A cell competition system with one gene expression from a single-copy gene in one cell.

Even with advanced plasmid and viral vectors, attaining copy numbers of multiple genes among different transfected cells is challenging. We achieved one gene expression from a single-copy gene in one cell using a transgene competition system, a combination of the Kazusa cDNA clones and our dual recombinase-mediated cassette exchange system. All 48 nuclear receptors were simultaneously expressed in one dish at the same expression level in HEK293 using this system, and the cell proliferation rate was compared. Significant differences were observed between cells transfected with CMV- or EF1 promoter-driven expression of the 48 nuclear receptors after 8 weeks. The EF1-NR1I2 cell line, which exhibited the highest increase from 2 to 8 weeks, showed 1.13-fold higher proliferation than the EF1-DsRed line. On the other hand, the EF1-NR4A1 cell line, which showed the maximum decrease at 8 weeks, showed 0.88-fold lower proliferation than the EF1-DsRed line. The results were confirmed in both our transgene competition system and long-term growth experiments. Our transgene competition system offers a wide-range, simple, and accurate cell competition method.

The role of bile acids in the therapy of selected diseases / Znaczenie kwasów zólciowych w terapii wybranych chorób.

The main function of bile acids (BA) is participation in the emulsification of dietary fats. Recently it has been discovered that BAs can also act as signaling molecules regulating the processes of their own synthesis and metabolism, as well as glucose and lipid metabolism. In addition, they affect the motility of the digestive tract and food intake. BA also interacts with the gut microbiota, a major player in their metabolism. Most of the regulatory actions of BAs are mediated by their receptors, the most important of which are the farnesoid X receptor (FXR) and the G protein-coupled receptor -TGR5, found in large amounts in the intestine, liver, adipose tissue and other tissues of the body. Recently, much attention has been paid to the influence of BA on various diseases and the possibility of using them in the treatment of e.g. inflammatory bowel disease, liver diseases, type 2 diabetes and obesity.

Bacteroides fragilis alleviates necrotizing enterocolitis through restoring bile acid metabolism balance using bile salt hydrolase and inhibiting FXR-NLRP3 signaling pathway.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants with no specific treatments available. We aimed to identify the molecular mechanisms underlying NEC and investigate the therapeutic effects of Bacteroides fragilis on NEC. Clinical samples of infant feces, bile acid-targeted metabolomics, pathological staining, bioinformatics analysis, NEC rat model, and co-immunoprecipitation were used to explore the pathogenesis of NEC. Taxonomic characterization of the bile salt hydrolase (bsh) gene, enzyme activity assays, 16S rRNA sequencing, and organoids were used to explore the therapeutic effects of B. fragilis on NEC-related intestinal damage. Clinical samples, NEC rat models, and in vitro experiments revealed that total bile acid increased in the blood but decreased in feces. Moreover, the levels of FXR and other bile acid metabolism-related genes were abnormal, resulting in disordered bile acid metabolism in NEC. Taurochenodeoxycholic acid accelerated NEC pathogenesis and taurodeoxycholate alleviated NEC. B. fragilis displayed bsh genes and enzyme activity and alleviated intestinal damage by restoring gut microbiota dysbiosis and bile acid metabolism abnormalities by inhibiting the FXR-NLRP3 signaling pathway. Our results provide valuable insights into the therapeutic role of B. fragilis in NEC. Administering B. fragilis may substantially alleviate intestinal damage in NEC.

Raspberry Ketone Attenuates Hepatic Fibrogenesis and Inflammation via Regulating the Crosstalk of FXR and PGC-1α Signaling.

