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1.
Ecotoxicol Environ Saf ; 202: 110960, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32800232

RESUMO

Zinc oxide nanoparticles (ZnO NPs) have been extensively used in various industries and reported to inhibit spermatogenesis, however, ZnO NPs-induced spermatogenesis failure is yet to be fully elucidated. Herein, mouse-derived spermatogonia cell line GC-1 spg cells were treated with ZnO NPs for 24 h in the presence or absence of radical scavenger N-acetyl-L-cysteine (NAC) or autophagy inhibitor 3-methyladenine (3-MA), then cell viability was observed by MTT assay; apoptosis was observed by western blotting analysis and AnnexinV-FITC/PI assay, respectively; autophagy was detected by western blotting analysis and transmission electron microscopy, respectively; and the contents of MDA and GSH and the activities of SOD and GSH-PX were measured by oxidative stress kits. The present study showed that ZnO NPs exposure inhibited viability and induced apoptosis of mouse GC-1 spg cells. Intriguingly, ZnO NPs markedly increased the protein content of LC3-II, the ratio of LC3-II/LC3-I, and the protein levels of ATG 5 and Beclin 1 in the cells. Furthermore, transmission electron microscopy (TEM) showed that autophagic vesicles in the cytoplasm increased significantly in the ZnO NPs-treated cells, indicating that ZnO NPs could induce autophagy of the cells. Oxidative stress could be induced by ZnO NPs; moreover, inhibition of oxidative stress could alleviate the induction of apoptosis and autophagy by ZnO NPs. Inhibition of autophagy by 3-MA could rescue the inhibition of cell viability and induction of apoptosis by ZnO NPs, which indicated that autophagy might have cytotoxic effect on ZnO NPs-induced apoptosis. In summary, oxidative stress was involved in ZnO NPs-induced apoptosis and autophagy of mouse GC-1 spg cells, and autophagy might play a cytotoxic role in ZnO NPs-induced apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Nanopartículas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Óxido de Zinco/toxicidade , Animais , Proteína Beclina-1/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Espécies Reativas de Oxigênio/metabolismo , Espermatogênese/efeitos dos fármacos
2.
Chemosphere ; 259: 127448, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32593828

RESUMO

Ivermectin (IVM), a broad-spectrum antiparasitic drug, is widely used in agriculture and animal husbandry. Due to widespread use and little metabolism in animals, the toxicity of IVM has received increasing attention. The accumulation of IVM in animal tissues and the excretion of urine and feces in the environment is the major source of potential toxicity. Human consumption of meat or milk contaminated with livestock can result in exposure to high levels of IVM exposure. The aim of this study was to reveal the cytotoxic mechanism of IVM in model cell HeLa in vitro, in order to provide a theoretical basis for the safe and rational use of IVM. Here we observed the γH2AX and 8-oxodG foci to detect the DNA damage in HeLa cells. As expected, we found that IVM can induce oxidative double-stranded damage in HeLa cells, indicating that IVM has potential genotoxicity to human health. In addition, we observed the formation of LC3-B in HeLa cells, the accumulation of Beclin1, the degradation of p62 and the activation of the AMPK/mTOR signal transduction pathway. This suggests that IVM confers cytotoxicity through autophagy mediated by the AMPK/mTOR signaling pathway. We conclude that IVM produces genotoxicity and cytotoxicity by inducing DNA damage and AMPK/mTOR-mediated autophagy, thereby posing a potential risk to human health.


Assuntos
Inseticidas/toxicidade , Ivermectina/toxicidade , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Antiparasitários/farmacologia , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Dano ao DNA/efeitos dos fármacos , Células HeLa , Humanos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
3.
Life Sci ; 256: 117911, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32504756

RESUMO

AIMS: To explore the potential regulatory mechanism of differentially expressed mRNAs in Hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). MAIN METHODS: Patients with HCV-related HCC and age- and gender-matched healthy subjects were enrolled. Differentially expressed mRNAs in the plasma were detected by digital gene expression (DGE) profile analysis. HepG2 and SMMC7721 cells stably transfected with HCV-core protein and the control plasmid were established. And small interfering RNA (siRNA) was used to knockdown the target gene in HCV core-expressing HCC cell lines. mRNA expression was determined by qRT-PCR. Protein expression was measured by Western blot and immunohistochemistry staining. KEY FINDINGS: DGE profile data showed aberrant mRNA expression contributed to the progression of HCV-HCC, and clusterin (CLU), which was significantly highly expressed, was chosen as a candidate gene. Further evidence showed CLU was highly expressed in tumor tissues of HCV-HCC patients and HCV core-expressing HCC cell lines, accompanied with enhanced autophagy and upregulation of pro-autophagy genes. And knockdown of CLU in HCC cell lines suppressed cell autophagy, which was indicated by decreased expression of autophagy marker light chain 3B (LC3B) ІІ/І ratio, and downregulated pro-autophagy genes like Beclin1, autophagy-related protein 7 (Atg7) and Lamp2. On the other hand, anti-autophagy genes or regulators, including p62 and phosphorylated mammalian target of rapamycin (p-mTOR), were notably upregulated. SIGNIFICANCE: CLU could promote the progression of HCV-related HCC by regulating autophagy, which might be a potential therapeutic target of HCV-HCC.


