This study aims to explore the specific mechanism by which puerarin inhibits ferroptosis and improves the myocardial contractile function in myocardial hypertrophy through the nuclear factor erythroid 2-related factor 2(Nrf2)/antioxidant response element(ARE)/heme oxygenase-1(HO-1) signaling pathway. The hypertrophic cardiomyocyte model was established using phenylephrine, and H9c2 cells were divided into control group, model group, puerarin group, and puerarin+ML385 group. Cell viability and surface area were detected by cell counting kit-8(CCK-8) and immunofluorescence experiments. The mitochondrial membrane potential and Ca~(2+) concentration were measured. The ferroptosis-related indicators were detected by biochemical and fluorescence staining methods. The expression of proteins related to ferroptosis and the Nrf2/ARE/HO-1 signaling pathway was detected by Western blot. A myocardial hypertrophy model was established, and 40 rats were randomly divided into sham group, model group, puerarin group, and puerarin+Nrf2 inhibitor(ML385) group, with 10 rats in each group. Echocardiogram, hemodynamic parameters, and myocardial hypertrophy parameters were measured. Histopathological changes of myocardial tissues were observed by hematoxylin and eosin(HE) staining and Masson staining. Biochemical methods, enzyme-linked immunosorbent assay(ELISA), and fluorescence staining were used to detect inflammatory factors and ferroptosis-related indicators. Immunohistochemistry was used to detect the expression of proteins related to ferroptosis and the Nrf2/ARE/HO-1 signaling pathway. Cell experiments showed that puerarin intervention significantly enhanced the viability of hypertrophic cardiomyocytes, reduced their surface area, and restored mitochondrial membrane potential and Ca~(2+) homeostasis. Mechanism studies revealed that puerarin promoted Nrf2 nuclear translocation, upregulated the expression of HO-1, solute carrier family 7 member 11(SLC7A11), and glutathione peroxidase 4(GPX4), and decreased malondialdehyde(MDA), reactive oxygen species(ROS), and iron levels. These protective effects were reversed by ML385. In animal experiments, puerarin improved cardiac function in rats with myocardial hypertrophy, alleviated myocardial hypertrophy and fibrosis, inhibited inflammatory responses and ferroptosis, and promoted nuclear Nrf2 translocation and HO-1 expression. However, combined intervention with ML385 led to deterioration of hemodynamics and a rebound in ferroptosis marker levels. In conclusion, puerarin may inhibit cardiomyocyte ferroptosis through the Nrf2/ARE/HO-1 signaling pathway, thereby improving myocardial contractile function in myocardial hypertrophy.
Animals ; NF-E2-Related Factor 2/genetics* ; Rats ; Ferroptosis/drug effects* ; Signal Transduction/drug effects* ; Isoflavones/pharmacology* ; Male ; Rats, Sprague-Dawley ; Cardiomegaly/genetics* ; Myocytes, Cardiac/metabolism* ; Antioxidant Response Elements/drug effects* ; Myocardial Contraction/drug effects* ; Heme Oxygenase-1/genetics* ; Cell Line

OBJECTIVE: To explore the protective effects and underlying mechanisms of H ₂S against lipid peroxidation-mediated carbonyl stress in the uranium-treated NRK-52E cells. METHODS: Cell viability was evaluated using CCK-8 assay. Apoptosis was measured using flow cytometry. Reagent kits were used to detect carbonyl stress markers malondialdehyde, 4-hydroxynonenal, thiobarbituric acid reactive substances, and protein carbonylation. Aldehyde-protein adduct formation and alcohol dehydrogenase, aldehyde dehydrogenase 2, aldo-keto reductase, nuclear factor E2-related factor 2 (Nrf2), and cystathionine β-synthase (CBS) expression were determined using western blotting or real-time PCR. Sulforaphane (SFP) was used to activate Nrf2. RNA interference was used to inhibit CBS expression. RESULTS: GYY4137 (an H ₂S donor) pretreatment significantly reversed the uranium-induced increase in carbonyl stress markers and aldehyde-protein adducts. GYY4137 effectively restored the uranium-decreased Nrf2 expression, nuclear translocation, and ratio of nuclear to cytoplasmic Nrf2, accompanied by a reversal of the uranium-decreased expression of CBS and aldehyde-metabolizing enzymes. The application of CBS siRNA efficiently abrogated the SFP-enhanced effects on the expression of CBS, Nrf2 activation, nuclear translocation, and ratio of nuclear to cytoplasmic Nrf2 and concomitantly reversed the SFP-enhanced effects of the uranium-induced mRNA expression of aldehyde-metabolizing enzymes. Simultaneously, CBS siRNA reversed the SFP-mediated alleviation of the uranium-induced increase in reactive aldehyde levels, apoptosis rates, and uranium-induced cell viability. CONCLUSION H ₂S induces Nrf2 activation and nuclear translocation, which modulates the expression of aldehyde-metabolizing enzymes and the CBS/H ₂S axis. Simultaneously, the Nrf2-controlled CBS/H ₂S axis may at least partially promote Nrf2 activation and nuclear translocation. These events form a cycle-regulating mode through which H ₂S attenuates the carbonyl stress-mediated NRK-52E cytotoxicity triggered by uranium.
NF-E2-Related Factor 2/genetics* ; Animals ; Hydrogen Sulfide/pharmacology* ; Rats ; Signal Transduction/drug effects* ; Lipid Peroxidation/drug effects* ; Cell Line ; Uranium/toxicity* ; Antioxidant Response Elements ; Kidney/metabolism* ; Oxidative Stress/drug effects* ; Cell Survival/drug effects* ; Apoptosis/drug effects*