Hepatic fibrosis is a compensatory response to chronic liver injury and inflammation, and dietary intervention is recommended as one of the fundamental prevention strategies. Raspberry ketone (RK) is an aromatic compound first isolated from raspberry and widely used to prepare food flavors. The current study investigated the hepatoprotection and potential mechanism of RK against hepatic fibrosis. In vitro, hepatic stellate cell (HSC) activation was stimulated with TGF-ß and cultured with RK, farnesoid X receptor (FXR), or peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) agonist or inhibitor, respectively. In vivo, C57BL/6 mice were injected intraperitoneally with thioacetamide (TAA) at 100/200 mg/kg from the first to the fifth week. Mice were intragastrically administrated with RK or Cur once a day from the second to the fifth week. In activated HSCs, RK inhibited extracellular matrix (ECM) accumulation, inflammation, and epithelial-mesenchymal transition (EMT) process. RK both activated FXR/PGC-1α and regulated their crosstalk, which were verified by their inhibitors and agonists. Deficiency of FXR or PGC-1α also attenuated the effect of RK on the reverse of activated HSCs. RK also decreased serum ALT/AST levels, liver histopathological change, ECM accumulation, inflammation, and EMT in mice caused by TAA. Double activation of FXR/PGC-1α might be the key targets for RK against hepatic fibrosis. Above all, these discoveries supported the potential of RK as a novel candidate for the dietary intervention of hepatic fibrosis.

Sensitivity of Mouse Lung Nuclear Receptors to Electronic Cigarette Aerosols and Influence of Sex Differences: A Pilot Study.

The emerging concern about chemicals in electronic cigarettes, even those without nicotine, demands the development of advanced criteria for their exposure and risk assessment. This study aims to highlight the sensitivity of lung nuclear receptors (NRs) to electronic cigarette e-liquids, independent of nicotine presence, and the influence of the sex variable on these effects. Adult male and female C57BL/6J mice were exposed to electronic cigarettes with 0%, 3%, and 6% nicotine daily (70 mL, 3.3 s, 1 puff per min/30 min) for 14 days, using the inExpose full body chamber (SCIREQ). Following exposure, lung tissues were harvested, and RNA extracted. The expression of 84 NRs was determined using the RT2 profiler mRNA array (Qiagen). Results exhibit a high sensitivity to e-liquid exposure irrespective of the presence of nicotine, with differential expression of NRs, including one (females) and twenty-four (males) in 0% nicotine groups compared to non-exposed control mice. However, nicotine-dependent results were also significant with seven NRs (females), fifty-three NRs (males) in 3% and twenty-three NRs (female) twenty-nine NRs (male) in 6% nicotine groups, compared to 0% nicotine mice. Sex-specific changes were significant, but sex-related differences were not observed. The study provides a strong rationale for further investigation.

Modulatory Roles of AHR, FFAR2, FXR, and TGR5 Gene Expression in Metabolic-Associated Fatty Liver Disease and COVID-19 Outcomes.

Metabolic-associated fatty liver disease (MAFLD) is a risk factor for severe COVID-19. This study explores the potential influence of gut hormone receptor and immune response gene expression on COVID-19 outcomes in MAFLD patients. METHODS: We investigated gene expression levels of AHR, FFAR2, FXR, and TGR5 in patients with MAFLD and COVID-19 compared to controls. We examined associations between gene expression and clinical outcomes. RESULTS: COVID-19 patients displayed altered AHR expression, potentially impacting immune response and recovery. Downregulated AHR in patients with MAFLD correlated with increased coagulation parameters. Elevated FFAR2 expression in patients with MAFLD was linked to specific immune cell populations and hospital stay duration. A significantly lower FXR expression was observed in both MAFLD and severe COVID-19. CONCLUSION: Our findings suggest potential modulatory roles for AHR, FFAR2, and FXR in COVID-19 and MAFLD.

Nuclear receptors for epidermal lipid barrier: Advances in mechanisms and applications.

The skin plays an essential role in preventing the entry of external environmental threats and the loss of internal substances, depending on the epidermal permeability barrier. Nuclear receptors (NRs), present in various tissues and organs including full-thickness skin, have been demonstrated to exert significant effects on the epidermal lipid barrier. Formation of the lipid lamellar membrane and the normal proliferation and differentiation of keratinocytes (KCs) are crucial for the development of the epidermal permeability barrier and is regulated by specific NRs such as PPAR, LXR, VDR, RAR/RXR, AHR, PXR and FXR. These receptors play a key role in regulating KC differentiation and the entire process of epidermal lipid synthesis, processing and secretion. Lipids derived from sebaceous glands are influenced by NRs as well and participate in regulation of the epidermal lipid barrier. Furthermore, intricate interplay exists between these receptors. Disturbance of barrier function leads to a range of diseases, including psoriasis, atopic dermatitis and acne. Targeting these NRs with agonists or antagonists modulate pathways involved in lipid synthesis and cell differentiation, suggesting potential therapeutic approaches for dermatosis associated with barrier damage. This review focuses on the regulatory role of NRs in the maintenance and processing of the epidermal lipid barrier through their effects on skin lipid synthesis and KC differentiation, providing novel insights for drug targets to facilitate precision medicine strategies.

