GSK-126 Enhances All-Trans-Retinoic Acid (ATRA) Response in Hepatocellular Carcinoma (HCC) by Upregulating RARG Expression.

BACKGROUND: Hepatocellular carcinoma (HCC) stands out as one of the most prevalent malignant tumors globally. The combination of all-trans-retinoic acid (ATRA) with FOLFOX chemotherapy has shown promise in enhancing the prognosis of HCC patients. ATRA, serving as a chemosensitizing agent, presents novel possibilities for therapeutic applications. Nevertheless, the responsiveness of HCC cells to ATRA varies. The epigenetic modifier-GSK-126 is currently under investigation as a potential antitumor drug. Our aim is to explore the molecular mechanisms underlying the diverse sensitivity of HCC patients to ATRA, and to propose a new combination regimen. This research aims to lay the groundwork for personalized medication approaches for individuals with HCC. METHODS: A cell model with low expression of retinoic acid receptor Alfa (RARA), retinoic acid receptor belta (RARB), and retinoic acid receptor gamma (RARG) was established through siRNA interference. The impact of reduced expression of RARA, RARB, and RARG on the half maximal inhibitory concentration (IC50) of ATRA in Hep3B cells was assessed using the 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl Tetrazolium Bromide (MTT) cytotoxicity assay. Flow cytometry revealed that RARG emerged as the key receptor influencing the combination's sensitivity. Conducting ChIP-qPCR analysis on genomic DNA from HCC cells through relevant websites demonstrated enrichment of the trimethylation modification of lysine 27 on histone H3 (H3K27me3) upstream of the RARG promoter. ChIP-PCR assay confirmed that GSK-126 could diminish H3K27me3 levels on the RARG promoter, subsequently elevating RARG expression. The synergistic efficacy of GSK-126 and ATRA was validated through MTT assay, flow cytometry apoptosis assay, cell cycle assay, and cell scratch assay. RESULTS: Our study unveiled that the insensitivity of HCC cells to ATRA could be linked to the low expression of RARG. ChIP-qPCR analysis illuminated that GSK-126 activated RARG expression by diminishing H3K27me3 enrichment in the RARG promoter region. Consequently, the concurrent administration of ATRA and GSK-126 to hepatoma cells exhibited a synergistic effect, inhibiting cell proliferation, inducing cell apoptosis, and reducing the proportion of cells in the S-phase. CONCLUSION: Our findings emphasize that the synergistic action of GSK-126 and ATRA enhances the sensitivity of HCC cells by upregulating the expression of RARG. This presents a potential foundation for personalized HCC treatment.

Retinoic acid receptor activation reprograms senescence response and enhances anti-tumor activity of natural killer cells.

Cellular senescence can exert dual effects in tumors, either suppressing or promoting tumor progression. The senescence-associated secretory phenotype (SASP), released by senescent cells, plays a crucial role in this dichotomy. Consequently, the clinical challenge lies in developing therapies that safely enhance senescence in cancer, favoring tumor-suppressive SASP factors over tumor-promoting ones. Here, we identify the retinoic-acid-receptor (RAR) agonist adapalene as an effective pro-senescence compound in prostate cancer (PCa). Reactivation of RARs triggers a robust senescence response and a tumor-suppressive SASP. In preclinical mouse models of PCa, the combination of adapalene and docetaxel promotes a tumor-suppressive SASP that enhances natural killer (NK) cell-mediated tumor clearance more effectively than either agent alone. This approach increases the efficacy of the allogenic infusion of human NK cells in mice injected with human PCa cells, suggesting an alternative therapeutic strategy to stimulate the anti-tumor immune response in "immunologically cold" tumors.

Report of IRF2BP1 as a novel partner of RARA in variant acute promyelocytic leukemia.

IRF2BP1 breaked in the middle of exon 1 at the c.322 position and fused with RARA intron 2 which is located at 3717 bp upstream of its exon 3. The fusion produced a new intron by forming a paired splicing donor GT at 9 bp downstream of RARA breakpoint and acceptor AG at the 5' end of RARA exon 3. The IRF2BP1::RARA fusion gene leads a fusion transcript involving IRF2BP1 exon 1 and RARA exon 3, linked by a 9-bp fragment derived from RARA intron 2. The patient with IRF2BP1::RARA has same clinical features of APL.

