Single-cell RNA-Seq analysis of molecular changes during radiation-induced skin injury: the involvement of Nur77.

Introduction: Ionizing radiation has been widely used in industry, medicine, military and agriculture. Radiation-induced skin injury is a significant concern in the context of radiotherapy and accidental exposure to radiation. The molecular changes at the single-cell level and intercellular communications during radiation-induced skin injury are not well understood. Methods: This study aims to illustrate this information in a murine model and human skin samples from a radiation accident using single-cell RNA sequencing (scRNA-Seq). We further characterize the functional significance of key molecule, which may provide a potential therapeutic target. ScRNA-Seq was performed on skin samples from a nuclear accident patient and rats exposed to ionizing radiation. Bioinformatic tools were used to analyze the cellular heterogeneity and preferential mRNAs. Comparative analysis was performed to identify dysregulated pathways, regulators, and ligand-receptor interactions in fibroblasts. The function of key molecule was validated in skin cells and in three mouse models of radiation-induced skin injury. Results: 11 clusters in human skin and 13 clusters of cells in rat skin were depicted respectively. Exposure to ionizing radiation caused changes in the cellular population (upregulation of fibroblasts and endothelial cells, downregulation of keratinocytes). Fibroblasts and keratinocytes possessed the most interaction pairs with other cell lineages. Among the five DEGs common to human and rat skins, Nur77 was highly expressed in fibroblasts, which mediated radiosensitivity by cell apoptosis and modulated crosstalk between macrophages, keratinocytes and endothelial cells in radiation-induced skin injury. In animal models, Nur77 knock-out mice (Nur77 -/-) showed more severe injury after radiation exposure than wild-type counterparts in three models of radiation-induced skin injury with complex mechanisms. Conclusion: The study reveals a single-cell transcriptional framework during radiation-induced skin injury, which provides a useful resource to uncover key events in its progression. Nur77 is a novel target in radiation-induced skin injury, which provides a potential therapeutic strategy against this disease.

The phthalide compound tokinolide B from Angelica sinensis exerts anti-inflammatory effects through Nur77 binding.

BACKGROUND: Nur77, an orphan member of the nuclear receptor superfamily, regulates inflammatory diseases and is a therapeutic target for treating inflammation. Phthalides in Angelica sinensis exhibit anti-inflammatory activity. PURPOSE: This study aimed to screen compounds from A. sinensis phthalide extract that could exert anti-inflammatory activity by targeting Nur77. To provide new theoretical support for better elucidation of Chinese medicine targeting mitochondria to achieve multiple clinical efficacies. METHODS: The anti-inflammatory capacity of phthalides was assessed in tumor necrosis factor-alpha (TNF-α)-stimulated HepG2 cells using western blotting. The interaction between phthalides and Nur77 was verified by molecular docking, surface plasmon resonance, and cellular thermal shift assay. Co-immunoprecipitation, western blotting, and immunostaining were performed to determine the molecular mechanisms. The in vivo anti-inflammatory activity of the phthalides was evaluated in a lipopolysaccharide (LPS)/d-galactosamine (d-GalN)-induced acute hepatitis and liver injury mouse model of acute hepatitis and liver injury. Finally, the toxicity of phthalide toxicity was assessed in zebrafish experiments. RESULTS: Among the 27 phthalide compounds isolated from A. sinensis, tokinolide B (TB) showed the best Nur77 binding capacity and, the best anti-inflammatory activity, which was induced without apoptosis. In vivo and in vitro experiments showed that TB promoted Nur77 translocation from the nucleus to the mitochondria and interacted with tumor necrosis factor receptor-associated factor 2 (TRAF2) and sequestosome 1 (p62) to induce mitophagy for anti-inflammatory functions. TB substantially inhibited LPS/d-GalN-induced acute hepatitis and liver injury in mice. TB also exhibited significantly lower toxicity than celastrol in zebrafish experiments. CONCLUSION: These findings suggested that TB inhibits inflammation by promoting Nur77 interaction with TRAF2 and p62, thereby inducing mitophagy. These findings offer promising directions for developing novel anti-inflammatory agents, enhance the understanding of phthalide compounds, and highlight the therapeutic potential of traditional Chinese herbs.

[Effect of Csn-B Combined with Imatinib on Apoptosis of Chronic Myeloid Leukemia Cells through Nur77/Pim-1/Drp1 Pathway].