The mRNA-capping enzyme localizes to stress granules in the cytoplasm and maintains cap homeostasis of target mRNAs.

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.

Synergistic Steatosis Induction in Mice: Exploring the Interactions and Underlying Mechanisms between PFOA and Tributyltin.

This study explores the impact of environmental pollutants on nuclear receptors (CAR, PXR, PPARα, PPARγ, FXR, and LXR) and their heterodimerization partner, the Retinoid X Receptor (RXR). Such interaction may contribute to the onset of non-alcoholic fatty liver disease (NAFLD), which is initially characterized by steatosis and potentially progresses to steatohepatitis and fibrosis. Epidemiological studies have linked NAFLD occurrence to the exposure to environmental contaminants like PFAS. This study aims to assess the simultaneous activation of nuclear receptors via perfluorooctanoic acid (PFOA) and RXR coactivation via Tributyltin (TBT), examining their combined effects on steatogenic mechanisms. Mice were exposed to PFOA (10 mg/kg/day), TBT (5 mg/kg/day) or a combination of them for three days. Mechanisms underlying hepatic steatosis were explored by measuring nuclear receptor target gene and lipid metabolism key gene expressions, by quantifying plasma lipids and hepatic damage markers. This study elucidated the involvement of the Liver X Receptor (LXR) in the combined effect on steatosis and highlighted the permissive nature of the LXR/RXR heterodimer. Antagonistic effects of TBT on the PFOA-induced activation of the Pregnane X Receptor (PXR) and Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) were also observed. Overall, this study revealed complex interactions between PFOA and TBT, shedding light on their combined impact on liver health.

Chemogenomics for NR1 nuclear hormone receptors.

Nuclear receptors (NRs) regulate transcription in response to ligand binding and NR modulation allows pharmacological control of gene expression. Although some NRs are relevant as drug targets, the NR1 family, which comprises 19 NRs binding to hormones, vitamins, and lipid metabolites, has only been partially explored from a translational perspective. To enable systematic target identification and validation for this protein family in phenotypic settings, we present an NR1 chemogenomic (CG) compound set optimized for complementary activity/selectivity profiles and chemical diversity. Based on broad profiling of candidates for specificity, toxicity, and off-target liabilities, sixty-nine comprehensively annotated NR1 agonists, antagonists and inverse agonists covering all members of the NR1 family and meeting potency and selectivity standards are included in the final NR1 CG set. Proof-of-concept application of this set reveals effects of NR1 members in autophagy, neuroinflammation and cancer cell death, and confirms the suitability of the set for target identification and validation.

The effect of selinexor on prostaglandin synthesis in virus-positive Merkel cell carcinoma cell lines.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with high rates of metastasis and mortality. In vitro studies suggest that selinexor (KPT-330), an inhibitor of exportin 1, may be a targeted therapeutic option for MCC. This selective inhibitor prevents the transport of oncogenic mRNA out of the nucleus. Of note, 80% of MCC tumors are integrated with Merkel cell polyomavirus (MCPyV), and virally encoded tumor-antigens, small T (sT) and large T (LT) mRNAs may require an exportin transporter to relocate to the cytoplasm and modulate host tumor-suppressing pathways. To explore selinexor as a targeted therapy for MCC, we examine its ability to inhibit LT and sT antigen expression in vitro and its impact on the prostaglandin synthesis pathway. Protein expression was determined through immunoblotting and quantified by densitometric analysis. Statistical significance was determined with t-test. Treatment of MCPyV-infected cell lines with selinexor resulted in a significant dose-dependent downregulation of key mediators of the prostaglandin synthesis pathway. Given the role of prostaglandin synthesis pathway in MCC, our findings suggest that selinexor, alone or in combination with immunotherapy, could be a promising treatment for MCPyV-infected MCC patients who are resistant to chemotherapy and immunotherapy.

