Resmetirom Attenuates Atherosclerosis in ApoE ⁻/⁻ Mice by Suppressing the NF- κB/ROS–NLRP3 Inflammasome Axis

Resmetirom Attenuates Atherosclerosis in ApoE ⁻/⁻ Mice by Suppressing the NF- κB/ROS–NLRP3 Inflammasome Axis | 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 Research Article Resmetirom Attenuates Atherosclerosis in ApoE ⁻/⁻ Mice by Suppressing the NF- κB/ROS–NLRP3 Inflammasome Axis Xuedong Bai, Wenjie Fei, Jingzhou Fang, Chaomin Kong, Yaqi Xiang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8902352/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Atherosclerosis is a chronic inflammatory vascular disease in which the NLRP3 inflammasome and NF-κB signaling play central pathogenic roles. Resmetirom, a liver-selective thyroid hormone receptor β (THR-β) agonist approved for metabolic dysfunction-associated steatohepatitis (MASH), exhibits potent lipid-lowering effects, yet its anti-inflammatory actions in atherosclerosis remain unexplored. We aimed to investigate whether resmetirom attenuates atherogenesis through suppression of the NF-κB/ROS–NLRP3 inflammasome axis. Male ApoE ⁻/⁻ mice fed a high-fat diet for 8 weeks received vehicle, low-dose (3 mg/kg/day), or high-dose (10 mg/kg/day) resmetirom by oral gavage for an additional 8 weeks (n = 10/group). High-dose resmetirom reduced body weight gain by 50.8%, improved atherogenic dyslipidemia with approximate decreases of 35% in total cholesterol and 45% in LDL-cholesterol, and diminished aortic plaque burden as assessed by Oil Red O staining. Circulating inflammatory cytokines were markedly suppressed, with reductions in IL-1β (59%), IL-18 (62%), TNF-α (59%), and CRP (70%) compared with model controls. Western blot analysis revealed that aortic NLRP3, ASC, and caspase-1 protein levels decreased by 70–80%, findings corroborated by RT-qPCR and immunohistochemistry. Mechanistically, resmetirom reduced NF-κB p65 phosphorylation by approximately 50%, increased IκBα expression by 68%, and lowered aortic reactive oxygen species by 36%. Flow cytometry demonstrated that resmetirom repolarized lesional macrophages from a pro-inflammatory M1 toward an anti-inflammatory M2 phenotype, shifting the M1/M2 ratio from 2.28 to 0.62. These findings demonstrate that resmetirom exerts atheroprotective effects beyond lipid lowering by coordinately suppressing the NF-κB/ROS–NLRP3 inflammasome axis and reprogramming vascular macrophages, providing a mechanistic rationale for its therapeutic repurposing in atherosclerotic cardiovascular disease, particularly in patients with comorbid metabolic dysfunction. Atherosclerosis NLRP3 inflammasome Resmetirom Thyroid hormone receptor β NF-κB signaling Macrophage polarization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Atherosclerosis, once considered primarily a lipid-storage disorder, is now firmly established as a chronic inflammatory disease of the arterial wall [ 1 , 2 ]. Despite significant advances in lipid-lowering therapies, particularly statins, substantial residual cardiovascular risk persists, underscoring the need to target inflammatory pathways that drive plaque progression independently of cholesterol levels [ 3 , 4 ]. The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome has emerged as a central mediator of vascular inflammation in atherosclerosis [ 5 , 6 ]. Upon activation by danger signals such as oxidized low-density lipoprotein (ox-LDL) and cholesterol crystals, NLRP3 assembles with the adaptor protein ASC and pro-caspase-1 to form a multiprotein complex that cleaves pro-IL-1β and pro-IL-18 into their mature, bioactive forms [ 7 ]. These cytokines propagate inflammatory cascades within the vessel wall, promoting endothelial dysfunction, monocyte recruitment, and foam cell formation [ 8 ]. The Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) provided landmark clinical evidence that IL-1β neutralization reduces recurrent cardiovascular events independent of lipid modification, validating the inflammasome–IL-1β axis as a therapeutic target [ 9 ]. Upstream of NLRP3 activation, nuclear factor-κB (NF-κB) signaling serves as a master regulator of proinflammatory gene transcription, including NLRP3 itself, adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), and chemokines including monocyte chemoattractant protein-1 (MCP-1) [ 10 ]. Reactive oxygen species (ROS) further amplify this inflammatory circuit by triggering NF-κB nuclear translocation and directly priming NLRP3 [ 11 ]. Additionally, macrophage polarization toward the classically activated M1 phenotype sustains plaque inflammation and instability, whereas a shift toward the alternatively activated M2 phenotype promotes resolution and tissue repair [ 12 ]. Interventions capable of simultaneously suppressing NF-κB/ROS signaling and rebalancing macrophage phenotypes therefore hold considerable therapeutic promise. Thyroid hormone receptor β (THR-β), predominantly expressed in the liver, regulates cholesterol metabolism, lipoprotein clearance, and hepatic lipogenesis [ 13 ]. Resmetirom is a first-in-class, liver-directed THR-β agonist recently approved for metabolic dysfunction-associated steatohepatitis (MASH) with moderate-to-advanced fibrosis [ 14 ]. Beyond its metabolic effects, emerging evidence suggests that thyroid hormone signaling modulates inflammatory responses; however, whether resmetirom exerts direct anti-inflammatory actions relevant to atherosclerosis remains unexplored [ 15 ]. Given the mechanistic overlap between MASH and atherosclerosis—both characterized by dyslipidemia, oxidative stress, and NLRP3-driven inflammation—we hypothesized that resmetirom may attenuate atherogenesis through dual lipid-lowering and anti-inflammatory mechanisms. The present study employed high-fat diet (HFD)-fed apolipoprotein E-deficient (ApoE ⁻/⁻ ) mice, a well-validated model of accelerated atherosclerosis [ 16 ], to investigate the effects of resmetirom on plaque burden and to delineate its modulatory actions on the NF-κB/ROS–NLRP3 inflammasome axis and macrophage polarization. Materials and Methods Animals and Experimental Design Male C57BL/6J wild-type (WT) and ApoE ⁻/⁻ mice (8 weeks old, Beijing Vital River, China) were housed under SPF conditions with ad libitum access to food and water. All procedures were approved by the Ethics Committee of Hebei General Hospital (No. 202385). Forty-five mice were used: 10 WT (normal control, NC) fed standard chow, and 35 ApoE ⁻/⁻ mice fed a high-fat diet (HFD; 21% fat, 0.15% cholesterol; D12079B, Research Diets) for 8 weeks. After confirming model establishment (n = 5), the remaining 30 ApoE ⁻/⁻ mice were randomized into: model control (MC, vehicle), low-dose resmetirom (LD, 3 mg/kg/day), and high-dose resmetirom (HD, 10 mg/kg/day) groups (n = 10 each). Resmetirom (MGL-3196; MedChemExpress) suspended in 0.5% carboxymethylcellulose was administered orally for 8 weeks under continued HFD. Body weight was recorded weekly. Serum Biochemistry After 12-hour fasting, blood was collected by cardiac puncture under isoflurane anesthesia. Serum lipids (TC, TG, HDL-C, LDL-C) were measured enzymatically (Nanjing Jiancheng), and inflammatory cytokines (IL-1β, IL-18, TNF-α, CRP) by ELISA (Abcam). Histology and Immunohistochemistry Aortic roots were cryosectioned (8 µm) for H&E and Oil Red O staining, or paraffin-embedded (4 µm) for immunohistochemistry. Sections were incubated with antibodies against NLRP3, caspase-1, IL-1β, IL-18, MCP-1, and VCAM-1 (Cell Signaling Technology), followed by HRP-conjugated secondary antibodies and DAB visualization. Positive areas were quantified using Image-Pro Plus. Western Blotting Aortic lysates (30 µg protein) were separated by SDS-PAGE and immunoblotted for NLRP3, ASC, caspase-1, IL-1β, IL-18, MCP-1, total and phospho-p65 (Ser536), IκBα, and GAPDH (Cell Signaling Technology). Band intensities were quantified by densitometry (ImageJ) and normalized to GAPDH. The p-p65/total p65 ratio indicated NF-κB activation. RT-qPCR Total RNA extracted with TRIzol (Invitrogen) was reverse-transcribed (Takara) and analyzed by SYBR-based qPCR (Roche LightCycler 480) for NLRP3, caspase-1, IL-1β, IL-18, MCP-1, and VCAM-1. Expression was normalized to β-actin using the 2⁻ΔΔCt method. Primer sequences are available in the GitHub repository ( https://github.com/ghitamaclaurin-ops/codes.git , primer folder). Flow Cytometry Single-cell aortic suspensions were prepared by collagenase/DNase digestion and stained for CD45, F4/80, CD11b, CD86 (M1 marker), and CD206 (M2 marker) (BioLegend). M1 and M2 macrophages were defined as CD45⁺F4/80⁺CD11b⁺CD86⁺ and CD45⁺F4/80⁺CD11b⁺CD206⁺, respectively. ROS was detected using CellROX Deep Red (Thermo Fisher). Intracellular p-p65 and total p65 were quantified after permeabilization. Data were acquired on a Cytek Aurora and analyzed with FlowJo v10.8. Statistical Analysis Data are mean ± SD. Sample size (n = 10 per group) was determined based on prior atherosclerosis studies showing adequate power (> 0.80) to detect 30% differences in plaque burden and inflammatory markers with α = 0.05. Statistical analyses were performed using Python 3.9 with SciPy (v1.9.0), NumPy (v1.23.0), and Pandas (v1.5.0) libraries. Group comparisons used one-way ANOVA with Tukey’s post-hoc test (scipy.stats.f_oneway and statsmodels.stats.multicomp.pairwise_tukeyhsd) for normally distributed data, or Kruskal–Wallis test with Dunn’s correction (scipy.stats.kruskal and scikit-posthocs.posthoc_dunn) for non-parametric data. Normality was assessed by Shapiro-Wilk test. Visualizations were generated with Matplotlib (v3.6.0) and Seaborn (v0.12.0). P < 0.05 was considered significant. Results Resmetirom attenuates HFD‑induced body‑weight gain Baseline body weight after 8 weeks of HFD did not differ materially among ApoE ⁻/⁻ groups (MC 22.83 ± 1.14 g vs. NC 22.59 ± 1.14 g; p = 0.946; Fig. 1 A). After 8 weeks of treatment under continued HFD, MC mice exhibited marked weight gain (final weight 28.73 ± 0.90 g; gain 5.90 ± 0.60 g), whereas resmetirom reduced both final weight and weight gain in a dose‑dependent manner. LD lowered final body weight to 25.78 ± 0.92 g and weight gain to 3.50 ± 0.50 g (both p < 0.001 vs. MC), while HD further reduced final weight to 26.05 ± 0.63 g and weight gain to 2.90 ± 0.30 g (both p < 0.001 vs. MC), corresponding to an approximate 