Rational design and virtual screening identify mimetics of the RXR agonist valerenic acid.

The ligand-sensing transcription factor retinoid X receptor (RXR) is the universal heterodimer partner of nuclear receptors and involved in multiple physiological processes. Its pharmacological modulation holds therapeutic potential in cancer and neurodegeneration but many available RXR ligands lack specificity. The sesquiterpenoid valerenic acid has been identified as RXR agonist with unprecedented subtype and homodimer preference. Here, we identified simplified mimetics of the complex natural product by rational design and virtual screening that exhibited similar activity profiles on RXR and informed about structural elements contributing to the favorable activity.

Retinoic acid signaling in development and differentiation commitment and its regulatory topology.

Retinoic acid (RA), the derivative of vitamin A/retinol, is a signaling molecule with important implications in health and disease. It is a well-known developmental morphogen that functions mainly through the transcriptional activity of nuclear RA receptors (RARs) and, uncommonly, through other nuclear receptors, including peroxisome proliferator-activated receptors. Intracellular RA is under spatiotemporally fine-tuned regulation by synthesis and degradation processes catalyzed by retinaldehyde dehydrogenases and P450 family enzymes, respectively. In addition to dictating the transcription architecture, RA also impinges on cell functioning through non-genomic mechanisms independent of RAR transcriptional activity. Although RA-based differentiation therapy has achieved impressive success in the treatment of hematologic malignancies, RA also has pro-tumor activity. Here, we highlight the relevance of RA signaling in cell-fate determination, neurogenesis, visual function, inflammatory responses and gametogenesis commitment. Genetic and post-translational modifications of RAR are also discussed. A better understanding of RA signaling will foster the development of precision medicine to improve the defects caused by deregulated RA signaling.

RAR-Dependent and RAR-Independent RXR Signaling in Stem-like Glioma Cells.

Retinoic acid (RA) exerts pleiotropic effects during neural development and regulates homeostasis in the adult human brain. The RA signal may be transduced through RXR (retinoid-X receptor)-non-permissive RA receptor/RXR heterodimers or through RXR-permissive RXR heterodimers. The significance of RA signaling in malignant brain tumors such as glioblastoma multiforme (GBM) and gliosarcoma (GS) is poorly understood. In particular, the impact RA has on the proliferation, survival, differentiation, or metabolism of GBM- or GS-derived cells with features of stem cells (SLGCs) remains elusive. In the present manuscript, six GBM- and two GS-derived SLGC lines were analyzed for their responsiveness to RAR- and RXR-selective agonists. Inhibition of proliferation and initiation of differentiation were achieved with a RAR-selective pan-agonist in a subgroup of SLGC lines, whereas RXR-selective pan-agonists (rexinoids) supported proliferation in most SLGC lines. To decipher the RAR-dependent and RAR-independent effects of RXR, the genes encoding the RAR or RXR isotypes were functionally inactivated by CRISPR/Cas9-mediated editing in an IDH1-/p53-positive SLGC line with good responsiveness to RA. Stemness, differentiation capacity, and growth behavior were preserved after editing. Taken together, this manuscript provides evidence about the positive impact of RAR-independent RXR signaling on proliferation, survival, and tumor metabolism in SLGCs.

Heparin-binding epidermal growth factor and fibroblast growth factor 2 rescue Müller glia-derived progenitor cell formation in microglia- and macrophage-ablated chick retinas.