Assuntos
Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Clusterina/metabolismo , Hepacivirus/metabolismo , Neoplasias Hepáticas/metabolismo , Idoso , Apoptose/efeitos dos fármacos , Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Biblioteca Genômica , Humanos , Fígado/metabolismo , Neoplasias Hepáticas/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Masculino , Pessoa de Meia-Idade , Fosforilação , RNA Mensageiro/efeitos dos fármacos , RNA Interferente Pequeno/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
4.
Life Sci ; 256: 118000, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32585246

RESUMO

AIMS: Hsp90 is regarded as an important therapeutic target in cancer treatment. Client proteins of Hsp90 like Beclin-1, PI3K, and AKT, are associated with tumor development, poor prognosis, and resistance to cancer therapies. This study aims to analyze the role of Gedunin, an Hsp-90 inhibitor, in mediation of crosstalk between apoptosis and autophagy by targeting Beclin-1:Bcl-2 interaction, and ER stress. MAIN METHODS: A549 cells were treated with different concentrations of gedunin, and inhibitory rate was evaluated by MTT assay. Effect of gedunin on generation of reactive oxygen species, mitochondrial membrane potential, and chromatin condensation was studied by staining methods like DCFH-DA, MitoTracker, and DAPI. Expression of EGFR, PIK3CA, AKT, marker genes for apoptosis and autophagy were studied using semi-quantitative RT-PCR. Interaction study of Hsp90:Beclin-1:Bcl-2 was done by immunoprecipitation analysis. Protein expression of autophagy and apoptosis markers along with Grp78, Hsp70, and Hsp90 was analyzed by immunoblotting. KEY FINDINGS: Gedunin exerts cytotoxic effects, causes increase in ROS generation, downregulates mitochondrial membrane potential and induces loss in DNA integrity. mRNA expression analysis revealed that gedunin sensitized A549 cells towards apoptosis by downregulating EGFR, PIK3CA, AKT, and autophagy. Gedunin also inhibited interaction between Hsp90:Beclin-1:Bcl-2, leading to downregulation of autophagy (Beclin-1, Atg5-12 complex, and LC3) and antiapoptotic protein Bcl-2, which may result in ER stress-induced apoptosis. Moreover, Hsp90 inhibition by gedunin did not cause upregulation of Hsp70 expression. SIGNIFICANCE: Gedunin induces apoptosis in lung cancer cells by disrupting Hsp90:Beclin-1:Bcl-2 interaction and autophagy downregulation, thus making gedunin a good drug lead for targeting lung cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Limoninas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Células A549 , Antineoplásicos Fitogênicos/administração & dosagem , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Limoninas/administração & dosagem , Neoplasias Pulmonares/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo
5.
Gene ; 754: 144775, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32428696

RESUMO

Myocardial ischemia/reperfusion (I/R) injury is a common consequence of restored blood supply after acute myocardial infarction (AMI), but its underlying mechanisms remain largely elusive. In this study, we aimed to investigate the functional role of long non-coding RNA PVT1 in hypoxia/reoxygenation (H/R)-treated AC16 cardiomyocytes. Our experimental results demonstrated that H/R treatment impaired the viability and increased the apoptosis of AC16 cells, and knockdown of PVT1 blocked the H/R injury. Besides, PVT1 knockdown also reduced excessive autophagy in H/R-treated AC16 cells. Furthermore, we confirmed that PVT1 might serve as a ceRNA for miR-186 in AC16 cells, and rescue experiments showed that miR-186 inhibition blocked the effects of PVT1 knockdown in H/R-treated AC16 cells. In summary, this study implied that PVT1 might be a promising therapeutic target for treating myocardial I/R injury.


