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1.
Medicine (Baltimore) ; 98(40): e17313, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31577725

RESUMO

To investigate the impact of carriage of single nucleotide polymorphisms (SNPs) of the Toll-like receptor-4 (TLR4) and of autophagy-related gene 16-like-1 (ATG16L1) in preterm delivery (PTD).A prospective cohort of 145 pregnant women was studied. Women were prospectively followed-up until delivery. Genotyping for rs4986790 (Asp299Gly transition) and rs4986791 (Thr399Ile transition) of TLR4 and for rs2241880 of ATG16L1 was done by PCR-restriction fragment length polymorphism. The primary study endpoint was the impact of carriage of minor alleles of TLR4 on early PTD before gestational week 32. Associations with human chorionic gonadotrophin (hCG) were also analyzed. Peripheral blood mononuclear cells were isolated from 15 healthy women and stimulated for cytokine production.No difference in clinical characteristics was observed between women delivering full term and preterm. The frequency of early PTD was 25% among women carrying minor alleles of TLR4 and 6.8% among women carrying major alleles (P: .032). Odds ratios for PTD were 3.85 among women carrying the GG genotype of rs2241880 and major alleles of TLR4 and 0.26 among carriers of GG genotype and minor alleles of TLR4 (P: .030). The co-presence of GG genotype of rs2241880 and hCG above 70 U/L was an independent variable for PTD. Stimulated production of interleukin-6 was greater among women with GG genotypes of rs2241880.Minor alleles of SNPs of TLR4 predispose to early PTD. The GG genotype of rs2241880 of ATG16L1 is associated with PTD when hCG is supra-elevated.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Nascimento Prematuro/genética , Receptor 4 Toll-Like/genética , Adulto , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Autofagia/fisiologia , Gonadotropina Coriônica/sangue , Citocinas/biossíntese , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Idade Gestacional , Humanos , Polimorfismo de Nucleotídeo Único , Gravidez , Estudos Prospectivos
2.
Chemosphere ; 235: 1134-1145, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31561304

RESUMO

Particulate matter (PM) from layer house has adverse effect on people and chicken respiratory health, which can further influence animal performance and reduce production efficiency. However, little study focus on the respiratory inflammation induced by PM2.5 from layer house and the underlying mechanism also unclear. In this study, human adenocarcinoma alveolar basal epithelial cells (A549 cell) was subjected to the PM2.5 from layer house to evaluate the inflammation reaction caused by PM2.5 and explore the role of Nrf2 and autophagy in regulating the inflammation. Results showed that the viability of A549 cell decreased in a time - and concentration - dependent manner after PM2.5 treatment. TNFα, IL6, and IL8 increased significantly treated with PM2.5 at 12 h. RNA sequencing indicated differentially expressed genes were enriched in immune system process, oxidative stress (OS), endoplasmic reticulum stress (ERS), and autophagy. Further studies showed TLR4 - NFκB p65 signal pathway involved in the inflammation reaction caused by PM2.5. The overexpression of Nrf2 decreased the level of TNFα, IL6, IL8 markedly as well as the level of NFκB p65 and NFκB pp65. OS and ERS were also limited under overactivation of Nrf2 in PM2.5 treated cells. Autophagy induced by PM2.5 promoted the inflammation through increasing the level of NFκB p65 and NFκB pp65. Autophagy deficient strengthened the expression of Nrf2. Collectively, our study revealed Nrf2 prevents inflammation caused by layer house PM2.5 stimulation, however, autophagy exerts a promotive role in TLR4 - NFκB p65 mediating inflammation in A549 cell.


Assuntos
Autofagia/fisiologia , Inflamação/etiologia , Fator 2 Relacionado a NF-E2/fisiologia , Material Particulado/efeitos adversos , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Células A549 , Animais , Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Humanos , Inflamação/induzido quimicamente , Inflamação/prevenção & controle , Fator 2 Relacionado a NF-E2/farmacologia , Estresse Oxidativo/genética , Transdução de Sinais
3.
J Dairy Sci ; 102(9): 8264-8272, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31255277

