Nuclear Respiratory Factor-1 Expression Research Articles

Secondary infections may cause host mortality by inhibiting NRF1-mediated antiviral immune responses

Pathogen infection does not directly cause host death. Secondary infection is the main factor that leads to severe damage. Nuclear respiratory factor 1 (NRF1) is a nuclear transcription factor that regulate mitochondrial biogenesis. However, the function of NRF1 in antiviral immunity, especially its role in the process of co-infection is largely unknown. NRF1 inhibited virus infection by mediating apoptosis in arthropod, Procambarus clarkii. Notably, NRF1 expression was significantly induced in the early stage of white spot syndrome virus (WSSV) infection and reduced during the later stage of infection. NRF1 mediated apoptosis, phagocytosis, and reactive oxygen species (ROS) production 24 h after WSSV infection. The number of culturable bacteria was significantly increased and induced hypoxia in vivo 72 h after WSSV infection. Hypoxia exposure resulted in a decrease in NRF1 expression led to a decrease in body immunity and a vicious cycle until the crayfish died. This study demonstrated that secondary infections may be a major cause of biological mortality in crustaceans.

Immunometabolic features of natural killer cells are associated with infection outcomes in critical illness.

Immunosuppression increases the risk of nosocomial infection in patients with chronic critical illness. This exploratory study aimed to determine the immunometabolic signature associated with nosocomial infection during chronic critical illness. We prospectively recruited patients who were admitted to the respiratory care center and who had received mechanical ventilator support for more than 10 days in the intensive care unit. The study subjects were followed for the occurrence of nosocomial infection until 6 weeks after admission, hospital discharge, or death. The cytokine levels in the plasma samples were measured. Single-cell immunometabolic regulome profiling by mass cytometry, which analyzed 16 metabolic regulators in 21 immune subsets, was performed to identify immunometabolic features associated with the risk of nosocomial infection. During the study period, 37 patients were enrolled, and 16 patients (43.2%) developed nosocomial infection. Unsupervised immunologic clustering using multidimensional scaling and logistic regression analyses revealed that expression of nuclear respiratory factor 1 (NRF1) and carnitine palmitoyltransferase 1a (CPT1a), key regulators of mitochondrial biogenesis and fatty acid transport, respectively, in natural killer (NK) cells was significantly associated with nosocomial infection. Downregulated NRF1 and upregulated CPT1a were found in all subsets of NK cells from patients who developed a nosocomial infection. The risk of nosocomial infection is significantly correlated with the predictive score developed by selecting NK cell-specific features using an elastic net algorithm. Findings were further examined in an independent cohort of COVID-19-infected patients, and the results confirm that COVID-19-related mortality is significantly associated with mitochondria biogenesis and fatty acid oxidation pathways in NK cells. In conclusion, this study uncovers that NK cell-specific immunometabolic features are significantly associated with the occurrence and fatal outcomes of infection in critically ill population, and provides mechanistic insights into NK cell-specific immunity against microbial invasion in critical illness.

Vaporization of perfluorocarbon attenuates sea-water-drowning-induced acute lung injury by deactivating the NLRP3 inflammasomes in canines.

Seawater-drowning-induced acute lung injury (SD-ALI) is a life-threatening disorder characterized by increased alveolar-capillary permeability, an excessive inflammatory response, and refractory hypoxemia. Perfluorocarbons (PFCs) are biocompatible compounds that are chemically and biologically inert and lack toxicity as oxygen carriers, which could reduce lung injury in vitro and in vivo. The aim of our study was to explore whether the vaporization of PFCs could reduce the severity of SD-ALI in canines and investigate the underlying mechanisms. Eighteen beagle dogs were randomly divided into three groups: the seawater drowning (SW), perfluorocarbon (PFC), and control groups. The dogs in the SW group were intratracheally administered seawater to establish the animal model. The dogs in the PFC group were treated with vaporized PFCs. Probe-based confocal laser endomicroscopy (pCLE) was performed at 3h. The blood gas, volume air index (VAI), pathological changes, and wet-to-dry (W/D) lung tissue ratios were assessed. The expression of heme oxygenase-1 (HO-1), nuclear respiratory factor-1 (NRF1), and NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasomes was determined by means of quantitative real-time polymerase chain reaction (qRT-PCR) and immunological histological chemistry. The SW group showed higher lung injury scores and W/D ratios, and lower VAI compared to the control group, and treatment with PFCs could reverse the change of lung injury score, W/D ratio and VAI. PFCs deactivated NLRP3 inflammasomes and reduced the release of caspase-1, interleukin-1β (IL-1β), and interleukin-18 (IL-18) by enhancing the expression of HO-1 and NRF1. Our results suggest that the vaporization of PFCs could attenuate SD-ALI by deactivating NLRP3 inflammasomes via the HO-1/NRF1 pathway.