Assuntos
Apoptose , Autofagia , Proteína Beclina-1/metabolismo , MicroRNAs/genética , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , Proteína Beclina-1/genética , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Hipóxia/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/patologia , Oxigênio/metabolismo , Substâncias Protetoras/metabolismo , RNA Longo não Codificante/genética
6.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 34(3): 318-322, 2020 Mar 15.
Artigo em Chinês | MEDLINE | ID: mdl-32174076

RESUMO

Objective: To investigate the expression and correlation of hypoxia inducible factor 1α (HIF-1α) and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells under hypoxia in vitro. Methods: The nucleus pulposus cells were extracted from the nucleus pulposus of healthy adult Sprague Dawley rats and passaged. The 3rd generation cells were identified by HE staining and collagenase type Ⅱ immunofluorescence staining and randomly divided into 4 groups. The cells in group A were cultured for 8 hours under normal oxygen condition (37℃, 5%CO 2, 20%O 2); the cells in group B were cultured for 8 hours under hypoxia condition (37℃, 5%CO 2, 1%O 2); the cells in group C were transfected with HIF-1α-small interfering RNA and cultured for 8 hours under hypoxia condition; and the cells in group D were cultured with autophagy inhibitor 3-MA for 8 hours under hypoxia condition. Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to detect the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in all groups. Results: HE staining of the 3rd generation nucleus pulposus cells showed that the cytoplasm was light pink and the nucleus was blue black, and the collagenase type Ⅱ immunofluorescence staining was positive. Western blot and qRT-PCR results showed that the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group B were significantly higher than those in group A ( P<0.05); the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group C were significantly lower than those in group B ( P<0.05). There was no significant difference in the relative expression of HIF-1α protein and gene between groups B and D ( P>0.05); while the relative expressions of Beclin1 and LC3B proteins and genes in group D were significant lower than those in group B ( P<0.05). Conclusion: Hypoxia can induce the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells, and HIF-1α in rat nucleus pulposus cells under hypoxia is related to the expression of autophagy related molecules, that is, down-regulation of HIF-1α can significantly reduce the expression of autophagy related molecules, while the down-regulation of autophagy levels under hypoxia has no or little effect on the expression of HIF-1α.


Assuntos
Autofagia , Proteína Beclina-1/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Núcleo Pulposo/citologia , Animais , Hipóxia Celular , Células Cultivadas , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley
7.
Life Sci ; 247: 117425, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32057904

RESUMO

AIMS: Glioma is the most common type of malignant tumor of the nervous system, and aggressiveness and recurrence are major obstacles for treatment. This study is designed to explore the effects of amentoflavone (AF) on glioma, and to investigate the underlying mechanism of the anti-cancer activities of AF. METHODS: Cell morphology was recorded under microscopy. Cell viability and cell death ratio were determined by CCK-8 assay and lactate dehydrogenase (LDH) release assay, respectively. Cell cycle progression was assessed by flow cytometry. The levels of iron, MDA (malondialdehyde), lipid ROS, and GSH (reduced glutathione) were assessed by ELISA kit. The cycle-related proteins, ferroptosis-related protein, autophagy-related protein, and the phosphorylation of AMPK, mTOR and p70S6K were analyzed by western blotting. The autophagic flux was observed by transfecting cells with mRFP-GFP-LC3 plasmids. The xenograft murine models were established to analyze the effects of amentoflavone in vivo. The immunohistochemistry assay was performed to analyze the expression of LC3B, Beclin1, ATG5, ATG7, and ferritin heavy chain (FTH). RESULTS: Our results showed that AF treatment led to reduction in cell viability and cell death. In addition, AF was found to block cell cycle progression in a dose-dependent manner in vitro. Following treatment with AF, the intracellular levels of iron, MDA, and lipid OS were increased, and the levels of GSH and the mitochondrial membrane potential were reduced. In addition, our results showed that AF promoted the autophagic by regulating autophagy-relevant proteins. Our results also showed that the autophagy-induction by AF was associated with regulation of AMPK/mTOR signaling. Mechanistically, the inhibition effects of AF on glioma cell were reversed by DFO, ferreostatin-1 as well as upregulation of FTH. Meanwhile, the FTH levels were increased by compound C and knockdown of ATG7. Moreover, both autophagy inhibitor Baf A1 and knockdown of ATG7 were able to compromising AF-induce ferroptosis and cell death. In vivo, the tumor growth was suppressed by AF in a dose-dependent manner. The level of MDA in the tumor tissue was increased while the level of GSH in tumor tissue was decreased by AF in a dose-dependent manner. Furthermore, the expression of LC3B, Beclin1, ATG5, ATG7 were increased, and the expression of FTH were decreased by AF in a dose-dependent manner in vivo. Conclusion These results demonstrate that AF triggered ferroptosis in autophagy-dependent manner. Our results suggest that AF has the potential to be considered as a novel treatment agent in glioma.


