scholarly journals The TBX1/miR-193a-3p/TGF-β2 Axis Mediates CHD by Promoting Ferroptosis

2022 ◽  
Vol 2022 ◽  
pp. 1-13
Author(s):  
Li Zhong ◽  
Huiqin Yang ◽  
Binlu Zhu ◽  
Xueqi Zhao ◽  
Meijun Xie ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Deletion Syndrome ◽  
H9c2 Cell ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Messenger Ribonucleic Acid ◽  
Nadph Oxidase 4

Congenital heart disease (CHD) is the most common noninfectious cause of death during the neonatal stage. T-box transcription factor 1 (TBX1) is the main genetic determinant of 22q11.2 deletion syndrome (22q11.2DS), which is a common cause of CHD. Moreover, ferroptosis is a newly discovered kind of programmed cell death. In this study, the interaction among TBX1, miR-193a-3p, and TGF-β2 was tested using quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and dual-luciferase reporter assays. TBX1 silencing was found to promote TGF-β2 messenger ribonucleic acid (mRNA) and protein expression by downregulating the miR-193a-3p levels in H9c2 cells. In addition, the TBX1/miR-193a-3p/TGF-β2 axis was found to promote ferroptosis based on assessments of lipid reactive oxygen species (ROS) levels, Fe2+ concentrations, mitochondrial ROS levels, and malondialdehyde (MDA) contents; Cell Counting Kit-8 (CCK-8) assays and transmission electron microscopy; and Western blotting analysis of glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), NADPH oxidase 4 (NOX4), and acyl-CoA synthase long-chain family member 4 (ACSL4) protein expression. The protein expression of NRF2, GPX4, HO-1, NOX4, and ACSL4 and the level of MDA in human CHD specimens were also detected. In addition, TBX1 and miR-193a-3p expression was significantly downregulated and TGF-β2 levels were high in human embryonic CHD tissues, as indicated by the H9c2 cell experiments. In summary, the TBX1/miR-193a-3p/TGF-β2 axis mediates CHD by inducing ferroptosis in cardiomyocytes. TGF-β2 may be a target gene for CHD diagnosis and treatment in children.

scholarly journals Oxysophocarpine Retards the Growth and Metastasis of Oral Squamous Cell Carcinoma by Targeting the Nrf2/HO-1 Axis

2018 ◽  
Vol 49 (5) ◽  
pp. 1717-1733 ◽  
Author(s):  
Rui Liu ◽  
Jia Peng ◽  
Huili Wang ◽  
Lei Li ◽  
Xiujie Wen ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Western Blotting ◽  
Squamous Cell ◽  
Cell Counting ◽  
Heme Oxygenase 1 ◽  
Related Factor

Background/Aims: Nuclear factor erythroid 2-related factor 2 (Nrf2) is an oncogene in various types of cancers, including oral squamous cell carcinoma (OSCC). Oxysophocarpine (OSC) is a natural alkaloid that has multiple pharmacological activities. However, the biological functions and molecular mechanism underlying the effects of OSC on the growth and metastasis of OSCC are unclear. Methods: Nrf2 levels were determined in OSCC tissues and non-cancerous specimens by quantitative real-time PCR, western blotting, and immunohistochemistry (IHC) assays. The effects of OSC on OSCC cell growth and metastasis were explored (1) using 5-ethynyl-20-deoxyuridine staining and Cell Counting Kit-8, colony formation, flow cytometry, wound-healing, Transwell, and tube formation assays in vitro; and (2) by establishing a xenograft nude mouse model in vivo. The molecular mechanisms underlying the effects of OSC on the growth and metastasis of OSCC were investigated in vitro by western blotting, caspase-3 activity, and enzyme-linked immunosorbent assays, and in vivo by western blotting and IHC assays. Results: The expression levels of Nrf2 in OSCC tissues and in cell lines were much higher than in non-cancerous tissues and normal oral keratinocytes. The upregulation of Nrf2 was positively correlated with a high incidence of lymph node metastasis and advanced histological grade and TNM stage, but inversely associated with differentiation and survival of OSCC patients. OSC reduced the expression of Nrf2 and heme oxygenase 1 (HO-1) in OSCC cells. OSC also inhibited proliferation, migration, invasion, and pro-angiogenesis of OSCC cells. Moreover, OSC induced cell cycle arrest, enhanced apoptosis of OSCC cells in vitro, and decreased OSCC tumor growth in vivo. Mechanically, OSC reduced the aggressive behavior of OSCC cells by inactivation of the Nrf2/HO-1 signaling pathway. Conclusion: Our findings provide evidence that OSC inhibits the growth and metastasis of OSCC by targeting the Nrf2/ HO-1 axis, suggesting that OSC may be a potential therapeutic agent for OSCC.