Andrographolide attenuated MCT-induced HSOS via regulating NRF2-initiated mitochondrial biogenesis and antioxidant response.

Hepatic sinusoidal obstruction syndrome (HSOS) is a death-dealing liver disease with a fatality rate of up to 67%. In the study present, we explored the efficacy of andrographolide (Andro), a diterpene lactone from Andrographis Herba, in ameliorating the monocrotaline (MCT)-induced HSOS and the underlying mechanism. The alleviation of Andro on MCT-induced rats HSOS was proved by biochemical index detection, electron microscope observation, and liver histological evaluation. Detection of hepatic ATP content, mitochondrial DNA (mtDNA) copy number, and protein expression of nuclear respiratory factor-1 (NRF1) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) demonstrated that Andro strengthened mitochondrial biogenesis in livers from MCT-treated rats. Chromatin immunoprecipitation assay exhibited that Andro enhanced the occupation of nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) in the promoter regions of both PPARGC1A and NRF1. Andro also activated the NRF2-dependent anti-oxidative response and alleviated liver oxidative injury. In Nrf2 knock-out mice, MCT induced more severe liver damage, and Andro showed no alleviation in it. Furthermore, the Andro-activated mitochondrial biogenesis and anti-oxidative response were reduced in Nrf2 knock-out mice. Contrastingly, knocking out Kelch-like ECH-associated protein 1 (Keap1), a NRF2 repressor, reduced MCT-induced liver damage. Results from co-immunoprecipitation, molecular docking analysis, biotin-Andro pull-down, cellular thermal shift assay, and surface plasmon resonance assay showed that Andro hindered the NRF2-KEAP1 interaction via directly binding to KEAP1. In conclusion, our results revealed that NRF2-dependent liver mitochondrial biogenesis and anti-oxidative response were essential for the Andro-provided alleviation of the MCT-induced HSOS. Graphical Headlights: 1. Andro alleviated MCT-induced HSOS via activating antioxidative response and promoting mitochondrial biogenesis. 2. Andro-activated antioxidative response and mitochondrial biogenesis were NRF2-dependent. 3. Andro activated NRF2 via binding to KEAP1.

Inhibitor of DNA Binding Protein 3 (ID3) and Nuclear Respiratory Factor 1 (NRF1) Mediated Transcriptional Gene Signatures are Associated with the Severity of Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is a degenerative vasculopathy. We have previously shown that transcription regulating proteins- inhibitor of DNA binding protein 3 (ID3) and the nuclear respiratory factor 1 (NRF1) contribute to vascular dysregulation. In this study, we have identified sex specific ID3 and NRF1-mediated gene networks in CAA patients diagnosed with Alzheimer's Disease (AD). High expression of ID3 mRNA coupled with low NRF1 mRNA levels was observed in the temporal cortex of men and women CAA patients. Low NRF1 mRNA expression in the temporal cortex was found in men with severe CAA. High ID3 expression was found in women with the genetic risk factor APOE4. Low NRF1 expression was also associated with APOE4 in women with CAA. Genome wide transcriptional activity of both ID3 and NRF1 paralleled their mRNA expression levels. Sex specific differences in transcriptional gene signatures of both ID3 and NRF1 were observed. These findings were further corroborated by Bayesian machine learning and the GeNIe simulation models. Dynamic machine learning using a Monte Carlo Markov Chain (MCMC) gene ordering approach revealed that ID3 was associated with disease severity in women. NRF1 was associated with CAA and severity of this disease in men. These findings suggest that aberrant ID3 and NRF1 activity presumably plays a major role in the pathogenesis and severity of CAA. Further analyses of ID3- and NRF1-regulated molecular drivers of CAA may provide new targets for personalized medicine and/or prevention strategies against CAA.

The Regulatory Role of Nuclear Respiratory Factor 1 in Bladder Cancer Cells.