Assuntos
Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Biflavonoides/uso terapêutico , Inibidores do Citocromo P-450 CYP3A/uso terapêutico , Ferroptose/efeitos dos fármacos , Glioma/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Biflavonoides/farmacologia , Linhagem Celular Tumoral , Inibidores do Citocromo P-450 CYP3A/farmacologia , Glioma/metabolismo , Glioma/patologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo
8.
Life Sci ; 248: 117466, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32101760

RESUMO

AIMS: Nanoparticles (NPs)-based drugs have been recently introduced to improve the efficacy of current therapeutic strategies for the treatment of cancer; however, the molecular mechanisms by which a NP interacts with cellular systems still need to be delineated. Here, we utilize the autophagic potential of TiO2 NPs for improving chemotherapeutic effects of 5-fluorouracil (5-FU) in human AGS gastric cells. MATERIALS AND METHODS: Cell growth and viability were determined by trypan blue exclusion test and MTT assay, respectively. Vesicular organelles formation was evaluated by acridine orange staining of cells. Cell cycle and apoptosis were monitored by flow cytometry. Reactive oxygen species (ROS) level were measured by DCHF-DA staining. Autophagy was examined by q-PCR and western blotting. Molecular docking was used for studying NP interaction with autophagic proteins. KEY FINDINGS: TiO2 NPs increase ROS production, impair lysosomal function and subsequently block autophagy flux in AGS cells. In addition, the autophagy blockade induced by non-toxic concentrations of TiO2 NPs (1 µg/ml) can promote cytotoxic and apoptotic effects of 5-FU in AGS cells. SIGNIFICANCE: These results confirm the beneficial effects of TiO2 NPs in combination with chemotherapy in in vitro model of gastric cancer, which may pave the way to develop a possible solution to circumvent chemoresistance in cancer.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Nanopartículas/química , Titânio/farmacologia , Antimetabólitos Antineoplásicos/química , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/genética , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Sinergismo Farmacológico , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fluoruracila/química , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Simulação de Acoplamento Molecular , Nanopartículas/ultraestrutura , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/antagonistas & inibidores , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Titânio/química , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
9.
Org Biomol Chem ; 18(10): 1978-1986, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32104826

RESUMO

Development of an intracellular delivery method for functional peptides via cell-penetrating peptides (CPPs) expands peptide use in basic research and therapeutic applications. Although direct conjugation of a functional peptide with a CPP is the simplest method for delivery, this method has not always been reliable. CPPs usually contain several positively charged amino acids that potentially interact non-specifically with negatively charged molecules in cells and subsequently interfere with conjugated functional peptide function. Here we demonstrate a new intracellular delivery method for peptides in which a functional peptide is released from a positively charged CPP via peptide nucleic acids (PNAs). We prepared an 8-mer PNA conjugated to octa-arginine in tandem (PNA1-CPP) and linked its complementary PNA to an autophagy inducing peptide (PNA2-AIP) by solid-phase peptide synthesis. PNA1-CPP and PNA2-AIP formed a 1 : 1 hybrid via PNA1/PNA2 interaction, thereby indirectly but stably connecting the AIP to the CPP. PNA2-AIP was successfully delivered into cells in a hybrid formation-dependent manner and at least some portion of the PNA1-CPP/PNA2-AIP hybrids dissociated into PNA2-AIP and PNA1-CPP inside the cells. Notably, PNA2-AIP delivered to cells induced more autophagy than AIP directly conjugated to CPP (CPP-AIP). Further, the PNA hybrid did not induce significant cell death. These findings indicate that the PNA1/PNA2 hybrid can function as a molecular glue enabling the delivery of functional peptides into cells.


Assuntos
Proteína Beclina-1/farmacologia , Peptídeos Penetradores de Células/metabolismo , Portadores de Fármacos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ácidos Nucleicos Peptídicos/metabolismo , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Proteína Beclina-1/toxicidade , Peptídeos Penetradores de Células/toxicidade , Portadores de Fármacos/toxicidade , Liberação Controlada de Fármacos , Células HeLa , Humanos , Zíper de Leucina , Oligopeptídeos/metabolismo , Oligopeptídeos/toxicidade , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Ácidos Nucleicos Peptídicos/toxicidade , Ligação Proteica
10.
Environ Toxicol ; 35(7): 758-767, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32061152

RESUMO

Increased deposition of silica dust in pulmonary interstitial tissues leads to silicosis, in which autophagy plays a defensive role in silica dust-associated stress response and cell death. Our previous studies revealed that silica dust exposure contributed to autophagy in pulmonary macrophages in vivo, while the specific regulatory mechanism is still unclear. This study aimed to figure out the regulatory mechanism as well as the role of autophagy in the pathogenesis of experimental silicosis. We used 3-methyladenine (3-MA) and ABT-737 to suppress the expression of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) and B cell leukemia/lymphoma 2 (Bcl-2), two critical initiators of autophagy, and detected and evaluated the autophagy in NR8383 cells with or without silica dust exposure. We found that exposure of silica dust increased autophagy in NR8383 cells and elevated the expression of Beclin1 and PIK3C3, but it reduced the expression of Bcl-2. The relationship among Beclin1, PIK3C3, and Bcl-2 were then investigated using immunoprecipitation analysis, and we found that suppression of PIK3C3 and/or Bcl-2 using 3-MA and/or ABT-737 could alter the autophagy induced by silica dust in NR8383 cells, and the complexes of Beclin1/PIK3C3 and Beclin1/Bcl-2 were both downregulated, which may be that inhibition of PIK3C3 and Bcl-2 altered the affinity of Beclin1 with PIK3C3 and Bcl-2 and lead to the silence of PIK3C3 signaling. These findings indicate that silica dust exposure induces autophagy via changing the connectivity of Beclin1 from Bcl-2 to PIK3C3.


