless, we identified TRX2 as implicated in the protective mechanism dependent on E2 in our experimental setups. TRX2, localized within mitochondria, serves as a crucial antioxidant defense system, scavenging ROS and maintaining mitochondrial redox balance (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). Dysregulation of TRX2 could exacerbate mitochondrial dysfunction, perpetuating oxidative stress and contributing to MASLD progression. Indeed, strategies aimed at enhancing TRX2 activity or expression hold the potential to mitigate oxidative stress and subsequent liver injury in MASLD (\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eAn aspect not investigated in our study pertained to the potential interaction between E2, ER, and TRX2, particularly behind the E2-dependent transcriptional regulation of TRX2. The interplay between TRX2 and estrogens can occur at multiple levels: as shown in our study, TRX2 appears to be an E2-dependent gene. However, estrogens can influence ROS level regulating mitochondrial biogenesis and subsequent function. Since TRX2 is primarily located in the mitochondria, E2 effects on mitochondrial function may also impact TRX2 activity. These effects can be reverted by the administration of the TRX2 inhibitor, namely auranofin, which resulted in an impairment of the antioxidant effects of estrogens in our MASLD-models. Indeed, auranofin showed to induce oxidative damage and modifications of cellular redox status, resulting in the overproduction of ROS and apoptosis of gastric cancer cells (\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e). Nonetheless, treatment with auranofin decreased palmitic acid-induced steatosis in HepG2 and lipogenesis and fibrosis in western diet-induced MASLD model mice (\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eTherefore, estrogens, through their antioxidant properties (\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e), can modulate redox signaling pathways hence, shaping the overall antioxidant cellular response: TRX2 may play a role and exert compensative antioxidant function and therefore, the role we have described in our \\u003cem\\u003ein vitro\\u003c/em\\u003e study has to be related to the MASLD pathogenesis. Further studies are needed to address the specific mechanisms underlying TRX2 and ER interaction and potential therapeutic implications, including also the importance of stromal and immune cells in this crosstalk, which will certainly participate in the progression of MASLD disease.\\u003c/p\\u003e \\u003cp\\u003eIn conclusion, understanding the gender-specific disparities, the influence of estrogens, the significance of redox balance, and the role of the TRX2 system in MASLD onset and progression provides valuable insights into the pathophysiology of this complex disorder. Targeted therapeutic interventions aimed at restoring hormonal balance, preserving redox homeostasis, and enhancing mitochondrial function hold promise in mitigating MASLD burden and improving clinical outcomes. Collaborative efforts integrating basic research and clinical translation are imperative in advancing our understanding and management of MASLD in both sexes.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgment\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank Giulia Mariottini that, during their BSc internship, discussed this study and helped with cell culturing and Alice Guida and Tommaso Mello that helped with AML12 cell lines.\\u003c/p\\u003e\\n\\u003cp\\u003eThe work was funded by by Ministero dell\\u0026rsquo;Istruzione dell\\u0026rsquo;Universit\\u0026agrave; e della Ricerca \\u0026ndash; Progetto di Ricerca di Rilevante Interesse Nazionale (PRIN) 2022RCFZZ3 to A.M., by Associazione Italiana Ricerca sul Cancro (AIRC) and Fondazione Cassa di Risparmio di Firenze (grant Multiuser 19515 and grant IG 22941 to A.M.). M.B. was supported by Fondazione Pezcoller/SIC Prof.ssa De Gasperi Ronc. N.L. was supported by an AIRC fellowship.\\u0026nbsp;We thank Fondazione Annastaccatolisa ODV for supporting A.Su. fellowship. The data presented in the current study were in part generated using the equipment of the Facility di Medicina Molecolare, funded by \\u0026ldquo;Ministero dell\\u0026rsquo;Istruzione dell\\u0026rsquo;Universit\\u0026agrave; e della Ricerca \\u0026ndash; Bando Dipartimenti di Eccellenza 2018-2022\\u0026rdquo;. The illustration was created with BioRender.com.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors contributed to this work in different capacities, described as follows. A.Sm. performed cell-based and biochemistry experiments, data acquisition, analysis, and interpretation and wrote the original draft of the manuscript; N.L., M.B., A.Su., G.C., F.B., M.A.K, and A.P performed cell-based and biochemistry experiments and participated in the interpretation of the data;, performed the data analysis and interpretation and revised the manuscript; A.M. conceived, designed, and supervised the study, was responsible for the financial support, analyzed and interpreted the data, wrote the original draft of the manuscript. All the authors reviewed the prepared manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll the other authors declare no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eRinella ME, Lazarus JV, Ratziu V, Francque SM, Sanyal AJ, Kanwal F, et al. 