RESUMO

Staphylococcus aureus is an important pathogen causing chronic and subclinical mastitis of cows. Autophagy is an important regulatory mechanism that participates in the elimination of invading pathogenic organisms. Here, we hypothesize that autophagy is involved in the process of Staph. aureus survival in bovine mammary epithelial cells (BMEC). In this study, we detected the expression of autophagy-related proteins during infection and assessed the effect of autophagosome formation and degradation on the proliferation of intracellular Staph. aureus. Infection with Staph. aureus increased the protein expression of microtubule-associated protein 1 light chain 3-II (MAP1LC3, also called LC3-II) and sequestosome-1 (SQSTM1, also called p62) in BMEC. After infection, the formation of the autophagosomes increased but the autophagosomes and lysosomes could not fuse normally to form autolysosomes. When the formation of the autophagosomes was enhanced or the degradation of the autolysosomes was inhibited, the number of Staph. aureus in the BMEC increased. However, the intracellular proliferation of Staph. aureus was slowed when formation of autophagosomes was inhibited. Therefore, autophagy was induced in BMEC challenged by Staph. aureus but the autophagic flux was obstructed. Inhibiting the formation of autophagosomes in BMEC facilitated the clearance of intracellular Staph. aureus, which may offer a new strategy for the treatment of mastitis in cows.


Assuntos
Autofagossomos/fisiologia , Autofagia/fisiologia , Células Epiteliais/fisiologia , Glândulas Mamárias Animais/citologia , Mastite Bovina/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Bovinos , Contagem de Células , Feminino , Proteína Sequestossoma-1/análise , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/fisiologia
4.
Life Sci ; 232: 116639, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31295472

RESUMO

AIMS: Sirtuins have been implicated in the aging process, however, the functions of SIRT2 in post-maturation aging of oocytes are not fully understood. The purpose of the present investigation was to assess the roles of SIRT2 in aged oocytes and mechanisms involved. MAIN METHODS: The fresh MII oocytes were aging in vitro, and treated with SIRT2 inhibitor (SirReal2), autophagy activator (Rapamycin), and autophagy inhibitor (3-Ma) for 24 h, respectively. Oocyte activation, cytoplasmic fragmentation, and spindle defects, mitochondrial distribution, ROS levels, ATP production, mitochondrial membrane potential, and early apoptosis were investigated. Western blotting was performed to determine LC3-II accumulation, SQSTM1 degradation, and caspase-3 activity. KEY FINDINGS: SIRT2 expression gradually decreased in a time-dependent manner during oocyte aging. Treatment with SirReal2 significantly increased the rates of oocyte activation, cytoplasmic fragmentation, and spindle defects. In particular, the high ROS levels, abnormal mitochondrial distribution, low ATP production, and lost ΔΨm were observed in SirReal2-exposed oocytes. Further analysis revealed that LC3-II accumulation and SQSTM1 degradation were induced by SIRT2 inhibition. By performing early apoptosis analysis showed that oocyte aging was accompanied with cellular apoptosis, and SIRT2 inhibition increased apoptosis rates of aged oocytes. Importantly, upregulating autophagy with Rapamycin could mimic the effects of SIRT2 inhibition on apoptosis by increasing caspase-3 activation, whereas downregulating autophagy with 3-MA could abolish those effects by blocking caspase-3 activation. SIGNIFICANCE: Our results suggest that SIRT2 inactivation is a key mechanism underlying of cellular aging, and SIRT2 inhibition contributes to autophagy-dependent cellular apoptosis in post-maturation oocytes.


Assuntos
Oócitos/fisiologia , Sirtuína 2/fisiologia , Acetamidas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Bovinos , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Feminino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Oócitos/classificação , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Sirolimo/farmacologia , Sirtuína 2/antagonistas & inibidores , Sirtuína 2/metabolismo , Tiazóis/farmacologia
5.
BMC Plant Biol ; 19(1): 326, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324141