OBJECTIVE: To investigate the anti- chronic myelogenous leukemia (CML) activity of Nur77-specific agonist Csn-B combined with imatinib by promoting Nur77 expression, and explore the potential role of its signaling pathway. METHODS: Firstly, CCK-8 and Transwell assay were used to detect the inhibitory effects of Csn-B, imatinib, and their combination on the proliferation and migration of K562 cells. Furthermore, the apoptosis rate of K562 cells treated with Csn-B, imatinib, and their combination was detected by flow cytometry. The expression levels of Nur77, Pim-1, Drp1, p-Drp1 S616, Bcl-2 and Bax in K562 cells were detected by Western blot. Finally, the expression levels of reactive oxygen species (ROS) in K562 cells treated with Csn-B, imatinib and their combination were detected by immunofluorescence assay. RESULTS: The level of Nur77 in CML patients decreased significantly compared with normal population in dataset of GSE43754 (P < 0.001). Csn-B combined with imatinib could significantly inhibit the proliferation and migration of K562 cells (both P < 0.001), and induce apoptosis (P < 0.001). Csn-B promoted Nur77 expression in K562 cells, and synergistically enhanced imatinib sensitivity when combined with imatinib. Csn-B combined with imatinib could significantly enhanced ROS levels in K562 cells and mitochondria compared with single-drug treatment (both P < 0.001). CONCLUSION: Csn-B combined with imatinib can enhance ROS expression and induce apoptosis of K562 cells through Nur77/Pim-1/Drp1 pathway.

Nur77 improves ovarian function in reproductive aging mice by activating mitophagy and inhibiting apoptosis.

Reproductive aging not only affects the fertility and physical and mental health of women but also accelerates the aging process of other organs. There is an urgent need newfor novel mechanisms, targets, and drugs to break the vicious cycle of mitochondrial dysfunction, redox imbalance, and germ cell apoptosis associated with ovarian aging. Autophagy, recognized as a longevity mechanism, has recently become a focal point in anti-aging research. Although mitophagy is a type of autophagy, its role and regulatory mechanisms in ovarian aging, particularly in age-related ovarian function decline, remain unclear. Nerve growth factor inducible gene B (Nur77) is an early response gene that can be stimulated by oxidative stress, DNA damage, metabolism, and inflammation. Recent evidence recommends that decreased expression of Nur77 is associated with age-related myocardial fibrosis, renal dysfunction, and Parkinson's disease; however, its association with ovarian aging has not been studied yet. We herein identified Nur77 as a regulator of germ cell senescence, apoptosis, and mitophagy and found that overexpression of Nur77 can activate mitophagy, improve oxidative stress, reduce apoptosis, and ultimately enhance ovarian reserve in aged mice ovaries. Furthermore, we discovered an association between Nur77 and the AKT pathway through String and molecular docking analyses. Experimental confirmation revealed that the AKT/mTOR signaling pathway is involved in the regulation of Nur77 in ovarian function. In conclusion, our results suggest Nur77 as a promising target for preventing and treating ovarian function decline related to reproductive aging.

Targeting KPNB1 with genkwadaphnin suppresses gastric cancer progression through the Nur77-mediated signaling pathway.

Gastric cancer (GC) remains a global challenge due to the lack of early detection and precision therapies. Genkwadaphnin (DD1), a natural diterpene isolated from the bud of Flos GenkWa (Thymelaeaceae), serves as a Karyopherin ß1 (KPNB1) inhibitor. In this study, we investigated the anti-tumor effect of DD1 in both cell culture and animal models. Our findings reveal that KPNB1, a protein involved in nuclear import, was highly expressed in GC tissues and associated with a poor prognosis in patients. We demonstrated that DD1, alongside the established KPNB1 inhibitor importazole (IPZ), inhibited GC cell proliferation and tumor growth by enhancing both genomic and non-genomic activity of Nur77. DD1 and IPZ reduced the interaction between KPNB1 and Nur77, resulting in Nur77 cytoplasmic accumulation and triggering mitochondrial apoptosis. The inhibitors also increased the expression of the Nur77 target apoptotic genes ATF3, RB1CC1 and PMAIP1, inducing apoptosis in GC cell. More importantly, loss of Nur77 effectively rescued the inhibitory effect of DD1 and IPZ on GC cells in both in vitro and in vivo experiments. In this study, we for the first time explored the relationship between KPNB1 and Nur77, and found KPNB1 inhibition could significantly increase the expression of Nur77. Moreover, we investigated the function of KPNB1 in GC for the first time, and the results suggested that KPNB1 could be a potential target for cancer therapy, and DD1 might be a prospective therapeutic candidate.