Determination of OTNE-induced thyroid effects within an adverse outcome pathway (AOP) network.

Octahydro-tetramethyl-naphthalenyl-ethanone (OTNE) is a synthetic fragrance ingredient. OTNE was evaluated in repeated-dose toxicological studies. Target organs via oral and dermal routes were the liver and skin/liver, respectively. Effects were observed on the thyroid and thyroid hormones, suggesting hypothalamic-pituitary-thyroid axis perturbation. We investigated the molecular initiating event(s) (MIEs), key events (KEs), and adverse outcomes of OTNE-induced thyroid perturbation within an adverse outcome pathway (AOP). Data were generated using new approach methodologies (NAMs) on human, mouse, and/or rat receptors exploring MIEs using in vitro receptor ligand-binding assays for androstane receptor variant 3 (CAR), farnesoid X receptor (FXR), liver X receptor alpha (LXRα), peroxisome proliferator-activated receptors alpha, delta, and gamma (PPARα, δ, and γ), pregnane X receptor (PXR), and aryl hydrocarbon receptor (AhR). These data inform an AOP network where CAR, FXR, and PXR activation serve as MIEs with thyroid perturbation occurring as secondary effects. These data represent a robust evaluation using NAMs for mapping OTNE-induced thyroid effects and identifying activation of receptor-ligand binding as MIEs in lieu of additional in vivo experimentation. These data indicate the observed thyroid effects are secondary to liver effects and the thyroid effects, therefore, should not be the basis for assessing potential OTNE-induced human health hazards.

Farnesol prevents chlorpyrifos nephrotoxicity by modulating inflammatory mediators, Nrf2 and FXR and attenuating oxidative stress.

Chlorpyrifos (CPF) is a broad-spectrum insecticide widely employed in agricultural field for pest control. Exposure to CPF is associated with serious effects to the main organs, including kidneys. Significant evidence denotes that oxidative stress (OS) and inflammation are implicated in CPF toxicity. This study aimed to evaluate the potential of farnesol (FAR) to modulate inflammatory mediators and farnesoid-X-receptor (FXR) and Nrf2 in a rat model of CPF nephrotoxicity. CPF and FAR were orally supplemented for 28 days and blood and kidney samples were collected for investigations. CPF administration elevated blood creatinine and urea, kidney MDA and NO, and upregulated NF-κB p65, IL-1ß, TNF-α, iNOS, and caspase-3. In addition, CPF upregulated kidney Keap1, and decreased GSH, antioxidant enzymes, and Nrf2, FXR, HO-1 and NQO-1. FAR ameliorated creatinine and urea, prevented histopathological alterations, decreased MDA and NO, and enhanced antioxidants in CPF-administered rats. FAR modulated NF-κB p65, iNOS, TNF-α, IL-1ß, caspase-3, Keap1, HO-1, NQO-1, Nrf2 and FXR. In silico investigations revealed the binding affinity of FAR towards Keap1 and FXR, as well as NF-κB, caspase-3, iNOS, and HO-1. In conclusion, FAR prevents CPF-induced kidney injury by attenuating OS, inflammation, and apoptosis, effects associated with modulation of FXR, Nrf2/HO-1 signaling and antioxidants.

Reconstitution of nuclear envelope subdomain formation on mitotic chromosomes in semi-intact cells.

In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation has been extensively studied using live-cell imaging. At the early step of NE reassembly in human cells, specific pattern-like localization of inner nuclear membrane (INM) proteins, connected to the nuclear pore complex (NPC), was observed in the so-called "core" region and "noncore" region on telophase chromosomes, which corresponded to the "pore-free" region and the "pore-rich" region, respectively, in the early G1 interphase nucleus. We refer to these phenomena as NE subdomain formation. To biochemically investigate this process, we aimed to develop an in vitro NE reconstitution system using digitonin-permeabilized semi-intact mitotic human cells coexpressing two INM proteins, emerin and lamin B receptor, which were labeled with fluorescent proteins. The targeting and accumulation of INM proteins to chromosomes before and after anaphase onset in semi-intact cells were observed using time-lapse imaging. Our in vitro NE reconstitution system recapitulated the formation of the NE subdomain, as in living cells, although chromosome segregation and cytokinesis were not observed. This in vitro NE reconstitution required the addition of a mitotic cytosolic fraction supplemented with a cyclin-dependent kinase inhibitor and energy sources. The cytoplasmic soluble factor(s) dependency of INM protein targeting differed among the segregation states of chromosomes. Furthermore, the NE reconstituted on segregated chromosomes exhibited active nucleocytoplasmic transport competency. These results indicate that the chromosome status changes after anaphase onset for recruiting NPC components.

