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1.
Ann Palliat Med ; 10(8): 8827-8836, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488371

RESUMO

BACKGROUND: Lipopolysaccharide (LPS) is one of the main causes of myocardial injury. Dioscin has a protective effect on myocardial injury induced by LPS; however, the biological function and mechanism remain unclear. The purpose of this study was to investigate the effect of dioscin on myocardial injury induced by LPS. METHODS: The myocardial injury model was constructed through LPS treatment of primary rat cardiomyocytes. Cardiomyocytes were treated with different concentrations of dioscin (50, 100, and 200 ng/mL). MTT was used to detect the activity of cardiomyocytes; flow cytometry and TUNEL assay were used to detect apoptosis; and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6). The release of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) was detected according to the kit instructions. The levels of apoptosis-related proteins (Bax, caspase-3, and Bcl 2) and the Nrf2-Keap1 pathway proteins were detected by western blot. RESULTS: Dioscin significantly reduced LPS-induced cardiomyocyte injury in neonatal rats in a concentration- and time-dependent manner. Dioscin also significantly inhibited cardiomyocyte inflammation and apoptosis induced by LPS. With the increase of dioscin concentration, reactive oxygen species (ROS) and MDA were downregulated, and SOD and GSH were upregulated. Moreover, dioscin inhibited LPS-induced myocardial injury by inhibiting the Nrf2-Keap1 pathway. CONCLUSIONS: Our study suggests that dioscin attenuates LPS-induced myocardial injury through oxidative stress-related pathways.


Assuntos
Lipopolissacarídeos , Fator 2 Relacionado a NF-E2 , Animais , Diosgenina/análogos & derivados , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Ratos
2.
Chem Biol Interact ; 347: 109616, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34363818

RESUMO

The reproductive toxicity of endocrine-disrupting chemicals has become a matter of great concern. However, the potential toxicological mechanism of typical environmental estrogens, bisphenol A (BPA) and genistein (GEN), on adult ovary remains ambiguous. In this study, we used laying hens as the experimental model and aimed to clarify the effect of long-term exposure to safe reference doses of BPA and GEN on adult ovary. Results demonstrated that 1/10 no-observable-adverse effect-level dose (1/10 NOAEL, 500 µg/kg body weight [bw]/day) of BPA significantly reduced the production performance and caused the degeneration of follicles and stromal cells and the increase of atretic follicles. Moreover, 1/10 NOAEL dose of BPA undermined the redox homeostasis of the ovary through activating Keap1 and suppressing the Nrf2-signaling pathway (Nrf2, NQO1, and HO-1). On the contrary, GEN (20, 40 mg/kg bw/day) dramatically improved the antioxidant capacity of the ovary by regulating the Nrf2-Keap1 pathway, enhancing the activities of antioxidant-related enzymes (CAT, GSH-Px, and T-SOD), and inhibiting the excessive accumulation of lipid peroxidation products (MDA). Parallel in vitro studies confirmed that the differential role of BPA and GEN on ovarian redox balance was directly mediated by Nrf2-Keap1 antioxidant system. And GEN could ameliorate BPA-induced oxidative stress. Importantly, our research found that exposure to BPA and GEN altered estrogen receptor alpha (ERα) expression in the ovary. And the use of specific ERα agonist/antagonist confirmed that BPA and GEN have opposite regulatory effects on the Nrf2-Keap1 pathway by targeting ERα.


Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Genisteína/toxicidade , Ovário/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fenóis/toxicidade , Transdução de Sinais/efeitos dos fármacos , Animais , Galinhas , Receptor alfa de Estrogênio/metabolismo , Feminino , Homeostase/efeitos dos fármacos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ovário/metabolismo , Ovário/patologia
3.
J Agric Food Chem ; 69(33): 9671-9683, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34388345

RESUMO

Binge alcohol consumption is a serious health concern. Ferroptosis is an iron-dependent lipid peroxidation mediated cell death. Activation of the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway has been shown to exert a protective effect by blunting the responses to ferroptosis inducers. The autophagy substrate p62 was demonstrated to modulate Nrf2 and contribute to the suppression of ferroptosis. Furthermore, autophagy inhibition resulted in the accumulation of p62, which is a specific substrate for this process. Therefore, we aimed to explore the protective effect of autophagy inhibition against alcohol-induced ferroptosis through activating the p62-Keap1-Nrf2 pathway. Our results demonstrated that alcohol induced ferroptosis, which could be significantly reduced by ferrostatin-1. Additionally, we found that autophagy inhibition could protect HepG2 cells against alcohol-induced ferroptosis by activating the p62-Keap1-Nrf2 pathway. Furthermore, inhibition of autophagy increased the expression of p62, which interacted with Keap1 to promote Nrf2 translocation into the nucleus and upregulation its target proteins ferritin heavy (FTH), ferroportin (FPN), and heme oxygenase-1 (HO-1). This study provides a theoretical basis for further elucidation of the relationship between autophagy and ferroptosis and lays a preliminary foundation for further research concerning dietary guidance in the prevention and treatment of diseases related to alcohol-induced ferroptosis.