Nuclear receptor Nur77 regulates immunomechanics of macrophages.

Nuclear receptor Nur77 plays a pivotal role in immune regulation across various tissues, influencing pro-inflammatory signaling pathways and cellular metabolism. While cellular mechanics have been implicated in inflammation, the contribution of Nur77 to these mechanical processes remains elusive. Macrophages exhibit remarkable plasticity in their morphology and mechanics, enabling them to adapt and execute essential inflammatory functions, such as navigating through inflamed tissue and pathogen engulfment. However, the precise regulatory mechanisms governing these dynamic changes in macrophage mechanics during inflammation remain poorly understood. To establish the potential correlation of Nur77 with cellular mechanics, we compared bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Nur77-deficient (Nur77-KO) mice and employed cytoskeletal imaging, single-cell acoustic force spectroscopy (AFS), migration and phagocytosis assays, and RNA-sequencing. Our findings reveal that Nur77-KO BMDMs exhibit changes to their actin networks compared to WT BMDMs, which is associated with a stiffer and more rigid phenotype. Subsequent in vitro experiments validated our observations, showcasing that Nur77 deficiency leads to enhanced migration, reduced adhesion, and increased phagocytic activity. The transcriptomics data confirmed altered mechanics-related pathways in Nur77-deficient macrophage that are accompanied by a robust pro-inflammatory phenotype. Utilizing previously obtained ChIP-data, we revealed that Nur77 directly targets differentially expressed genes associated with cellular mechanics. In conclusion, while Nur77 is recognized for its role in reducing inflammation of macrophages by inhibiting the expression of pro-inflammatory genes, our study identifies a novel regulatory mechanism where Nur77 governs macrophage inflammation through the modulation of expression of genes involved in cellular mechanics. Our findings suggest that immune regulation by Nur77 may be partially mediated through alterations in cellular mechanics, highlighting a potential avenue for therapeutic targeting.

Nur77 Mediates Anaphylaxis by Regulating miR-21a.

Nur77 belongs to the NR4A subfamily of orphan nuclear hormone receptors. It has been shown to play important roles in metabolism, cancer progression, cellular differentiation, and the regulation of immune process. However, there has yet to be research reporting on the role of Nur77 in allergic inflammations such as anaphylaxis. This study aimed to identify molecules that could mediate allergic inflammations. To this end, we performed RNA sequencing analysis employing bone marrow-derived mast cells (BMMCs). Antigen (DNP-HSA) stimulation increased the expression levels of transcription factors such as Nr4a3 (NOR1), Nr4a1 (Nur77), and Nr4a2 (Nurr1). We focused our study on Nur77. Antigen stimulation increased the expression of Nur77 in a time- and dose-dependent manner in rat basophilic leukemia cells (RBL2H3). The downregulation of Nur77 prevented both antigen-induced increase in ß-hexosaminidase activity as well as hallmarks of allergic reactions such as HDAC3, COX2, and MCP1 in RBL2H3 cells. Nur77 was necessary for both passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA). TargetScan analysis predicted that miR-21a would be a negative regulator of Nur77. miR-21a mimic negatively regulated PCA and PSA by inhibiting the hallmarks of allergic reactions. ChIP assays showed that c-JUN could bind to the promoter sequences of Nur77. Antigen stimulation increased the expression of c-JUN in RBL2H3 cells. Altogether, our findings demonstrate the regulatory role played by Nur77-miR-21a loop in allergic inflammations such as anaphylaxis, making this the first report to present the role played by Nur77 in an allergic inflammation. Our results suggest that Nur77 and miR-21 might serve as targets for developing anti-allergy drugs.

In Silico Discovery of Stapled Peptide Inhibitor Targeting the Nur77-PPARγ Interaction and Its Anti-Breast-Cancer Efficacy.

The binding of peroxisome proliferator-activated receptor γ (PPARγ) to the orphan nuclear receptor Nur77 facilitates the ubiquitination and degradation of Nur77, and leads to aberrant fatty acid uptake for breast cancer progression. Because of its crucial role in clinical prognosis, the interaction between Nur77 and PPARγ is an attractive target for anti-breast-cancer therapy. However, developing an inhibitor of the Nur77-PPARγ interaction poses a technical challenge due to the absence of the crystal structure of PPARγ and its corresponding interactive model with Nur77. Here, ST-CY14, a stapled peptide, is identified as a potent modulator of Nur77 with a KD value of 3.247 × 10-8 M by in silico analysis, rational design, and structural modification. ST-CY14 effectively increases Nur77 protein levels by blocking the Nur77-PPARγ interaction, thereby inhibiting lipid metabolism in breast tumor cells. Notably, ST-CY14 significantly suppresses breast cancer growth and bone metastasis in mice. The findings demonstrate the feasibility of exploiting directly Nur77-PPARγ interaction in breast cancer, and generate what to the best knowledge is the first direct inhibitor of the Nur77-PPARγ interaction available for impeding fatty acid uptake and therapeutic development.