Butylparaben induces glycolipid metabolic disorders in mice via disruption of gut microbiota and FXR signaling.

Butylparaben, a common preservative, is widely used in food, pharmaceuticals and personal care products. Epidemiological studies have revealed the close relationship between butylparaben and diabetes; however the mechanisms of action remain unclear. In this study, we administered butylparaben orally to mice and observed that exposure to butylparaben induced glucose intolerance and hyperlipidemia. RNA sequencing results demonstrated that the enrichment of differentially expressed genes was associated with lipid metabolism, bile acid metabolism, and inflammatory response. Western blot results further validated that butylparaben promoted hepatic lipogenesis, inflammation, gluconeogenesis, and insulin resistance through the inhibition of the farnesoid X receptor (FXR) pathway. The FXR agonists alleviated the butylparaben-induced metabolic disorders. Moreover, 16 S rRNA sequencing showed that butylparaben reduced the abundance of Bacteroidetes, S24-7, Lactobacillus, and Streptococcus, and elevated the Firmicutes/Bacteroidetes ratio. The gut microbiota dysbiosis caused by butylparaben led to decreased bile acids (BAs) production and increased inflammatory response, which further induced hepatic glycolipid metabolic disorders. Our results also demonstrated that probiotics attenuated butylparaben-induced disturbances of the gut microbiota and hepatic metabolism. Taken collectively, the findings reveal that butylparaben induced gut microbiota dysbiosis and decreased BAs production, which further inhibited FXR signaling, ultimately contributing to glycolipid metabolic disorders in the liver.

On the Cholesterol Raising Effect of Coffee Diterpenes Cafestol and 16-O-Methylcafestol: Interaction with Farnesoid X Receptor.

The diterpene cafestol represents the most potent cholesterol-elevating compound known in the human diet, being responsible for more than 80% of the effect of coffee on serum lipids, with a mechanism still not fully clarified. In the present study, the interaction of cafestol and 16-O-methylcafestol with the stabilized ligand-binding domain (LBD) of the Farnesoid X Receptor was evaluated by fluorescence and circular dichroism. Fluorescence quenching was observed with both cafestol and 16-O-methylcafestol due to an interaction occurring in the close environment of the tryptophan W454 residue of the protein, as confirmed by docking and molecular dynamics. A conformational change of the protein was also observed by circular dichroism, particularly for cafestol. These results provide evidence at the molecular level of the interactions of FXR with the coffee diterpenes, confirming that cafestol can act as an agonist of FXR, causing an enhancement of the cholesterol level in blood serum.

Antibiotic-Induced Gut Microbial Dysbiosis Reduces the Growth of Weaning Rats via FXR-Mediated Hepatic IGF-2 Inhibition.

The gut microbiota plays a crucial role in postnatal growth, particularly in modulating the development of animals during their growth phase. In this study, we investigated the effects of antibiotic-induced dysbiosis of the gut microbiota on the growth of weaning rats by administering a non-absorbable antibiotic cocktail (ABX) in water for 4 weeks. ABX treatment significantly reduced body weight and feed intake in rats. Concurrently, ABX treatment decreased microbial abundance and diversity in rat ceca, predominantly suppressing microbes associated with bile salt hydrolase (BSH) activity. Furthermore, decreased appetite may be attributed to elevated levels of glucagon-like peptide-1 (GLP-1) in the serum, along with reduced neuropeptide Y (NPY) and increased cocaine and amphetamine-regulated transcript (CART) in the hypothalamus at the mRNA level. Importantly, concentrations of insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2) were decreased in the serum and liver of antibiotic-treated rats. These alterations were associated with significant down-regulation of IGF-2 mRNA in the liver and significantly decreased farnesoid X receptor (FXR) protein expression and binding to the IGF-2 promoter. These results indicate that antibiotic-induced gut microbial dysbiosis not only impacts bile acid metabolism but also diminishes rat growth through the FXR-mediated IGF-2 pathway.