DcCDPK8 involved in abiotic stress such as low temperature and signal transduction of hormones ABA and MeJA,but the transcriptional regulation is still unclear. In order to study the core promoter region of DcCDPK8 gene in Dendrobium catenatum and explore its transcriptional regulation mechanism,the DcCDPK8 gene promoter sequence was cloned by PCR from D. catenatum. Promoter sequence function was studied by fusion of 5 'terminal deletion and GUS gene. The results showed that the promoter sequence of DcCDPK8 gene has a low-temperature responsive element( LTR) between~(-1) 749 bp and-614 bp,two MeJA responsive elements between~(-1) 749 bp and-230 bp,and one ABA responsive elements between-614 bp and-230 bp. Three 5'-end different deletion fragments were constructed to fuse the eukaryotic expression vectors p BI121 with GUS,which were transformed into tobacco leaves. The GUS activity under cold stress treatment was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. GUS activity under exogenous ABA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3,and GUS activity under exogenous MeJA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. It is speculated that the ABA response element( ARE) in the promoter sequences of DcCDPK8 is positive regulatory role in response to exogenous ABA,the MeJA cis-acting element plays a negative role in response to exogenous MeJA.
Abscisic Acid ; Acetates ; Cloning, Molecular ; Cold Temperature ; Cyclopentanes ; Dendrobium ; genetics ; Gene Expression Regulation, Plant ; Oxylipins ; Plant Proteins ; genetics ; Plants, Genetically Modified ; Promoter Regions, Genetic ; Response Elements ; Stress, Physiological ; Tobacco

OBJECTIVESTo identify a NFB responsive element within the dimethylarginine dimethylaminohydrolase 2 gene (DDAH) promoter and demonstrate its role in DDAH2 transactivation.

METHODSDDAH2 promoter was analyzed with software to identify potential binding sites of transcription factors. A series of truncated DDAH2 promoter luciferase reporter plasmids were constructed and transfected into human embryonic kidney derived HEK293 cells. Luciferase assays were carried out to analyze the activity of the promoter. Electrophoresis mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were used to identify the NFB responsive element in vitro and in vivo. DDAH2 promoter luciferase reporter plasmid with mutated NFB site was constructed and transfected into cells, and its activity was compared with that of the wild-type plasmid.

RESULTSPotential bindings sites of many transcription factors were found within the DDAH2 promoter. The transcription activity of the DDAH2 promoter was high, and -530 to -437 was a positive regulating region. -476 to -469 of the DDAH2 promoter was a NFB responsive element, to which NFB can specifically bind. Mutation of the NFB element could significantly decrease the DDAH2 promoter activity.

CONCLUSION-476 to -469 of the DDAH2 promoter was a NFB responsive element and is important for the transactivation of DDAH2.

Amidohydrolases ; genetics ; metabolism ; Base Sequence ; Gene Expression Regulation, Enzymologic ; Humans ; Molecular Sequence Data ; NF-kappa B ; metabolism ; Promoter Regions, Genetic ; Protein Binding ; Response Elements

OBJECTIVETo investigate the protective effect of extracts from Cichorium endivia (CEE) in H2O2-induced HepG2 cell oxidative stress injury, and explore the antioxidant mechanism of CEE in HepG2 cells.

METHODThe viability of H2O2-induced HepG2 cells and the intracellular ROS level were measured by MTT assay and DCFH-DA fluorescence staining assay. The antioxidant-response element (ARE)-Luciferase activity was tested in HepG2 cells stably transected by ARE reporter gene. The fluorescence quantitative RT-PCR was adopted to determine the mRNA expressions of genes containing ARE sequence in HepG2 cells.