Regulation of the Nur77-P2X7r Signaling Pathway by Nodakenin: A Potential Protective Function against Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is the main factor that induces liver-related death worldwide and represents a common chronic hepatopathy resulting from binge or chronic alcohol consumption. This work focused on revealing the role and molecular mechanism of nodakenin (NK) in ALD associated with hepatic inflammation and lipid metabolism through the regulation of Nur77-P2X7r signaling. In this study, an ALD model was constructed through chronic feeding of Lieber-DeCarli control solution with or without NK treatment. Ethanol (EtOH) or NK was administered to AML-12 cells, after which Nur77 was silenced. HepG2 cells were exposed to ethanol (EtOH) and subsequently treated with recombinant Nur77 (rNur77). Mouse peritoneal macrophages (MPMs) were treated with lipopolysaccharide/adenosine triphosphate (LPS/ATP) and NK, resulting in the generation of conditioned media. In vivo, histopathological alterations were markedly alleviated by NK, accompanied by reductions in serum triglyceride (TG), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels and the modulation of Lipin-1, SREBP1, and Nur77 levels in comparison to the EtOH-exposed group (p < 0.001). Additionally, NK reduced the production of P2X7r and NLRP3. NK markedly upregulated Nur77, inhibited P2X7r and Lipin-1, and promoted the function of Cytosporone B, a Nur77 agonist (p < 0.001). Moreover, Nur77 deficiency weakened the regulatory effect of NK on P2X7r and Lipin-1 inhibition (p < 0.001). In NK-exposed MPMs, cleaved caspase-1 and mature IL-1ß expression decreased following LPS/ATP treatment (p < 0.001). NK also decreased inflammatory-factor production in primary hepatocytes stimulated with MPM supernatant. NK ameliorated ETOH-induced ALD through a reduction in inflammation and lipogenesis factors, which was likely related to Nur77 activation. Hence, NK is a potential therapeutic approach to ALD.

Phase separation of Nur77 mediates XS561-induced apoptosis by promoting the formation of Nur77/Bcl-2 condensates.

The orphan nuclear receptor Nur77 is a critical regulator of the survival and death of tumor cells. The pro-death effect of Nur77 can be regulated by its interaction with Bcl-2, resulting in conversion of Bcl-2 from a survival to killer. As Bcl-2 is overexpressed in various cancers preventing them from apoptosis and promoting their resistance to chemotherapy, targeting the apoptotic pathway of Nur77/Bcl-2 may lead to new cancer therapeutics. Here, we report our identification of XS561 as a novel Nur77 ligand that induces apoptosis of tumor cells by activating the Nur77/Bcl-2 pathway. In vitro and animal studies revealed an apoptotic effect of XS561 in a range of tumor cell lines including MDA-MB-231 triple-negative breast cancer (TNBC) and MCF-7/LCC2 tamoxifen-resistant breast cancer (TAMR) in a Nur77-dependent manner. Mechanistic studies showed XS561 potently induced the translocation of Nur77 from the nucleus to mitochondria, resulting in mitochondria-related apoptosis. Interestingly, XS561-induced accumulation of Nur77 at mitochondria was associated with XS561 induction of Nur77 phase separation and the formation of Nur77/Bcl-2 condensates. Together, our studies identify XS561 as a new activator of the Nur77/Bcl-2 apoptotic pathway and reveal a role of phase separation in mediating the apoptotic effect of Nur77 at mitochondria.

An engineering strategy to target activated EGFR with CAR T cells.