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Bone. 2007;40(3):674\\u0026ndash;84.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"cell-death-and-disease\",\"isNatureJournal\":false,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"cddis\",\"sideBox\":\"Learn more about [Cell Death \\u0026 Disease](http://www.nature.com/cddis/)\",\"snPcode\":\"41419\",\"submissionUrl\":\"https://mts-cddis.nature.com/cgi-bin/main.plex\",\"title\":\"Cell Death \\u0026 Disease\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Nature AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4259782/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4259782/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eMetabolic disfunction-associated steatotic liver disease (MASLD) encompasses a plethora of hepatic disorders ranging from steatosis to steatohepatitis with the worst clinical outcome represented by cirrhosis, liver failure, and hepatocellular carcinoma. According to the lower MASLD prevalence reported in pre-menopausal women compared to men, we identified a potential protective role of estrogens in counteracting the oxidative stress during disease induction and progression. We have used preclinical relevant \\u003cem\\u003ein vitro\\u003c/em\\u003e models [i.e., immortalized cells and hepatocyte-like cells (HLC) derived from human embryonic stem cells (hESC)], exposed to sodium lactate, sodium pyruvate, and octanoic acid (LPO) to induce hepatic steatosis. This established practice of MASLD induction resulted in lipid droplet (LD) accumulation and increased mitochondrial and cytosolic reactive oxygen species (ROS) levels, paralleled by the reduction of several markers of hepatocyte function and differentiation. Here we found that estrogen replacement reduced ROS levels and LD content through the upregulation of mitochondrial thioredoxin 2 (TRX2), an antioxidant system that is under the control of the estrogen receptor alpha (hereafter referred as ER). Last, disrupting the TRX2 system using auranofin was sufficient to revert the scavenging effects exerted by estrogens, thus identifying a potential mechanism that could prevent or delay the progression of the disease.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Estrogen-dependent TRX2 activation reverts oxidative stress and metabolic dysfunction associated to steatotic disease\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-04-27 13:34:32\",\"doi\":\"10.21203/rs.3.rs-4259782/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"revise\",\"date\":\"2024-05-17T10:51:25+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"This content is not available.\",\"date\":\"2024-05-14T14:30:47+00:00\",\"index\":2,\"fulltext\":\"This content is not available.\"},{\"type\":\"editorInvitedReview\",\"content\":\"This content is not available.\",\"date\":\"2024-04-26T19:58:14+00:00\",\"index\":1,\"fulltext\":\"This content is not available.\"},{\"type\":\"reviewerAgreed\",\"content\":\"This content is not available.\",\"date\":\"2024-04-26T05:38:52+00:00\",\"index\":2,\"fulltext\":\"This content is not available.\"},{\"type\":\"reviewerAgreed\",\"content\":\"This content is not available.\",\"date\":\"2024-04-23T09:33:07+00:00\",\"index\":1,\"fulltext\":\"This content is not available.\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-04-23T07:03:44+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-04-15T11:15:25+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Cell Death \\u0026 Disease\",\"date\":\"2024-04-12T22:07:06+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-04-12T22:07:06+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"cell-death-and-disease\",\"isNatureJournal\":false,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"cddis\",\"sideBox\":\"Learn more about [Cell Death \\u0026 Disease](http://www.nature.com/cddis/)\",\"snPcode\":\"41419\",\"submissionUrl\":\"https://mts-cddis.nature.com/cgi-bin/main.plex\",\"title\":\"Cell Death \\u0026 Disease\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Nature AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"ed1e1d06-13c3-4c6c-b849-fcdcc9630ae2\",\"owner\":[],\"postedDate\":\"April 27th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":31032207,\"name\":\"Biological sciences/Cell biology\"},{\"id\":31032208,\"name\":\"Biological sciences/Molecular biology\"},{\"id\":31032209,\"name\":\"Health sciences/Diseases/Metabolic disorders\"}],\"tags\":[],\"updatedAt\":\"2025-02-01T08:05:59+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-4259782\",\"link\":\"https://doi.org/10.1038/s41419-025-07331-7\",\"journal\":{\"identity\":\"cell-death-and-disease\",\"isVorOnly\":false,\"title\":\"Cell Death \\u0026 Disease\"},\"publishedOn\":\"2025-01-31 05:00:00\",\"publishedOnDateReadable\":\"January 31st, 2025\"},\"versionCreatedAt\":\"2024-04-27 13:34:32\",\"video\":\"\",\"vorDoi\":\"10.1038/s41419-025-07331-7\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41419-025-07331-7\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4259782\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4259782\",\"identity\":\"rs-4259782\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}