Recent studies have demonstrated the impact of pro-inflammatory signaling and reactive microglia/macrophages on the formation of Müller glial-derived progenitor cells (MGPCs) in the retina. In chick retina, ablation of microglia/macrophages prevents the formation of MGPCs. Analyses of single-cell RNA-sequencing chick retinal libraries revealed that quiescent and activated microglia/macrophages have a significant impact upon the transcriptomic profile of Müller glia (MG). In damaged monocyte-depleted retinas, MG fail to upregulate genes related to different cell signaling pathways, including those related to Wnt, heparin-binding epidermal growth factor (HBEGF), fibroblast growth factor (FGF) and retinoic acid receptors. Inhibition of GSK3ß, to simulate Wnt signaling, failed to rescue the deficit in MGPC formation, whereas application of HBEGF or FGF2 completely rescued the formation of MGPCs in monocyte-depleted retinas. Inhibition of Smad3 or activation of retinoic acid receptors partially rescued the formation of MGPCs in monocyte-depleted retinas. We conclude that signals produced by reactive microglia/macrophages in damaged retinas stimulate MG to upregulate cell signaling through HBEGF, FGF and retinoic acid, and downregulate signaling through TGFß/Smad3 to promote the reprogramming of MG into proliferating MGPCs.

A novel retinoic acid receptor-γ agonist antagonizes immune checkpoint resistance in lung cancers by altering the tumor immune microenvironment.

All-trans-retinoic acid (ATRA), the retinoic acid receptors (RARs) agonist, regulates cell growth, differentiation, immunity, and survival. We report that ATRA-treatment repressed cancer growth in syngeneic immunocompetent, but not immunodeficient mice. The tumor microenvironment was implicated: CD8+ T cell depletion antagonized ATRA's anti-tumorigenic effects in syngeneic mice. ATRA-treatment with checkpoint blockade did not cooperatively inhibit murine lung cancer growth. To augment ATRA's anti-tumorigenicity without promoting its pro-tumorigenic potential, an RARγ agonist (IRX4647) was used since it regulates T cell biology. Treating with IRX4647 in combination with an immune checkpoint (anti-PD-L1) inhibitor resulted in a statistically significant suppression of syngeneic 344SQ lung cancers in mice-a model known for its resistance to checkpoints and characterized by low basal T cell and PD-L1 expression. This combined treatment notably elevated CD4+ T-cell presence within the tumor microenvironment and increased IL-5 and IL-13 tumor levels, while simultaneously decreasing CD38 in the tumor stroma. IL-5 and/or IL-13 treatments increased CD4+ more than CD8+ T-cells in mice. IRX4647-treatment did not appreciably affect in vitro lung cancer growth, despite RARγ expression. Pharmacokinetic analysis found IRX4647 plasma half-life was 6 h in mice. Yet, RARα antagonist (IRX6696)-treatment with anti-PD-L1 did not repress syngeneic lung cancer growth. Together, these findings provide a rationale for a clinical trial investigating an RARγ agonist to augment check point blockade response in cancers.

Downregulation of cellular retinoic acid binding protein 1 fosters epithelial-mesenchymal transition in thyroid cancer.

Cellular retinoic acid binding protein 1 (CRABP1) participates in the regulation of retinoid signaling. Previous studies showed conflicting results regarding the role of CRABP1 in tumor biology, including protumorigenic and tumor-suppressive effects in different types of cancer. Our bioinformatics analyses suggested that CRABP1 expression was downregulated in thyroid cancer. Ectopic expression of CRABP1 in thyroid cancer cells suppressed migratory and invasive activity without affecting cell growth or cell cycle distribution. In transformed normal thyroid follicular epithelial cells, silencing of CRABP1 expression increased invasiveness. Additionally, CRABP1 overexpression was associated with downregulation of the mesenchymal phenotype. Kinase phosphorylation profiling indicated that CRABP1 overexpression was accompanied by a decrease in phosphorylation of epidermal growth factor (EGF) receptor and downstream phosphorylation of Akt, STAT3, and FAK, which were reversed by exogenous EGF treatment. Immunohistochemical analysis of our tissue microarrays revealed an inverse association between CRABP1 expression and disease stage of differentiated thyroid cancer. Taken together, our results suggest that CRABP1 expression is aberrantly lost in thyroid cancer, and this downregulation promotes the epithelial-mesenchymal transition at least partly through modulating EGF receptor signaling.