50.8% reduction in body‑weight gain compared with MC (Fig. 1 B,C). Resmetirom improves serum lipid profile Consistent with successful model induction, MC mice displayed a pronounced atherogenic lipid profile relative to NC, with higher total cholesterol (TC; 8.60 ± 0.52 vs. 4.52 ± 0.29 mmol/L), triglycerides (TG; 3.89 ± 0.34 vs. 1.20 ± 0.22 mmol/L), and LDL‑cholesterol (LDL‑C; 5.33 ± 0.32 vs. 2.06 ± 0.11 mmol/L), alongside reduced HDL‑cholesterol (HDL‑C; 0.80 ± 0.13 vs. 1.17 ± 0.09 mmol/L; all p < 0.001; Fig. 2 A–D). Resmetirom significantly improved this dyslipidaemia. LD reduced TC, TG and LDL‑C to 6.83 ± 0.37, 3.04 ± 0.28 and 4.12 ± 0.14 mmol/L, respectively (all p < 0.001 vs. MC), with a partial increase in HDL‑C (0.95 ± 0.13 mmol/L; p < 0.05 vs. MC). HD produced a more pronounced correction, lowering TC, TG and LDL‑C to 5.60 ± 0.16, 2.13 ± 0.20 and 2.94 ± 0.15 mmol/L (all p < 0.001 vs. MC), corresponding to approximate reductions of 35.0%, 45.3% and 44.9%, and increasing HDL‑C to 1.11 ± 0.07 mmol/L (+ 38.5% vs. MC; p < 0.001). Together, these data indicate robust dose‑dependent lipid‑lowering effects of resmetirom in ApoE ⁻/⁻ mice (Fig. 2 A–D). Resmetirom reduces aortic lipid deposition and plaque burden Oil Red O staining of aortic root cross-sections revealed marked differences in lipid deposition and plaque burden among groups (Fig. 3 A–D). NC mice showed minimal lipid accumulation with preserved aortic wall structure (Fig. 3 A). In contrast, MC animals exhibited extensive fibro‑fatty plaques with luminal narrowing and abundant lipid cores (Fig. 3 B). LD and, more prominently, HD resmetirom treatment markedly reduced plaque size and lipid content in a dose-dependent manner (Fig. 3 C,D). LD mice displayed thinner intimal lesions with reduced lipid cores, while HD mice showed significantly thinner plaques and better preserved lumen caliber compared with MC. These morphological findings provide structural evidence that resmetirom ameliorates aortic lipid accumulation and plaque burden in ApoE ⁻/⁻ mice. Resmetirom reduces systemic inflammatory cytokines Systemic inflammatory cytokines were markedly elevated in MC mice compared with NC (Fig. 4 A–D). MC serum IL‑1β, IL‑18, TNF‑α and CRP levels were 152.1 ± 10.4, 101.8 ± 6.9 and 82.2 ± 5.3 pg/mL, and 5.0 ± 0.5 mg/L, respectively, versus 50.4 ± 4.8, 30.0 ± 3.2 and 20.6 ± 1.1 pg/mL, and 1.0 ± 0.1 mg/L in NC (all p < 0.001). Resmetirom reduced these cytokines in a dose‑dependent fashion. LD decreased IL‑1β, IL‑18, TNF‑α and CRP to 96.6 ± 7.4, 70.6 ± 4.7 and 58.4 ± 2.8 pg/mL, and 2.9 ± 0.4 mg/L (all p < 0.001 vs. MC), whereas HD further lowered them to 61.9 ± 3.2, 38.5 ± 4.0 and 34.0 ± 2.2 pg/mL, and 1.5 ± 0.1 mg/L, corresponding to reductions of approximately 59.3%, 62.1%, 58.6% and 70.0% compared with MC (all p < 0.001). These findings demonstrate that resmetirom substantially attenuates systemic vascular inflammation associated with HFD‑induced atherosclerosis. Aortic NLRP3 inflammasome and adhesion molecule expression Immunohistochemistry (IHC) in the aortic wall showed increased staining of caspase‑1, IL‑1β, MCP‑1, IL‑18, NLRP3 and VCAM‑1 in MC mice compared with NC, consistent with activation of the NLRP3 inflammasome and endothelial activation (Fig. 5 A–F). Although most IHC indices in LD and HD groups showed clear numerical reductions versus MC, only NLRP3 staining in the HD group reached statistical significance (MC 4.10 vs. HD 1.00; p < 0.05 vs. MC), while changes in caspase‑1, IL‑1β, MCP‑1, IL‑18 and VCAM‑1 did not achieve the pre‑specified significance threshold (all p ≥ 0.05 vs. MC). At the mRNA level, RT‑qPCR confirmed partial suppression of inflammasome and adhesion pathway components (Fig. 5 G–L). MC mice exhibited robust up‑regulation of caspase‑1, IL‑1β, MCP‑1, IL‑18, NLRP3 and VCAM‑1 transcripts relative to NC. HD resmetirom significantly reduced caspase‑1 (MC 8.13 vs. HD 1.80‑fold; p < 0.05), IL‑1β (5.11 vs. 0.58‑fold; p < 0.01), NLRP3 (4.67 vs. 1.00‑fold; p < 0.05) and VCAM‑1 expression (3.14 vs. 0.52‑fold; p < 0.01 vs. MC), with LD also significantly lowering caspase‑1 (1.76‑fold; p < 0.05) and VCAM‑1 (1.37‑fold; p < 0.05 vs. MC). MCP‑1 and IL‑18 transcripts showed consistent downward trends in LD and HD groups, but did not reach statistical significance under the current sample size (p ≥ 0.05 vs. MC). Overall, these data indicate that resmetirom partially suppresses aortic NLRP3 inflammasome signalling and endothelial adhesion molecule expression at the transcript level, with more modest effects detectable by semi‑quantitative IHC. The observed discrepancy between robust molecular assay results and more modest IHC findings likely reflects the inherently lower sensitivity of semi‑quantitative immunohistochemistry compared to quantitative Western blotting and qPCR, as well as potential spatial heterogeneity in protein expression within atherosclerotic plaques. Western blot analysis of aortic lysates further corroborated down‑regulation of the NLRP3–ASC–caspase‑1 axis (Fig. 6 ). Compared with NC, MC mice displayed markedly increased protein abundance of NLRP3, ASC, caspase‑1, IL‑1β, IL‑18 and MCP‑1, whereas LD and especially HD resmetirom visibly reduced the intensity of these bands. Densitometric quantification revealed that HD resmetirom reduced NLRP3 protein by 78.3 ± 4.2%, ASC by 74.6 ± 5.1%, and caspase-1 by 71.2 ± 6.3% compared to MC controls (all p < 0.001), in line with the qPCR and cytokine data, indicating coordinated suppression of inflammasome activation at both transcriptional and protein levels. Resmetirom modulates NF‑κB activation, ROS and macrophage polarization Mechanistic analyses in aortic tissue further supported a direct anti‑inflammatory action of resmetirom on the NF‑κB/ROS–NLRP3 axis (Fig. 7 A–F). Compared with NC, MC mice displayed increased NF‑κB activation, as reflected by a higher p‑p65/total p65 ratio, elevated ROS intensity and an increased proportion of pro‑inflammatory M1 macrophages, accompanied by reduced IκBα and anti‑inflammatory M2 macrophages. Resmetirom attenuated these changes in a dose‑dependent manner. LD reduced the p‑p65/total p65 ratio to 1.44 ± 0.19 vs. 2.26 ± 0.21 in MC, and HD further suppressed it to 1.14 ± 0.17 (both p < 0.001 vs. MC), corresponding to an approximate 49.6% reduction with HD. In parallel, HD significantly increased IκBα expression (MC 0.55 ± 0.12 vs. HD 0.92 ± 0.08; +67.7%; p < 0.001), indicating reinforcement of NF‑κB inhibition. Consistent with reduced NF‑κB activity, HD resmetirom markedly decreased ROS levels (MC 186.36 ± 14.04 vs. HD 120.09 ± 9.25; −35.6%; p < 0.001) and shifted macrophage polarization toward an anti‑inflammatory phenotype. M1 macrophage frequency fell from 69.07 ± 7.97% in MC to 54.92 ± 4.15% in LD (p < 0.01) and 38.18 ± 4.87% in HD (p < 0.001), while M2 macrophages increased from 31.51 ± 5.72% in MC to 45.46 ± 6.30% in LD (p < 0.01) and 62.22 ± 5.84% in HD (p < 0.001). Consequently, the M1/M2 ratio decreased from 2.28 ± 0.61 in MC to 1.24 ± 0.29 in LD and 0.62 ± 0.13 in HD (both p < 0.001 vs. MC). Together, these data demonstrate that resmetirom not only improves lipid metabolism and systemic inflammatory cytokine profiles, but also directly dampens NF‑κB/ROS–NLRP3 signalling and repolarizes vascular macrophages toward an M2 phenotype, providing a mechanistic basis for its anti‑atherosclerotic efficacy in ApoE ⁻/⁻ mice. Discussion This study represents the first demonstration that resmetirom—a clinically approved, liver-selective THR-β agonist—exerts potent atheroprotective effects through coordinated suppression of the NF-κB/ROS–NLRP3 inflammasome axis. Whereas resmetirom was developed and approved for MASH based on its hepatic metabolic effects, our findings reveal a previously unrecognized dual mechanism encompassing both lipid-lowering and direct anti-inflammatory actions in the vessel wall. In HFD-fed ApoE ⁻/⁻ mice, resmetirom not only improved atherogenic dyslipidemia and reduced plaque burden, but also profoundly attenuated systemic inflammatory cytokines (IL-1β, IL-18, TNF-α, CRP), suppressed aortic NLRP3 inflammasome activation, and repolarized lesional macrophages toward a reparative M2 phenotype. These converging metabolic and anti-inflammatory mechanisms position resmetirom as a promising therapeutic repurposing candidate for atherosclerotic cardiovascular disease, particularly in patients with comorbid MASH who face elevated cardiovascular risk and would benefit from integrated hepato-vascular protection. NLRP3 inflammasome suppression: a central anti-inflammatory mechanism A key finding of this study is that resmetirom coordinately suppressed multiple components of the NLRP3 inflammasome cascade at transcriptional, translational, and functional levels. The NLRP3 inflammasome serves as a critical sensor of metabolic danger signals in atherosclerosis, including oxidized LDL, cholesterol crystals, and mitochondrial dysfunction-derived ROS [ 17 , 18 ]. Upon activation, the NLRP3–ASC–caspase-1 complex processes pro-IL-1β and pro-IL-18 into their mature forms, which amplify vascular inflammation and promote plaque vulnerability [ 6 , 19 ]. Our data demonstrated that high-dose resmetirom reduced NLRP3, ASC, and caspase-1 protein abundance by approximately 70–80% compared with model controls, with corresponding decreases in circulating IL-1β (59%) and IL-18 (62%). These reductions are comparable in magnitude to those achieved by direct NLRP3 inhibitors such as MCC950 in experimental atherosclerosis models [ 20 ]. The clinical relevance of targeting the IL-1β pathway in atherosclerosis was established by CANTOS, which demonstrated that canakinumab, a monoclonal antibody against IL-1β, reduced recurrent cardiovascular events by 15% independent of lipid lowering [ 9 ]. Similarly, the COLCOT trial showed that colchicine, which inhibits NLRP3 inflammasome assembly, decreased cardiovascular events in post-myocardial infarction patients [ 21 ]. Our findings suggest that resmetirom may represent an orally bioavailable alternative for inflammasome suppression, with the added advantage of concurrent lipid-lowering effects. NF-κB inhibition and ROS reduction: upstream regulatory mechanisms The NF-κB signaling pathway functions as the master transcriptional regulator of NLRP3 inflammasome priming, controlling the expression of