Nuclear respiratory factor 1 (NRF1) is a key mediator of genes involved in mitochondrial biogenesis and the respiratory chain; however, its role in bladder cancer remains unknown. Transitional cell carcinoma, also known as urothelial cell carcinoma, is the most common type of bladder cancer resistant to chemotherapy. An established high-grade and invasive transitional cell carcinoma line from patients with urinary bladder cancer, known as T24, has been extensively used in cancer research. In this study, we aimed to investigate the mechanisms through which NRF1 regulates proliferation and cell migration of bladder cancer cells using the T24 cell line. Cells were transfected with plasmid cloning DNA for NRF1 to evaluate the effect of NRF1 overexpression on bladder cancer cells. Western blot was used to examine epithelial and mesenchymal markers (E-cadherin and α-smooth muscle actin), transcriptional regulators for epithelial-mesenchymal transition (snail family transcriptional repressors), components of transforming growth factor-β1/SMADs signaling, high-mobility group box 1 (HMGB1), and receptor for advanced glycation end-products (RAGE). The in situ expression of E-cadherin, α-smooth muscle actin and SMAD7 was determined using immunofluorescence staining. Cell migration capacity was assessed by wound-healing assay. Transfection with NRF1 expression vector repressed the migration capacity of bladder cancer cells, diminishing HMGB1/RAGE expression and reducing transforming growth factor β-associated epithelial-mesenchymal transition in T24 cells. Therapeutic avenues that increase NRF1 expression may serve as an adjunct to conventional treatments for bladder cancer.

Nuclear respiratory factor 1 promotes the progression of EBV-associated gastric cancer and maintains EBV latent infection.

This study aimed to investigate the association of Epstein-Barr virus (EBV) with nuclear respiratory factor 1 (NRF1) and the biological function of NRF1 in EBV-associated gastric cancer (EBVaGC). Western blot and qRT-PCR were used to assess the effect of latent membrane protein 2A (LMP2A) on NRF1 expression after transfection with LMP2A plasmid or siLMP2A. The effects of NRF1 on the migration and apoptosis ability of GC cells were investigated by transwell assay and flow cytometry apoptosis analysis in vitro, respectively. In addition, we determined the regulatory role of NRF1 in EBV latent infection by western blot and droplet digital PCR (ddPCR). LMP2A upregulated NRF1 expression by activating the NF-κB pathway. Moreover, NRF1 upregulated the expression of N-Cadherin and ZEB1 to promote cell migration. NRF1 promoted the expression of Bcl-2 to increase the anti-apoptotic ability of cells. In addition, NRF1 maintained latent infection of EBV by promoting the expression of the latent protein Epstein-Barr nuclear antigen 1 (EBNA1) and inhibiting the expression of the lytic proteins. Our data indicated the role of NRF1 in EBVaGC progression and the maintenance of EBV latent infection. This provided a new theoretical basis for further NRF1-based anti-cancer therapy.

NRF1 Alleviated Oxidative Stress of Glioblastoma Cells by Regulating NOR1.

Oxidored-nitro domain-containing protein 1 (NOR1) is a critical tumour suppressor gene, though its regulatory mechanism in oxidative stress of glioblastoma (GBM) remains unclear. Hence, further study is needed to unravel the function of NOR1 in the progression of oxidative stress in GBM. In this study, we evaluated the expression of NOR1 and nuclear respiratory factor 1 (NRF1) in GBM tissue and normal brain tissue (NBT) using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB), and investigated their relationship. We then induced oxidative stress in U251 cells through H2O2 treatment and conducted Cell Count-ing Kit-8, Transwell and wound healing assays to analyse cell proliferation, invasion and migration. Cell apoptosis was assessed by flow cytometry and TUNEL staining. We also measured the activities of superoxide dismutase and catalase, as well as the level of reactive oxygen species (ROS) using biochemical techniques. Via qRT-PCR and WB, the mRNA and protein expression levels of NOR1 and NRF1 were determined. Chromatin immunoprecipitation (ChIP) assays were applied to validate NRF1's interaction with NOR1. Our results showed that the expression of NOR1 and NRF1 was low in GBM, and their expression levels were positively correlated. H2O2-induced oxidative stress reduced NRF1 and NOR1 expression levels and increased the ROS level. The ChIP assay confirmed the binding of NRF1 to NOR1. Over-expression of NRF1 attenuated the inhibitory effect of oxidative stress on the proliferation, migration and invasion of U251 cells, which was reversed by knockdown of NOR1.

NRF1 Regulates the Epithelial Mesenchymal Transition of Breast Cancer by Modulating ROS Homeostasis.

Introduction: Nuclear respiratory factor 1 (NRF1) is an important regulator involved in mitochondrial biogenesis and energy metabolism. However, the specific mechanism of NRF1 in anoikis and epithelial-mesenchymal transition (EMT) remains unclear. Methods: We examined the effect of NRF1 on mitochondria and identified the specific mechanism through transcriptome sequencing, and explored the relationships among NRF1, anoikis, and EMT. Results: We found that upregulated NRF1 expression led to increased mitochondrial oxidative phosphorylation (OXPHOS) and ATP generation. Simultaneously, a significant amount of ROS is generated during OXPHOS. Alternatively, NRF1 upregulates the expression of ROS-scavenging enzymes, allowing tumor cells to maintain low ROS levels and promoting anoikis resistance and EMT. We also found that exogenous ROS was maintained at a low level by NRF1 in breast cancer cells. Conclusion: our study provides mechanistic insight into the function of NRF1 in breast cancer, indicating that NRF1 may serve as a therapeutic target for breast cancer treatment.