Discovery of novel RARα agonists using pharmacophore-based virtual screening, molecular docking, and molecular dynamics simulation studies.

Nuclear retinoic acid receptors (RARs) are ligand-dependent transcription factors involved in various biological processes, such as embryogenesis, cell proliferation, differentiation, reproduction, and apoptosis. These receptors are regulated by retinoids, i.e., retinoic acid (RA) and its analogs, as receptor agonists. RAR agonists are promising therapeutic agents for the treatment of serious dermatological disorders, including some malignant conditions. By inducing apoptosis, they are able to inhibit the proliferation of diverse cancer cell lines. Also, RAR agonists have recently been identified as therapeutic options for some neurodegenerative diseases. These features make retinoids very attractive molecules for medical purposes. Synthetic selective RAR agonists have several advantages over endogenous ones, but they suffer poor pharmacokinetic properties. These compounds are normally lipophilic acids with unfavorable drug-like features such as poor oral bioavailability. Recently, highly selective, potent, and less toxic RAR agonists with proper lipophilicity, thus, good oral bioavailability have been developed for some therapeutic applications. In the present study, ligand and structure-based virtual screening technique was exploited to introduce some novel RARα agonists. Pharmacokinetic assessment was also performed in silico to suggest those compounds which have optimized drug-like features. Finally, two compounds with the best in silico pharmacological features are proposed as lead molecules for future development of RARα agonists.

Retinoic Acid Receptor-Related Orphan Receptors (RORs) in Eye Development and Disease.

The retinoic acid receptor-related orphan receptors (RORs) are ligand-mediated transcription factors with important biological roles in regulating circadian rhythms, metabolism, immunity, angiogenesis, inflammation, and development. They belong to the superfamily of nuclear receptors and include three family members: RORα, RORß, and RORγ. Currently identified ROR ligands include cholesterol and cholesterol derivatives for RORα and RORγ, and stearic acid and all-trans retinoic acid for RORß. Aberrant signaling of the RORs is involved in the pathogenesis of several human diseases including autoimmune diseases, metabolic disorders, and certain cancers. In the eye, RORs regulate normal development of the lens and the retina, and also contribute to potentially blinding eye diseases, especially retinal vascular diseases. Here, we review the role of RORs in eye development and disease to highlight their potential as druggable targets for therapeutic development in retinal vascular and degenerative diseases.

Am80, a retinoic acid receptor agonist, activates the cardiomyocyte cell cycle and enhances engraftment in the heart.

Human induced pluripotent stem cell-derived (hiPSC) cardiomyocytes are a promising source for regenerative therapy. To realize this therapy, however, their engraftment potential after their injection into the host heart should be improved. Here, we established an efficient method to analyze the cell cycle activity of hiPSC cardiomyocytes using a fluorescence ubiquitination-based cell cycle indicator (FUCCI) system. In vitro high-throughput screening using FUCCI identified a retinoic acid receptor (RAR) agonist, Am80, as an effective cell cycle activator in hiPSC cardiomyocytes. The transplantation of hiPSC cardiomyocytes treated with Am80 before the injection significantly enhanced the engraftment in damaged mouse heart for 6 months. Finally, we revealed that the activation of endogenous Wnt pathways through both RARA and RARB underlies the Am80-mediated cell cycle activation. Collectively, this study highlights an efficient method to activate cell cycle in hiPSC cardiomyocytes by Am80 as a means to increase the graft size after cell transplantation into a damaged heart.

The G protein-coupled receptor GPRC5A-a phorbol ester and retinoic acid-induced orphan receptor with roles in cancer, inflammation, and immunity.