Assuntos
Poluentes Atmosféricos/toxicidade , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Dióxido de Silício/toxicidade , Animais , Compostos de Bifenilo/farmacologia , Linhagem Celular , Poeira/análise , Humanos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Ratos , Transdução de Sinais , Silicose/metabolismo , Silicose/patologia , Sulfonamidas/farmacologia
11.
Nature ; 578(7796): 605-609, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32051584

RESUMO

The activation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle coordinates systemic metabolic responses to exercise1. Autophagy-a lysosomal degradation pathway that maintains cellular homeostasis2-is upregulated during exercise, and a core autophagy protein, beclin 1, is required for AMPK activation in skeletal muscle3. Here we describe a role for the innate immune-sensing molecule Toll-like receptor 9 (TLR9)4, and its interaction with beclin 1, in exercise-induced activation of AMPK in skeletal muscle. Mice that lack TLR9 are deficient in both exercise-induced activation of AMPK and plasma membrane localization of the GLUT4 glucose transporter in skeletal muscle, but are not deficient in autophagy. TLR9 binds beclin 1, and this interaction is increased by energy stress (glucose starvation and endurance exercise) and decreased by a BCL2 mutation3,5 that blocks the disruption of BCL2-beclin 1 binding. TLR9 regulates the assembly of the endolysosomal phosphatidylinositol 3-kinase complex (PI3KC3-C2)-which contains beclin 1 and UVRAG-in skeletal muscle during exercise, and knockout of beclin 1 or UVRAG inhibits the cellular AMPK activation induced by glucose starvation. Moreover, TLR9 functions in a muscle-autonomous fashion in ex vivo contraction-induced AMPK activation, glucose uptake and beclin 1-UVRAG complex assembly. These findings reveal a heretofore undescribed role for a Toll-like receptor in skeletal-muscle AMPK activation and glucose metabolism during exercise, as well as unexpected crosstalk between this innate immune sensor and autophagy proteins.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Beclina-1/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/fisiologia , Receptor Toll-Like 9/metabolismo , Animais , Autofagia , Ativação Enzimática , Exercício Físico , Glucose/metabolismo , Humanos , Masculino , Camundongos , Modelos Animais , Músculo Esquelético/enzimologia , Fosfatidilinositol 3-Quinase/metabolismo , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genética , Proteínas Supressoras de Tumor/metabolismo
12.
Vet Res ; 51(1): 22, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32093773

RESUMO

Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs) for defense against numerous viral infections, including classical swine fever virus (CSFV). However, the mechanisms underlying the effect of ISGs on CSFV infection are rarely reported. In this study, we demonstrate that IFN-α treatment induces upregulation of ISG15 and thus attenuates CSFV replication. To determine whether ISG15 is critical for controlling CSFV replication, we established porcine alveolar macrophages (PAMs) with stable overexpression or knockdown of ISG15. Overexpression of Flag-ISG15 significantly prevented CSFV replication, whereas loss of ISG15 led to abnormal proliferation of CSFV. Furthermore, upregulated ISG15 promoted beclin-1 (BECN1) ISGylation and dysfunction and subsequently inhibited autophagy, which is indispensable for CSFV replication. In addition, HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), which functions to catalyze conjugation of ISG15 protein, was confirmed to interact with BECN1. Collectively, these results indicate that IFN-α restricts CSFV replication through ISG15-mediated BECN1 ISGylation and autophagy inhibition, providing insight into the mechanism of CSFV replication control by type I IFN. This mechanism may not be the only antiviral mechanism of ISG15; nonetheless, this study may contribute to the development of CSFV treatment and prevention strategies.