Agrimol B inhibits colon carcinoma progression by blocking mitochondrial function through the PGC-1α/NRF1/TFAM signaling pathway.

The activation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) stimulates the transcription of the downstream target proteins, mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF1), which induces mitochondrial biogenesis and promotes colorectal tumorigenesis. Agrimol B (Agr) is a constituent of Agrimonia pilosa Ledeb. that exerts anticancer effects. Herein, we aimed to investigate the antitumor activity of Agr and its mechanism of action. The interaction between Agr and PGC-1α was predicted by molecular docking. After the treatment with different concentrations of Agr (0, 144, 288, and 576 nM), the cell viability, migration rate, proliferation rate, and apoptosis rate of human colon cancer HCT116 cells were determined. Mitochondrial activity, cellular reactive oxygen species (ROS), and mitochondrial membrane potential were assessed to measure the regulatory effect of Agr on mitochondrial function. Western blotting (WB) assay was used to examine the expression of PGC-1α, NRF1, and TFAM, as well as of the pro-apoptotic proteins, Bax and Caspase-3, and the antiapoptotic protein (Bcl-2). Finally, subcutaneous tumor xenograft model mice were used to evaluate the effect of Agr on colorectal cancer (CRC) in vivo. The molecular docking results revealed a high likelihood of Agr interacting with PGC-1α. Agr inhibited the proliferation and migration of HCT116 cells, promoted ROS production and mitochondrial oxidative stress, inhibited mitochondrial activity, and decreased mitochondrial membrane potential. Agr induced cell apoptosis and, in combination with PGC-1α, impaired mitochondrial biogenesis and suppressed the expression of NRF1 and TFAM. Agr also suppressed the expression of Bcl-2 and Cleaved-Caspase-3 and increased the expression of Bax and Caspase-3. In addition, the in vivo antitumor effect and mechanism of Agr were confirmed by using a subcutaneous tumor xenograft mouse model. Our findings demonstrated that Agr regulates the expression of PGC-1α, thereby inducing mitochondrial dysfunction and promoting tumor cell apoptosis. This work highlights the potential of Agr as a promising therapeutic candidate in CRC.

Caveolin-1 accelerates hypoxia-induced endothelial dysfunction in high-altitude cerebral edema

BackgroundHigh-altitude cerebral edema (HACE) is a serious and potentially fatal brain injury that is caused by acute hypobaric hypoxia (HH) exposure. Vasogenic edema is the main pathological factor of this condition. Hypoxia-induced disruptions of tight junctions in the endothelium trigger blood‒brain barrier (BBB) damage and induce vasogenic edema. Nuclear respiratory factor 1 (NRF1) acts as a major regulator of hypoxia-induced endothelial cell injury, and caveolin-1 (CAV-1) is upregulated as its downstream gene in hypoxic endothelial cells. This study aimed to investigate whether CAV-1 is involved in HACE progression and the underlying mechanism.MethodsC57BL/6 mice were exposed to HH (7600 m above sea level) for 24 h, and BBB injury was assessed by brain water content, Evans blue staining and FITC-dextran leakage. Immunofluorescence, transmission electron microscope, transendothelial electrical resistance (TEER), transcytosis assays, and western blotting were performed to confirm the role and underlying mechanism of CAV-1 in the disruption of tight junctions and BBB permeability. Mice or bEnd.3 cells were pretreated with MβCD, a specific blocker of CAV-1, and the effect of CAV-1 on claudin-5 internalization under hypoxic conditions was detected by immunofluorescence, western blotting, and TEER. The expression of NRF1 was knocked down, and the regulation of CAV-1 by NRF1 under hypoxic conditions was examined by qPCR, western blotting, and immunofluorescence.ResultsThe BBB was severely damaged and was accompanied by a significant loss of vascular tight junction proteins in HACE mice. CAV-1 was significantly upregulated in endothelial cells, and claudin-5 explicitly colocalized with CAV-1. During the in vitro experiments, hypoxia increased cell permeability, CAV-1 expression, and claudin-5 internalization and downregulated tight junction proteins. Simultaneously, hypoxia induced the upregulation of CAV-1 by activating NRF1. Blocking CAV-1-mediated intracellular transport improved the integrity of TJs in hypoxic endothelial cells and effectively inhibited the increase in BBB permeability and brain water content in HH animals.ConclusionsHypoxia upregulated CAV-1 transcription via the activation of NRF1 in endothelial cells, thus inducing the internalization and autophagic degradation of claudin-5. These effects lead to the destruction of the BBB and trigger HACE. Therefore, CAV-1 may be a potential therapeutic target for HACE.E5r9nDKChFa7F_nrUkRwh3Video abstract