Chimeric antigen receptor (CAR) T cells have shown remarkable response rates in hematological malignancies. In contrast, CAR T cell treatment of solid tumors is associated with several challenges, in particular the expression of most tumor-associated antigens at lower levels in vital organs, resulting in on-target/off-tumor toxicities. Thus, innovative approaches to improve the tumor specificity of CAR T cells are urgently needed. Based on the observation that many human solid tumors activate epidermal growth factor receptor (EGFR) on their surface through secretion of EGFR ligands, we developed an engineering strategy for CAR-binding domains specifically directed against the ligand-activated conformation of EGFR. We show, in several experimental systems, that the generated binding domains indeed enable CAR T cells to distinguish between active and inactive EGFR. We anticipate that this engineering concept will be an important step forward to improve the tumor specificity of CAR T cells directed against EGFR-positive solid cancers.

Nur77 inhibition of ß-catenin expression mediates Hepatoblastoma progression and enhances cisplatin's therapeutic effect.

Hepatoblastoma (HB) is the most common malignant tumor in children under 5 years old, but its pathogenesis remains unclear. Nur77 has been reported to be an important regulator for cancer progression in various cancer types. This study found that Nur77 was downregulated in HB tumors, compared with paracancer tissue. Knockout or overexpression of Nur77 in HB tumor cell line HepG2 and HuH6 could significantly enhance or inhibit the proliferation, migration and invasion of tumor cells both in vitro and in vivo. Further studies illustrated that Nur77 regulated the proliferation of tumor cells by affecting the expression of ß-catenin. Nur77 agonist Csn-B effectively enhanced the therapeutic effect of cisplatin on HB tumors both in vitro and in vivo. This study confirms that Nur77 may act as an oncogene in HB tumors and mediate the progression of HB by inhibiting the expression of ß-catenin, which provides a new targeted therapy for the clinical treatment of HB patients; meanwhile, the combination of Nur77 agonist and cisplatin treatment may improve the chemotherapeutic efficacy of HB patients, which provides a new idea for the improvement of the clinical prognosis of HB patients.

Nur77 increases mitophagy and decreases aggregation of α-synuclein by modulating the p-c-Abl/p-PHB2 Y121 in α-synuclein PFF SH-SY5Y cells and mice.

Parkinson's disease (PD) is characterized by the progressive death of dopamine (DA) neurons and the pathological accumulation of α-synuclein (α-syn) fibrils. In our previous study, simulated PHB2 phosphorylation was utilized to clarify the regulatory role of c-Abl in PHB2-mediated mitophagy in PD models. In this investigation, we employed an independently patented PHB2Y121 phosphorylated antibody in the PD model to further verify that the c-Abl inhibitor STI571 can impede PHB2Y121 phosphorylation, decrease the formation of α-Syn polymers, and improve autophagic levels. The specific involvement of Nur77 in PD pathology has remained elusive. We also investigate the contribution of Nur77, a nuclear transcription factor, to α-syn and mitophagy in PD. Our findings demonstrate that under α-syn, Nur77 translocates from the cytoplasm to the mitochondria, improving PHB-mediated mitophagy by regulating c-Abl phosphorylation. Moreover, Nur77 overexpression alleviates the expression level of pS129-α-syn and the loss of DA neurons in α-syn PFF mice, potentially associated with the p-c-Abl/p-PHB2 Y121 axis. This study provides initial in vivo and in vitro evidence that Nur77 protects PD DA neurons by modulating the p-c-Abl/p-PHB2 Y121 axis, and STI571 holds promise as a treatment for PD.

Exploring the impact of the PNPLA3 I148M variant on primary human hepatic stellate cells using 3D extracellular matrix models.