NLRP3, pro-IL-1β, and adhesion molecules including VCAM-1 and MCP-1 [ 10 , 22 ]. In atherosclerotic lesions, persistent NF-κB activation sustains a feed-forward inflammatory loop that perpetuates plaque progression [ 23 ]. Our observation that resmetirom reduced NF-κB p65 phosphorylation by approximately 50% while increasing IκBα expression by 68% indicates effective interruption of this proinflammatory cascade at its proximal signaling node. ROS serve dual roles in inflammasome regulation: as direct activators of NLRP3 through thioredoxin-interacting protein (TXNIP) dissociation, and as amplifiers of NF-κB signaling via IκB kinase activation [ 11 , 24 ]. The 36% reduction in aortic ROS levels observed with high-dose resmetirom likely contributes to both diminished NLRP3 activation and reduced NF-κB nuclear translocation. Thyroid hormones have been shown to modulate mitochondrial function and oxidative stress through effects on uncoupling proteins and antioxidant enzyme expression [ 25 ]. Whether resmetirom’s ROS-lowering effects reflect direct hepatic actions, improved systemic metabolism, or secondary consequences of reduced inflammatory burden warrants further investigation. Macrophage repolarization: implications for plaque stability Macrophage phenotypic heterogeneity profoundly influences atherosclerotic plaque fate [ 12 , 26 ]. Classically activated M1 macrophages predominate in unstable plaques, secreting proinflammatory cytokines and matrix metalloproteinases that promote plaque rupture, whereas alternatively activated M2 macrophages facilitate inflammation resolution, efferocytosis, and tissue repair [ 27 ]. The dramatic shift in M1/M2 ratio from 2.28 in model controls to 0.62 with high-dose resmetirom represents a fundamental reprogramming of the lesional inflammatory milieu. This macrophage repolarization likely results from the combined effects of reduced NF-κB–dependent M1 polarizing signals, decreased ROS-mediated inflammatory priming, and attenuated IL-1β/IL-18 autocrine amplification loops [ 28 ]. Notably, thyroid hormone signaling has been implicated in modulating inflammatory responses and immune cell function [ 29 ]. Whether resmetirom exerts direct effects on vascular macrophages or influences polarization primarily through hepatic-derived factors such as altered lipoprotein composition or secreted anti-inflammatory mediators remains to be elucidated. Hepatic-vascular inflammatory crosstalk: mechanistic considerations The liver occupies a central position in the metabolic-inflammatory network linking MASH and atherosclerosis [ 30 , 31 ]. Both conditions share common pathogenic drivers including dyslipidemia, insulin resistance, oxidative stress, and NLRP3-mediated inflammation. Hepatic inflammation in MASH generates circulating proinflammatory mediators—including IL-1β, IL-6, and CRP—that promote systemic endothelial dysfunction and accelerate atherogenesis [ 32 ]. Conversely, atherogenic lipoproteins exacerbate hepatic lipotoxicity and inflammasome activation, establishing a bidirectional pathogenic axis [ 33 ]. Resmetirom’s liver-selective THR-β agonism positions it uniquely to interrupt this hepatic-vascular inflammatory crosstalk. By improving hepatic lipid handling, reducing hepatocyte lipotoxic stress, and suppressing hepatic NF-κB/NLRP3 activation, resmetirom may attenuate the systemic inflammatory burden that drives vascular disease [ 14 ]. The pronounced reductions in circulating CRP (70%) and TNF-α (59%) observed in our study support this hepatocentric anti-inflammatory mechanism. Future studies employing tissue-specific knockouts or hepatocyte-targeted interventions will be essential to dissect the relative contributions of hepatic versus extrahepatic THR-β signaling to vascular protection. Comparison with other anti-inflammatory approaches in atherosclerosis Several anti-inflammatory strategies have demonstrated efficacy in experimental and clinical atherosclerosis. Direct IL-1β neutralization with canakinumab (CANTOS) and NLRP3 inhibition with colchicine (COLCOT, LoDoCo2) have validated inflammation as a therapeutic target [ 9 , 21 , 34 ]. However, these approaches carry limitations: canakinumab is expensive and immunosuppressive, while colchicine has modest efficacy and gastrointestinal tolerability concerns [ 35 ]. Resmetirom offers a differentiated profile as an orally administered agent with dual lipid-lowering and anti-inflammatory properties. In MASH clinical trials, resmetirom achieved LDL-C reductions of 13–16% alongside improvements in hepatic inflammation and fibrosis, with a favorable safety profile [ 14 , 36 ]. The anti-atherosclerotic effects observed in our preclinical study—encompassing both metabolic correction and inflammasome suppression—suggest potential for additive or synergistic benefits in patients with combined metabolic and inflammatory cardiovascular risk. Limitations and future directions Several limitations merit consideration. First, the ApoE ⁻/⁻ mouse model, while well-validated, may not fully recapitulate human atherosclerosis heterogeneity, particularly regarding plaque rupture and thrombotic complications. Second, our 8-week treatment duration assessed plaque burden but not long-term stability or regression. Third, although resmetirom is a liver-selective THR-β agonist, we did not directly measure hepatic inflammasome activity, hepatic cytokine production, or circulating hepatokines that might mediate vascular effects. This limits our ability to definitively distinguish hepatic-mediated systemic anti-inflammatory effects from potential direct actions on vascular THR-β signaling. The pronounced reductions in circulating CRP and cytokines support a hepatocentric mechanism, but future studies employing hepatocyte-specific THR-β knockout models will be essential to dissect tissue-specific contributions. Fourth, semi-quantitative IHC showed more modest changes than molecular assays, reflecting inherent methodological limitations in detecting graded protein expression differences, as discussed above. Future investigations should address several priorities: (1) longer-term studies assessing plaque composition, stability markers (collagen content, fibrous cap thickness), and vulnerability indices; (2) mechanistic studies in hepatocyte-specific THR-β knockout models to define the contribution of hepatic versus systemic effects; (3) evaluation of combination approaches with statins, PCSK9 inhibitors, or colchicine to assess additive anti-inflammatory efficacy; and (4) dedicated cardiovascular outcome trials in MASH patients, who harbor elevated atherosclerotic risk and may derive particular benefit from resmetirom’s dual hepatic and vascular protection. Conclusions This study establishes resmetirom as a potent suppressor of vascular inflammation in experimental atherosclerosis, acting through coordinated inhibition of the NF-κB/ROS–NLRP3 inflammasome axis and favorable reprogramming of lesional macrophage phenotypes. These anti-inflammatory effects, combined with robust lipid-lowering activity, provide a compelling mechanistic rationale for investigating resmetirom as an atheroprotective therapy, particularly in patients with metabolic dysfunction-associated steatotic liver disease who face elevated cardiovascular risk. Our findings expand the therapeutic potential of THR-β agonism beyond hepatic steatosis to encompass the broader spectrum of metabolic-inflammatory cardiovascular disease. Declarations Funding This work was supported by 2025 Hebei Province Medical Science Research Project Plan (Grant No. 20250296) and the 2021 Government-funded Clinical Medicine Outstanding Talent Training Program (“Study on the regulation of thyroid hormone receptor β1 on glucose and lipid metabolism and its mechanisms”). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Ethics Approval All animal experimental procedures were conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Hebei General Hospital (Approval No. 202385). The study was performed in line with ethical standards for animal research. All efforts were made to minimize animal suffering and reduce the number of animals used. Clinical trial number Not applicable. Consent to Participate Not applicable (animal study). Consent to Publish Not applicable. Data Availability The datasets generated during the current study are available from the corresponding author upon reasonable request. All Python statistical analysis scripts are publicly available at https://github.com/ghitamaclaurin-ops/codes.git Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Xuedong Bai, Wenjie Fei, Jingzhou Fang, Chaomin Kong, Yaqi Xiang, and Yuele Tian. The first draft of the manuscript was written by Xuedong Bai and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability The datasets generated during the current study are available from the corresponding author upon reasonable request. All Python statistical analysis scripts are publicly available at https://github.com/ghitamaclaurin-ops/codes.git References Libby, P. 2021. The changing landscape of atherosclerosis. Nature 592:524–533. https://doi.org/10.1038/s41586-021-03392-8 Ridker, P. M. 2016. From C-reactive protein to interleukin-6 to interleukin-1: moving upstream to identify novel targets for atheroprotection. Circulation Research 118:145–156. https://doi.org/10.1161/CIRCRESAHA.115.306656 Sampson, U. K., S. Fazio, and M. F. Linton. 2012. 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PLoS One 5:e11765. https://doi.org/10.1371/journal.pone.0011765 Hendrikx, T., M. L. Jeurissen, and P. J. van Gorp et al. 2015. Bone marrow-specific caspase-1/11 deficiency inhibits atherosclerosis development in Ldlr(-/-) mice. FEBS Journal 282:2327–2338. https://doi.org/10.1111/febs.13279 Shi, J., Y. Zhao, and K. Wang et al. 2015. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526:660–665. https://doi.org/10.1038/nature15514 van der Heijden, T., E. Kritikou, and W. Venema et al. 2017. NLRP3 inflammasome inhibition by MCC950 reduces atherosclerotic lesion development in apolipoprotein E-deficient mice. Arteriosclerosis Thrombosis and Vascular Biology 37:1457–1461. https://doi.org/10.1161/ATVBAHA.117.309575 Tardif, J. C., S. Kouz, and D. D. Waters et al. 2019. Efficacy and safety of low-dose colchicine after myocardial infarction. New England Journal of Medicine 381:2497–2505. https://doi.org/10.1056/NEJMoa1912388 Liu, T., L. Zhang, D. Joo, and S. C. Sun. 2017. NF-κB signaling in inflammation. Signal Transduction and Targeted Therapy 2:17023. https://doi.org/10.1038/sigtrans.2017.23 Brand, K., S. Page, and G. Rogler et al. 1996. Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. Journal of Clinical Investigation 97:1715–1722. https://doi.org/10.1172/JCI118598 Heid, M. E., P. A. Keyel, and C. Kamga et al. 2013. Mitochondrial reactive oxygen species induces NLRP3-dependent lysosomal damage and inflammasome activation. Journal of Immunology 191:5230–5238. https://doi.org/10.4049/jimmunol.1301490 Venditti, P., and S. Di Meo. 2006. Thyroid hormone-induced oxidative stress. Cellular and Molecular Life Sciences 63:414–434. https://doi.org/10.1007/s00018-005-5457-9 Chinetti-Gbaguidi, G., S. Colin, and B. Staels. 2015. Macrophage subsets in atherosclerosis. Nature Reviews Cardiology 12:10–17. https://doi.org/10.1038/nrcardio.2014.173 Tabas, I., and K. E. Bornfeldt. 2016. Macrophage phenotype and function in different stages of atherosclerosis. Circulation Research 118:653–667. https://doi.org/10.1161/CIRCRESAHA.115.306256 Saha, S., I. N. Shalova, and S. K. Biswas. 2017. Metabolic regulation of macrophage phenotype and function. Immunological Reviews 280:102–111. https://doi.org/10.1111/imr.12603 Jabbar, A., A. Pingitore, and S. H. Pearce et al. 2017. Thyroid hormones and cardiovascular disease. Nature Reviews Cardiology 14:39–55. https://doi.org/10.1038/nrcardio.2016.174 Bieghs, V., S. M. Walenbergh, and T. Hendrikx et al. 2013. Trapping of oxidized LDL in lysosomes of Kupffer cells is a trigger for hepatic inflammation. Liver International 33:1056–1061. https://doi.org/10.1111/liv.12170 Kasper, P., A. Martin, and S. Lang et al. 2021. NAFLD and cardiovascular diseases: a clinical review. Clinical Research in Cardiology 110:921–937. https://doi.org/10.1007/s00392-020-01709-7 Targher, G., C. D. Byrne, and H. Tilg. 2020. NAFLD and increased risk of cardiovascular disease: clinical associations, pathophysiological mechanisms and pharmacological implications. Gut 69:1691–1705. https://doi.org/10.1136/gutjnl-2020-320622 Bieghs, V., F. Verheyen, and P. J. van Gorp et al. 2012. Internalization of modified lipids by CD36 and SR-A leads to hepatic inflammation and lysosomal cholesterol storage in Kupffer cells. PLoS One 7:e34378. https://doi.org/10.1371/journal.pone.0034378 Nidorf, S. M., A. T. L. Fiolet, and A. Mosterd et al. 2020. Colchicine in patients with chronic coronary disease. New England Journal of Medicine 383:1838–1847. https://doi.org/10.1056/NEJMoa2021372 Ridker, P. M., and M. Rane. 2021. Interleukin-6 signaling and anti-interleukin-6 therapeutics in cardiovascular disease. Circulation Research 128:1728–1746. https://doi.org/10.1161/CIRCRESAHA.121.319077 Harrison, S. A., M. R. Bashir, and C. D. Guy et al. 2019. Resmetirom (MGL-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial. Lancet 394:2012–2024. https://doi.org/10.1016/S0140-6736(19)32517-6 Additional Declarations No competing interests reported. Supplementary Files figuresepsRAW.zip Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8902352","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":595083018,"identity":"ed8f6c68-3ae6-4dcc-8318-2af4089950cb","order_by":0,"name":"Xuedong Bai","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xuedong","middleName":"","lastName":"Bai","suffix":""},{"id":595083019,"identity":"c2247078-bf7f-49b1-924c-639a24956b5c","order_by":1,"name":"Wenjie Fei","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenjie","middleName":"","lastName":"Fei","suffix":""},{"id":595083020,"identity":"37671162-6238-4e6a-9362-75ca84df89b3","order_by":2,"name":"Jingzhou Fang","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingzhou","middleName":"","lastName":"Fang","suffix":""},{"id":595083021,"identity":"b58846b1-39bd-4466-a2d6-9d7515af306c","order_by":3,"name":"Chaomin Kong","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chaomin","middleName":"","lastName":"Kong","suffix":""},{"id":595083022,"identity":"3effe803-5d6c-40f6-b131-e3db1c40d6b7","order_by":4,"name":"Yaqi Xiang","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yaqi","middleName":"","lastName":"Xiang","suffix":""},{"id":595083023,"identity":"77e1e252-8d22-48cb-95a7-051641c7677c","order_by":5,"name":"Yuele Tian","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuele","middleName":"","lastName":"Tian","suffix":""},{"id":595083024,"identity":"13c462cd-3feb-4900-a5bb-63a196a4135e","order_by":6,"name":"Limin Wei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIie3RsQ6CMBCA4ZomdWlkPSKBV7iEhOehMXHShJGBoUYDg3H3MdjUTZZOdXeE+AKwOThIwqgB3Bz6zf1z7ZUQw/hDzCu2VY0Jt7xHUYZxMpzMgKY+RMq15XKBpVbDiQtTCbymPspVYFc7OuJi841EQCZycg1iIRmxsn3YnziFLCN0xGkil3dxdgjoW96fENFNuWyJugvNCMJ6OAGOVORqkkYipSMS6BIfNW3zUQkv2iVju+QjoxBqxQff4mXZo6pf7VeC1TTPOHGt7NCffOC/HTcMwzC+egMlSkjX6EmIkwAAAABJRU5ErkJggg==","orcid":"","institution":"Hebei Medical University","correspondingAuthor":true,"prefix":"","firstName":"Limin","middleName":"","lastName":"Wei","suffix":""}],"badges":[],"createdAt":"2026-02-17 15:09:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8902352/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8902352/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103347816,"identity":"3c75a55e-3779-4772-bd4c-0cd58ba82e9c","added_by":"auto","created_at":"2026-02-24 16:29:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":57444,"visible":true,"origin":"","legend":"\u003cp\u003eResmetirom attenuates HFD-induced body weight gain in ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mice. A Baseline body weight after 8 weeks of HFD acclimation, prior to treatment initiation. B Terminal body weight following 8 weeks of vehicle or resmetirom treatment under continued HFD. C Absolute weight gain calculated as the difference between terminal and baseline body weight. Bars represent mean ± SD (n=10 per group). One-way ANOVA with Tukey’s post-hoc test. P\u0026lt;0.05, P\u0026lt;0.01, P\u0026lt;0.001 vs MC.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8902352/v1/052b00913dc06003a12e0f34.png"},{"id":103507054,"identity":"11ec53e5-d50e-47f3-8f3f-9fc09c37a5ca","added_by":"auto","created_at":"2026-02-26 13:40:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99078,"visible":true,"origin":"","legend":"\u003cp\u003eDose-dependent improvement of atherogenic dyslipidemia by resmetirom in ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mice. A Serum total cholesterol (TC) concentrations. B Serum triglyceride (TG) levels. C Low-density lipoprotein cholesterol (LDL-C). D High-density lipoprotein cholesterol (HDL-C). Bars show mean ± SD (n=10 per group). One-way ANOVA with Tukey’s post-hoc test. P\u0026lt;0.05, P\u0026lt;0.01, P\u0026lt;0.001 vs MC.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8902352/v1/ee1968f80cf02930bad18b24.png"},{"id":103347817,"identity":"8b154941-2fd1-4604-8f9d-c6b01ee7bc1f","added_by":"auto","created_at":"2026-02-24 16:29:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":640740,"visible":true,"origin":"","legend":"\u003cp\u003eResmetirom attenuates aortic lipid accumulation and atherosclerotic plaque burden in ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mice. Representative Oil Red O-stained aortic root cross-sections from A Normal Control (NC), B Model Control (MC), C Low-dose resmetirom (LD), and D High-dose resmetirom (HD) groups. Each panel shows progressive magnifications (left to right; scale bars: 200 μm, 100 μm, 50 μm). Red staining indicates lipid deposition and atherosclerotic plaques. NC mice show minimal lipid accumulation with preserved aortic wall structure. MC mice exhibit extensive fibro-fatty plaques with large lipid cores and marked luminal narrowing. LD and HD groups demonstrate dose-dependent reduction in plaque burden, with HD showing significantly thinner intimal lesions and better-preserved lumen caliber compared to MC.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8902352/v1/02ae39112399604352842dd9.png"},{"id":103347822,"identity":"73c5f057-3158-4275-b5ff-ce78b80b40d5","added_by":"auto","created_at":"2026-02-24 16:29:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":92096,"visible":true,"origin":"","legend":"\u003cp\u003eResmetirom suppresses circulating inflammatory mediators in HFD-fed ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mice. A Serum IL-1β concentrations measured by ELISA. B Serum IL-18 concentrations measured by ELISA. C Serum TNF-α concentrations measured by ELISA. D Serum C-reactive protein (CRP) levels. Bars show mean ± SD (n=10 per group). One-way ANOVA with Tukey’s post-hoc test. P\u0026lt;0.05, P\u0026lt;0.01, P\u0026lt;0.001 vs MC.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8902352/v1/1e797c09f335749606cd2452.png"},{"id":103506972,"identity":"46a175b0-d093-4c1a-a216-7315fa83f4b4","added_by":"auto","created_at":"2026-02-26 13:40:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":221856,"visible":true,"origin":"","legend":"\u003cp\u003eResmetirom suppresses NLRP3 inflammasome components and endothelial adhesion molecules at protein and mRNA levels. A-F Immunohistochemistry staining and quantification of (A) caspase-1, (B) IL-1β, (C) MCP-1, (D) IL-18, (E) NLRP3, and (F) VCAM-1 in aortic wall sections. G-L RT-qPCR quantification of mRNA expression (fold change relative to NC) for (G) caspase-1, (H) IL-1β, (I) MCP-1, (J) IL-18, (K) NLRP3, and (L) VCAM-1. Bars represent mean ± SD (n=10 per group). IHC: positive staining area quantified using Image-Pro Plus software. RT-qPCR: fold change normalized to β-actin using 2⁻ΔΔCt method. One-way ANOVA with Tukey’s post-hoc test. P\u0026lt;0.05, P\u0026lt;0.01, P\u0026lt;0.001 vs MC.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8902352/v1/86e7cb3c4664a57d0964875f.png"},{"id":103347819,"identity":"bcbde41d-edc8-43bc-8894-f7311a3c115e","added_by":"auto","created_at":"2026-02-24 16:29:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":114782,"visible":true,"origin":"","legend":"\u003cp\u003eResmetirom suppresses aortic protein expression of NLRP3 inflammasome components in ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mice. Representative Western blot images showing protein levels of NLRP3, ASC, caspase-1, IL-1β, IL-18, and MCP-1 in aortic lysates from NC (Normal Control), MC (Model Control), LD (Low-dose resmetirom), and HD (High-dose resmetirom) groups. GAPDH serves as loading control. MC mice display markedly increased protein abundance of the NLRP3–ASC–caspase-1 axis and associated inflammatory cytokines compared to NC. LD and HD resmetirom treatment produces dose-dependent reduction in all target proteins, with HD showing the most pronounced suppression of inflammasome components.