RESULTThe cell viability reduced, while the ROS level increased after HepG2 cells were treated by H2O2. Different concentrations of CEE could be added to significantly improve the above results. After HepG2 cells transected by ARE reporter gene were treated with different concentrations of CEE, the intracellular ARE activity could increase in a concentration-dependent manner. In addition, the mRNA expressions of regulatory genesGCLC, GCLM and HMOX-1 containing ARE sequence in HepG2 cells were up-regulated in a concentration-dependent manner by CEE.

CONCLUSIONCEE inhibited the H2O2-injured HepG2 cells by reducing the ROS level. CEE's antioxidant mechanism for HepG2 cells may be closely related to the antioxidant defense system associated with its effect of activating Nrf2-ARE pathway in HepG2 cells.

Antioxidants ; isolation & purification ; pharmacology ; Asteraceae ; chemistry ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Hep G2 Cells ; Humans ; Hydrogen Peroxide ; pharmacology ; Reactive Oxygen Species ; metabolism ; Response Elements ; genetics

PURPOSE: SNF2L belongs to Imitation Switch family and plays an essential role in neural tissues and gonads. In our previous studies, we have demonstrated that the basal transcription of human SNF2L gene is regulated by two cis-elements, cAMP response element (CRE)- and Sp1-binding sites. Recent studies suggested that cyclic adenosine monophosphate (cAMP) stimulation significantly up-regulated SNF2L expression in ovarian granulose cells. These data suggested that protein kinase-mediated signal pathways might also regulate SNF2L expression in neural cells. We therefore investigated the effects of agents that activate protein kinases A on SNF2L gene expression in neural cells. MATERIALS AND METHODS: To increase intracellular cAMP levels, all neural cells were treated with forskolin and dbcAMP, two cAMP response activators. We exmined the effects of cAMP on the promoter activity of human SNF2L gene by luciferase reporter gene assays, and further examined the effects of cAMP on endogenous SNF2L mRNA levels by qPCR. RESULTS: Transient expression of a luciferase fusion gene under the control of the SNF2L promoter was significantly increased by treatment of rat primary neurons with forskolin or dbcAMP, but not PC12, C6 and SH-SY5Y cells. Consistently, treatment with forskolin or dbcAMP could enhance endogenous SNF2L mRNA levels also only in rat primary neurons. CONCLUSION: These results suggest that the CRE consensus sequence in the SNF2L proximal promoter most likely confers constitutive activation and regulation by cAMP in neural cells.
Animals ; Bucladesine/pharmacology ; Cell Line ; Colforsin/pharmacology ; Cyclic AMP/*metabolism ; DNA-Binding Proteins/chemistry/*genetics/metabolism ; *Gene Expression Regulation ; Humans ; Luciferases/analysis ; Neurons/*metabolism ; PC12 Cells ; Promoter Regions, Genetic ; RNA, Messenger/metabolism ; Rats ; Rats, Wistar ; Recombinant Fusion Proteins/analysis ; *Response Elements ; Transcription Factors/chemistry/*genetics/metabolism

The aim of this study is to investigate the protection effect of tanshinone IIA (Tan) against triptolide (TP)-induced liver injury and the mechanisms involved. Acute liver injury was induced by intraperitoneal injection of TP (1 mg x kg(-1)) in mice. The activities of AST, ALT and LDH in serum and the levels of GSH, GST, GSH-PX, SOD, CAT and MDA in liver tissue were detected. The histopathological changes of liver tissues were observed after HE staining. Nrf2 translocation in liver tissue was detected by Western blotting, and real-time PCR was used to measure the expression levels of GCLC, NQO1 and HO-1 mRNA. The results showed that pretreatment with Tan significantly prevented the TP induced liver injury as indicated by reducing the activities of AST, ALT and LDH (P < 0.01). Tan pretreatment also prevented TP-induced oxidative stress in the mice liver by inhibiting MDA and restoring the levels of GSH, GST, SOD and CAT (P < 0.05). Parallel to these changes, pretreatment with Tan could attenuate histopathologic changes induced by TP. Furthermore, the results indicated that Tan pretreatment caused nuclear accumulation of Nrf2 as well as induction of mRNA expression of antioxidant response element (ARE)-driven genes such as GCLC, NQO1 and HO-1. These results indicated that Tan could protect against TP-induced acute liver injury via the activation of Nrf2/ARE pathway.
Animals ; Antioxidant Response Elements ; drug effects ; Chemical and Drug Induced Liver Injury ; metabolism ; pathology ; Diterpenes ; toxicity ; Diterpenes, Abietane ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Epoxy Compounds ; toxicity ; Glutamate-Cysteine Ligase ; genetics ; metabolism ; Heme Oxygenase-1 ; genetics ; metabolism ; Liver ; metabolism ; pathology ; Male ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL ; NAD(P)H Dehydrogenase (Quinone) ; genetics ; metabolism ; NF-E2-Related Factor 2 ; metabolism ; Phenanthrenes ; toxicity ; RNA, Messenger ; metabolism ; Signal Transduction ; drug effects