Assuntos
Ferroptose , Fator 2 Relacionado a NF-E2 , Autofagia , Células Hep G2 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais
4.
Zhongguo Zhong Yao Za Zhi ; 46(13): 3402-3409, 2021 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-34396761

RESUMO

This study aimed to investigate the effect and the possible mechanism of Shenlian( SL) extract on tumor necrosis factor-α( TNF-α)-induced ECV304 injury. After the establishment of TNF-α-induced ECV304 cells injure model,MTT assay was used to detect cell viability and the level of reactive oxygen species( ROS) was measured by flow cytometry. The contents of superoxide dismutase( SOD),malondialdehyde( MDA),nitric oxide( NO),endothelin-1( ET-1) and interleukin-1ß( IL-1ß) in the supernatant were detected by biochemical method and enzyme linked immunosorbent assay( ELISA). The expression levels of apoptosis-related proteins B-lymphoma-2 gene( Bcl-2),Bcl-2 associated X protein( Bax),caspase-3,caspase-9 and nuclear factor E2 associated factor2( Nrf2)/Kelch like epichlorohydrin associated protein-1( Keap1) signaling pathway related proteins Nrf2,Keap1,quinone oxidoreductase( NQO1) and heme oxygenase 1( HO-1) were detected by Western blot. The results showed that 50 µg·L-1 TNF-α significantly damaged ECV304 cells,induced the impairment of cell viability( P<0. 01),the increase of ROS production,the decrease of SOD activity,and the increase of MDA,NO,ET-1 and IL-1ß( P<0. 01),meanwhile,it caused the up-regulation of Keap1,caspase-9 and Bax protein expression,and down-regulation of NQO1 and Bcl-2 protein expression( P<0. 05) compared with the control group.Compared with the model group,SL extract reduced the damage of ECV304 cells induced by TNF-α,improved cell viability,reduced ROS production,increased SOD activity and decreased MDA,NO,ET-1,IL-1ß content( P<0. 01 or P<0. 05). In addition,SL extract also down-regulated the protein expression levels of Keap1,caspase-3,caspase-9 and Bax,and increased the protein expressions of Nrf2,NQO1,HO-1 and Bcl-2( P<0. 01 or P<0. 05). The above results indicate that SL extract can provide protective effect on ECV304 cells injury induced by TNF-α,alleviate oxidative stress injury,inflammation and apoptosis,and its mechanism may be related to regulating Nrf2/Keap1 signaling pathway.


Assuntos
Fator 2 Relacionado a NF-E2 , Fator de Necrose Tumoral alfa , Apoptose , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Extratos Vegetais , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética
5.
Sci Total Environ ; 795: 148646, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34247093

RESUMO

Acute hypoxia can aggravate the oxidation metabolism of fish muscle tissue. However, the molecular mechanism of oxidative metabolism in fish muscle under acute hypoxia is not very clear. We carried out effects of a typical oxidative metabolism pathway Keap1/Nrf2 (MafG)-GST on muscle oxidative metabolism of Japanese flounder (Paralichthys olivaceus) during acute hypoxia stimulation (1.65 ± 0.05 mg/L; 1 h, 3 h, 6 h, 12 h, 24 h) and reoxygenation (7.30 ± 0.08 mg/L; R12 h, R24 h, R48 h). The mRNAs of Nrf2 and GST in skeletal muscle were found co-existent, and their expressions were significant increase in 3 h and 6 h. The methylation level of CpG island1 in Nrf2 promoter, whose minimum value appeared at 3 h hypoxia treatment group, was affected by acute hypoxia, and it was negatively correlated with Nrf2 expression. The result suggests that environmental factors may regulate gene expression by epigenetic modification. Dual-luciferase reporter assay showed that GST gene was activated by transcription factor Nrf2, whose transcriptional activation binding region in GST promoter was antioxidant response element located near -980 and -852 sites, and Keap1 and MafG were Nrf2 antagonistic and synergistic factor, respectively. Furthermore, the GST activity changed with hypoxia and reoxygenation treatment in muscle, where other oxidative stress factor (MDA), antioxidant factors (T-AOC, GSH) and antioxidant enzyme activities (GST, SOD, CAT) were also changed. The results of MDA and T-AOC being further different between its hypoxia and normoxia groups (P < 0.05) at 6 h demonstrated that hypoxia stimulation lasting for 6 h would deeply affect Japanese flounder. The study illustrated that Japanese flounder responded to acute hypoxia in multiple metabolic levels by changing methylation status and transcription factor activation. It is significant to understand oxidative metabolic mechanism, analyze organism stress response and promote the scientific development of aquaculture.


