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1.
Biochemistry (Mosc) ; 84(4): 441-451, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228936

RESUMO

Prions are proteins that can exist under the same conditions in two or more conformations, at least one of them is infectious. Usually, acquisition of infectious prion conformation is associated with the formation of amyloids - protein aggregates with a characteristic spatial structure. About 10 prions have been identified in the yeast Saccharomyces cerevisiae. The Gln3 protein, which is one of the key regulators of nitrogen metabolism in S. cerevisiae, contains an amyloidogenic region manifesting prion-like properties. The prion properties of the full-length Gln3 have not been studied. We have found that the amyloidogenic region of Gln3 acts as a template and initiates aggregation of the full-length Gln3 in the presence of the [PIN+] prion when Gln3 is overexpressed. Full-length Gln3 in its aggregated form manifests prion-like properties, including infectivity and dependence on the anti-prion agents; however, unlike other known yeast prions, prion-like state of Gln3 is observed only upon the protein overproduction. Here, we suggest the term "conditional prions" for proteins, whose prion state is maintained exclusively under non-physiological conditions.


Assuntos
Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Metalotioneína/genética , Microscopia Confocal , Agregados Proteicos/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Fatores de Transcrição/genética
2.
J Mol Histol ; 50(3): 273-283, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31049797

RESUMO

Recent studies have demonstrated that IGF-1 modulates the pluripotent differentiation of dental pulp stem cells (DPSCs). Although mTOR pathway activation has been showed as responsible for IGF-1 induced pluripotent differentiation, the mechanism that the IGF-1-mTOR pathway induces the neural differentiation of DPSCs is still unclear. In our research, we have demonstrated that 0-10 ng/mL IGF-1 had no obvious effect on the proliferation of DPSCs, but IGF-1 nonetheless enhances the neural differentiation of DPSCs in a dose-dependent manner. Simultaneously, we found that phosphorylated mTOR was up-regulated, which indicated the involvement of mTOR in the process. Rapamycin, an inhibitor of mTOR activity, can reverse the effect of DPSCs stimulated by IGF-1. Next, we studied the role of mTORC1 and mTORC2, two known mTOR complexes, in the neural differentiation of DPSCs. We found that inhibition of mTORC1 can severely restricts the neural differentiation of DPSCs. However, inhibition of mTORC2 has the opposite effect. This latter effect disappears when both rictor and mTOR are inhibited, showing that the mTORC2 effect is mTORC1 dependent. This study has expanded the role of mTOR in DPSCs neural differentiation regulated by IGF-1.


Assuntos
Diferenciação Celular/genética , Polpa Dentária/enzimologia , Fator de Crescimento Insulin-Like I/genética , Células-Tronco/efeitos dos fármacos , Adolescente , Adulto , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/crescimento & desenvolvimento , Feminino , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Adulto Jovem
3.
Biomed Res Int ; 2019: 1854323, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30956975

RESUMO

To evaluate the effect of Tanreqing injection on the pharmacokinetics of sirolimus in rats, a high performance liquid chromatography tandem mass spectrometry method was developed for sirolimus assay in whole blood. Calibration curve of sirolimus was acquired over a concentration ranging from 2.5 to 100 ng/mL with r2= 0.9955. The matrix effects and extraction recoveries of sirolimus ranged from 144% to 152% and from 80% to 96%, respectively. The inter- and intraday relative standard deviations were both <10%. The stability investigation showed that the blood samples were stable for 30-day-storage at -20°C, for 8 h storage at room temperature, for 24 h storage in the auto-sampler at 4°C, and for three freeze-thaw cycle process. The pharmacokinetic results demonstrated that the C max, AUC, and AUMC of sirolimus in rats (7.5 mg/kg, i.g.) were increased after beincoadministration with Tanreqing Injection at 2.5, 5.0, and 7.5 mL/kg (i.v.), respectively, or at 5 min, 2 h, and 4 h (5.0 mL/kg, i.v.) after SRL dosing, respectively. For the first time, the results proved the herb-drug interaction between Tanreqing Injection and sirolimus and accordingly suggested avoiding concurrent reception of those two drugs for patients.