GPRC5A is the first member of a new class of orphan receptors coupled to G proteins, which also includes GPRC5B, GPRC5C, and GPRC5D. Since its cloning and identification in the 1990s, substantial progress has been made in understanding the possible functions of this receptor. GPRC5A has been implicated in a variety of cellular events, such as cytoskeleton reorganization, cell proliferation, cell cycle regulation, migration, and survival. It appears to be a central player in different pathological processes, including tumorigenesis, inflammation, immune response, and tissue damage. The levels of GPRC5A expression differ depending on the type of cancer, with increased expression in colon, pancreas, and prostate cancers; decreased expression in lung cancer; and varied results in breast cancer. In this review, we discuss the early discovery of GPRC5A as a phorbol ester-induced gene and later as a retinoic acid-induced gene, its regulation, and its participation in important canonical pathways related to numerous types of tumors and inflammatory processes. GPRC5A represents a potential new target for cancer, inflammation, and immunity therapies.

Investigation of Flavonoid Scaffolds as DAX1 Inhibitors against Ewing Sarcoma through Pharmacoinformatic and Dynamic Simulation Studies.

Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX1) is an orphan nuclear receptor encoded by the NR0B1 gene. The functional study showed that DAX1 is a physiologically significant target for EWS/FLI1-mediated oncogenesis, particularly Ewing Sarcoma (ES). In this study, a three-dimensional DAX1 structure was modeled by employing a homology modeling approach. Furthermore, the network analysis of genes involved in Ewing Sarcoma was also carried out to evaluate the association of DAX1 and other genes with ES. Moreover, a molecular docking study was carried out to check the binding profile of screened flavonoid compounds against DAX1. Therefore, 132 flavonoids were docked in the predicted active binding pocket of DAX1. Moreover, the pharmacogenomics analysis was performed for the top ten docked compounds to evaluate the ES-related gene clusters. As a result, the five best flavonoid-docked complexes were selected and further evaluated by Molecular Dynamics (MD) simulation studies at 100 ns. The MD simulation trajectories were evaluated by generating RMSD, hydrogen bond plot analysis, and interaction energy graphs. Our results demonstrate that flavonoids showed interactive profiles in the active region of DAX1 and can be used as potential therapeutic agents against DAX1-mediated augmentation of ES after in-vitro and in-vivo evaluations.

The Endogenous Dual Retinoid Receptor Agonist Alitretinoin Exhibits Immunoregulatory Functions on Antigen-Presenting Cells.

Retinoids are a frequently used class of drugs in the treatment of inflammatory as well as malignant skin diseases. Retinoids have differential affinity for the retinoic acid receptor (RAR) and/or the retinoid X receptor (RXR). The endogenous dual RAR and RXR agonist alitretinoin (9-cis retinoic acid) demonstrated remarkable efficacy in the treatment of chronic hand eczema (CHE) patients; however, detailed information on the mechanisms of action remains elusive. Here, we used CHE as a model disease to unravel immunomodulatory pathways following retinoid receptor signaling. Transcriptome analyses of skin specimens from alitretinoin-responder CHE patients identified 231 significantly regulated genes. Bioinformatic analyses indicated keratinocytes as well as antigen presenting cells as cellular targets of alitretinoin. In keratinocytes, alitretinoin interfered with inflammation-associated barrier gene dysregulation as well as antimicrobial peptide induction while markedly inducing hyaluronan synthases without affecting hyaluronidase expression. In monocyte-derived dendritic cells, alitretinoin induced distinct morphological and phenotypic characteristics with low co-stimulatory molecule expression (CD80 and CD86), the increased secretion of IL-10 and the upregulation of the ecto-5'-nucleotidase CD73 mimicking immunomodulatory or tolerogenic dendritic cells. Indeed, alitretinoin-treated dendritic cells demonstrated a significantly reduced capacity to activate T cells in mixed leukocyte reactions. In a direct comparison, alitretinoin-mediated effects were significantly stronger than those observed for the RAR agonist acitretin. Moreover, longitudinal monitoring of alitretinoin-responder CHE patients could confirm in vitro findings. Taken together, we demonstrate that the dual RAR and RXR agonist alitretinoin targets epidermal dysregulation and demonstrates strong immunomodulatory effects on antigen presenting cell functions.

Exploring the interactions of antihistamine with retinoic acid receptor beta (RARB) by molecular dynamics simulations and genome-wide meta-analysis.