RESUMO

BACKGROUND: Autophagy is a conserved, highly-regulated catabolic process that plays important roles in growth, development and innate immunity in plants. In this study, we compared the rate of autophagy induction in Nicotiana benthamiana plants infected with Tobacco mosaic virus or the TMV 24A + UPD mutant variant, which replicates at a faster rate and induces more severe symptoms. Using a BirA* tag and proximity-dependent biotin identification (BioID) analysis, we identified host proteins that interact with the core autophagy protein, ATG8 in TMV 24A + UPD infected plants. By combining the use of a fast replicating TMV mutant and an in vivo protein-protein screening technique, we were able to gain functional insight into the role of autophagy in a compatible virus-host interaction. RESULTS: Our study revealed an increased autophagic flux induced by TMV 24A + UPD, as compared to TMV in N. benthamiana. Analysis of the functional proteome associated with ATG8 revealed a total of 67 proteins, 16 of which are known to interact with ATG8 or its orthologs in mammalian and yeast systems. The interacting proteins were categorized into four functional groups: immune system process, response to ROS, sulphur amino acid metabolism and calcium signalling. Due to the presence of an ubiquitin-associated (UBA) domain, which is demonstrated to interact with ATG8, the Huntingtin-interacting protein K-like (HYPK) was selected for validation of the physical interaction and function. We used yeast two hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and subcellular localization to validate the ATG8-HYPK interaction. Subsequent down-regulation of ATG8 by virus-induced gene silencing (VIGS) showed enhanced TMV symptoms, suggesting a protective role for autophagy during TMV 24A + UPD infection. CONCLUSION: This study presents the use of BioID as a suitable method for screening ATG8 interacting proteins in planta. We have identified many putative binding partners of ATG8 during TMV 24A + UPD infection in N. benthamiana plants. In addition, we have verified that NbHYPK is an interacting partner of ATG8. We infer that autophagy plays a protective role in TMV 24A + UPD infected plants.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Tabaco/virologia , Autofagossomos/metabolismo , Autofagia/genética , Autofagia/fisiologia , Biotinilação , Imunidade Vegetal , Tabaco/metabolismo , Vírus do Mosaico do Tabaco
6.
Nat Chem Biol ; 15(8): 776-785, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285595

RESUMO

Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling.


Assuntos
Autofagia/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Autofagia/fisiologia , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Estrutura Molecular , Proteínas Proto-Oncogênicas c-akt
7.
Eur J Histochem ; 63(2)2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31189296

RESUMO

The Kölliker's organ is a transient epithelial structure during cochlea development that gradually degenerates and disappears at postnatal 12-14 days (P12-14). While apoptosis has been shown to play an essential role in the degeneration of the Kölliker's organ, the role of another programmed cell death, autophagy, remains unclear. In our study, autophagy markers including microtubule associated protein light chain 3-II (LC3-II), sequestosome 1 (SQSTM1/p62) and Beclin1 were detected in the supporting cells of the Kölliker's organ through immunohistochemistry staining. In addition, Western blot and real-time PCR revealed a gradually decreased expression of LC3-II and an increased expression of p62 during early postnatal development. Compared to apoptosis markers that peaks between P7 and P10, autophagy flux peaked earlier at P1 and decreased from P1 to P14. By transmission electron microscopy, we observed representative autophagosome and autolysosome that packaged various organelles in the supporting cells of the Kölliker's organ. During the degeneration, these organelles were digested via autophagy well ahead of the cellular apoptosis. These results suggest that autophagy plays an important role in transition and degeneration of the Kölliker's organ prior to apoptosis during the early postnatal development.


Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Cóclea/embriologia , Cóclea/metabolismo , Animais , Anticorpos/imunologia , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Proteína Beclina-1/metabolismo , Caspase 3/genética , Caspase 3/imunologia , Caspase 3/metabolismo , Cóclea/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Imuno-Histoquímica/métodos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/imunologia , Proteína Sequestossoma-1/metabolismo , Fatores de Tempo
8.
Nat Commun ; 10(1): 2602, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197136

RESUMO

Temperature is a key factor for determining the lifespan of both poikilotherms and homeotherms. It is believed that animals live longer at lower body temperatures. However, the precise mechanism remains largely unknown. Here, we report that autophagy serves as a boost mechanism for longevity at low temperature in the nematode Caenorhabditis elegans. The adiponectin receptor AdipoR2 homolog PAQR-2 signaling detects temperature drop and augments the biosynthesis of two ω-6 polyunsaturated fatty acids, γ-linolenic acid and arachidonic acid. These two polyunsaturated fatty acids in turn initiate autophagy in the epidermis, delaying an age-dependent decline in collagen contents, and extending the lifespan. Our findings reveal that the adiponectin receptor PAQR-2 signaling acts as a regulator linking low temperature with autophagy to extend lifespan, and suggest that such a mechanism may be evolutionally conserved among diverse organisms.