Assuntos
Linguado , Fator 2 Relacionado a NF-E2 , Animais , Linguado/genética , Linguado/metabolismo , Hipóxia , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Músculo Esquelético/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo
6.
Cell Death Dis ; 12(7): 679, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226516

RESUMO

An ultra-large structure-based virtual screening has discovered iKeap1 as a direct Keap1 inhibitor that can efficiently activate Nrf2 signaling. We here tested its potential effect against hydrogen peroxide (H2O2)-induced oxidative injury in osteoblasts. In primary murine and human osteoblasts, iKeap1 robustly activated Nrf2 signaling at micromole concentrations. iKeap1 disrupted Keap1-Nrf2 association, causing Nrf2 protein stabilization, cytosol accumulation and nuclear translocation in murine and human osteoblasts. The anti-oxidant response elements (ARE) activity and transcription of Nrf2-ARE-dependent genes (including HO1, NQO1 and GCLC) were increased as well. Significantly, iKeap1 pretreatment largely ameliorated H2O2-induced reactive oxygen species production, lipid peroxidation and DNA damage as well as cell apoptosis and programmed necrosis in osteoblasts. Moreover, dexamethasone- and nicotine-induced oxidative injury and apoptosis were alleviated by iKeap1. Importantly, Nrf2 shRNA or CRISPR/Cas9-induced Nrf2 knockout completely abolished iKeap1-induced osteoblast cytoprotection against H2O2. Conversely, CRISPR/Cas9-induced Keap1 knockout induced Nrf2 cascade activation and mimicked iKeap1-induced cytoprotective actions in murine osteoblasts. iKeap1 was ineffective against H2O2 in the Keap1-knockout murine osteoblasts. Collectively, iKeap1 activated Nrf2 signaling cascade to inhibit H2O2-induced oxidative injury and death of osteoblasts.


Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/metabolismo , Osteoblastos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Células Cultivadas , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Osteoblastos/metabolismo , Osteoblastos/patologia , Transdução de Sinais
7.
Cell Death Dis ; 12(7): 680, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226519

RESUMO

It has been recently reported that CD38 expressed on tumor cells of multiple murine and human origins could be upregulated in response to PD-L1 antibody therapy, which led to dysfunction of tumor-infiltrating CD8+ T immune cells due to increasing the production of adenosine. However, the role of tumor expressed-CD38 on neoplastic formation and progression remains elusive. In the present study, we aimed to delineate the molecular and biochemical function of the tumor-associated CD38 in lung adenocarcinoma progression. Our clinical data showed that the upregulation of tumor-originated CD38 was correlated with poor survival of lung cancer patients. Using multiple in vitro assays we found that the enzymatic activity of tumor expressed-CD38 facilitated lung cancer cell migration, proliferation, colony formation, and tumor development. Consistently, our in vivo results showed that inhibition of the enzymatic activity or antagonizing the enzymatic product of CD38 resulted in the similar inhibition of tumor proliferation and metastasis as CD38 gene knock-out or mutation. At biochemical level, we further identified that cADPR, the mainly hydrolytic product of CD38, was responsible for inducing the opening of TRPM2 iron channel leading to the influx of intracellular Ca2+ and then led to increasing levels of NRF2 while decreasing expression of KEAP1 in lung cancer cells. These findings suggested that malignant lung cancer cells were capable of using cADPR catalyzed by CD38 to facilitate tumor progression, and blocking the enzymatic activity of CD38 could be represented as an important strategy for preventing tumor progression.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Adenocarcinoma de Pulmão/enzimologia , ADP-Ribose Cíclica/metabolismo , Neoplasias Pulmonares/enzimologia , Glicoproteínas de Membrana/metabolismo , Células A549 , ADP-Ribosil Ciclase 1/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/secundário , Animais , Sinalização do Cálcio , Carcinoma Pulmonar de Lewis/enzimologia , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patologia , Movimento Celular , Proliferação de Células , Bases de Dados Genéticas , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/metabolismo , Invasividade Neoplásica , Canais de Cátion TRPM/metabolismo
8.
J Nutr Sci Vitaminol (Tokyo) ; 67(3): 145-152, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34193673