Assuntos
Medicamentos de Ervas Chinesas , Interações Ervas-Drogas , Sirolimo , Animais , Medicamentos de Ervas Chinesas/farmacocinética , Medicamentos de Ervas Chinesas/farmacologia , Humanos , Ratos , Ratos Sprague-Dawley , Sirolimo/farmacocinética , Sirolimo/farmacologia
4.
Nat Commun ; 10(1): 1685, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30976008

RESUMO

Neonatal sepsis is characterized by hyperinflammation causing enhanced morbidity and mortality compared to adults. This suggests differences in the response towards invading threats. Here we investigate activated cord blood macrophages (CBMΦ) in comparison to adult macrophages (PBMΦ), indicating incomplete interferon gamma (IFN-γ) and interleukin 10 (IL-10)-induced activation of CBMΦ. CBMΦ show reduced expression of phagocytosis receptors and cytokine expression in addition to altered energy metabolism. In particular, IFN-γ as well as IL-10-activated CBMΦ completely fail to increase glycolysis and furthermore show reduced activation of the mTOR pathway, which is important for survival in sepsis. MTOR inhibition by rapamycin equalizes cytokine production in CBMΦ and PBMΦ. Finally, incubation of PBMΦ with cord blood serum or S100A8/A9, which is highly expressed in neonates, suppresses mTOR activation, prevents glycolysis and the expression of an PBMΦ phenotype. Thus, a metabolic alteration is apparent in CBMΦ, which might be dependent on S100A8/A9 expression.


Assuntos
Citotoxicidade Imunológica , Metabolismo Energético/imunologia , Macrófagos/metabolismo , Adulto , Fatores Etários , Calgranulina A/imunologia , Calgranulina A/metabolismo , Calgranulina B/imunologia , Calgranulina B/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Sangue Fetal/citologia , Glicólise/imunologia , Voluntários Saudáveis , Humanos , Recém-Nascido , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Macrófagos/imunologia , Cultura Primária de Células , Sepse/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/imunologia , Serina-Treonina Quinases TOR/metabolismo
5.
Ren Fail ; 41(1): 267-277, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30982374

RESUMO

OBJECTIVE: To compare the anti-peritoneal fibrotic effects between a mammalian target of rapamycin complex 1-specific blocker and a phosphatidyl-inositol 3-kinase/mammalian target of rapamycin dual-blocker. METHODS: A total of 40 male Sprague-Dawley rats were randomly divided into five groups with eight animals per group. The normal group (N group) did not receive any intervention. The normal saline group (NS group) received an intraperitoneal injection of normal saline at 1 ml/100 g daily. The model group (3 W group), rapamycin (RAPA) group and BEZ235 (PI3K/mTOR dual-blocker) group all received an intraperitoneal injection of 0.1% chlorhexidine gluconate at 1 ml/100g daily. And the RAPA and BEZ235 groups also received a 0.5 mg/d RAPA or 2.5 mg/d BEZ235 gavage every day, respectively. Rats in each group were sacrificed after 3 weeks. RESULTS: Immunohistochemistry, real-time PCR and western blotting analysis of fibrosis-related indicators (FN, Col 1, and α-SMA) confirmed that RAPA and BEZ235 significantly inhibited peritoneal fibrosis and that these two drugs had similar effects. The p-Akt, p-mTOR, p-p70S6K expression levels were significantly up-regulated in the 3 W group compared to the NS group, confirming that the mTOR pathway was significantly activated during peritoneal fibrosis. RAPA significantly inhibited the phosphorylation of mTOR and p70S6K but did not have significant effects on p-Akt upstream of mTOR. BEZ235 had significant inhibitory effects on all signaling molecules (p-Akt, p-mTOR, and p-p70S6K) in the mTOR pathway. CONCLUSION: RAPA did not up-regulate p-Akt in a negative feedback fashion. Both drugs effectively inhibited peritoneal fibrosis.