Assuntos
Autofagia/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Longevidade/fisiologia , Proteínas de Membrana/fisiologia , Animais , Animais Geneticamente Modificados , Ácido Araquidônico/biossíntese , Temperatura Baixa , Colágeno/metabolismo , Epiderme/metabolismo , Ácidos Graxos Ômega-6/biossíntese , Interferência de RNA , Transdução de Sinais/fisiologia
9.
Infect Immun ; 87(8)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31160361

RESUMO

Brucella is an intracellular bacterial pathogen that causes chronic systemic infection in domesticated livestock and poses a zoonotic infectious risk to humans. The virulence of Brucella is critically dependent on its ability to replicate and survive within host macrophages. Brucella modulates host physiological pathways and cell biology in order to establish a productive intracellular replicative niche. Conversely, the host cell presumably activates pathways that limit infection. To identify host pathways contributing to this yin and yang during host cell infection, we performed a high-throughput chemical genetics screen of known inhibitors and agonists of host cell targets to identify host factors that contribute to intracellular growth of the model pathogen Brucella neotomae Using this approach, we identified the p38 mitogen-activated protein (MAP) kinase pathway and autophagy machinery as both a linchpin and an Achilles' heel in B. neotomae's ability to coopt host cell machinery and replicate within macrophages. Specifically, B. neotomae induced p38 MAP kinase phosphorylation and autophagy in a type IV secretion system-dependent fashion. Both p38 MAP kinase stimulation and an intact autophagy machinery in turn were required for phagosome maturation and intracellular replication. These findings contrasted with those for Legionella pneumophila, where chemical inhibition of the p38 MAP kinase pathway and autophagy factor depletion failed to block intracellular replication. Therefore, results from a chemical genetics screen suggest that intersections of the MAP kinase pathways and autophagy machinery are critical components of Brucella's intracellular life cycle.


Assuntos
Autofagia/fisiologia , Brucella/crescimento & desenvolvimento , Macrófagos/microbiologia , Fagossomos/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Linhagem Celular , Feminino , Humanos , Imidazóis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Piridinas/farmacologia , Proteínas rab de Ligação ao GTP/fisiologia
10.
New Microbiol ; 42(3): 161-165, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31157401

RESUMO

Human bocavirus 1 (HBoV1) refers to a human parvovirus causing acute respiratory tract infection in children. Bocaviruses encode an NP1 protein, which has 47% amino acid homology with NP1 of Minute Virus of Canines (MVC) and Bovine Parvovirus (BPV), but not with any protein of other parvoviruses. NP1 was found to induce apoptosis in Hela cells, which does not depend on viral replication and other protein expression. However, whether NP1 induces pulmonary cell death is unclear. In the present study, we investigate the impacts of NP1 on the autophagy and viability of A549 cells by expressing NP1. The plasmid containing NP1 gene was transfected into A549 cells. The apoptosis of A549 was evaluated by apoptosis detection kit and expression of caspase3. Cell viability and cell migration were detected by CCK8 kit and cell scratch test, respectively. The autophagy-related proteins and HMGB1 were detected by Western blot after NP1 expression in transfected cells. The real-time PCR was employed to detect HMGB1 mRNA. The secretory HMGB1 in supernatant of cell culture was measured by ELISA kit. The transient expression of NP1 did not induce apoptosis in A549 cells, but inhibited cell viability and migration. The expression of Beclin1 and LC3 II increased significantly and that of autophagy substrate P62 decreased dramatically upon transfection of NP1. The expression of NP1 reduced both levels of mRNA and protein HMGB1. The NP1 induced A549 autophagy was activated by STAT3 signaling pathway. HBoV1 NP1 induced autophagy in A549 cells by activating phosphorylation of STAT3 signaling pathway and inhibited A549 cell viability. This study provides insight into further elucidating the replication mechanism of HBoV1.