Functional implications of NHR-210 enrichment in C. elegans cephalic sheath glia: insights into metabolic and mitochondrial disruptions in Parkinson's disease models.

Glial cells constitute nearly half of the mammalian nervous system's cellular composition. The glia in C. elegans perform majority of tasks comparable to those conducted by their mammalian equivalents. The cephalic sheath (CEPsh) glia, which are known to be the counterparts of mammalian astrocytes, are enriched with two nuclear hormone receptors (NHRs)-NHR-210 and NHR-231. This unique enrichment makes the CEPsh glia and these NHRs intriguing subjects of study concerning neuronal health. We endeavored to assess the role of these NHRs in neurodegenerative diseases and related functional processes, using transgenic C. elegans expressing human alpha-synuclein. We employed RNAi-mediated silencing, followed by behavioural, functional, and metabolic profiling in relation to suppression of NHR-210 and 231. Our findings revealed that depleting nhr-210 changes dopamine-associated behaviour and mitochondrial function in human alpha synuclein-expressing strains NL5901 and UA44, through a putative target, pgp-9, a transmembrane transporter. Considering the alteration in mitochondrial function and the involvement of a transmembrane transporter, we performed metabolomics study via HR-MAS NMR spectroscopy. Remarkably, substantial modifications in ATP, betaine, lactate, and glycine levels were seen upon the absence of nhr-210. We also detected considerable changes in metabolic pathways such as phenylalanine, tyrosine, and tryptophan biosynthesis metabolism; glycine, serine, and threonine metabolism; as well as glyoxalate and dicarboxylate metabolism. In conclusion, the deficiency of the nuclear hormone receptor nhr-210 in alpha-synuclein expressing strain of C. elegans, results in altered mitochondrial function, coupled with alterations in vital metabolite levels. These findings underline the functional and physiological importance of nhr-210 enrichment in CEPsh glia.

Tailoring FXR Modulators for Intestinal Specificity: Recent Progress and Insights.

While FXR has shown promise in regulating bile acid synthesis and maintaining glucose and lipid homeostasis, undesired side effects have been observed in clinical trials. To address this issue, the development of intestinally restricted FXR modulators has gained attention as a new avenue for drug design with the potential for safer systematic effects. Our review examines all currently known intestinally restricted FXR ligands and provides insights into the steps taken to enhance intestinal selectivity.

Neoagarotetraose Alleviates Atherosclerosis via Modulating Cholesterol and Bile Acid Metabolism in ApoE-/- Mice.

Atherosclerosis is closely associated with metabolic disorders such as cholesterol accumulation, bile acid metabolism, and gut dysbiosis. Neoagarotetraose supplementation has been shown to inhibit obesity and alleviate type 2 diabetes, but its effects on modulating the development of atherosclerosis remain unexplored. Therefore, the present study was conducted to investigate the protective effects and potential mechanisms of neoagarotetraose on high-fat, high-cholesterol diet (HFHCD)-induced atherosclerosis in ApoE-/- mice. The results showed that neoagarotetraose supplementation decreased the atherosclerotic lesion area by 50.1% and the aortic arch lesion size by 80.4% compared to the HFHCD group. Furthermore, neoagarotetraose supplementation led to a significant reduction in hepatic lipid content, particularly non-high-density lipoprotein cholesterol. It also resulted in a substantial increase in total bile acid content in both urine and fecal samples by 3.0-fold and 38.7%, respectively. Moreover, neoagarotetraose supplementation effectively downregulated the intestinal farnesoid X receptor by 35.8% and modulated the expressions of its associated genes in both the liver and intestine. In addition, correlation analysis revealed strong associations between gut microbiota composition and fecal bile acid levels. These findings highlight the role of gut microbiota in neoagarotetraose-mitigating atherosclerosis in HFHCD-fed ApoE-/- mice. This study indicates the potential of neoagarotetraose as a functional dietary supplement for the prevention of atherosclerosis.