Oroxylin A exerts anti-inflammatory activity on lipopolysaccharide-induced mouse macrophage via Nrf2/ARE activation

2014 ◽  
Vol 92 (5) ◽  
pp. 337-348 ◽  
Author(s):  
Ming Ye ◽  
Qing Wang ◽  
Weifeng Zhang ◽  
Zhiyu Li ◽  
Yajing Wang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Inflammatory Diseases ◽  
Antioxidant Response ◽  
Raw264.7 Cells ◽  
Luciferase Reporter ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Oroxylin A ◽  
Quinone Oxidoreductase ◽  
Cox 2

Regulating inflammation could be an important measure for the effective treatment of cancer. Here we examine the mechanisms by which oroxylin A inhibits inflammation in RAW264.7 cells. The results demonstrate that pretreatment with oroxylin A (50, 100, and 150 μmol/L) inhibited lipopolysaccharide (LPS)-induced mRNA and protein expression of COX-2 and iNOS. In addition, oroxylin A significantly increased the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and NADP(H):quinone oxidoreductase (NQO1), induced Nrf2 translocation to the nucleus and up-regulated antioxidant response element (ARE)-luciferase reporter activity. Moreover, oroxylin A inhibited Nrf2 ubiquitination and proteasome activity. Transfection with Nrf2 siRNA knocked down Nrf2 expression and partially reversed oroxylin A-mediated inhibition of LPS-induced COX-2 and iNOS expression. Importantly, we showed for the first time that Nrf2 plays an important role in oroxylin A-suppressed inflammation in RAW264.7 cells. Uncovering the effect of oroxylin A on the regulation of Nrf2 signaling may be beneficial for developing new therapeutic strategies against inflammatory diseases.

scholarly journals Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 308
Author(s):  
Ying-Ray Lee ◽  
Chia-Ming Chang ◽  
Yuan-Chieh Yeh ◽  
Chi-Ying F. Huang ◽  
Feng-Mao Lin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Expression Profiles ◽  
Plaque Assay ◽  
Virus Titer ◽  
Enterovirus 71 ◽  
Luciferase Reporter ◽  
Pathogen Invasion

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

scholarly journals LncRNA PVT1 Promotes Hypoxia-Induced Cardiomyocyte Injury by Inhibiting miR-214-3p

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Chuanliang Liu ◽  
Jieqiong Zhang ◽  
Xuejie Lun ◽  
Lei Li
Keyword(s):  
Cell Viability ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Flow Cytometry Analysis ◽  
Chain Reaction ◽  
Cell Vitality ◽  
Gene Assay ◽  
Polymerase Chain

Objective. To explore the effect and related mechanism of LncRNA PVT1 on hypoxia-induced cardiomyocyte injury. Methods. PVT1RNA and miR-214-3p levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell vitality and apoptosis were, respectively, evaluated by Cell Counting Kit-8 (CCK-8) and flow cytometry analysis. Starbase and Dual luciferase reporter (DLR) gene assay was employed to validate the interaction between miR-214-3p and PVT1. Results. PVT1 was statistically upregulated, and miR-214-3p was statistically downregulated in hypoxia-induced H9c2 cells. The survival rate of H9c2 cells induced by hypoxia decreased statistically, while the apoptosis rate increased statistically ( P < 0.05 ). PVT1 knockdown upregulated the hypoxia-induced H9c2 cell viability and inhibited apoptosis. DLR assay verified the targeting relationship between PVT1 and miR-214-3p. In addition, miR-214-3p inhibitors reversed the viability of H9c2 cells with PVT1 knockout and promoted apoptosis. Conclusion. Silencing PVT1 can enhance the hypoxia-induced H9c2 cell viability and inhibit apoptosis, providing a potential target for the treatment of cardiovascular diseases.