Abstract 779: A NRF1-driven JRK-mediated β-catenin transcriptional activity contributes to the aggressive growth of ovarian cancer

Abstract Ovarian cancer is the most lethal gynecological cancer, accounting for thousands of cancer-related deaths in the United States every year. Though there has been progress towards understanding ovarian cancer pathogenesis, much is not yet understood about the molecular mechanisms that facilitate chemoresistance and metastasis. Our work has shown that nuclear respiratory factor 1 (NRF1) is highly active in several human cancers, though the role of NRF1 remains largely unexplored in ovarian carcinomas. Therefore, our aim was to examine the role of this transcription factor in ovarian cancer, since understanding its transcriptional landscape may reveal NRF1 or its target genes as possible therapeutic targets for this highly aggressive cancer. The Cancer Genome Atlas (TCGA) was utilized to collect RNA-Seq data from 379 serous cystadenocarcinoma samples, where we found that about 20% of ovarian cancer patients had altered NRF1 expression, showing either high expression or amplification of this transcription factor. Patients with high NRF1 expression were found to have decreased survival compared to patients with low NRF1 expression. NRF1 amplification was also correlated with the overamplification of one of its target genes - Jrk helix-turn-helix protein (JRK) in 26% of the samples. JRK regulates centrosome amplification, and through the production of mitotic spindle abnormalities, centrosome amplification contributes to genomic instability. Further investigation of the relationship between NRF1 and JRK uncovered that protein levels of JRK and β-catenin were significantly higher in NRF1-induced stem-like cells compared to the control group, and JRK is known to bind to β-catenin. Our NRF1 ChIP-Seq analyses showed that promoters of these two genes - JRK and CTNNB1 - were bound to NRF1. The Flow Assorted Cell Sorting (FACS), ChIP-Seq, RNA Seq and confocal microscopic studies suggest that through JRK, NRF1 regulates β-catenin transcriptional activity. Amplified gene expression is the one of the first steps towards oncogene activation, and our findings illustrate that altered NRF1 expression (i.e., overexpression or amplification) coupled with that of JRK influences the prognosis of patients diagnosed with ovarian cancer. The Wnt/β-catenin pathway is also highly active in the aggressive ovarian cancer. In addition to JRK, through the activation of β-catenin, NRF1may also affect the development of ovarian tumors. Taken together, our findings provide a novel insight into the molecular basis of the contribution of NRF1-driven JRK-mediated β-catenin transcriptional activity to the susceptibility of highly aggressive ovarian cancer. A better understanding of how ovarian neoplasm formation depends on NRF1-JRK-β-catenin pathway may open new avenues for therapeutic strategy against ovarian cancer. Citation Format: Ana Ruas, Quentin Felty, Jayanta K. Das, Alok Deoraj, Changwon Yoo, Deodutta Roy. A NRF1-driven JRK-mediated β-catenin transcriptional activity contributes to the aggressive growth of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 779.

Oxygen as an important factor modulating in vitro MeHgCl toxicity associated with mitochondrial genes in hiPSCs

Mitochondria are energy factories of cells and important targets for methylmercury chloride (MgHgCl). Methylmercury (MeHg) is a well-known environmental toxicant that bioaccumulates in fish and shellfish. It readily crosses the placental barrier, making it a threat to correct fetal development. Despite being comprehensively investigated for years, this compound has not been assessed for its in vitro mitochondrial toxicity under different oxygen conditions. In this study, human induced pluripotent stem cells (hiPSCs) were used to evaluate the dependence of the expression of genes associated with pluripotency and mitochondria on atmospheric (21% O2) and low (5% O2) oxygen concentrations upon MeHgCl treatment. We showed that the toxicity of MeHgCl was strongly related to an increased mtDNA copy number and downregulation of the expression of an mtDNA replication and damage repair-associated gene POLG1 (Mitochondrial Polymerase Gamma Catalytic Subunit) in both tested oxygen conditions. In addition, the viability and mitochondrial membrane potential of hiPSCs were significantly lowered by MeHgCl regardless of the oxygen concentration. However, reactive oxygen species accumulation significantly increased only under atmospheric oxygen conditions; what was associated with increased expression of TFAM (Transcription Factor A, Mitochondrial) and NRF1 (Nuclear Respiratory Factor 1) and downregulation of PARK2 (Parkin RBR E3 Ubiquitin Protein Ligase). Taken together, our results demonstrated that MeHgCl could induce in vitro toxicity in hiPSCs through altering mitochondria-associated genes in an oxygen level-dependent manner. Thus, our work suggests that oxygen should be considered a factor was modulating the in vitro toxicity of environmental pollutants. Typical atmospheric conditions of in vitro culture significantly lower the predictive value of studies of such toxicity.