Assuntos
Autofagia , Sobrevivência Celular , Bocavirus Humano , Proteínas Virais , Células A549 , Autofagia/fisiologia , Caspase 3/genética , Caspase 3/metabolismo , Expressão Gênica , Células HeLa , Bocavirus Humano/genética , Bocavirus Humano/metabolismo , Humanos , Pulmão/citologia , Fosforilação , Fatores de Transcrição STAT/metabolismo , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismo
11.
Life Sci ; 231: 116551, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31185236

RESUMO

Octreotide (OCT) shows clinical efficacies in the treatment of liver cirrhosis complicated with gastrointestinal hemorrhage. Experiments were designed to investigate its function mechanism associated with endoplasmic reticulum stress (ERS)-induced autophagy and microRNA (miR). Protein associated with ERS and autophagy was detected by western blot. miR-101 was examined by qRT-PCR. Besides, miR-101 or G protein-coupled receptor 78 (GPR78)-silenced Caco-2 cells were established by transfection. Furthermore, western blot was used to determine TGF-beta activated kinase 1 (TAK1), AMPK, mTOR, p70S6K as well as their phosphorylated forms. Lipopolysaccharide (LPS) enforced the expression of GPR78. Besides, LPS triggered the production of Beclin-1 and LC3-II while mitigated the accumulation of p62. Then all these above results were reversed by OCT pretreatment. Moreover, miR-101 expression was downregulated by LPS while upregulated by OCT. Further, miR-101 knockdown strengthened ERS and promoted autophagy. GPR78 silence retarded autophagy process. In the end, OCT mitigated phosphorylation of TAK1, AMPK while enhanced the phosphorylated expression of mTOR and p70S6K in LPS-treated Caco-2 cells. The anti-autophagy property of OCT was mediated by miR-101-induced suppression of GPR78 in LPS-treated Caco-2 cells.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , MicroRNAs/metabolismo , Octreotida/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Proteína Beclina-1/genética , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , Octreotida/metabolismo , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
12.
J Microbiol Biotechnol ; 29(6): 989-998, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154748

RESUMO

Autophagy is crucial for immune defense against Mycobacterium tuberculosis (Mtb) infection. Mtb can evade host immune attack and survival within macrophages by manipulating the autophagic process. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in regulating vital genes during Mtb infection. The precise role of miRNAs in autophagy with the exits of Mtb remains largely unknown. In this study, we found miR-1958, a new miRNA that could regulate autophagy by interacting with 3'UTR of autophagy-related gene 5 (Atg5). In addition, Mtb infection triggered miR-1958 expression in RAW264.7 cells. What's more, miR- 1958 overexpression blocked autophagic flux by impairing the fusion of autophagosomes and lysosomes. Overexpression of miR-1958 reduced Atg5 expression and LC3 puncta while inhibition of miR-1958 brought an increase of Atg5 and LC3 puncta; the opposite results were observed in detection of p62. The survival of Mtb in RAW264.7 cells transfected with mimic of miR-1958 was enhanced. Taken together, our research demonstrated that a novel miR-1958 could inhibit autophagy by interacting with Atg5 and favored intracellular Mtb survival in RAW264.7 cells.


Assuntos
Proteína 5 Relacionada à Autofagia/genética , Autofagia/fisiologia , MicroRNAs/metabolismo , Viabilidade Microbiana/genética , Mycobacterium tuberculosis/fisiologia , Regiões 3' não Traduzidas , Animais , Autofagossomos/metabolismo , Expressão Gênica , Evasão da Resposta Imune , Lisossomos/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , MicroRNAs/genética , Mycobacterium tuberculosis/imunologia , Células RAW 264.7
13.
Chemosphere ; 233: 261-272, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31176127