RESUMO

Acetaminophen (N-acetyl-p-aminophenol, APAP) overdose causes hepatotoxicity, even liver failure, and oxidative stress plays pivotal role in its pathogenesis. Nicotinic acid (NA) is one form of vitamin B3, which has been used to treat a series of diseases in clinic for decades. To date, several studies have evidenced that NA has anti-oxidative property. Therefore, NA may have the hepatoprotective potential against APAP-induced toxicity. Here, our aim was to investigate the beneficial effect of NA against hepatotoxicity induced by APAP and its mechanism in vivo. BALB/c mice were intraperitoneally injected with NA (100 mg/kg) 3 times at 24, 12 and 1 h before APAP (600 mg/kg or 400 mg/kg) challenge. The results showed that pretreatment of NA markedly improved the survival rate, alleviated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and mitigated the histopathological injuries compared to APAP-exposed mice. Furthermore, NA significantly elevated the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) content, while reduced malondialdehyde (MDA) level. Finally, the signaling pathway was probed. The western blot revealed that NA up-regulated Sirtuin1 (Sirt1), nuclear factor erythroid 2-related factor 2 (Nrf2) and NAD(P)H quinone dehydrogenase-1 (NQO-1) expression and down-regulated Kelch-like ECH-associated protein 1 (Keap1) level in liver followed APAP exposure, implying Sirt1/Nrf2 axis exerted an essential role in the protective mechanism of NA on APAP toxicity. In brief, pretreatment of NA effectively protects liver against hepatotoxicity due to overdose of APAP through an antioxidant dependent manner modulated by Sirt1/Nrf2 signaling pathway.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Niacina , Acetaminofen/metabolismo , Acetaminofen/toxicidade , Animais , Antioxidantes/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fator 2 Relacionado a NF-E2/metabolismo , Niacina/metabolismo , Estresse Oxidativo , Sirtuína 1/metabolismo
9.
Life Sci ; 282: 119791, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34229009

RESUMO

AIMS: Keap1-Nrf2 signaling pathway is one of the most important antioxidant signaling pathways, and its abnormal activation is related to cancer metastasis and drug resistance. Many studies have shown Keap1 and Nrf2 mutations are closely associated with cancer occurrence. However, few studies focus on Keap1-Nrf2 binding characteristics of cancer-associated mutations. The study investigated the molecular mechanism between Keap1/Nrf2 mutations and cancer. MAIN METHODS: We have determined the crystal structure of the Keap1-Kelch domain with Nrf2 25-mer peptide. What's more, we clarified the molecular effects of Nrf2Thr80 and Nrf2Pro85 on the binding of Keap1 by the method isothermal titration calorimetry (ITC), differential scanning fluorimetry (DSF) and electrophoretic mobility shift assay (EMSA). Especially, we confirmed the effect of Thr80 and Pro85 mutations on Keap1/Nrf2 signaling pathway in HEK293T cells by RT-PCR and western blot (WB). Finally, we verified the effect of six cancer-related high-frequency somatic mutations Keap1G364C, Keap1D422N, Keap1R470C, Keap1G480W, Keap1E493Q and Keap1R601L on binding with Nrf2 through ITC experiments. KEY FINDINGS: Nrf2Thr80 and Nrf2Pro85 play a vital role in the Keap1-Nrf2 interaction. Mutant or modification at position Thr80 will disrupt the interaction. Especially, Nrf2Thr80 and Nrf2Pro85 mutations activate the expression of cytoprotective genes in HEK293T cells. As for Keap1, except G364C, the binding affinity of other cancer-related mutants to Nrf2 hardly changed, which means that Keap1 mutants can activate Nrf2 without disrupting the binding to Nrf2. SIGNIFICANCE: The study provides new insight into Keap1/Nrf2 signaling pathway and cancer.


Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias/metabolismo , Animais , Células HEK293 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Camundongos , Modelos Moleculares , Mutação , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/genética , Neoplasias/genética , Ligação Proteica , Mapas de Interação de Proteínas
10.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(8): 687-692, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34236028

RESUMO

Objective To investigate the effect of N-acetylcysteine (NAC) on the proliferation of fibroblast-like synoviocytes (FLS) treated with low concentration of hydrogen peroxide (H2O2) in rats with adjuvant arthritis (AA) and its mechanism. Methods Twenty SD rats were divided into a normal group and a model group (10 rats in each group). The model group was established by subcutaneous injection of Freund's complete adjuvant into the toe of rats, and the rats were sacrificed 28 days later. The contents of serum malondialdehyde (MDA) were detected by thiobarbituric acid method; the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined by hydroxylamine method and colorimetry respectively; and Nrf2 and Keap1 proteins in ankle synovial tissues of AA rats were detected by immunohistochemistry. AA-FLS were isolated, cultured, and identified by digestion of ankle joint slides of AA rats in vitro. The effects of NAC at different concentrations (final concentration 0, 0.3, 0.9, 3, 10, 30, 90, 180 µmol/L) on the activity of AA-FLS treated with H2O2 at low concentration (5 µmol/L) were detected by CCK-8 assay. The content of mitochondrial reactive oxygen species (ROS) in AA-FLS was detected by MitoSOX fluorescent probe. The effects of NAC (final concentration 0, 3, 10, 30 µmol/L) on Nrf2 and Keap1 protein expressions in AA-FLS treated with H2O2 at low concentration were detected by Western blotting. Results Compared with those in the control group, in AA model, the MDA level increased and SOD and GSH-Px levels decreased in serum, and the Nrf2 protein increased and the Keap1 protein decreased in synovial tissue. Immunocytochemical staining confirmed that the isolated and cultured cells were AA-FLS; NAC inhibited the proliferation of AA-FLS treated with H2O2 in a concentration-dependent manner, and the mitochondrial ROS content and the protein expressions of Nrf2 and Keap1 decreased. Conclusion NAC can inhibit the proliferation of AA-FLS treated with H2O2, which may be related to blocking Nrf2/Keap1 pathway.