BACKGROUND & AIMS: The PNPLA3 rs738409 C>G (encoding for I148M) variant is a risk locus for the fibrogenic progression of chronic liver diseases, a process driven by hepatic stellate cells (HSCs). We investigated how the PNPLA3 I148M variant affects HSC biology using transcriptomic data and validated findings in 3D-culture models. METHODS: RNA sequencing was performed on 2D-cultured primary human HSCs and liver biopsies of individuals with obesity, genotyped for the PNPLA3 I148M variant. Data were validated in wild-type (WT) or PNPLA3 I148M variant-carrying HSCs cultured on 3D extracellular matrix (ECM) scaffolds from human healthy and cirrhotic livers, with/without TGFB1 or cytosporone B (Csn-B) treatment. RESULTS: Transcriptomic analyses of liver biopsies and HSCs highlighted shared PNPLA3 I148M-driven dysregulated pathways related to mitochondrial function, antioxidant response, ECM remodelling and TGFB1 signalling. Analogous pathways were dysregulated in WT/PNPLA3-I148M HSCs cultured in 3D liver scaffolds. Mitochondrial dysfunction in PNPLA3-I148M cells was linked to respiratory chain complex IV insufficiency. Antioxidant capacity was lower in PNPLA3-I148M HSCs, while reactive oxygen species secretion was increased in PNPLA3-I148M HSCs and higher in bioengineered cirrhotic vs. healthy scaffolds. TGFB1 signalling followed the same trend. In PNPLA3-I148M cells, expression and activation of the endogenous TGFB1 inhibitor NR4A1 were decreased: treatment with the Csn-B agonist increased total NR4A1 in HSCs cultured in healthy but not in cirrhotic 3D scaffolds. NR4A1 regulation by TGFB1/Csn-B was linked to Akt signalling in PNPLA3-WT HSCs and to Erk signalling in PNPLA3-I148M HSCs. CONCLUSION: HSCs carrying the PNPLA3 I148M variant have impaired mitochondrial function, antioxidant responses, and increased TGFB1 signalling, which dampens antifibrotic NR4A1 activity. These features are exacerbated by cirrhotic ECM, highlighting the dual impact of the PNPLA3 I148M variant and the fibrotic microenvironment in progressive chronic liver diseases. IMPACT AND IMPLICATIONS: Hepatic stellate cells (HSCs) play a key role in the fibrogenic process associated with chronic liver disease. The PNPLA3 genetic mutation has been linked with increased risk of fibrogenesis, but its role in HSCs requires further investigation. Here, by using comparative transcriptomics and a novel 3D in vitro model, we demonstrate the impact of the PNPLA3 genetic mutation on primary human HSCs' behaviour, and we show that it affects the cell's mitochondrial function and antioxidant response, as well as the antifibrotic gene NR4A1. Our publicly available transcriptomic data, 3D platform and our findings on NR4A1 could facilitate the discovery of targets to develop more effective treatments for chronic liver diseases.

Structure-based design and synthesis of anti-fibrotic compounds derived from para-positioned 3,4,5-trisubstituted benzene.

Liver fibrosis is an abnormal wound-healing response to liver injuries. It can lead to liver cirrhosis, and even liver cancer and liver failure. There is a lack of treatment for liver fibrosis and it is of great importance to develop anti-fibrotic drugs. A pivotal event in the process of developing liver fibrosis is the activation of hepatic stellate cells (HSCs), in which the nuclear receptor Nur77 plays a crucial role. This study aimed to develop novel anti-fibrotic agents with Nur77 as the drug target by modifying the structure of THPN, a Nur77-binding and anti-melanoma compound. Specifically, a series of para-positioned 3,4,5-trisubstituted benzene ring compounds with long-chain backbone were generated and tested for anti-fibrotic activity. Among these compounds, compound A8 was with the most potent and Nur77-dependent inhibitory activity against TGF-ß1-induced activation of HSCs. In a crystal structure analysis, compound A8 bound Nur77 in a peg-in-hole mode as THPN did but adopted a different conformation that could interfere the Nur77 interaction with AKT, which was previous shown to be important for an anti-fibrotic activity. In a cell-based assay, compound A8 indeed impeded the interaction between Nur77 and AKT leading to the stabilization of Nur77 without the activation of AKT. In a mouse model, compound A8 effectively suppressed the activation of AKT signaling pathway and up-regulated the cellular level of Nur77 to attenuate the HSCs activation and ameliorate liver fibrosis with no significant toxic side effects. Collectively, this work demonstrated that Nur77-targeting compound A8 is a promising anti-fibrotic drug candidate.

Nur77 mitigates endothelial dysfunction through activation of both nitric oxide production and anti-oxidant pathways.