Assuntos
Proteína Beclina-1/metabolismo , Vírus da Febre Suína Clássica/fisiologia , Peste Suína Clássica/imunologia , Citocinas/genética , Macrófagos Alveolares/imunologia , Ubiquitinas/genética , Animais , Autofagia , Citocinas/metabolismo , Suínos , Ubiquitinas/metabolismo , Replicação Viral
13.
Toxicology ; 435: 152408, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32057834

RESUMO

PURPOSE: To investigate the effects and mechanisms of NADPH on Kainic acid (KA)-induced excitotoxicity. METHODS: KA, a non-N-methyl-d-aspartate glutamate receptor agonist, was exposed to adult SD rats via intrastriatal injection and rat primary cortical neurons to establish excitotoxic models in vivo and in vitro, respectively. To determine the effects of NADPH on KA-induced excitotoxicity, neuronal survival, neurologically behavioral score and oxidative stress were evaluated. To explore the mechanisms of neuroprotective effects of NADPH, the autophagy-lysosome pathway related proteins were detected. RESULTS: In vivo, NADPH (1 mg/kg or 2 mg/kg) diminished KA (2.5 nmol)-induced enlargement of lesion size in striatum, improved KA-induced dyskinesia and reversed KA-induced activation of glial cells. Nevertheless, the neuroprotective effect of NADPH was not significant under the condition of autophagy activation. NADPH (2 mg/kg) inhibited KA (2.5 nmol)-induced down-regulation of TP-53 induced glycolysis and apoptosis regulator (TIGAR) and p62, and up-regulation of the protein levels of LC3-II/LC3-I, Beclin-1 and Atg5. In vitro, the excitotoxic neuronal injury was induced after KA (50 µM, 100 µM or 200 µM) treatment as demonstrated by decreased cell viability. Moreover, KA (100 µM) increased the intracellular levels of calcium and reactive oxygen species (ROS) and declined the levels of the reduced form of glutathione (GSH). Pretreatment of NADPH (10 µM) effectively reversed these changes. Meanwhile NADPH (10 µM) inhibited KA (100 µM)-induced down-regulation of TIGAR and p62, and up-regulation of the ratio of LC3-II/LC3-I, Beclin-1, Atg5, active-cathepsin B and active-cathepsin D. CONCLUSIONS: Our data provide a possible mechanism that NADPH ameliorates KA-induced excitotoxicity by blocking the autophagy-lysosome pathway and up-regulating TIGAR along with its antioxidant properties.


Assuntos
Autofagia/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/toxicidade , Ácido Caínico/toxicidade , Lisossomos/efeitos dos fármacos , NADP/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Comportamento Animal/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Lisossomos/metabolismo , Lisossomos/patologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Atividade Motora/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/metabolismo , Ratos Sprague-Dawley , Proteína Sequestossoma-1/metabolismo
14.
Nat Commun ; 11(1): 1032, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098961

RESUMO

The AMP-activated protein kinase (AMPK) is a master sensor of the cellular energy status that is crucial for the adaptive response to limited energy availability. AMPK is implicated in the regulation of many cellular processes, including autophagy. However, the precise mechanisms by which AMPK controls these processes and the identities of relevant substrates are not fully understood. Using protein microarrays, we identify Cyclin Y as an AMPK substrate that is phosphorylated at Serine 326 (S326) both in vitro and in cells. Phosphorylation of Cyclin Y at S326 promotes its interaction with the Cyclin-dependent kinase 16 (CDK16), thereby stimulating its catalytic activity. When expressed in cells, Cyclin Y/CDK16 is sufficient to promote autophagy. Moreover, Cyclin Y/CDK16 is necessary for efficient AMPK-dependent activation of autophagy. This functional interaction is mediated by AMPK phosphorylating S326 of Cyclin Y. Collectively, we define Cyclin Y/CDK16 as downstream effector of AMPK for inducing autophagy.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Análise Serial de Proteínas , Pironas/farmacologia , Serina/metabolismo , Tiofenos/farmacologia
15.
Biochem Biophys Res Commun ; 524(3): 629-635, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32029275

RESUMO

AIMS: Endothelial progenitor cells (EPCs) are widely accepted to be applied in ischemic diseases. However, the therapeutic potency is largely impeded because of its inviability in these ischemic conditions. Autophagy is recognized to be vital in cell activity. Therefore, we explore the role and the mechanism of autophagy in ischemic EPCs. METHODS AND RESULTS: We applied 7d-cultured bone marrow EPCs to investigate the autophagy status under the oxygen and glucose deprivation (OGD) conditions in vitro, mimicking the in-vivo harsh ischemia and anoxia microenvironment. We found increased EPC apoptosis, accompanied by an impaired autophagy activation. Intriguingly, mTOR inhibitor Rapamycin was incapable to reverse this damped autophagy and EPC damage. We further found that autophagy pathway downstream Vps34-Beclin1-Atg14 complex assembly and activity were impaired in OGD conditions, and an autophagy-inducing peptide Tat-Beclin1 largely recovered the impaired complex activity and attenuated OGD-stimulated EPC injury through restoring autophagy activation. CONCLUSIONS: The present study discovered that autophagy activation is inhibited when EPCs located in the ischemia and anoxia conditions. Restoration of Vps34 complex activity obtains sufficient autophagy, thus promoting EPC survival, which will provide a potential target and advance our understanding of autophagy manipulation in stem cell transplantation.