Salvianolic acid A promotes mitochondrial biogenesis and function via regulating the AMPK/PGC-1α signaling pathway in HUVECs.

Mitochondrial dysregulation is an important pathology that leads to endothelial dysfunction, and the occurrence and development of cardiovascular diseases. Salvianolic acid A (SAA) has been demonstrated to be effective in the treatment of vascular complications of type 2 diabetes mellitus. Limited information has been reported on the effects of SAA on mitochondrial function in endothelial cells. In the present study, the effects of SAA on mitochondrial biogenesis and the related underlying mechanisms were investigated in human umbilical vein endothelial cells (HUVECs). Mitotracker red staining and transmission electron microscopy were used to evaluate the effect of SAA on mitochondrial quality. The effect of SAA treatment on mitochondrial DNA/nuclear DNA ratio of HUVECs was detected by real-time quantitative PCR. Western blot was used to determine the protein expression levels of complex III and Complex IV of mitochondrial oxidative phosphorylation subunit, and ATP production was determined by ATP test kit. Real-time quantitative PCR and Western blot were used to determine the effects of SAA on the expression of peroxisome proliferator-activated receptor γ coactivator (PGC-1α) and its target genes nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) proteins and genes. Finally, in the presence of 5'AMP-activated protein kinase (AMPK) specific inhibitors, the expression of PGC-1α, NRF1 and TFAM proteins and the phosphorylation levels of AMPK and Acetyl CoA Carboxylase (ACC) were detected by Western blot or real-time quantitative PCR. The results showed that SAA treatment significantly promoted mitochondrial biogenesis and enhanced mitochondrial function of HUVECs. SAA significantly increased the expression levels of PGC-1α and its target genes NRF1 and (TFAM), a key regulator of mitochondrial biogenesis in HUVECs. These enhancements were accompanied by significantly increased phosphorylation of AMPK and ACC, and were significantly inhibited by specific AMPK inhibitors. These results suggest that SAA may promote mitochondrial biogenesis in endothelial cells by activating the AMPK-mediated PGC-1α/TFAM signaling pathway. These data provide new insights into the mechanism of action of SAA in treating diabetic vascular complications.

Nuclear respiratory factor 1 promotes the growth of liver hepatocellular carcinoma cells via E2F1 transcriptional activation

BackgroundRecent studies have shown that functional mitochondria are essential for cancer cells. Nuclear respiratory factor 1 (NRF1) is a transcription factor that activates mitochondrial biogenesis and the expression of the respiratory chain, but little is known about its role and underlying mechanism in liver hepatocellular carcinoma (LIHC).MethodsNRF1 expression was analyzed via public databases and 24 paired LIHC samples. Clinical-pathological information and follow-up data were collected from 165 patients with LIHC or online datasets. Furthermore, cellular proliferation and the cell cycle were analyzed by MTT, Clone-forming assay and flow cytometric analyses. NRF1 target genes were analyzed by Chromatin immunoprecipitation sequencing (ChIP-Seq). PCR and WB analysis was performed to detect the expression of related genes. ChIP and luciferase activity assays were used to identify NRF1 binding sites.ResultsOur results showed that NRF1 expression was upregulated in LIHC compared to normal tissues. NRF1 expression was associated with tumour size and poor prognosis in patients. Knockdown of NRF1 repressed cell proliferation and overexpression of NRF1 accelerated the G1/S phase transition. Additionally, data from ChIP-seq pointed out that some NRF1 target genes are involved in the cell cycle. Our findings indicated that NRF1 directly binds to the E2F1 promoter as a transcription factor and regulates its gene expression.ConclusionTherefore, this study revealed that NRF1 promotes cancer cell growth via the indirect transcriptional activation of E2F1 and is a potential biomarker in LIHC.