Assuntos
Artrite Experimental , Sinoviócitos , Acetilcisteína/farmacologia , Animais , Artrite Experimental/tratamento farmacológico , Proliferação de Células , Fibroblastos/metabolismo , Peróxido de Hidrogênio , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ratos , Ratos Sprague-Dawley , Sinoviócitos/metabolismo
11.
Int J Mol Sci ; 22(14)2021 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-34299054

RESUMO

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription regulator that plays a pivotal role in coordinating the cellular response to oxidative stress. Through interactions with other proteins, such as Kelch-like ECH-associated protein 1 (Keap1), CREB-binding protein (CBP), and retinoid X receptor alpha (RXRα), Nrf2 mediates the transcription of cytoprotective genes critical for removing toxicants and preventing DNA damage, thereby playing a significant role in chemoprevention. Dysregulation of Nrf2 is linked to tumorigenesis and chemoresistance, making Nrf2 a promising target for anticancer therapeutics. However, despite the physiological importance of Nrf2, the molecular details of this protein and its interactions with most of its targets remain unknown, hindering the rational design of Nrf2-targeted therapeutics. With this in mind, we used a combined bioinformatics and experimental approach to characterize the structure of full-length Nrf2 and its interaction with Keap1. Our results show that Nrf2 is partially disordered, with transiently structured elements in its Neh2, Neh7, and Neh1 domains. Moreover, interaction with the Kelch domain of Keap1 leads to protection of the binding motifs in the Neh2 domain of Nrf2, while the rest of the protein remains highly dynamic. This work represents the first detailed structural characterization of full-length Nrf2 and provides valuable insights into the molecular basis of Nrf2 activity modulation in oxidative stress response.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/metabolismo , Sítios de Ligação , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Modelos Moleculares , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Ligação Proteica , Estrutura Terciária de Proteína
12.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200377

RESUMO

High levels of aldosterone (Aldo) trigger oxidative stress and vascular dysfunction independent of effects on blood pressure. We sought to determine whether Aldo disrupts Nrf2 signaling, the main transcriptional factor involved in antioxidant responses that aggravate cell injury. Thoracic aorta from male C57Bl/6J mice and cultured human endothelial cells (EA.hy926) were stimulated with Aldo (100 nM) in the presence of tiron [reactive oxygen species (ROS) scavenger, eplerenone [mineralocorticoid receptor (MR) antagonist], and L-sulforaphane (SFN; Nrf2 activator). Thoracic aortas were also isolated from mice infused with Aldo (600 µg/kg per day) for 14 days. Aldo decreased endothelium-dependent vasorelaxation and increased ROS generation, effects prevented by tiron and MR blockade. Pharmacological activation of Nrf2 with SFN abrogated Aldo-induced vascular dysfunction and ROS generation. In EA.hy926 cells, Aldo increased ROS generation, which was prevented by eplerenone, tiron, and SFN. At short times, Aldo-induced ROS generation was linked to increased Nrf2 activation. However, after three hours, Aldo decreased the nuclear accumulation of Nrf2. Increased Keap1 protein expression, but not activation of p38 MAPK, was linked to Aldo-induced reduced Nrf2 activity. Arteries from Aldo-infused mice also exhibited decreased nuclear Nrf2 and increased Keap1 expression. Our findings suggest that Aldo reduces vascular Nrf2 transcriptional activity by Keap1-dependent mechanisms, contributing to mineralocorticoid-induced vascular dysfunction.