RESUMO

Aflatoxin B1 (AFB1), a potential endocrine disrupter, has been shown to induce hepatotoxicity in animal models, but the effects of AFB1 on Leydig cell function are unclear. In this study, in vivo exposure to AFB1 at 15 and 150 µg/kg/day lowered serum testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels, reduced Leydig cell number, and down-regulated the expression of testosterone biosynthesis-related genes. In vitro study showed that AFB1 (10 µM) significantly increased ROS levels, and decreased T production in Leydig cells by suppressing certain T-biosynthesis gene expressions. Moreover, AFB1 induced Leydig cell apoptosis through lowering pAMPK/AMPK ratio and increasing pmTOR/mTOR ratio, and then further up-regulating autophagy and apoptosis proteins, LC3, BECLIN 1, and BAX, as well as down-regulating autophagy flux protein P62 and anti-apoptosis protein BCL-2. AFB1-induced toxicity in Leydig cells was characterized by inhibiting T-biosynthesis gene expression, reducing Leydig cell number, promoting ROS production, and inducing cell apoptosis via suppressing AMPK/mTOR-mediated autophagy flux pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aflatoxina B1/toxicidade , Autofagia/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/fisiologia , Proteína Beclina-1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Hormônio Luteinizante/sangue , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Testosterona/sangue , Testosterona/genética , Testosterona/metabolismo
14.
Food Chem Toxicol ; 131: 110591, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31212009

RESUMO

Kidney ischemia reperfusion injury (IRI) is an acute kidney injury associated with high number of mortality. We have examined the molecular mechanism and found that oxidative stress and hypoxia leads to induction of autophagy. In IRI induced autophagy, TFEB translocated to nucleus in response to IRI and induced a number of target genes of Coordinated Lysosomal Expression and Regulation (CLEAR) network. Real-time PCR analyses result showed IRI dependent increase in mRNA level to lysosomal hydrolases (Ctsa, Psap), lysosomal membranes (Lamp1), lysosomal acidification (Atp6ap1) non-lysosomal proteins involved in lysosomal biogenesis (M6pr, Nagpa) and autophagy (Becn1, VPS11). Overall, both lysosomal biogenesis and autophagy pathways were induced. Two key players of TFEB dependent proteins in autophagy, LAMP1 and BECN1 were verified by protein analyses. Pretreatment with urolithin A promoted autophagy and attenuated renal injury in kidney IRI and thus inverse relationship existed between TFEB-CLEAR pathway and kidney injury. Urolithin A also attenuated IRI induced pro-inflammatory cytokines TNFα, IL1ß, MIP1α and MIP2 mRNA and associated kidney injury. Overall, our results explored the understanding of autophagy and CLEAR network to kidney IRI and those insights may help to develop new therapeutic strategies to protect against IRI.


Assuntos
Lesão Renal Aguda/prevenção & controle , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Cumarínicos/uso terapêutico , Substâncias Protetoras/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Lesão Renal Aguda/fisiopatologia , Animais , Autofagia/fisiologia , Núcleo Celular/metabolismo , Citocinas/metabolismo , Inflamação/prevenção & controle , Rim/patologia , Rim/fisiopatologia , Lisossomos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Traumatismo por Reperfusão/fisiopatologia
15.
BMC Vet Res ; 15(1): 173, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31126305

RESUMO

BACKGROUND: Avian reovirus (ARV) is an important pathogen that can cause serious disease in poultry. Though several in vitro studies revealed some molecular mechanisms that are responsible for ARV-induced autophagy, it is still largely unknown how ARV manipulates autophagy to promote its own propagation. RESULTS: In this study, we demonstrated that ARV infection triggered autophagy in chicken tissues, evident from the enhancement of LC3-I/-II conversion and the appearance of abundant autophagosomes. Moreover, viral replication and the expression of IL-1ß were coupled with the process of ARV-induced autophagy in the early stage of infection. Furthermore, regulation of autophagy affected the accumulation of LC3-II, the production of ARV and the expression of IL-1ß. CONCLUSIONS: Altogether, our data suggest that ARV induces autophagy, which benefits its replication and dissemination in chicken tissues at the early infection stage.


Assuntos
Autofagia/fisiologia , Orthoreovirus Aviário/fisiologia , Doenças das Aves Domésticas/virologia , Replicação Viral/fisiologia , Animais , Galinhas , Interleucina-1beta/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Infecções por Reoviridae/virologia
16.
J Biomed Sci ; 26(1): 40, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118030