Preparation and anti-fatigue effects of Vicatia thibertica polysaccharide

Background: Prior studies have revealed that crude Vicatia thibertica polysaccharide have the anti-fatigue effect, but the anti-fatigue effect and potential mechanism of purified polysaccharides endure blurred. Objectives: We intended to purify and gauge the anti-fatigue effects of a polysaccharide isolated from Vicatia thibertica polysaccharide 1 (VTP1). Materials and Methods: First, the exercise fatigue model was recognized in Kunming mice. After 14 days of continuous intragastric administration of VTP1 (50, 100, and 200 mg/kg/day), we assessed the anti-fatigue and antioxidant effects of VTP1 on fatigued mice. In addition, the effects of VTP1 on mitochondrial morphology of skeletal muscle of fatigue mice were detected by transmission electron microscopy. Meanwhile, the expression of mitochondrial DNA was noticed. Finally, using Western blot, immunohistochemistry, and real-time quantitative polymerase chain reaction, respectively, to sense the expression of sirttuin1, peroxisome proliferative activated receptor-γ coactivator 1α (PGC-1α), and nuclear respiratory factor 1 (NRF1). Results: VTP1 meaningly extends the time to exhaustion during weight-bearing swimming and improves the endurance and bodyweight of fatigued mice. Furthermore, VTP1 exerts antioxidant effects, reduces creatine kinase, blood urea nitrogen and lactate dehydrogenase in the serum, surges superoxide dismutase and cuts malondialdehyde in the liver. Meanwhile, VTP1 diminishes degeneration of mitochondria in the skeletal muscle of mice with exercise fatigue and improves the biosynthetic efficiency of mitochondria in skeletal muscle. Finally, VTP1 can upregulate the mRNA and protein expression of PGC-1α and NRF1. Conclusion: VTP1 has an anti-fatigue effect, and the effect may be mediated by the PGC-1α/NRF1 signaling pathway.

Environmentally relevant concentrations of fluconazole alter the embryonic development, oxidative status, and gene expression of NRF1, NRF2, WNT3A, WNT8A, NRD1, and NRD2 of Danio rerio embryos

Up to date, there is little information published concerning fluconazole (FCZ) toxicity at environmentally relevant concentrations. Bearing in mind the above background of FCZ, we aimed to evaluate the embryotoxic effects environmentally relevant concentrations of FCZ (800-1000 ng/L) may induce in Danio rerio. Moreover, we also wanted to prove whether these FCZ concentrations could generate oxidative stress and alter the expression of several genes related to the antioxidant mechanisms, sterol and retinol biosynthesis, and embryogenesis. Our findings demonstrate that FCZ, at all concentrations, induced pericardial edema, yolk sac deformation, scoliosis, and tail malformation in embryos. Moreover, we also demonstrated this drug altered the redox equilibrium of fish, promoting the production of lipoperoxidation level, hydroperoxide content, and protein carbonyl content in a concentration-dependent manner. Concerning gene expression, FCZ downregulated wingless-type MMTV integration site family member 3a (WNT3A), wingless-type MMTV integration site family member 8a (WNT8A), N-arginine dibasic convertase 1 (NRD1), and N-arginine dibasic convertase 2 (NRD2) and upregulated cytochrome P450 family 26 subfamily a member 1 (CYP26A1), cytochrome P450 family 26 subfamily a member 1B (CYP261B), nuclear respiratory factor 1 (NRF1), and nuclear respiratory factor 2 (NRF2) in D. rerio larvae. Collectively, our results point out that FCZ, at low concentrations, may alter the embryogenesis, oxidative status, and expression of several genes in D. rerio embryos via an impairment in sterol and retinol biosynthesis. Thus, our results provide some of the first evidence that FCZ, even at environmentally relevant concentrations, is harmful to aquatic species.

Nuclear Respiratory Factor 1 Overexpression Inhibits Proliferation and Migration of PC3 Prostate Cancer Cells.

The role of nuclear respiratory factor 1 (NRF1) on the prostate cancer progression is controversial. We aimed to investigate the effect of NRF1 overexpression on the metastasis potential of PC3 prostate cancer cells and the associated molecular mechanisms. Cell survival, migration capacity, mitochondrial biogenesis, the expression of TGF-β signaling and EMT markers were examined after overexpression and silencing of NRF1 in PC3 cells. We found that NRF1-overexpressing cells exhibited a decreased cell viability and proliferation ability as well as a reduced migration capacity compared to control cells. Moreover, ectopic expression of NRF1 increased the mitochondrial biogenesis and inhibited the EMT characteristics, including a decrease in the mesenchymal marker, α-SMA and an increase in the epithelial cell marker, E-cadherin. We also demonstrated that overexpression of NRF1 suppressed the expression of TGF-β signaling in PC3 cells. As expected, silencing of NRF1 reversed the abovementioned effects. This study demonstrated that upregulation of NRF1 holds the potential to inhibit the metastasis of prostate cancer, possibly through an elevation of mitochondrial biogenesis and the subsequent repression of TGF-β-associated EMT. Therapeutic avenues that increase NRF1 expression may serve as an adjunct to conventional treatments of prostate cancer.