Assuntos
Aldosterona/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptores de Mineralocorticoides/química , Doenças Vasculares/patologia , Animais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Fator 2 Relacionado a NF-E2/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Doenças Vasculares/induzido quimicamente , Doenças Vasculares/metabolismo
13.
Sci Total Environ ; 783: 146898, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34088152

RESUMO

Di-n-butylphthalate (DBP) has been listed as an environmental priority pollutant in China due to its distinct biotoxicity. Epidemiological studies have shown that exposure to DBP is closely related to a series of congenital and acquired defects in the male reproductive system. The oxidative stress injury caused by DBP plays an important role in these defects. Previous studies have demonstrated that the Keap1/Nrf2 antioxidative pathway plays a protective role in DBP-induced oxidative stress injury. However, the further molecular regulation mechanism of the activation of Nrf2 pathway remains unclear. Here, we demonstrate that DBP caused testicular oxidative stress injury and Nrf2 pathway was activated in response to the injury in vivo and in vitro. Moreover, we validated that reduced level of USP15 attenuates DBP-induced oxidative stress injury through restraining the ubiquitylation and degradation of Nrf2. Notably, USP15 is confirmed as a target of miR-135b-5p and miR-135b-5p mediated inhibition of USP15 is involved in the DBP-induced oxidative stress injury. Collectively, these findings indicated that decreased level of USP15 functions a significant protective effect on the oxidative stress injury of testis caused by DBP via regulating the Keap1/Nrf2 signaling pathway.


Assuntos
Endopeptidases/genética , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Transdução de Sinais , Testículo , Animais , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Masculino , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Ratos Sprague-Dawley , Testículo/metabolismo , Testículo/patologia
14.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071676

RESUMO

The docking program PLANTS, which is based on ant colony optimization (ACO) algorithm, has many advanced features for molecular docking. Among them are multiple scoring functions, the possibility to model explicit displaceable water molecules, and the inclusion of experimental constraints. Here, we add support of PLANTS to VirtualFlow (VirtualFlow Ants), which adds a valuable method for primary virtual screenings and rescoring procedures. Furthermore, we have added support of ligand libraries in the MOL2 format, as well as on the fly conversion of ligand libraries which are in the PDBQT format to the MOL2 format to endow VirtualFlow Ants with an increased flexibility regarding the ligand libraries. The on the fly conversion is carried out with Open Babel and the program SPORES. We applied VirtualFlow Ants to a test system involving KEAP1 on the Google Cloud up to 128,000 CPUs, and the observed scaling behavior is approximately linear. Furthermore, we have adjusted several central docking parameters of PLANTS (such as the speed parameter or the number of ants) and screened 10 million compounds for each of the 10 resulting docking scenarios. We analyzed their docking scores and average docking times, which are key factors in virtual screenings. The possibility of carrying out ultra-large virtual screening with PLANTS via VirtualFlow Ants opens new avenues in computational drug discovery.


Assuntos
Algoritmos , Inteligência Artificial , Biologia Computacional/métodos , Simulação de Acoplamento Molecular , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Ligantes , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/metabolismo , Ligação Proteica , Conformação Proteica , Reprodutibilidade dos Testes , Termodinâmica
15.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069882

RESUMO

Geographically, East Asia had the highest liver cancer burden in 2017. Besides this, liver cancer-related deaths were high in Japan, accounting for 3.90% of total deaths. The development of liver cancer is influenced by several factors, and genetic alteration is one of the critical factors among them. Therefore, the detailed mechanism driving the oncogenic transformation of liver cells needs to be elucidated. Recently, many researchers have focused on investigating the liver cancer genome and identified somatic mutations (MTs) of several transcription factors. In this line, next-generation sequencing of the cancer genome identified that oxidative stress-related transcription factor NRF2 (NFE2L2) is mutated in different cancers, including hepatocellular carcinoma (HCC). Here, we demonstrated that NRF2 DLG motif mutations (NRF2 D29A and L30F), found in Japanese liver cancer patients, upregulate the transcriptional activity of NRF2 in HCC cell lines. Moreover, the transcriptional activity of NRF2 mutations is not suppressed by KEAP1, presumably because NRF2 MTs disturb proper NRF2-KEAP1 binding and block KEAP1-mediated degradation of NRF2. Additionally, we showed that both MTs upregulate the transcriptional activity of NRF2 on the MMP9 promoter in Hepa1-6 and Huh7 cells, suggesting that MT derived gain-of-function of NRF2 may be important for liver tumor progression. We also found that ectopic overexpression of oncogenic BRAF WT and V600E increases the transcriptional activity of NRF2 WT on both the 3xARE reporter and MMP9 promoter. Interestingly, NRF2 D29A and L30F MTs with oncogenic BRAF V600E MT synergistically upregulate the transcription activity of NRF2 on the 3xARE reporter and MMP9 promoter in Hepa1-6 and Huh7 cells. In summary, our findings suggest that MTs in NRF2 have pathogenic effects, and that NRF2 MTs together with oncogenic BRAF V600E MT synergistically cause more aberrant transcriptional activity. The high activity of NRF2 MTs in HCC with BRAF MT warrants further exploration of the potential diagnostic, prognostic, and therapeutic utility of this pathway in HCC.