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8902352/v1/fd55e76933ff2de2b5e1ccd9.png"},{"id":103347821,"identity":"166e3d7d-1bbf-4219-8511-a3eb56c96fb9","added_by":"auto","created_at":"2026-02-24 16:29:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":121157,"visible":true,"origin":"","legend":"\u003cp\u003eResmetirom suppresses NF-κB/ROS signaling and repolarizes aortic macrophages toward anti-inflammatory M2 phenotype. A Ratio of phosphorylated p65 (Ser536) to total p65 protein, indicating NF-κB activation status. B IκBα protein expression levels (NF-κB inhibitor). C Reactive oxygen species (ROS) intensity measured by flow cytometry using CellROX Deep Red probe. D Percentage of M1 macrophages (CD45⁺F4/80⁺CD11b⁺CD86⁺) among total aortic macrophages. E Percentage of M2 macrophages (CD45⁺F4/80⁺CD11b⁺CD206⁺) among total aortic macrophages. F M1/M2 macrophage ratio. Bars represent mean ± SD (n=10 per group). Flow cytometry data acquired on Cytek Aurora and analyzed with FlowJo v10.8. One-way ANOVA with Tukey’s post-hoc test. P\u0026lt;0.05, P\u0026lt;0.01, P\u0026lt;0.001 vs MC.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8902352/v1/33f27f7b632cbbbb849f124b.png"},{"id":105901426,"identity":"9771e8f7-26d9-4a87-a3cc-de15cd0eec9e","added_by":"auto","created_at":"2026-04-01 09:28:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1746565,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8902352/v1/790a2d76-47e2-4be8-b647-c7cf2fa78ebb.pdf"},{"id":103347823,"identity":"51075169-9917-49f5-b3f0-f46f57562b79","added_by":"auto","created_at":"2026-02-24 16:29:33","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":60121175,"visible":true,"origin":"","legend":"","description":"","filename":"figuresepsRAW.zip","url":"https://assets-eu.researchsquare.com/files/rs-8902352/v1/80b4bc2a902b2e6cfa16600d.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Resmetirom Attenuates Atherosclerosis in ApoE ⁻/⁻ Mice by Suppressing the NF- κB/ROS–NLRP3 Inflammasome Axis","fulltext":[{"header":"Background","content":"\u003cp\u003eAtherosclerosis, once considered primarily a lipid-storage disorder, is now firmly established as a chronic inflammatory disease of the arterial wall [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite significant advances in lipid-lowering therapies, particularly statins, substantial residual cardiovascular risk persists, underscoring the need to target inflammatory pathways that drive plaque progression independently of cholesterol levels [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome has emerged as a central mediator of vascular inflammation in atherosclerosis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Upon activation by danger signals such as oxidized low-density lipoprotein (ox-LDL) and cholesterol crystals, NLRP3 assembles with the adaptor protein ASC and pro-caspase-1 to form a multiprotein complex that cleaves pro-IL-1β and pro-IL-18 into their mature, bioactive forms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These cytokines propagate inflammatory cascades within the vessel wall, promoting endothelial dysfunction, monocyte recruitment, and foam cell formation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) provided landmark clinical evidence that IL-1β neutralization reduces recurrent cardiovascular events independent of lipid modification, validating the inflammasome\u0026ndash;IL-1β axis as a therapeutic target [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUpstream of NLRP3 activation, nuclear factor-κB (NF-κB) signaling serves as a master regulator of proinflammatory gene transcription, including NLRP3 itself, adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), and chemokines including monocyte chemoattractant protein-1 (MCP-1) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Reactive oxygen species (ROS) further amplify this inflammatory circuit by triggering NF-κB nuclear translocation and directly priming NLRP3 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, macrophage polarization toward the classically activated M1 phenotype sustains plaque inflammation and instability, whereas a shift toward the alternatively activated M2 phenotype promotes resolution and tissue repair [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Interventions capable of simultaneously suppressing NF-κB/ROS signaling and rebalancing macrophage phenotypes therefore hold considerable therapeutic promise.\u003c/p\u003e \u003cp\u003eThyroid hormone receptor β (THR-β), predominantly expressed in the liver, regulates cholesterol metabolism, lipoprotein clearance, and hepatic lipogenesis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Resmetirom is a first-in-class, liver-directed THR-β agonist recently approved for metabolic dysfunction-associated steatohepatitis (MASH) with moderate-to-advanced fibrosis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Beyond its metabolic effects, emerging evidence suggests that thyroid hormone signaling modulates inflammatory responses; however, whether resmetirom exerts direct anti-inflammatory actions relevant to atherosclerosis remains unexplored [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the mechanistic overlap between MASH and atherosclerosis\u0026mdash;both characterized by dyslipidemia, oxidative stress, and NLRP3-driven inflammation\u0026mdash;we hypothesized that resmetirom may attenuate atherogenesis through dual lipid-lowering and anti-inflammatory mechanisms. The present study employed high-fat diet (HFD)-fed apolipoprotein E-deficient (ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e) mice, a well-validated model of accelerated atherosclerosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], to investigate the effects of resmetirom on plaque burden and to delineate its modulatory actions on the NF-κB/ROS\u0026ndash;NLRP3 inflammasome axis and macrophage polarization.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eAnimals and Experimental Design\u003c/p\u003e \u003cp\u003eMale C57BL/6J wild-type (WT) and ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mice (8 weeks old, Beijing Vital River, China) were housed under SPF conditions with \u003cem\u003ead libitum\u003c/em\u003e access to food and water. All procedures were approved by the Ethics Committee of Hebei General Hospital (No. 202385). Forty-five mice were used: 10 WT (normal control, NC) fed standard chow, and 35 ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mice fed a high-fat diet (HFD; 21% fat, 0.15% cholesterol; D12079B, Research Diets) for 8 weeks. After confirming model establishment (n\u0026thinsp;=\u0026thinsp;5), the remaining 30 ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mice were randomized into: model control (MC, vehicle), low-dose resmetirom (LD, 3 mg/kg/day), and high-dose resmetirom (HD, 10 mg/kg/day) groups (n\u0026thinsp;=\u0026thinsp;10 each). Resmetirom (MGL-3196; MedChemExpress) suspended in 0.5% carboxymethylcellulose was administered orally for 8 weeks under continued HFD. Body weight was recorded weekly.\u003c/p\u003e \u003cp\u003eSerum Biochemistry\u003c/p\u003e \u003cp\u003eAfter 12-hour fasting, blood was collected by cardiac puncture under isoflurane anesthesia. Serum lipids (TC, TG, HDL-C, LDL-C) were measured enzymatically (Nanjing Jiancheng), and inflammatory cytokines (IL-1β, IL-18, TNF-α, CRP) by ELISA (Abcam).\u003c/p\u003e \u003cp\u003eHistology and Immunohistochemistry\u003c/p\u003e \u003cp\u003eAortic roots were cryosectioned (8 \u0026micro;m) for H\u0026amp;E and Oil Red O staining, or paraffin-embedded (4 \u0026micro;m) for immunohistochemistry. Sections were incubated with antibodies against NLRP3, caspase-1, IL-1β, IL-18, MCP-1, and VCAM-1 (Cell Signaling Technology), followed by HRP-conjugated secondary antibodies and DAB visualization. Positive areas were quantified using Image-Pro Plus.\u003c/p\u003e \u003cp\u003eWestern Blotting\u003c/p\u003e \u003cp\u003eAortic lysates (30 \u0026micro;g protein) were separated by SDS-PAGE and immunoblotted for NLRP3, ASC, caspase-1, IL-1β, IL-18, MCP-1, total and phospho-p65 (Ser536), IκBα, and GAPDH (Cell Signaling Technology). Band intensities were quantified by densitometry (ImageJ) and normalized to GAPDH. The p-p65/total p65 ratio indicated NF-κB activation.\u003c/p\u003e \u003cp\u003eRT-qPCR\u003c/p\u003e \u003cp\u003eTotal RNA extracted with TRIzol (Invitrogen) was reverse-transcribed (Takara) and analyzed by SYBR-based qPCR (Roche LightCycler 480) for NLRP3, caspase-1, IL-1β, IL-18, MCP-1, and VCAM-1. Expression was normalized to β-actin using the 2⁻ΔΔCt method. Primer sequences are available in the GitHub repository (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/ghitamaclaurin-ops/codes.git\u003c/span\u003e\u003cspan address=\"https://github.com/ghitamaclaurin-ops/codes.git\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, primer folder).\u003c/p\u003e \u003cp\u003eFlow Cytometry\u003c/p\u003e \u003cp\u003eSingle-cell aortic suspensions were prepared by collagenase/DNase digestion and stained for CD45, F4/80, CD11b, CD86 (M1 marker), and CD206 (M2 marker) (BioLegend). M1 and M2 macrophages were defined as CD45⁺F4/80⁺CD11b⁺CD86⁺ and CD45⁺F4/80⁺CD11b⁺CD206⁺, respectively. ROS was detected using CellROX Deep Red (Thermo Fisher). Intracellular p-p65 and total p65 were quantified after permeabilization. Data were acquired on a Cytek Aurora and analyzed with FlowJo v10.8.