Anti-oxidative and anti-hyperglycemic properties of Agastache foeniculum essential oil and oily fraction in hyperglycemia-stimulated and lipopolysaccharide-stimulated macrophage cells: In vitro and in silico studies

Ethnopharmacological relevanceHyperglycemia (HG) and lipopolysaccharide (LPS) often promote superoxide accumulation, which may increase oxidative stress. Reducing superoxide production in hyperglycemia and the inflammatory condition is an emerging way to reduce protein and lipid oxidation and diabetes complication. Aim of studyTo examine the effect of Agastache foeniculum essential oil (AFEO) and oil fraction (AFoil) on HG- and LPS-stimulated oxidative stress, the pathogenicity of AFEO and AFoil on oxidative stress was assessed. MethodsThe stimulatory effects of AFEO and AFoil on the activity and expression of NADH oxide (NOX), catalase (CAT), superoxide dismutase (SOD), and the expression of nuclear respiratory factor 2 (NRF2) and nuclear factor-kappa B (NF-kB) in the stimulated macrophage cell line, J774.A1, was studied. The interaction patterns of AFEO and AFoil components with NOX, SOD, CAT, NRF2, and NF-kB proteins were also deduced using molecular docking. ResultsEstragole was the main ingredient in AFEO (97%). Linolenic acid (32.10%), estragole (16.22%), palmitic acid (12.62%), linoleic acid (12.04%), and oleic acid (8.73%) were the major chemical components of the AFoil. NOX activation was stimulated in macrophage cells by HG and LPS. At 20 μg/mL, AFEO and AFoil decreased NOX activity while increased SOD and CAT activities in stimulated macrophages. AFoil with estragole and omega-3 fatty acids was better than AFEO with estragole in anti-hyperglycemic and anti-oxidative activity. According to molecular docking research, estragole, linoleic acid, and linolenic acid bind to different hydrophobic pockets of NOX, SOD, CAT, NFR2, and NF-kB using hydrogen bonds, van der Waals bonds, pi-alkyl, and pi-anion interactions, with different binding energies. ConclusionAFEO and AFoil showed antioxidant and anti-diabetic activity. The mechanisms in lowering oxidative stress markers depended on down-regulating superoxide-producing enzymes and up-regulating superoxide-removing enzymes at gene and protein levels. The AFoil emulsion can be used to reduce the detrimental impacts of hyperglycemia and oxidative stress.

Gynostemma pentaphyllum extract and its active component gypenoside L improve the exercise performance of treadmill-trained mice.

BACKGROUND/OBJECTIVESThe effectiveness of natural compounds in improving athletic ability has attracted attention in both sports and research. Gynostemma pentaphyllum (Thunb.) leaves are used to make traditional herbal medicines in Asia. The active components of G. pentaphyllum, dammarane saponins, or gypenosides, possess a range of biological activities. On the other hand, the anti-fatigue effects from G. pentaphyllum extract (GPE) and its effective compound, gypenoside L (GL), remain to be determined.MATERIALS/METHODSThis study examined the effects of GPE on fatigue and exercise performance in ICR mice. GPE was administered orally to mice for 6 weeks, with or without treadmill training. The biochemical analysis in serum, glycogen content, mRNA, and protein expressions of the liver and muscle were analyzed.RESULTSThe ExGPE (exercise with 300 mg/kg body weight/day of GPE) mice decreased the fat mass percentage significantly compared to the ExC mice, while the ExGPE showed the greatest lean mass percentage compared to the ExC group. The administration of GPE improved the exercise endurance and capacity in treadmill-trained mice, increased glucose and triglycerides, and decreased the serum creatine kinase and lactate levels after intensive exercise. The muscle glycogen levels were higher in the ExGPE group than the ExC group. GPE increased the level of mitochondrial biogenesis by enhancing the phosphorylation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) protein and the mRNA expression of nuclear respiratory factor 1, mitochondrial DNA, peroxisome proliferator-activated receptor-δ, superoxide dismutase 2, and by decreasing the lactate dehydrogenase B level in the soleus muscle (SOL). GPE also improved PGC-1α activation in the SOL significantly through AMPK/p38 phosphorylation.CONCLUSIONSThese results showed that GPE supplementation enhances exercise performance and has anti-fatigue activity. In addition, the underlying molecular mechanism was elucidated. Therefore, GPE is a promising candidate for developing functional foods and enhancing the exercise capacity and anti-fatigue activity.