Assuntos
Autofagia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Isquemia/patologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Células Progenitoras Endoteliais/efeitos dos fármacos , Glucose/deficiência , Masculino , Camundongos Endogâmicos C57BL , Oxigênio , Sirolimo/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
16.
Life Sci ; 243: 117279, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31926245

RESUMO

AIMS: Treadmill exercise is a beneficial treatment following childhood stroke. Thus, studies focusing on the neuroprotective mechanism of exercise training during postischemic treatment in children with ischemic stroke are urgently needed. We evaluated the effects of treadmill exercise on autophagy after cerebral ischemia in young rats. MAIN METHODS: Rats (23-25 days old) underwent cerebral ischemia-reperfusion (CI/R) surgery. The experimental animals were divided into 5 groups, and some groups received either treadmill exercise, a rapamycin (RAPA) injection or combination therapy for 3 or 7 days. We performed a series of experimental tests including neurological scoring, hematoxylin-eosin staining (H&E), Nissl staining, triphenyl tetrazolium chloride (TTC) staining, Western blot analysis (WB), immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), transmission electron microscopy (TEM) and Terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) fluorescence. KEY FINDINGS: The experimental data indicated that treadmill exercise inhibited autophagy in the ischemic penumbra, inhibited high mobility group box 1 (HMGB1) translocation and binding to Beclin1, reduced apoptosis, reduced infarct volumes, and aided in functional recovery. However, RAPA promoted the opposite effects of treadmill exercise. SIGNIFICANCE: We found that treadmill exercise improves the neurological deficits induced by CI/R by inhibiting autophagy and HMGB1 binding to Beclin1.


Assuntos
Autofagia , Proteína Beclina-1/metabolismo , Encéfalo/fisiopatologia , Proteína HMGB1/metabolismo , Fármacos Neuroprotetores , Condicionamento Físico Animal , Animais , Apoptose , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/metabolismo , Masculino , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo
17.
Nat Commun ; 11(1): 260, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937766

RESUMO

Transmission from an infected mosquito to a host is an essential process in the life cycle of mosquito-borne flaviviruses. Numerous studies have demonstrated that mosquito saliva facilitates viral transmission. Here we find that a saliva-specific protein, named Aedes aegypti venom allergen-1 (AaVA-1), promotes dengue and Zika virus transmission by activating autophagy in host immune cells of the monocyte lineage. The AG6 mice (ifnar1-/-ifngr1-/-) bitten by the virus-infected AaVA-1-deficient mosquitoes present a lower viremia and prolonged survival. AaVA-1 intracellularly interacts with a dominant negative binder of Beclin-1, known as leucine-rich pentatricopeptide repeat-containing protein (LRPPRC), and releases Beclin-1 from LRPPRC-mediated sequestration, thereby enabling the initialization of downstream autophagic signaling. A deficiency in Beclin-1 reduces viral infection in mice and abolishes AaVA-1-mediated enhancement of ZIKV transmission by mosquitoes. Our study provides a mechanistic insight into saliva-aided viral transmission and could offer a potential prophylactic target for reducing flavivirus transmission.


Assuntos
Aedes/metabolismo , Autofagia , Infecções por Flavivirus/transmissão , Flavivirus/fisiologia , Proteínas de Insetos/metabolismo , Mosquitos Vetores/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Aedes/virologia , Animais , Proteína Beclina-1/deficiência , Proteína Beclina-1/metabolismo , Vírus da Dengue/fisiologia , Infecções por Flavivirus/virologia , Humanos , Proteínas de Insetos/deficiência , Proteínas de Insetos/genética , Camundongos , Mosquitos Vetores/virologia , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Proteínas e Peptídeos Salivares/deficiência , Proteínas e Peptídeos Salivares/genética , Células THP-1 , Replicação Viral , Zika virus/fisiologia
18.
J Photochem Photobiol B ; 203: 111748, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31918235

RESUMO

Nanotechnology is an emerged field to develop the plant mediated metal based nanodrugs by green method. In this current study, the zinc oxide metal based nanoparticles were developed using (Clausena lansium (Lour.) Skeels) Peel aqueous extracts and zinc nitrate. The C.L extract zinc nanoparticleswere indicated by the sharp peak seen at 350 nm utilizing the Ultraviolet-Visible spectroscopy (UV-Vis). The high peaks indicate the presence of phytochemicals and its functional groups in ZnONPs were studied by the Fourier Transform Infrared Spectroscopy (FT-IR). The X-Ray Diffraction analysis (XRD) explores the pattern and structure of ZnONPs as spherical and base-centered monoclinic crystalline shapes. The C.L extract with Zn nanoparticles were spherical in nature and the size of the synthesized particles were about 28.42 nm respectively. The autophagy (Beclin-1, LC3-I, LC3-II and ATG4B) and apoptotic (Bax, Bcl-2 and Caspase-3) proteins were regulated by the treatment with ZnONPs in SH-SY5Y neuroblastoma cells. The DNA loss or damage was occurred in the ZnONPs treatment and it was performed using Comet assay. The ZnONPs treatment generates the ROS in the cells and decreased its stability and viability. Addition of NAC prevents ROS in the cultured SH-SY5Y cells and prevents the cells from the apoptosis. We concluded that the ZnONPs potentially kills the neuroblastoma cells by producing the intracellular ROS.