Kaposi sarcoma (KS) is one of the most common AIDS-related malignant neoplasms, which can leave lesions on the skin among HIV patients. These lesions can be treated with 9-cis-retinoic acid (9-cis-RA), an endogenous ligand of retinoic acid receptors that has been FDA-approved for treatment of KS. However, topical application of 9-cis-RA can induce several unpleasant side effects, like headache, hyperlipidemia, and nausea. Hence, alternative therapeutics with less side effects are desirable. There are case reports associating over-the-counter antihistamine usage with regression of KS. Antihistamines competitively bind to H1 receptor and block the action of histamine, best known for being released in response to allergens. Furthermore, there are already dozens of antihistamines that are FDA-approved with less side effects than 9-cis-RA. This led our team to conduct a series of in-silico assays to determine whether antihistamines can activate retinoic acid receptors. First, we utilized high-throughput virtual screening and molecular dynamics simulations to model high-affinity interactions between antihistamines and retinoic acid receptor beta (RARß). We then performed systems genetics analysis to identify a genetic association between H1 receptor itself and molecular pathways involved in KS. Together, these findings advocate for exploration of antihistamines against KS, starting with our two promising hit compounds, bepotastine and hydroxyzine, for experimental validation study in the future.

Vitamin A and retinoid signaling in the kidneys.

Vitamin A (VA, retinol) and its metabolites (commonly called retinoids) are required for the proper development of the kidney during embryogenesis, but retinoids also play key roles in the function and repair of the kidney in adults. Kidneys filter 180-200 liters of blood per day and each kidney contains approximately 1 million nephrons, which are often referred to as the 'functional units' of the kidney. Each nephron consists of a glomerulus and a series of tubules (proximal tubule, loop of Henle, distal tubule, and collecting duct) surrounded by a network of capillaries. VA is stored in the liver and converted to active metabolites, most notably retinoic acid (RA), which acts as an agonist for the retinoic acid receptors ((RARs α, ß, and γ) to regulate gene transcription. In this review we discuss some of the actions of retinoids in the kidney after injury. For example, in an ischemia-reperfusion model in mice, injury-associated loss of proximal tubule (PT) differentiation markers occurs, followed by re-expression of these differentiation markers during PT repair. Notably, healthy proximal tubules express ALDH1a2, the enzyme that metabolizes retinaldehyde to RA, but transiently lose ALDH1a2 expression after injury, while nearby myofibroblasts transiently acquire RA-producing capabilities after injury. These results indicate that RA is important for renal tubular injury repair and that compensatory mechanisms exist for the generation of endogenous RA by other cell types upon proximal tubule injury. ALDH1a2 levels also increase in podocytes, epithelial cells of the glomeruli, after injury, and RA promotes podocyte differentiation. We also review the ability of exogenous, pharmacological doses of RA and receptor selective retinoids to treat numerous kidney diseases, including kidney cancer and diabetic kidney disease, and the emerging genetic evidence for the importance of retinoids and their receptors in maintaining or restoring kidney function after injury. In general, RA has a protective effect on the kidney after various types of injuries (eg. ischemia, cytotoxic actions of chemicals, hyperglycemia related to diabetes). As more research into the actions of each of the three RARs in the kidney is carried out, a greater understanding of the actions of vitamin A is likely to lead to new insights into the pathology of kidney disorders and the development of new therapies for kidney diseases.

Liraglutide ameliorates hepatic steatosis via retinoic acid receptor-related orphan receptor α-mediated autophagy pathway.