Assuntos
Imidazóis/farmacologia , Falência Renal Crônica/terapia , Diálise Peritoneal/efeitos adversos , Fibrose Peritoneal/prevenção & controle , Quinolinas/farmacologia , Sirolimo/farmacologia , Animais , Clorexidina/administração & dosagem , Clorexidina/análogos & derivados , Clorexidina/toxicidade , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Imidazóis/uso terapêutico , Injeções Intraperitoneais , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fibrose Peritoneal/induzido quimicamente , Fibrose Peritoneal/patologia , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinolinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
6.
Nat Commun ; 10(1): 1897, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015486

RESUMO

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 binds to its target loci, where it opens chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closes these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal is constitutively activated, RUNX3 inhibits cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Cromatina/química , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Animais , Butadienos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Subunidade alfa 3 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imidazóis/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nitrilos/farmacologia , Piperazinas/farmacologia , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
7.
J Cancer Res Ther ; 15(2): 398-403, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30964118

RESUMO

Objective: The objective of this study was to investigate the molecular mechanisms involved in rapamycin-induced inhibition of tumor growth. Materials and Methods: Murine S180 sarcoma cells were subcutaneously injected into mice, and the tumor-bearing mice were randomly divided into three groups (vehicle control, 2 mg/kg rapamycin, and 4 mg/kg rapamycin). The effect of rapamycin on tumor growth was determined by measuring tumor volume. Mammalian target of rapamycin (mTOR), Beclin1, ULK1, LC3, Notch1, CD133, and CD90 expressions was confirmed using confocal microscopy and Western blotting. Results: The tumor growth inhibition rates induced by high-dose and low-dose rapamycin were 48.8% and 30.1%, respectively. Beclin1 and ULK1 expressions and the LC3-II/LC3-I ratio in tumor tissues were altered by rapamycin, whereas mTOR, Notch1, CD133, and CD90 expressions were significantly inhibited by rapamycin in immunofluorescence assays. Western blotting also showed similar results. Conclusion: Tumor growth delay induced by rapamycin may be associated with the suppression of the cancer stem cell phenotype (Notch1, CD133, and CD90) and promotion of autophagy (mTOR, Beclin1, ULK1, and LC3-II/LC3-I ratio) in the murine S180 sarcoma model.


Assuntos
Autofagia/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Sarcoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Animais , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Imunofenotipagem , Masculino , Camundongos
8.
Oncol Rep ; 41(5): 2833-2843, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864729

RESUMO

Programmed death­ligand 1 (PD­L1), an immune co­stimulatory molecule, is expressed on various cancer cells and the surface of immune cells. Its overexpression on tumor cells suppresses the immune response to promote tumor cell immune escape. The present study demonstrated that PD­L1 was critical in head and neck squamous cell carcinoma (HNSCC) carcinogenesis. Immunohistochemical analysis of HNSCC tissue microarrays revealed that PD­L1 was overexpressed in tumor tissue, and its expression increased as tumor malignancy progressed (from grade I to IV). Subsequently, the expression of PD­L1 was knocked down or overexpressed in the HNSCC cell lines Cal­27 and Fadu. It was demonstrated that PD­L1 significantly induced HNSCC cell proliferation and colony forming ability. Cell proliferation was also promoted in Cal­27 cell xenograft BALB/c nude mice. In addition, it was determined by western blotting that the PD­L1­mediated increase in HNSCC cell proliferation may have been associated with the activation of mammalian target of rapamycin (mTOR) signaling pathway. Furthermore, mTOR inhibitor (rapamycin) prevented the increase in proliferation. Based on these results, it was concluded that PD­L1 promoted cell proliferation of HNSCC cells through mTOR signaling, and blocking PD­L1 may be conducive in HNSCC therapy.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Serina-Treonina Quinases TOR/metabolismo , Adulto , Idoso , Animais , Apoptose/efeitos dos fármacos , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Clin Interv Aging ; 14: 525-534, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30880933

RESUMO

Background: Ischemia-reperfusion (I/R) injury is the most common cause of acute kidney injury (AKI). Numerous therapeutic approaches for I/R injury have been studied, including autophagy, particularly in animal models of renal I/R injury derived from young or adult animals. However, the precise role of autophagy in renal ischemia-reperfusion in the aged animal model remains unclear. The purpose of this study was to demonstrate whether autophagy has similar effects on renal I/R injury in young and aged rats. Materials and methods: All rats were divided into two age groups (3 months and 24 months) with each group being further divided into four subgroups (sham, I/R, I/R+Rap (rapamycin, an activator of autophagy), I/R+3-MA (3-methyladenine, an inhibitor of autophagy)). The I/R+Rap and I/R+3-MA groups were intraperitoneally injected with rapamycin and 3-MA prior to ischemia. We then measured serum levels of urea nitrogen, creatinine and assessed damage in the renal tissue. Immunohistochemistry was used to assess LC3-II and caspase-3, and Western blotting was used to evaluate the autophagy-related proteins LC3-II, Beclin-1 and P62. Apoptosis and autophagosomes were evaluated by TUNEL and transmission electron microscopy, respectively. Results: Autophagy was activated in both young and aged rats by I/R and enhanced by rapamycin, although the level of autophagy was lower in the aged groups. In young rats, the activation of autophagy markedly improved renal function, reduced apoptosis in the renal tubular epithelial cells and the injury score in the renal tissue, thereby exerting protective effects on renal I/R injury. However, this level of protection was not present in aged rats. Conclusion: Our data indicated that the activation of autophagy was ineffective in aged rat kidneys. These discoveries may have major implications in that severe apoptosis in aged kidneys might be refractory to antiapoptotic effect induced by the activation of autophagy.