Regulation of heme oxygenase-1 expression by demethoxy curcuminoids through Nrf2 by a PI3-kinase/Akt-mediated pathway in mouse β-cells

2007 ◽  
Vol 293 (3) ◽  
pp. E645-E655 ◽  
Author(s):  
Subbiah Pugazhenthi ◽  
Leonid Akhov ◽  
Gopalan Selvaraj ◽  
Maorong Wang ◽  
Jawed Alam
Keyword(s):  
Signaling Pathways ◽  
Heme Oxygenase ◽  
Nuclear Translocation ◽  
Regulatory Subunit ◽  
Luciferase Reporter ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Mrna Levels ◽  
Phase 2 ◽  
Β Cells

Curcumin (diferuloylmethane), a component of turmeric, has been shown to have therapeutic properties. Induction of phase 2 detoxifying enzymes is a potential mechanism through which some of the actions of curcumin could proceed. Heme oxygenase-1 (HO-1), an antioxidant phase 2 enzyme, has been reported to have cytoprotective effects in pancreatic β-cells. Curcumin on further purification yields demethoxy curcumin (DMC) and bisdemethoxy curcumin (BDMC). The objective of the present study was to determine the mechanism by which these purified curcuminoids induce HO-1 in MIN6 cells, a mouse β-cell line. Demethoxy curcuminoids induced HO-1 promoter linked to the luciferase reporter gene more effectively than curcumin. The induction was dependent on the presence of antioxidant response element (ARE) sites containing enhancer regions (E1 and E2) in HO-1 promoter and nuclear translocation of nuclear factor-E2-related factor (Nrf2), the transcription factor that binds to ARE. Curcuminoids stimulated multiple signaling pathways that are known to induce HO-1. Inhibition of specific signaling pathways with pharmacological inhibitors and cotransfection experiments suggested the involvement of phosphotidylinositol 3-kinase and Akt. Real-time quantitative RT-PCR analysis showed significant elevation in the mRNA levels of HO-1 and two other phase 2 enzymes, the regulatory subunit of glutamyl cysteine ligase, which is needed for the synthesis of glutathione, and NAD(P)H:quinone oxidoreductase, which detoxifies quinones. DMC and BDMC induced the expression of HO-1 and translocated Nrf2 to nucleus in β-cells of mouse islets. Our observations suggest that demethoxy curcuminoids could be used to induce a cellular defense mechanism in β-cells under conditions of stress as seen in diabetes.

scholarly journals Effects of Four Compounds from Gentianella acuta (Michx.) Hulten on Hydrogen Peroxide-Induced Injury in H9c2 Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Kai Ren ◽  
He Su ◽  
Li-juan Lv ◽  
Le-tai Yi ◽  
Xue Gong ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Protein Expression ◽  
Treated Group ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Protective Effects ◽  
H9c2 Cells ◽  
Mtt Method ◽  
Bioassay Guided Fractionation ◽  
Induced Apoptosis

In previous studies, Gentianella acuta (Michx.) Hulten was reported to contain xanthones, iridoids, terpenoids, and sterols and is mainly used to cure hepatitis, jaundice, fever, headache, and angina pectoris. In this study, we used bioassay guided fractionation to identify compounds from G. acuta and investigated their activity against hydrogen peroxide (H2O2)-induced apoptosis of H9c2 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and glutamate-cysteine ligase catalytic (GCLC) expression were assessed using quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was evaluated using western blot. The results showed that all four compounds had protective effects on H9c2 cells. The transcription levels of HO-1 and GCLC significantly increased in H9c2 cells pretreated with norswertianolin (1), swetrianolin (2), demethylbellidifolin (3), and bellidifolin (4). However, compared to the model group, the transcription levels of Nrf2 were not enhanced by pretreatment with compounds 1, 2, and 4. The protein expression levels of HO-1 and GCLC in H9c2 cells were greater than that in the H2O2-treated group, and the expression of Nrf2 was not significantly changed except by swetrianolin treatment; inhibitors can reverse the protective effect by ZnPP (15 μM), BSO (10 μM), and brusatol (10 μM). The results indicated that the four compounds isolated from G. acuta inhibited the oxidative injury induced by H2O2 by activating the Nrf2/ARE pathway in H9c2 cells and provide evidence that G. acuta may be a potential therapeutic agent for the treatment of cardiovascular diseases.