Assuntos
Carcinoma Hepatocelular/genética , Fator 2 Relacionado a NF-E2/genética , Motivos de Aminoácidos/genética , Carcinogênese/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Japão , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Metaloproteinase 9 da Matriz/metabolismo , Mutação , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/genética , Prognóstico , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética
16.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073079

RESUMO

Sulforaphane (SFN) is a natural glucosinolate found in cruciferous vegetables that acts as a chemopreventive agent, but its mechanism of action is not clear. Due to antioxidative mechanisms being thought central in preventing cancer progression, SFN could play a role in oxidative processes. Since redox imbalance with increased levels of reactive oxygen species (ROS) is involved in the initiation and progression of bladder cancer, this mechanism might be involved when chemoresistance occurs. This review summarizes current understanding regarding the influence of SFN on ROS and ROS-related pathways and appraises a possible role of SFN in bladder cancer treatment.


Assuntos
Anticarcinógenos , Antioxidantes , Carcinoma/tratamento farmacológico , Isotiocianatos , Sulfóxidos , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Anticarcinógenos/farmacologia , Anticarcinógenos/uso terapêutico , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Linhagem Celular Tumoral , Humanos , Isotiocianatos/farmacologia , Isotiocianatos/uso terapêutico , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sulfóxidos/farmacologia , Sulfóxidos/uso terapêutico
17.
FASEB J ; 35(7): e21710, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34143548

RESUMO

Injury to the blood-brain barrier (BBB) plays a vital role in sepsis-associated encephalopathy (SAE), which is one of the most common complications of sepsis. GYY4137, a new synthetic compound of hydrogen sulfide (H2 S), has extensive biological benefits. In this study, we focused on the protective effects of GYY4137 on the BBB in septic mice and the underlying mechanisms. The results suggested that whether administrated at the same time or 3 hours after LPS injection, GYY4137 both significantly alleviated the clinical symptoms and the long-term prognosis. Besides, GYY4137 improved the pathological abnormalities of septic mice. Moreover, the degradation of tight junctions in the BBB was considerably inhibited by GYY4137. In addition, GYY4137 significantly attenuated inflammation and apoptosis in the brain. Furthermore, GYY4137 activated the Nrf2/ARE pathway through the sulfhydrylation of Keap1 and inhibited oxidative stress. ML385, the specific inhibitor of Nrf2, significantly reversed the protective effects of GYY4137 in sepsis mice. In conclusion, this study indicated that through the sulfhydrylation of Keap1, GYY4137 activated the Nrf2/ARE pathway and exerted anti-inflammatory, anti-apoptotic and antioxidant effects in septic mice that consequently protected the integrity of the BBB and improved the clinical outcome of sepsis. Our findings suggest that GYY4137 might be a promising agent for the treatment of SAE.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Morfolinas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Compostos Organotiofosforados/farmacologia , Sepse/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Modelos Animais de Doenças , Sulfeto de Hidrogênio/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Sepse/metabolismo
18.
Eur J Med Chem ; 222: 113599, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34119834

RESUMO

Blocking the Kelch-like epichlorohydrin-related protein 1 (Keap1)-nuclear factor-erythroid 2 related factor 2 (Nrf2) pathway represents as a promising strategy to reduce oxidative stress and related-inflammation, including acute lung injury (ALI). NXPZ-2, a naphthalensulfonamide derivative, was previously reported to effectively inhibit the Keap1-Nrf2 protein-protein interaction (PPI) by our group. In the present work, a series of novel isothiocyanate-containing naphthalensulfonamides with the thioether, sulfoxide and sulfone moieties were designed by a structure-based molecular hybridization strategy using NXPZ-2 and the Nrf2 activator sulforaphane. They possessed good Keap1-Nrf2 PPI inhibitory activity and low cytotoxicity. The molecular docking study was performed to further explain the different activity of the thioether-, sulfoxide- and sulfone-containing naphthalensulfonamides. Among these new derivatives, 2-((N-(4-((N-(2-amino-2-oxoethyl)-4-((3-isothiocyanatopropyl)sulfinyl)phenyl)sulfonamido) naphthalen-1-yl)-4-methoxyphenyl)sulfonamido)acetamide (SCN-16) showed a good KD2 value of 0.455 µM to disrupt the PPI. In an LPS-induced peritoneal macrophage cell model, this compound could cause a significant increase in the nuclear Nrf2 protein, decrease in the cytosolic Nrf2 protein, and further elevate the downstream protective enzymes HO-1 and NQO-1, which were better than the lead compound NXPZ-2 and sulforaphane. What's more, the production of ROS and NO and the expression of pro-inflammatory cytokine TNF-α were also suppressed. In the LPS-induced ALI model, SCN-16 could significantly reduce LPS-induced inflammations and alleviate lung injuries by triggering Nrf2 nuclear translocation. Collectively, our results suggested that SCN-16 could be a novel lead compound targeting Keap1-Nrf2 protective pathway for clinical treatment of ALI.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Naftalenos/farmacologia , Substâncias Protetoras/farmacologia , Sulfonamidas/farmacologia , Lesão Pulmonar Aguda/metabolismo , Animais , Relação Dose-Resposta a Droga , Desenho de Fármacos , Feminino , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Fator 2 Relacionado a NF-E2/metabolismo , Naftalenos/síntese química , Naftalenos/química , Substâncias Protetoras/síntese química , Substâncias Protetoras/química , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química
19.
Free Radic Biol Med ; 172: 82-89, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34089788