RESUMO

BACKGROUND: Oxidative stress is a major factor in retinal pigment epithelium (RPE) cells injury that contributes to age-related macular degeneration (AMD). NaIO3 is an oxidative toxic agent and its selective RPE cell damage makes it as a reproducible model of AMD. Although NaIO3 is an oxidative stress inducer, the roles of ROS in NaIO3-elicited signaling pathways and cell viability have not been elucidated, and the effect of NaIO3 on autophagy in RPE cells remains elusive. METHODS: In human ARPE-19 cells, we used Annexin V/PI staining to determine cell viability, immunoblotting to determine protein expression and signaling cascades, confocal microscopy to determine mitochondrial dynamics and mitophagy, and Seahorse analysis to determine mitochondrial oxidative phosphorylation. RESULTS: We found that NaIO3 can dramatically induce cytosolic but not mitochondrial ROS production. NaIO3 can also activate ERK, p38, JNK and Akt, increase LC3II expression, induce Drp-1 phosphorylation and mitochondrial fission, but inhibit mitochondrial respiration. Confocal microscopic data indicated a synergism of NaIO3 and bafilomycin A1 on LC3 punctate formation, indicating the induction of autophagy. Using cytosolic ROS antioxidant NAC, we found that p38 and JNK are downstream signals of ROS and involve in NaIO3-induced cytotoxicity but not in mitochondrial dynamics, while ROS is also involved in LC3II expression. Unexpectedly NAC treatment upon NaIO3 stimulation leads to an enhancement of mitochondrial fragmentation and cell death. Moreover, inhibition of autophagy and Akt further enhances cell susceptibility to NaIO3. CONCLUSIONS: We conclude that NaIO3-induced oxidative stress and cytosolic ROS production exert multiple signaling pathways that coordinate to control cell death in RPE cells. ROS-dependent p38 and JNK activation lead to cytotoxicity, while ROS-mediated autophagy and mitochondrial dynamic balance counteract the cell death mechanisms induced by NaIO3 in RPE cells.


Assuntos
Autofagia/fisiologia , Iodatos/toxicidade , Degeneração Macular/fisiopatologia , Dinâmica Mitocondrial/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/fisiopatologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Estresse Oxidativo/fisiologia , Epitélio Pigmentado da Retina/efeitos dos fármacos
17.
Biomed Res Int ; 2019: 5171602, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31111057

RESUMO

Chondrocytes are the sole cellular constituents of normal cartilage. The degeneration and apoptosis of these cells are considered the main cause of osteoarthritis (OA). Previous studies have suggested that the enhancement of autophagy in chondrocytes can delay the progression of osteoarthritis by affecting intracellular metabolic activity, i.e., by regulating the metabolism of nutrients, which can delay cell aging and death. In this review, we explored the relationship between autophagy and chondrocyte metabolism and provided new ideas for the prevention and treatment of OA.


Assuntos
Autofagia/fisiologia , Condrócitos/metabolismo , Osteoartrite/metabolismo , Proteínas/metabolismo , Morte Celular , Senescência Celular , Metabolismo Energético , Humanos , Metabolismo dos Lipídeos , Biossíntese de Proteínas
18.
Life Sci ; 231: 116459, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31075234

RESUMO

AIM: Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent types of cancer worldwide with unfavorable patient outcomes and relatively low survival rates. Long non-coding RNAs (lncRNAs) have been demonstrated to participate in the progression of HNSCC. The present study aimed to investigate the functional mechanism of lncRNA LINC00460 in HNSCC by mediating microRNA-206 (miR-206)/stanniocalcin-2 (STC2) axis. METHODS: The interactions among miR-206, LINC00460 and STC2 were identified, and the expression of LINC00460, miR-206 and STC2 in tissues and cells was determined. Gain- and loss-of function experiments were conducted to analyze effects of LINC00460, miR-206 and STC2 on the expression of apoptosis-related proteins, autophagy-related proteins, and the extents of AKT, ERK phosphorylation. Cell cycle distribution, apoptosis and the production of autophagosomes after transfection were evaluated to further explore the role of LINC00460/miR-206/STC2 axis in HNSCC. RESULTS: LINC00460 and STC2 were highly expressed while miR-206 was poorly expressed in HNSCC. Besides, miR-206 was found to bind to both LINC00460 and STC2. After the transfection of HNSCC cells with miR-206 mimic or si-LINC00460, the expression of STC2, AKT, ERK, as well as the extent of AKT, ERK phosphorylation all decreased, which facilitated the apoptosis and autophagy of HNSCC cells. CONCLUSION: Collectively, the apoptosis and autophagy of HNSCC can be facilitated by downregulating LINC00460, which highlights a novel target in the treatment of HNSCC.