scholarly journals Discovery of 4-Anilinoquinolinylchalcone Derivatives as Potential NRF2 Activators

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3133
Author(s):  
Yu-Tse Kao ◽  
Yi-Siao Chen ◽  
Kai-Wei Tang ◽  
Jin-Ching Lee ◽  
Chih-Hua Tseng ◽  
...  
Keyword(s):  
Target Genes ◽  
Hacat Cell ◽  
Effective Means ◽  
Mrna Level ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Stable Cell Line ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Nrf2 Activator

Activation of nuclear factor erythroid-2-related factor 2 (NRF2) has been proven to be an effective means to prevent the development of cancer, and natural curcumin stands out as a potent NRF2 activator and cancer chemopreventive agent. In this study, we have synthesized a series of 4-anilinoquinolinylchalcone derivatives, and used a NRF2 promoter-driven firefly luciferase reporter stable cell line, the HaCaT/ARE cells, to screen a panel of these compounds. Among them, (E)-3-{4-[(4-acetylphenyl)amino]quinolin-2-yl}-1-(4-fluorophenyl)prop-2-en-1-one (13b) significantly increased NRF2 activity in the HaCaT cell with a half maximal effective concentration (EC50) value of 1.95 μM. Treatment of compound 13b upregulated HaCaT cell NRF2 expression at the protein level. Moreover, the mRNA level of NRF2 target genes, heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), and glucose-6-phosphate dehydrogenase (G6PD) were significantly increased in HaCaT cells upon the compound 13b treatment. The molecular docking results exhibited that the small molecule 13b is well accommodated by the bound region of Kelch-like ECH-associated protein 1 (Keap1)-Kelch and NRF2 through stable hydrogen bonds and hydrophobic interaction, which contributed to the enhancement of affinity and stability between the ligand and receptor. Compound 13b has been identified as the lead compound for further structural optimization.

scholarly journals Protective effects of upregulated HO-1 gene against the apoptosis of human retinal pigment epithelial cells in vitro

2021 ◽  
Vol 14 (5) ◽  
pp. 649-655
Author(s):  
Yu Hong ◽  
◽  
Wei-Qi Chen ◽  
Liu-Xia You ◽  
Qing-Feng Ni ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Lentiviral Vector ◽  
Retinal Pigment ◽  
B Cell Lymphoma ◽  
Retinal Pigment Epithelial Cells ◽  
Cell Counting ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Protective Effects

AIM: To investigate the protective effect of heme oxygenase-1 (HO-1) against H2O2-induced apoptosis in human ARPE-19 cells. METHODS: The lentiviral vector expressing HO-1 was prepared and transfected into apoptotic ARPE-19 cells induced by H2O2. Functional experiments including cell counting kit-8 (CCK-8) assay, flow cytometry (FCM) and mitochondrial membrane potential assay were conducted. RESULTS: The ultrastructure of ARPE-19 cells was observed using transmission electron microscope (TEM). It was found that exogenous HO-1 significantly ameliorated H2O2-induced loss of cell viability, apoptosis and intracellular levels of reactive oxygen species (ROS) in ARPE-19 cells. The overexpression of HO-1 facilitated the transfer of nuclear factor erythroid-2-related factor 2 (Nrf2) from cytoplasm to nucleus, which in turn upregualted expressions HO-1 and B-cell lymphoma-2 (Bcl-2). Furthermore, HO-1 upregulation further inhibited H2O2-induced release of cysteinyl aspartate specific proteinase-3 (caspase-3). CONCLUSION: Exogenous HO-1 protect ARPE-19 cells against H2O2-induced oxidative stress by regulating the expressions of Nrf2, HO-1, Bcl-2, and caspase-3.