Liraglutide, an analog of human glucagon-like peptide-1 (GLP-1), has been found to improve hepatic steatosis in clinical practice. However, the underlying mechanism remains to be fully defined. Increasing evidence suggests that retinoic acid receptor-related orphan receptor α (RORα) is involved in hepatic lipid accumulation. In the current study, we investigated whether the ameliorating impact of liraglutide on lipid-induced hepatic steatosis is dependent on RORα activity and examined the underlying mechanisms. Cre-loxP-mediated, liver-specific Rorα knockout (Rora LKO) mice, and littermate controls with a Roraloxp/loxp genotype were established. The effects of liraglutide on lipid accumulation were evaluated in mice challenged with a high-fat diet (HFD) for 12 weeks. Moreover, mouse AML12 hepatocytes expressing small interfering RNA (siRNA) of Rora were exposed to palmitic acid to explore the pharmacological mechanism of liraglutide. The results showed that liraglutide treatment significantly alleviated HFD-induced liver steatosis, marked by reduced liver weight and triglyceride accumulation, improved glucose tolerance and serum levels of lipid profiles and aminotransferase. Consistently, liraglutide also ameliorated lipid deposits in a steatotic hepatocyte model in vitro. In addition, liraglutide treatment reversed the HFD-induced downregulation of Rora expression and autophagic activity in mouse liver tissues. However, the beneficial effect of liraglutide on hepatic steatosis was not observed in Rora LKO mice. Mechanistically, the ablation of Rorα in hepatocytes diminished liraglutide-induced autophagosome formation and the fusion of autophagosomes and lysosomes, resulting in weakened autophagic flux activation. Thus, our findings suggest that RORα is essential for the beneficial impact of liraglutide on lipid deposition in hepatocytes and regulates autophagic activity in the underlying mechanism.

Retinoic acid protects against lipopolysaccharide-induced ferroptotic liver injury and iron disorders by regulating Nrf2/HO-1 and RARß signaling.

Acute liver injury (ALI) can progress to severe liver diseases, making its prevention and treatment a focus of research. Retinoic acid (RA) has been shown to have anti-oxidative and iron-regulatory effects on organs. In this study, we investigated the effect of RA on lipopolysaccharide (LPS)-induced ALI in both in vivo and in vitro experiments. We found that RA significantly reduced LPS-induced serum iron and red blood cell-associated disorders, as well as decreased serum ALT and AST levels. RA also reversed the accumulation of non-heme iron and labile iron in LPS-induced mice and hepatocytes by increasing the expression of FTL/H and Fpn. Furthermore, RA inhibited tissue reactive oxygen species (ROS) and malondialdehyde (MDA) production and improved the expression of Nrf2/HO-1/GPX4 in mice and Nrf2 signaling in hepatocytes. In vitro experiments employing RAR agonists and antagonists have revealed that retinoic acid (RA) can effectively inhibit cell ferroptosis induced by lipopolysaccharide (LPS), erastin, and RSL3. The mechanism underlying this inhibition may involve the activation of retinoic acid receptors beta (RARß) and gamma (RARγ). Knocking down the RARß gene in Hepatocytes cells significantly diminished the RA's protective effect, indicating that the anti-ferroptotic role of RA was partially mediated by RARß signaling. Overall, our study demonstrated that RA inhibited ferroptosis-induced liver damage by regulating Nrf2/HO-1/GPX4 and RARß signaling.

Discovery, Synthesis, and In Vitro Characterization of 2,3 Derivatives of 4,5,6,7-Tetrahydro-Benzothiophene as Potent Modulators of Retinoic Acid Receptor-Related Orphan Receptor γt.

Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor that is expressed in a variety of tissues and is a potential drug target for the treatment of inflammatory and auto-immune diseases, metabolic diseases, and resistant cancer types. We herein report the discovery of 2,3 derivatives of 4,5,6,7-tetrahydro-benzothiophene modulators of RORγt. We also report the solubility in acidic/neutral pH, mouse/human/dog/rat microsomal stability, Caco-2, and MDR1-MDCKII permeabilities of a set of these derivatives. For this group of modulators, inverse agonism by steric clashes and push-pull mechanisms induce greater instability to protein conformation compared to agonist lock hydration. Independent of the two mechanisms, we observed a basal modulatory activity of the tested 2,3 derivatives of 4,5,6,7-tetrahydro-benzothiophene toward RORγt due to the interactions with the Cys320-Glu326 and Arg364-Phe377 hydrophilic regions. The drug discovery approach reported in the current study can be employed to discover modulators of nuclear receptors and other globular protein targets.