BACKGROUND: Nur77 belongs to the member of orphan nuclear receptor 4A family that plays critical roles in maintaining vascular homeostasis. This study aims to determine whether Nur77 plays a role in attenuating vascular dysfunction, and if so, to determine the molecular mechanisms involved. METHODS: Both Nur77 knockout (Nur77 KO) and Nur77 endothelial specific transgenic mice (Nur77-Tg) were employed to examine the functional significance of Nur77 in vascular endothelium in vivo. Endothelium-dependent vasodilatation to acetylcholine (Ach) and reactive oxygen species (ROS) production was determined under inflammatory and high glucose conditions. Expression of genes was determined by real-time PCR and western blot analysis. RESULTS: In response to tumor necrosis factor alpha (TNF-α) treatment and diabetes, the endothelium-dependent vasodilatation to Ach was significantly impaired in aorta from Nur77 KO as compared with those from the wild-type (WT) mice. Endothelial specific overexpression of Nur77 markedly prevented both TNF-α- and high glucose-induced endothelial dysfunction. Compared with WT mice, after TNF-α and high glucose treatment, ROS production in aorta was significantly increased in Nur77 KO mice, but it was inhibited in Nur77-Tg mice, as determined by dihydroethidium (DHE) staining. Furthermore, we demonstrated that Nur77 overexpression substantially increased the expression of several key enzymes involved in nitric oxide (NO) production and ROS scavenging, including endothelial nitric oxide synthase (eNOS), guanosine triphosphate cyclohydrolase 1 (GCH-1), glutathione peroxidase-1 (GPx-1), and superoxide dismutases (SODs). Mechanistically, we found that Nur77 increased GCH1 mRNA stability by inhibiting the expression of microRNA-133a, while Nur77 upregulated SOD1 expression through directly binding to the human SOD1 promoter in vascular endothelial cells. CONCLUSION: Our results suggest that Nur77 plays an essential role in attenuating endothelial dysfunction through activating NO production and anti-oxidant pathways in vascular endothelium. Targeted activation of Nur77 may provide a novel therapeutic approach for the treatment of cardiovascular diseases associated with endothelial dysfunction.

Tasquinimod enhances the sensitivity of ovarian cancer cells to cisplatin by regulating the Nur77-Bcl-2 apoptotic pathway.

BACKGROUND: Resistance to cisplatin (DDP) in ovarian cancer therapy has been a major clinical barrier. Drug-resistant cancers have been shown to downregulate the proapoptotic protein B-cell lymphoma-2 (Bcl-2) to inhibit apoptosis. Therefore, we explored whether tasquinimod could modulate resistance to DDP through apoptotic pathways. OBJECTIVES: We aimed to explore the relationship between tasquinimod, Nur77-Bcl-2 apoptosis pathway and sensitivity of the ovarian carcinoma cell line SKOV3 and the DDP-resistant strain SKOV3/DDP cells to DDP. MATERIAL AND METHODS: First, SKOV3 and SKOV3/DDP cells were treated with 2 µg/mL DDP or 40 µM tasquinimod. Western blot and quantitative real-time polymerase chain reaction (qPCR) were then used to analyze the expression of histone deacetylase 4 (HDAC4), Nur77, Bcl-2 (BH3 domain-specific), and caspase-3. Flow cytometry, scratch-wound assay and immunofluorescence were used to detect apoptosis, migration rate, and related expression of Nur77 and Bcl-2 (BH3 domain-specific). Subsequently, 5×107 SKOV3 or SKOV3/DDP cells cultured with 2 µg/mL DDP were injected into 4-week-old female BALB/c nude mice. Then, the mice were administered 4 mg/kg DDP and 50 mg/kg tasquinimod every 3 days. Finally, the changes in tumor diameter and weight were measured. RESULTS: After treatment of SKOV3 and SKOV3/DDP cells with tasquinimod, cell migration and HDAC4 expression levels were significantly reduced, while Nur77 expression was increased. Tasquinimod treatment enhanced the expression of Nur77 and caspase-3, and cells transfected with si-Nur77 showed the opposite result. Transfection of si-Nur77 reduced the expression of caspase-3 and Nur77 in the SKOV3/DDP cells that were treated with both DDP and tasquinimod. After injection of SKOV3/DDP cells into the mice, the tumor diameter, mass and in vivo HDAC4 level were significantly decreased by tasquinimod. Meanwhile, the levels of Nur77 and Bcl-2 (BH3 domain-specific) were increased. CONCLUSIONS: Tasquinimod upregulated the Nur77/Bcl-2 pathway to induce apoptosis in SKOV3/DDP cells and enhanced the anti-tumor effect of DDP in SKOV3/DDP xenografts. Therefore, tasquinimod can be expected to find clinical applications in enhancing DDP resistance.

Honokiol ameliorates angiotensin II-induced cardiac hypertrophy by promoting dissociation of the Nur77-LKB1 complex and activating the AMPK pathway.