scholarly journals miR-92a-3p Promoted EMT via Targeting LATS1 in Cervical Cancer Stem Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Shuangyue Liu ◽  
Liping Chu ◽  
Mingzhu Xie ◽  
Lisha Ma ◽  
Hongmei An ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Stem Cells ◽  
Western Blotting ◽  
Cyclin E ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Gene Assay ◽  
Cell Cycle Transition ◽  
E Cadherin

miR-92a-3p (microRNA-92a-3p) has been reported to be dysregulated in several cancers, and as such, it is considered to be a cancer-related microRNA. However, the influence of miR-92a-3p on biological behaviors in cervical cancer (CC) still remains unclear. Quantitative real-time PCR was used to detect miR-92a-3p levels in CC stem cells. Here, Cell Counting Kit-8 (CCK8) assay, Transwell cell invasion assay and flow cytometry assay were used to characterize the effects that miR-92a-3p and large tumor suppressor l (LATS1) had on proliferation, invasion and cell cycle transition. The luciferase reporter gene assay was used to verify the targeting relationship between miR-92a-3p and LATS1. Western Blotting was used to investigate the related signaling pathways and proteins. Data from The Cancer Genome Atlas (TCGA) showed that miR-92a-3p was upregulated in CC tissues and closely associated with overall survival. miR-92a-3p promoted proliferation, invasion and cell cycle transition in CC stem cells. The luciferase reporter assay showed that miR-92a-3p bound to the 3′-untranslated region (3′-UTR) of the LATS1 promoter. LATS1 inhibited proliferation, invasion and cell cycle transition. Results measured by Western Blotting showed that LATS1 downregulated expressions of transcriptional co-activator with PDZ-binding motif (TAZ), vimentin and cyclin E, but upregulated the expression of E-cadherin. Re-expression of LATS1 partly reversed the effects of miR-92a-3p on proliferation, invasion and cell cycle transition, as well as on TAZ, E-cadherin, vimentin, and cyclin E. miR-92a-3p promoted the malignant behavior of CC stem cells by targeting LATS1, which regulated TAZ and E-cadherin.

Protective role of Salvia miltiorrhiza Polysaccharides on Florfenicol Induced Oxidative and Apoptotic Injury of Liver in Broilers

2021 ◽  
Author(s):  
Xiao Wang ◽  
Yuqing Cui ◽  
Chao Han ◽  
Yumeng Geng ◽  
Di Zhang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Liver Injury ◽  
Protein Expression ◽  
Liver Tissue ◽  
Salvia Miltiorrhiza ◽  
Protective Role ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Mrna And Protein Expression ◽  
Liver Tissues

Abstract Salvia miltiorrhiza Polysaccharides (SMPs) can alleviate liver injury in mice, but there are few reports on liver injury of broilers, especially the liver injury caused by antibiotics. To explore the hepatoprotective effects of SMPs against florfenicol (FFC) induced broilers liver injury, the broilers were treated with FFC and SMPs. The results showed SMPs could significantly inhibit the decrease of weight gain and the increase of liver index induced by FFC (P < 0.05). SMPs could significantly reduce the contents of Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) in serum and the malondialdehyde (MDA), nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in liver tissues (P < 0.05), also significantly increased the content of total protein (TP) in serum and superoxide dismutase (SOD), catalase (CAT) in liver tissues (P < 0.05). QPCR and western bolt results showed that SMPs significantly increased the mRNA and protein expression of cytochrome P4501A1 (CYP1A1), cytochrome P4502H1 (CYP2H1), nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase quinone-1 (NQO-1) in liver tissue, also significantly reduced the rate of hepatocyte apoptosis and the mRNA and protein expression of p53, cytochrome-C (CytC), caspase-3 in liver tissue (P < 0.05). The results demonstrated that SMPs can inhibit the oxidative stress in hepatocytes by regulating the related proteins in Nrf2 pathway, thereby reducing the apoptosis of hepatocytes, and protecting liver injury.

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