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Sample size (n\u0026thinsp;=\u0026thinsp;10 per group) was determined based on prior atherosclerosis studies showing adequate power (\u0026gt;\u0026thinsp;0.80) to detect 30% differences in plaque burden and inflammatory markers with α\u0026thinsp;=\u0026thinsp;0.05. Statistical analyses were performed using Python 3.9 with SciPy (v1.9.0), NumPy (v1.23.0), and Pandas (v1.5.0) libraries. Group comparisons used one-way ANOVA with Tukey\u0026rsquo;s post-hoc test (scipy.stats.f_oneway and statsmodels.stats.multicomp.pairwise_tukeyhsd) for normally distributed data, or Kruskal\u0026ndash;Wallis test with Dunn\u0026rsquo;s correction (scipy.stats.kruskal and scikit-posthocs.posthoc_dunn) for non-parametric data. Normality was assessed by Shapiro-Wilk test. Visualizations were generated with Matplotlib (v3.6.0) and Seaborn (v0.12.0). P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eResmetirom attenuates HFD‑induced body‑weight gain\u003c/p\u003e \u003cp\u003eBaseline body weight after 8 weeks of HFD did not differ materially among ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e groups (MC 22.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14 g vs. NC 22.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14 g; p\u0026thinsp;=\u0026thinsp;0.946; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). After 8 weeks of treatment under continued HFD, MC mice exhibited marked weight gain (final weight 28.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90 g; gain 5.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60 g), whereas resmetirom reduced both final weight and weight gain in a dose‑dependent manner. LD lowered final body weight to 25.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92 g and weight gain to 3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 g (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. MC), while HD further reduced final weight to 26.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 g and weight gain to 2.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 g (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. MC), corresponding to an approximate 50.8% reduction in body‑weight gain compared with MC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB,C).\u003c/p\u003e \u003cp\u003eResmetirom improves serum lipid profile\u003c/p\u003e \u003cp\u003eConsistent with successful model induction, MC mice displayed a pronounced atherogenic lipid profile relative to NC, with higher total cholesterol (TC; 8.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 vs. 4.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 mmol/L), triglycerides (TG; 3.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 vs. 1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 mmol/L), and LDL‑cholesterol (LDL‑C; 5.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 vs. 2.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 mmol/L), alongside reduced HDL‑cholesterol (HDL‑C; 0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 vs. 1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 mmol/L; all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;D). Resmetirom significantly improved this dyslipidaemia. LD reduced TC, TG and LDL‑C to 6.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37, 3.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 and 4.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 mmol/L, respectively (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. MC), with a partial increase in HDL‑C (0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mmol/L; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. MC). HD produced a more pronounced correction, lowering TC, TG and LDL‑C to 5.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16, 2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 and 2.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 mmol/L (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. MC), corresponding to approximate reductions of 35.0%, 45.3% and 44.9%, and increasing HDL‑C to 1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 mmol/L (+\u0026thinsp;38.5% vs. MC; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Together, these data indicate robust dose‑dependent lipid‑lowering effects of resmetirom in ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;D).\u003c/p\u003e \u003cp\u003eResmetirom reduces aortic lipid deposition and plaque burden\u003c/p\u003e \u003cp\u003eOil Red O staining of aortic root cross-sections revealed marked differences in lipid deposition and plaque burden among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;D). NC mice showed minimal lipid accumulation with preserved aortic wall structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In contrast, MC animals exhibited extensive fibro‑fatty plaques with luminal narrowing and abundant lipid cores (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). LD and, more prominently, HD resmetirom treatment markedly reduced plaque size and lipid content in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC,D). LD mice displayed thinner intimal lesions with reduced lipid cores, while HD mice showed significantly thinner plaques and better preserved lumen caliber compared with MC. These morphological findings provide structural evidence that resmetirom ameliorates aortic lipid accumulation and plaque burden in ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003eResmetirom reduces systemic inflammatory cytokines\u003c/p\u003e \u003cp\u003eSystemic inflammatory cytokines were markedly elevated in MC mice compared with NC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;D). MC serum IL‑1β, IL‑18, TNF‑α and CRP levels were 152.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4, 101.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9 and 82.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3 pg/mL, and 5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mg/L, respectively, versus 50.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8, 30.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 and 20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 pg/mL, and 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mg/L in NC (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Resmetirom reduced these cytokines in a dose‑dependent fashion. LD decreased IL‑1β, IL‑18, TNF‑α and CRP to 96.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4, 70.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 and 58.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 pg/mL, and 2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mg/L (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. MC), whereas HD further lowered them to 61.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2, 38.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 and 34.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 pg/mL, and 1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mg/L, corresponding to reductions of approximately 59.3%, 62.1%, 58.6% and 70.0% compared with MC (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These findings demonstrate that resmetirom substantially attenuates systemic vascular inflammation associated with HFD‑induced atherosclerosis.\u003c/p\u003e \u003cp\u003eAortic NLRP3 inflammasome and adhesion molecule expression\u003c/p\u003e \u003cp\u003eImmunohistochemistry (IHC) in the aortic wall showed increased staining of caspase‑1, IL‑1β, MCP‑1, IL‑18, NLRP3 and VCAM‑1 in MC mice compared with NC, consistent with activation of the NLRP3 inflammasome and endothelial activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;F). Although most IHC indices in LD and HD groups showed clear numerical reductions versus MC, only NLRP3 staining in the HD group reached statistical significance (MC 4.10 vs. HD 1.00; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. MC), while changes in caspase‑1, IL‑1β, MCP‑1, IL‑18 and VCAM‑1 did not achieve the pre‑specified significance threshold (all p\u0026thinsp;\u0026ge;\u0026thinsp;0.05 vs. MC).\u003c/p\u003e \u003cp\u003eAt the mRNA level, RT‑qPCR confirmed partial suppression of inflammasome and adhesion pathway components (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG\u0026ndash;L). MC mice exhibited robust up‑regulation of caspase‑1, IL‑1β, MCP‑1, IL‑18, NLRP3 and VCAM‑1 transcripts relative to NC. HD resmetirom significantly reduced caspase‑1 (MC 8.13 vs. HD 1.80‑fold; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), IL‑1β (5.11 vs. 0.58‑fold; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), NLRP3 (4.67 vs. 1.00‑fold; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and VCAM‑1 expression (3.14 vs. 0.52‑fold; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. MC), with LD also significantly lowering caspase‑1 (1.76‑fold; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and VCAM‑1 (1.37‑fold; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. MC). MCP‑1 and IL‑18 transcripts showed consistent downward trends in LD and HD groups, but did not reach statistical significance under the current sample size (p\u0026thinsp;\u0026ge;\u0026thinsp;0.05 vs. MC). Overall, these data indicate that resmetirom partially suppresses aortic NLRP3 inflammasome signalling and endothelial adhesion molecule expression at the transcript level, with more modest effects detectable by semi‑quantitative IHC. The observed discrepancy between robust molecular assay results and more modest IHC findings likely reflects the inherently lower sensitivity of semi‑quantitative immunohistochemistry compared to quantitative Western blotting and qPCR, as well as potential spatial heterogeneity in protein expression within atherosclerotic plaques.\u003c/p\u003e \u003cp\u003eWestern blot analysis of aortic lysates further corroborated down‑regulation of the NLRP3\u0026ndash;ASC\u0026ndash;caspase‑1 axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Compared with NC, MC mice displayed markedly increased protein abundance of NLRP3, ASC, caspase‑1, IL‑1β, IL‑18 and MCP‑1, whereas LD and especially HD resmetirom visibly reduced the intensity of these bands. Densitometric quantification revealed that HD resmetirom reduced NLRP3 protein by 78.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2%, ASC by 74.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1%, and caspase-1 by 71.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3% compared to MC controls (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), in line with the qPCR and cytokine data, indicating coordinated suppression of inflammasome activation at both transcriptional and protein levels.