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Nanopartículas Metálicas/química , Óxido de Zinco/química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Clausena/química , Clausena/metabolismo , Dano ao DNA/efeitos dos fármacos , Química Verde , Humanos , Nanopartículas Metálicas/toxicidade , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo
19.
PLoS One ; 15(1): e0227603, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31940411

RESUMO

In-vitro studies with different Fanconi anemia (FA) cell lines and FANC gene silenced cell lines indicating involvement of mitochondria function in pathogenesis of FA have been reported. However, in-vivo studies have not been studied so far to understand the role of mitochondrial markers in pathogenesis of FA. We have carried out a systematic set of biomarker studies for elucidating involvement of mitochondrial dysfunction in disease pathogenesis for Indian FA patients. We report changes in the mtDNA number in 59% of FA patients studied, a high frequency of mtDNA variations (37.5% of non-synonymous variations and 62.5% synonymous variations) and downregulation of mtDNA complex-I and complex-III encoding genes of OXPHOS (p<0.05) as strong biomarkers for impairment of mitochondrial functions in FA. Deregulation of expression of mitophagy genes (ATG; p>0.05, Beclin-1; p>0.05, and MAP1-LC3, p<0.05) has also been observed, suggesting inability of FA cells to clear off impaired mitochondria. We hypothesize that accumulation of such impaired mitochondria in FA cells therefore may be the principal cause for bone marrow failure (BMF) and a plausible effect of inefficient clearance of impaired mitochondria in FA.


Assuntos
DNA Mitocondrial/genética , Anemia de Fanconi/genética , Adolescente , Adulto , Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Criança , DNA Mitocondrial/metabolismo , Anemia de Fanconi/metabolismo , Feminino , Variação Genética/genética , Humanos , Índia , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitofagia , Espécies Reativas de Oxigênio/metabolismo
20.
Am J Physiol Renal Physiol ; 318(3): F772-F792, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31984794

RESUMO

Klotho- and beclin 1-driven autophagy extends life. We examined the role of beclin 1 in modifying acute kidney injury (AKI) and whether beclin 1 mediates Klotho's known renoprotective action in AKI. AKI was induced by ischemia-reperfusion injury in mice with different levels of autophagy activity by genetic manipulation: wild-type (WT) mice with normal beclin 1 expression and function, mice with normal beclin 1 levels but high activity through knockin of gain-of-function mutant beclin 1 (Becn1F121A), mice with low beclin 1 levels and activity caused by heterozygous global deletion of beclin 1 (Becn1+/-), or mice with extremely low beclin 1 activity from knockin of the mutant constitutively active beclin 1 inhibitor Bcl-2 (Bcl2AAA). Klotho was increased by transgenic overexpression (Tg-Kl) or recombinant Klotho protein administration. After ischemia-reperfusion injury, Becn1F121A mice (high autophagy) had milder AKI and Becn1+/- and Bcl2AAA mice (low autophagy) had more severe AKI than WT mice. Tg-Kl mice had milder AKI, but its renoprotection was partially attenuated in Becn1+/-;Tg-Kl mice and was significantly reduced, although not completely abolished, in Bcl2AAA;Tg-Kl mice. Recombinant Klotho protein conferred more renoprotection from AKI in WT mice than in Becn1+/- or Bcl2AAA mice. Klotho reduced beclin 1/Bcl-2 protein complexes and increased autophagy activity, but this effect was less prominent in mice or cells with Bcl2AAA. Transfected Bcl2AAA or Becn1F123A decreased or increased autophagy activity and rendered cells more susceptible or more resistant to oxidative cytotoxicity, respectively. In conclusion, beclin 1 confers renoprotection by activating autophagy. Klotho protects the kidney partially via disruption of beclin 1/Bcl-2 interactions and enhancement of autophagy activity.


Assuntos
Autofagia/fisiologia , Proteína Beclina-1/metabolismo , Glucuronidase/metabolismo , Nefropatias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Traumatismo por Reperfusão , Animais , Proteína Beclina-1/genética , Linhagem Celular , Regulação da Expressão Gênica , Genótipo , Glucuronidase/genética , Peróxido de Hidrogênio , Nefropatias/etiologia , Camundongos , Gambás , Proteínas Proto-Oncogênicas c-bcl-2/genética
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