OBJECTIVETo construct a luciferase reporter vector containing the response element of transcription protein AP2α for screening the effect of bone morphogenetic proteins (BMPs) on the transcriptional activity of AP2α.

METHODSFour tandem-linked response elements of AP2α were cloned to the pBGLuc luciferase reporter gene plasmid, which was digested with Bam HI and Mlu I to construct pBGLuc-AP2α-RE vector. The recombinant adenovirus Ad-AP2α and its dominant negative mutant Ad-dnAP2α were used to infect mouse mesenchymal stem cells C3H10; the changes in cellular AP2α mRNA and protein expressions were detected by real-time PCR and Western blotting, and electrophoretic mobility shift assay (EMSA) was carried out to assess the DNA-binding ability of AP2α. C3H10 cells were transfected with pBGLuc-AP2α-RE vector, and AP2α transcriptional activity was measured using luciferase reporter gene assay. In pBGLuc-AP2α-RE vector-transfected C3H10 cells infected with Ad-BMPs, luciferase reporter gene assay was performed to screen the effect of BMPs on AP2α transcriptional activity.

RESULTSThe results of PCR, enzyme digestion and sequencing all confirmed correct cloning of AP2α-RE into pBGLuc-AP2α-RE luciferase reporter vector, and Ad-AP2α infection significantly increased AP2α expression and its DNA binding ability. The dominant negative mutants expressed the corresponding mutants, and EMSA results showed that Ad-dnAP2α-δbHLH significantly lowered while Ad-dnAP2α-δTAD enhanced the DNA-binding ability of AP2α. AP2α over-expression promoted AP2α transcriptional activity, which was suppressed by the two dominant negative mutants. AP2α transcriptional activity increased in the cells infected with the recombinant adenovirus BMPs, especially in cells with BMP9 infection.

CONCLUSIONSThe luciferase reporter vector containing the response element of AP2α we constructed allows detection of AP2α transcriptional activity. BMP9 can significantly enhance AP2α transcriptional activity.

Adenoviridae ; Animals ; Bone Morphogenetic Proteins ; genetics ; metabolism ; Genes, Reporter ; Genetic Vectors ; Growth Differentiation Factor 2 ; genetics ; metabolism ; Luciferases ; genetics ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Osteogenesis ; Protein Binding ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Response Elements ; Transcription Factor AP-2 ; genetics ; metabolism ; Transcriptional Activation ; Transfection

Gastrointestinal tract carcinoma is one of the leading causes of cancer-related death in China. Chemoprevention has been considered as a potential approach to control this type of disease. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that protects cells from oxidative/electrophilic stresses by activating the expression of a battery of cytoprotective genes through the antioxidant response element (ARE). Recently, Nrf2 has emerged as a novel target for chemoprevention. Several natural or synthetic chemicals, which activate Nrf2/ARE signaling pathway, have showed effect in animal models, and promises in many ongoing clinical trials. This review summarizes the recent findings on the regulation of Nrf2/ARE signaling pathway, and the developments in both preclinical and clinical studies.
Animals ; Anticarcinogenic Agents ; pharmacology ; Antioxidants ; metabolism ; Chemoprevention ; Digestive System Neoplasms ; genetics ; metabolism ; prevention & control ; Humans ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Oxidative Stress ; drug effects ; Response Elements ; genetics ; Signal Transduction ; drug effects

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.
Anemia/blood/*therapy ; Animals ; Base Sequence ; Blood Urea Nitrogen ; Cell Hypoxia ; Creatinine/blood ; Erythropoietin/biosynthesis/*genetics/secretion ; Gene Expression Regulation ; Genes, Reporter ; Genetic Therapy ; HeLa Cells ; Humans ; Injections, Intramuscular ; Kidney/pathology ; Luciferases, Firefly/biosynthesis/genetics ; Molecular Sequence Data ; Plasmids/*genetics ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/biosynthesis/genetics/secretion ; Response Elements ; Transcriptional Activation ; Uremia/blood/*therapy