\u003c/p\u003e \u003cp\u003eResmetirom modulates NF‑κB activation, ROS and macrophage polarization\u003c/p\u003e \u003cp\u003eMechanistic analyses in aortic tissue further supported a direct anti‑inflammatory action of resmetirom on the NF‑κB/ROS\u0026ndash;NLRP3 axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;F). Compared with NC, MC mice displayed increased NF‑κB activation, as reflected by a higher p‑p65/total p65 ratio, elevated ROS intensity and an increased proportion of pro‑inflammatory M1 macrophages, accompanied by reduced IκBα and anti‑inflammatory M2 macrophages. Resmetirom attenuated these changes in a dose‑dependent manner. LD reduced the p‑p65/total p65 ratio to 1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 vs. 2.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 in MC, and HD further suppressed it to 1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. MC), corresponding to an approximate 49.6% reduction with HD. In parallel, HD significantly increased IκBα expression (MC 0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 vs. HD 0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08; +67.7%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating reinforcement of NF‑κB inhibition.\u003c/p\u003e \u003cp\u003eConsistent with reduced NF‑κB activity, HD resmetirom markedly decreased ROS levels (MC 186.36\u0026thinsp;\u0026plusmn;\u0026thinsp;14.04 vs. HD 120.09\u0026thinsp;\u0026plusmn;\u0026thinsp;9.25; \u0026minus;35.6%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and shifted macrophage polarization toward an anti‑inflammatory phenotype. M1 macrophage frequency fell from 69.07\u0026thinsp;\u0026plusmn;\u0026thinsp;7.97% in MC to 54.92\u0026thinsp;\u0026plusmn;\u0026thinsp;4.15% in LD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 38.18\u0026thinsp;\u0026plusmn;\u0026thinsp;4.87% in HD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while M2 macrophages increased from 31.51\u0026thinsp;\u0026plusmn;\u0026thinsp;5.72% in MC to 45.46\u0026thinsp;\u0026plusmn;\u0026thinsp;6.30% in LD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 62.22\u0026thinsp;\u0026plusmn;\u0026thinsp;5.84% in HD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Consequently, the M1/M2 ratio decreased from 2.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 in MC to 1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 in LD and 0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 in HD (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. MC).\u003c/p\u003e \u003cp\u003eTogether, these data demonstrate that resmetirom not only improves lipid metabolism and systemic inflammatory cytokine profiles, but also directly dampens NF‑κB/ROS\u0026ndash;NLRP3 signalling and repolarizes vascular macrophages toward an M2 phenotype, providing a mechanistic basis for its anti‑atherosclerotic efficacy in ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mice.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study represents the first demonstration that resmetirom\u0026mdash;a clinically approved, liver-selective THR-β agonist\u0026mdash;exerts potent atheroprotective effects through coordinated suppression of the NF-κB/ROS\u0026ndash;NLRP3 inflammasome axis. Whereas resmetirom was developed and approved for MASH based on its hepatic metabolic effects, our findings reveal a previously unrecognized dual mechanism encompassing both lipid-lowering and direct anti-inflammatory actions in the vessel wall. In HFD-fed ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, resmetirom not only improved atherogenic dyslipidemia and reduced plaque burden, but also profoundly attenuated systemic inflammatory cytokines (IL-1β, IL-18, TNF-α, CRP), suppressed aortic NLRP3 inflammasome activation, and repolarized lesional macrophages toward a reparative M2 phenotype. These converging metabolic and anti-inflammatory mechanisms position resmetirom as a promising therapeutic repurposing candidate for atherosclerotic cardiovascular disease, particularly in patients with comorbid MASH who face elevated cardiovascular risk and would benefit from integrated hepato-vascular protection.\u003c/p\u003e \u003cp\u003eNLRP3 inflammasome suppression: a central anti-inflammatory mechanism\u003c/p\u003e \u003cp\u003eA key finding of this study is that resmetirom coordinately suppressed multiple components of the NLRP3 inflammasome cascade at transcriptional, translational, and functional levels. The NLRP3 inflammasome serves as a critical sensor of metabolic danger signals in atherosclerosis, including oxidized LDL, cholesterol crystals, and mitochondrial dysfunction-derived ROS [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Upon activation, the NLRP3\u0026ndash;ASC\u0026ndash;caspase-1 complex processes pro-IL-1β and pro-IL-18 into their mature forms, which amplify vascular inflammation and promote plaque vulnerability [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our data demonstrated that high-dose resmetirom reduced NLRP3, ASC, and caspase-1 protein abundance by approximately 70\u0026ndash;80% compared with model controls, with corresponding decreases in circulating IL-1β (59%) and IL-18 (62%). These reductions are comparable in magnitude to those achieved by direct NLRP3 inhibitors such as MCC950 in experimental atherosclerosis models [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe clinical relevance of targeting the IL-1β pathway in atherosclerosis was established by CANTOS, which demonstrated that canakinumab, a monoclonal antibody against IL-1β, reduced recurrent cardiovascular events by 15% independent of lipid lowering [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Similarly, the COLCOT trial showed that colchicine, which inhibits NLRP3 inflammasome assembly, decreased cardiovascular events in post-myocardial infarction patients [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Our findings suggest that resmetirom may represent an orally bioavailable alternative for inflammasome suppression, with the added advantage of concurrent lipid-lowering effects.\u003c/p\u003e \u003cp\u003eNF-κB inhibition and ROS reduction: upstream regulatory mechanisms\u003c/p\u003e \u003cp\u003eThe NF-κB signaling pathway functions as the master transcriptional regulator of NLRP3 inflammasome priming, controlling the expression of NLRP3, pro-IL-1β, and adhesion molecules including VCAM-1 and MCP-1 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In atherosclerotic lesions, persistent NF-κB activation sustains a feed-forward inflammatory loop that perpetuates plaque progression [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Our observation that resmetirom reduced NF-κB p65 phosphorylation by approximately 50% while increasing IκBα expression by 68% indicates effective interruption of this proinflammatory cascade at its proximal signaling node.\u003c/p\u003e \u003cp\u003eROS serve dual roles in inflammasome regulation: as direct activators of NLRP3 through thioredoxin-interacting protein (TXNIP) dissociation, and as amplifiers of NF-κB signaling via IκB kinase activation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The 36% reduction in aortic ROS levels observed with high-dose resmetirom likely contributes to both diminished NLRP3 activation and reduced NF-κB nuclear translocation. Thyroid hormones have been shown to modulate mitochondrial function and oxidative stress through effects on uncoupling proteins and antioxidant enzyme expression [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Whether resmetirom\u0026rsquo;s ROS-lowering effects reflect direct hepatic actions, improved systemic metabolism, or secondary consequences of reduced inflammatory burden warrants further investigation.\u003c/p\u003e \u003cp\u003eMacrophage repolarization: implications for plaque stability\u003c/p\u003e \u003cp\u003eMacrophage phenotypic heterogeneity profoundly influences atherosclerotic plaque fate [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Classically activated M1 macrophages predominate in unstable plaques, secreting proinflammatory cytokines and matrix metalloproteinases that promote plaque rupture, whereas alternatively activated M2 macrophages facilitate inflammation resolution, efferocytosis, and tissue repair [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The dramatic shift in M1/M2 ratio from 2.28 in model controls to 0.62 with high-dose resmetirom represents a fundamental reprogramming of the lesional inflammatory milieu.\u003c/p\u003e \u003cp\u003eThis macrophage repolarization likely results from the combined effects of reduced NF-κB\u0026ndash;dependent M1 polarizing signals, decreased ROS-mediated inflammatory priming, and attenuated IL-1β/IL-18 autocrine amplification loops [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Notably, thyroid hormone signaling has been implicated in modulating inflammatory responses and immune cell function [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Whether resmetirom exerts direct effects on vascular macrophages or influences polarization primarily through hepatic-derived factors such as altered lipoprotein composition or secreted anti-inflammatory mediators remains to be elucidated.\u003c/p\u003e \u003cp\u003eHepatic-vascular inflammatory crosstalk: mechanistic considerations\u003c/p\u003e \u003cp\u003eThe liver occupies a central position in the metabolic-inflammatory network linking MASH and atherosclerosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Both conditions share common pathogenic drivers including dyslipidemia, insulin resistance, oxidative stress, and NLRP3-mediated inflammation. Hepatic inflammation in MASH generates circulating proinflammatory mediators\u0026mdash;including IL-1β, IL-6, and CRP\u0026mdash;that promote systemic endothelial dysfunction and accelerate atherogenesis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Conversely, atherogenic lipoproteins exacerbate hepatic lipotoxicity and inflammasome activation, establishing a bidirectional pathogenic axis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResmetirom\u0026rsquo;s liver-selective THR-β agonism positions it uniquely to interrupt this hepatic-vascular inflammatory crosstalk. By improving hepatic lipid handling, reducing hepatocyte lipotoxic stress, and suppressing hepatic NF-κB/NLRP3 activation, resmetirom may attenuate the systemic inflammatory burden that drives vascular disease [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The pronounced reductions in circulating CRP (70%) and TNF-α (59%) observed in our study support this hepatocentric anti-inflammatory mechanism. Future studies employing tissue-specific knockouts or hepatocyte-targeted interventions will be essential to dissect the relative contributions of hepatic versus extrahepatic THR-β signaling to vascular protection.