Pathological cardiac hypertrophy is a key contributor to heart failure, and the molecular mechanisms underlying honokiol (HNK)-mediated cardioprotection against this condition remain worth further exploring. This study aims to investigate the effect of HNK on angiotensin II (Ang II)-induced myocardial hypertrophy and elucidate the underlying mechanisms. Sprague-Dawley rats were exposed to Ang II infusion, followed by HNK or vehicle treatment for 4 weeks. Our results showed that HNK treatment protected against Ang II-induced myocardial hypertrophy, fibrosis and dysfunction in vivo and inhibited Ang II-induced hypertrophy in neonatal rat ventricular myocytes in vitro. Mechanistically, HNK suppressed the Ang II-induced Nur77 expression at the transcriptional level and promoted ubiquitination-mediated degradation of Nur77, leading to dissociation of the Nur77-LKB1 complex. This facilitated the translocation of LKB1 into the cytoplasm and activated the LKB1-AMPK pathway. Our findings suggest that HNK attenuates pathological remodelling and cardiac dysfunction induced by Ang II by promoting dissociation of the Nur77-LKB1 complex and subsequent activation of AMPK signalling. This study uncovers a novel role of HNK on the LKB1-AMPK pathway to protect against cardiac hypertrophy.

Mechanistic study of tripterygium glycosides in the treatment of ulcerative colitis through the Nur77-Traf2-P62 signaling pathway / 中国现代医生

@#Objective To investigated the effect of tripterygium glycosides(TG)on dextran sodium sulfate(DSS)-induced colonic mucosal damage in ulcerative colitis(UC)mice and its regulatory mechanism.Methods Forty C57BL/6J mice were randomly divided into a normal group,a model group,and a tretinoin low,medium,and high dose group(administered at concentrations of 9.00mg/kg,27.03mg/kg,and 81.09mg/kg,respectively).The mice in the normal group were free to drink distilled water,and the rest of the mice drank 5％DSS to induce UC modeling.After modeling,mice in the model group were given 0.4ml of saline by gavage daily,and the rest of the mice in the treatment group were given the corresponding dose of TG for gavage intervention.The mass and disease activity index of the mice in each group were compared,and the pathological and histological damage of the colon was observed.Tumor necrosis factor-α(TNF-α),malondialdehyde(MDA),and superoxide dismutase(SOD)levels were measured using the corresponding kits.Western blot Detection of Nur77,tumor necrosis factor receptor-associated factor 2(Traf2),nucleoporin 62(P62),autophagy protein-microtubule associated protein1 light chain 3(LC3)molecular expression.Results Compared with the blank group,the body weight,colon length,SOD,Nur77,Traf2,and LC3Ⅱ/LC3Ⅰ levels of mice in the model group were significantly decreased(P<0.05),and the DAI level,colon pathology score,TNF-α,MDA level,and P62 of the mice were significantly increased(P<0.05).Compared with mice in the UC model group,mice in the low,medium and high dose groups of tretinoin polyphenols showed significant increases in body weight,colon length,SOD,Nur77,Traf2,LC3Ⅱ/LC3Ⅰlevels(P<0.05),and mice with DAI scores,TNF-α,MDA levels in the colon,and P62 levels were significantly decreased(P<0.05).Mice in the medium and high dose groups of tretinoin polyphenols pathological scores were significantly reduced(P<0.05).Conclusion TG is able to treat ulcerative colitis through Nur77-Traf2-P62 signaling pathway.

Nur77 alleviates cardiac fibrosis by upregulating GSK-3ß transcription during aging.

Cardiac fibrosis is associated with aging, for which no targeted therapies are available. With aging, the levels of nerve growth factor-induced gene B (Nur77) are reduced during cardiac remodelling; however, its role in cardiac fibrosis in aging remains unclear. Here, we found that Nur77 knockout increased cardiac structure abnormalities, systolic and diastolic dysfunction, cardiac hypertrophy, and fibrotic marker expression in 15-month-old mice. Furthermore, Nur77 deficiency induced collagen type I (Col-1) and α-smooth muscle actin overproduction in transforming growth factor beta (TGF-ß) treated H9c2 cells, whereas Nur77 overexpression attenuated this effect. Nur77 deficiency in vivo and in vitro downregulated glycogen synthase kinase (GSK)-3ß expression and increased ß-catenin activity, while its overexpression increased GSK-3ß expression. GSK-3ß knockdown counteracted the anti-fibrotic effect of Nur77 on TGF-ß-treated H9c2 cells. Chromatin immunoprecipitation and luciferase reporter assay results suggested GSK-3ß as the direct target of Nur77. Our findings suggest that Nur77 directly initiates GSK-3ß transcription and age-related cardiac fibrosis partly through the GSK-3ß/ß-catenin pathway. This study proposes a novel mechanism for Nur77 regulating cardiac fibrosis and suggests Nur77 as a target for the prevention and treatment of aging-associated cardiac fibrosis and heart failure.