RESUMO

High intensity exercise is a popular mode of exercise to elicit similar or greater adaptive responses compared to traditional moderate intensity continuous exercise. However, the molecular mechanisms underlying these adaptive responses are still unclear. The purpose of this pilot study was to compare high and low intensity contractile stimulus on the Nrf2-mediated redox stress response in mouse skeletal muscle. An intra-animal design was used to control for variations in individual responses to muscle stimulation by comparing a stimulated limb (STIM) to the contralateral unstimulated control limb (CON). High Intensity (HI - 100Hz), Low Intensity (LI - 50Hz), and Naïve Control (NC - Mock stimulation vs CON) groups were used to compare these effects on Nrf2-ARE binding, Keap1 protein, and downstream gene and protein expression of Nrf2 target genes. Muscle stimulation significantly increased Nrf2-ARE binding in LI-STIM compared to LI-CON (p = 0.0098), while Nrf2-ARE binding was elevated in both HI-CON and HI-STIM compared to NC (p = 0.0007). The Nrf2-ARE results were mirrored in the downregulation of Keap1, where Keap1 expression in HI-CON and HI-STIM were both significantly lower than NC (p = 0.008) and decreased in LI-STIM compared to LI-CON (p = 0.015). In addition, stimulation increased NQO1 protein compared to contralateral control regardless of stimulation intensity (p = 0.019), and HO1 protein was significantly higher in high intensity compared to the Naïve control group (p = 0.002). Taken together, these data suggest a systemic redox signaling exerkine is activating Nrf2-ARE binding and is intensity gated, where Nrf2-ARE activation in contralateral control limbs were only seen in the HI group. Other research in exercise induced Nrf2 signaling support the general finding that Nrf2 is activated in peripheral tissues in response to exercise, however the specific exerkine responsible for the systemic signaling effects is not known. Future work should aim to delineate these redox sensitive systemic signaling mechanisms.


Assuntos
Músculo Esquelético , Fator 2 Relacionado a NF-E2 , Animais , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Projetos Piloto
20.
J Agric Food Chem ; 69(26): 7334-7343, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34170670

RESUMO

The clinical usage of doxorubicin (DOX), a potent anthracycline antineoplastic drug, is limited due to its cardiotoxicity. The aim of this study was to assess the possible cardioprotective effects of nerolidol (NERO) in a rat model of DOX-induced chronic cardiotoxicity and the underlying molecular mechanisms. DOX (2.5 mg/kg) was injected intraperitoneally once in a week for 5 weeks to induce chronic cardiotoxicity in male albino Wistar rats. The rats were treated with NERO (50 mg/kg, orally) 6 days a week for a duration of 5 weeks. DOX-injected rats showed a significant decline in cardiac function, elevated levels of serum cardiac marker enzymes, and enhanced oxidative stress markers along with altered PI3K/Akt and Nrf2/Keap1/HO-1 signaling pathways. DOX also triggered the activation of NF-κB/MAPK signaling and increased the levels/expression of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß) and expression of inflammatory mediators (iNOS and COX-2) in the heart. DOX activated NLRP3 inflammasome-mediated pyroptotic cell death along with fibrosis, mitochondrial dysfunction, DNA damage, and apoptosis in the myocardium. Additionally, histological studies, TUNEL staining, and myocardial lesions revealed structural alterations of the myocardium. NERO treatment showed considerable protective effects on the biochemical and molecular parameters studied. The findings demonstrate that NERO protects against DOX-induced chronic cardiotoxicity and the observed cardioprotective effects are attributed to its potent antioxidant and free radical scavenging properties.


Assuntos
Óleos Voláteis , Sesquiterpenos , Animais , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/toxicidade , Apoptose , Cardiotoxicidade/tratamento farmacológico , Cardiotoxicidade/metabolismo , Doxorrubicina/toxicidade , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Masculino , Miocárdio/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Óleos Voláteis/farmacologia , Estresse Oxidativo , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Ratos Wistar , Sesquiterpenos/metabolismo
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