Assuntos
Glicoproteínas/metabolismo , Neoplasias de Cabeça e Pescoço/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , MicroRNAs/genética , RNA Longo não Codificante/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Autofagia/fisiologia , Linfócitos T CD8-Positivos/metabolismo , Carcinogênese , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transformação Celular Neoplásica , Feminino , Glicoproteínas/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Ativação Transcricional , Regulação para Cima
19.
Invest Ophthalmol Vis Sci ; 60(6): 2380-2387, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31141609

RESUMO

Purpose: To determine whether tauopathies are associated with impaired autophagy and involved in the death of retinal ganglion cells (RGCs) of rats from an optic nerve crush (ONC). Methods: Short interfering RNA (siRNA) of the tau gene (si-Tau) or nontargeting siRNA (si-NC) was injected intravitreally 48 hours prior to ONC. The effects of silencing the tau gene on neuroprotection were determined by the number of Tuj-1-stained RGCs on days 7 and 14 after the ONC. The changes in the expressions of phosphorylated tau, P62, and LC3B were determined by immunoblots and immunohistochemistry on day 7. Results: Autophagy was impaired in the retina on day 7 after the ONC as the P62 level increased by 3.1-fold from the sham control level with a reduction in the ratio LC3B2/LC3B1. There was a 2.1-fold increase of phosphorylated tau (ser 396) in the retina, and si-Tau depressed the increase by 1.3-fold (n = 3 each). The expressions of tau and P62 were well colocalized. They were observed in the somas of RGCs and retinal nerve fibers (RNFs), and these expressions were increased after the ONC. Pretreatment by si-Tau showed significant protection in the number of RGCs after the ONC. Specifically, the density of RGCs was 540 ± 74.5 cells/mm2 on day 14 in the si-NC group, while the level was maintained at 1321 ± 192 cells/mm2 in the si-Tau group (n = 4 each). Conclusions: Silencing the tau gene is neuroprotective, and tauopathies may be involved in the death of RGCs after ONC. Impaired autophagy may be involved in ONC-induced tauopathies.


Assuntos
Autofagia/fisiologia , Compressão Nervosa , Neuroproteção/fisiologia , Traumatismos do Nervo Óptico/fisiopatologia , Células Ganglionares da Retina/fisiologia , Proteínas tau/fisiologia , Animais , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Inativação Gênica , Masculino , Ratos , Proteínas tau/metabolismo
20.
Nat Commun ; 10(1): 1566, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30952952

RESUMO

The class 3 phosphoinositide 3-kinase (PI3K) is required for lysosomal degradation by autophagy and vesicular trafficking, assuring nutrient availability. Mitochondrial lipid catabolism is another energy source. Autophagy and mitochondrial metabolism are transcriptionally controlled by nutrient sensing nuclear receptors. However, the class 3 PI3K contribution to this regulation is unknown. We show that liver-specific inactivation of Vps15, the essential regulatory subunit of the class 3 PI3K, elicits mitochondrial depletion and failure to oxidize fatty acids. Mechanistically, transcriptional activity of Peroxisome Proliferator Activated Receptor alpha (PPARα), a nuclear receptor orchestrating lipid catabolism, is blunted in Vps15-deficient livers. We find PPARα repressors Histone Deacetylase 3 (Hdac3) and Nuclear receptor co-repressor 1 (NCoR1) accumulated in Vps15-deficient livers due to defective autophagy. Activation of PPARα or inhibition of Hdac3 restored mitochondrial biogenesis and lipid oxidation in Vps15-deficient hepatocytes. These findings reveal roles for the class 3 PI3K and autophagy in transcriptional coordination of mitochondrial metabolism.


Assuntos
Autofagia/fisiologia , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , PPAR alfa/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Fenofibrato/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histona Desacetilases/fisiologia , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 1 de Receptor Nuclear/fisiologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteína VPS15 de Distribuição Vacuolar/genética , Proteína VPS15 de Distribuição Vacuolar/metabolismo , Proteína VPS15 de Distribuição Vacuolar/fisiologia
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