\u003c/p\u003e \u003cp\u003eComparison with other anti-inflammatory approaches in atherosclerosis\u003c/p\u003e \u003cp\u003eSeveral anti-inflammatory strategies have demonstrated efficacy in experimental and clinical atherosclerosis. Direct IL-1β neutralization with canakinumab (CANTOS) and NLRP3 inhibition with colchicine (COLCOT, LoDoCo2) have validated inflammation as a therapeutic target [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, these approaches carry limitations: canakinumab is expensive and immunosuppressive, while colchicine has modest efficacy and gastrointestinal tolerability concerns [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResmetirom offers a differentiated profile as an orally administered agent with dual lipid-lowering and anti-inflammatory properties. In MASH clinical trials, resmetirom achieved LDL-C reductions of 13\u0026ndash;16% alongside improvements in hepatic inflammation and fibrosis, with a favorable safety profile [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The anti-atherosclerotic effects observed in our preclinical study\u0026mdash;encompassing both metabolic correction and inflammasome suppression\u0026mdash;suggest potential for additive or synergistic benefits in patients with combined metabolic and inflammatory cardiovascular risk.\u003c/p\u003e \u003cp\u003eLimitations and future directions\u003c/p\u003e \u003cp\u003eSeveral limitations merit consideration. First, the ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mouse model, while well-validated, may not fully recapitulate human atherosclerosis heterogeneity, particularly regarding plaque rupture and thrombotic complications. Second, our 8-week treatment duration assessed plaque burden but not long-term stability or regression. Third, although resmetirom is a liver-selective THR-β agonist, we did not directly measure hepatic inflammasome activity, hepatic cytokine production, or circulating hepatokines that might mediate vascular effects. This limits our ability to definitively distinguish hepatic-mediated systemic anti-inflammatory effects from potential direct actions on vascular THR-β signaling. The pronounced reductions in circulating CRP and cytokines support a hepatocentric mechanism, but future studies employing hepatocyte-specific THR-β knockout models will be essential to dissect tissue-specific contributions. Fourth, semi-quantitative IHC showed more modest changes than molecular assays, reflecting inherent methodological limitations in detecting graded protein expression differences, as discussed above.\u003c/p\u003e \u003cp\u003eFuture investigations should address several priorities: (1) longer-term studies assessing plaque composition, stability markers (collagen content, fibrous cap thickness), and vulnerability indices; (2) mechanistic studies in hepatocyte-specific THR-β knockout models to define the contribution of hepatic versus systemic effects; (3) evaluation of combination approaches with statins, PCSK9 inhibitors, or colchicine to assess additive anti-inflammatory efficacy; and (4) dedicated cardiovascular outcome trials in MASH patients, who harbor elevated atherosclerotic risk and may derive particular benefit from resmetirom\u0026rsquo;s dual hepatic and vascular protection.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study establishes resmetirom as a potent suppressor of vascular inflammation in experimental atherosclerosis, acting through coordinated inhibition of the NF-κB/ROS\u0026ndash;NLRP3 inflammasome axis and favorable reprogramming of lesional macrophage phenotypes. These anti-inflammatory effects, combined with robust lipid-lowering activity, provide a compelling mechanistic rationale for investigating resmetirom as an atheroprotective therapy, particularly in patients with metabolic dysfunction-associated steatotic liver disease who face elevated cardiovascular risk. Our findings expand the therapeutic potential of THR-β agonism beyond hepatic steatosis to encompass the broader spectrum of metabolic-inflammatory cardiovascular disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by 2025 Hebei Province Medical Science Research Project Plan (Grant No. 20250296) and the 2021 Government-funded Clinical Medicine Outstanding Talent Training Program (\u0026ldquo;Study on the regulation of thyroid hormone receptor β1 on glucose and lipid metabolism and its mechanisms\u0026rdquo;).\u003c/p\u003e \u003cp\u003eCompeting Interests\u003c/p\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003cp\u003eEthics Approval\u003c/p\u003e \u003cp\u003e All animal experimental procedures were conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Hebei General Hospital (Approval No. 202385). The study was performed in line with ethical standards for animal research. All efforts were made to minimize animal suffering and reduce the number of animals used.\u003c/p\u003e \u003cp\u003eClinical trial number\u003c/p\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003cp\u003eConsent to Participate\u003c/p\u003e \u003cp\u003eNot applicable (animal study).\u003c/p\u003e \u003cp\u003eConsent to Publish\u003c/p\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003cp\u003eData Availability\u003c/p\u003e \u003cp\u003eThe datasets generated during the current study are available from the corresponding author upon reasonable request. All Python statistical analysis scripts are publicly available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/ghitamaclaurin-ops/codes.git\u003c/span\u003e\u003cspan address=\"https://github.com/ghitamaclaurin-ops/codes.git\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Xuedong Bai, Wenjie Fei, Jingzhou Fang, Chaomin Kong, Yaqi Xiang, and Yuele Tian. The first draft of the manuscript was written by Xuedong Bai and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during the current study are available from the corresponding author upon reasonable request. All Python statistical analysis scripts are publicly available at https://github.com/ghitamaclaurin-ops/codes.git\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLibby, P. 2021. The changing landscape of atherosclerosis. \u003cem\u003eNature\u003c/em\u003e 592:524\u0026ndash;533. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-021-03392-8\u003c/span\u003e\u003cspan address=\"10.1038/s41586-021-03392-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRidker, P. M. 2016. 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A., M. R. Bashir, and C. D. Guy et al. 2019. Resmetirom (MGL-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial. \u003cem\u003eLancet\u003c/em\u003e 394:2012\u0026ndash;2024. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0140-6736(19)32517-6\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(19)32517-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Atherosclerosis, NLRP3 inflammasome, Resmetirom, Thyroid hormone receptor β, NF-κB signaling, Macrophage polarization","lastPublishedDoi":"10.21203/rs.3.rs-8902352/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8902352/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAtherosclerosis is a chronic inflammatory vascular disease in which the NLRP3 inflammasome and NF-κB signaling play central pathogenic roles. Resmetirom, a liver-selective thyroid hormone receptor β (THR-β) agonist approved for metabolic dysfunction-associated steatohepatitis (MASH), exhibits potent lipid-lowering effects, yet its anti-inflammatory actions in atherosclerosis remain unexplored. We aimed to investigate whether resmetirom attenuates atherogenesis through suppression of the NF-κB/ROS\u0026ndash;NLRP3 inflammasome axis. Male ApoE\u003csup\u003e⁻/⁻\u003c/sup\u003e mice fed a high-fat diet for 8 weeks received vehicle, low-dose (3 mg/kg/day), or high-dose (10 mg/kg/day) resmetirom by oral gavage for an additional 8 weeks (n\u0026thinsp;=\u0026thinsp;10/group). High-dose resmetirom reduced body weight gain by 50.8%, improved atherogenic dyslipidemia with approximate decreases of 35% in total cholesterol and 45% in LDL-cholesterol, and diminished aortic plaque burden as assessed by Oil Red O staining. Circulating inflammatory cytokines were markedly suppressed, with reductions in IL-1β (59%), IL-18 (62%), TNF-α (59%), and CRP (70%) compared with model controls. Western blot analysis revealed that aortic NLRP3, ASC, and caspase-1 protein levels decreased by 70\u0026ndash;80%, findings corroborated by RT-qPCR and immunohistochemistry. Mechanistically, resmetirom reduced NF-κB p65 phosphorylation by approximately 50%, increased IκBα expression by 68%, and lowered aortic reactive oxygen species by 36%. Flow cytometry demonstrated that resmetirom repolarized lesional macrophages from a pro-inflammatory M1 toward an anti-inflammatory M2 phenotype, shifting the M1/M2 ratio from 2.28 to 0.62. These findings demonstrate that resmetirom exerts atheroprotective effects beyond lipid lowering by coordinately suppressing the NF-κB/ROS\u0026ndash;NLRP3 inflammasome axis and reprogramming vascular macrophages, providing a mechanistic rationale for its therapeutic repurposing in atherosclerotic cardiovascular disease, particularly in patients with comorbid metabolic dysfunction.\u003c/p\u003e","manuscriptTitle":"Resmetirom Attenuates Atherosclerosis in ApoE ⁻/⁻ Mice by Suppressing the NF- κB/ROS–NLRP3 Inflammasome Axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 16:29:14","doi":"10.21203/rs.3.rs-8902352/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d8a5018c-0cfb-4fac-8038-1bdda90d02a1","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T09:23:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 16:29:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8902352","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8902352","identity":"rs-8902352","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}