Assuntos
Lesão Renal Aguda/fisiopatologia , Adenina/análogos & derivados , Autofagia/efeitos dos fármacos , Traumatismo por Reperfusão/fisiopatologia , Sirolimo/farmacologia , Lesão Renal Aguda/etiologia , Lesão Renal Aguda/patologia , Adenina/farmacologia , Fatores Etários , Animais , Apoptose , Autofagossomos , Proteína Beclina-1/metabolismo , Nitrogênio da Ureia Sanguínea , Caspase 3/metabolismo , Creatinina/sangue , Modelos Animais de Doenças , Rim/fisiopatologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/complicações
10.
Acta Biochim Biophys Sin (Shanghai) ; 51(4): 393-401, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30908573

RESUMO

MicroRNAs (miRNAs) are short (20-23 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. In recent years, deep sequencing of the transcription is being increasingly utilized with the promise of higher sensitivity for the identification of differential expression patterns as well as the opportunity to discover new transcripts, including new alternative isoforms and miRNAs. In this study, miRNAs from A549 cells treated with/without rapamycin or starvation were subject to genome-wide deep sequencing. A total of 1534 miRNAs were detected from the rapamycin- and starvation-treated A549 cells. Among them, 31 miRNAs were consistently upregulated and 131 miRNAs were downregulated in the treated cells when compared with the untreated cells. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis of the predicted target genes of the most significantly differentially expressed miRNAs revealed that the autophagy-related miRNAs are involved in cancer pathway. Taken together, our findings indicate that the underlying mechanism responsible for autophagy is associated with dysregulation of miRNAs in rapamycin- or starvation-induced A549 cells.


Assuntos
Autofagia/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Sirolimo/farmacologia , Células A549 , Autofagia/genética , Perfilação da Expressão Gênica , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunossupressores/farmacologia
12.
J Immunol Res ; 2019: 8575407, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30915372

RESUMO

Objective: To investigate the potential therapeutic effect in a rheumatoid arthritis model of stable human CD8+ regulatory T cells (hCD8+Tregs) induced by TGF-ß1 and rapamycin (RAPA) in vitro. Methods: Human CD8+T cells were isolated from human peripheral blood mononuclear cells and induced/expanded with TGF-ß1 and RAPA along with anti-CD3/28 beads and IL-2 in vitro and harvested as hCD8+Tregs. The phenotypes, suppressive characteristics, and stability of the hCD8+Tregs in an inflammatory microenvironment were examined in vitro. Human CD8+Tregs were transfused into an acollagen-induced arthritis (CIA) mouse model, and their therapeutic effects and related mechanisms were investigated. Results: Human CD8+Tregs induced by TGF-ß1/RAPA showed high expression of Foxp3 and CD103, exhibited vigorous suppression ability, and were stable in inflammatory microenvironments. In CIA mice, the clinical scores, levels of anti-collagen IgG antibody, and cartilage destruction were significantly reduced after adoptive transfusion with hCD8+Tregs. Moreover, hCD8+Treg treatment significantly reduced the number of Th17 cells, increased the number of CD4+IFN-γ +T cells, and produced self CD4+Foxp3+Tregs in vivo. In an in vitro cell coculture assay, hCD8+Tregs significantly inhibited mouse CD4+ effector T cell proliferation, induced mouse CD4+Foxp3+Treg and CD4+IFN-γ +Th1 cell production, reduced Th17 cell development, and downregulated CD80/86 expression on mature DCs (mDCs). Conclusion: TGF-ß1/RAPA can induce hCD8+Tregs with stable suppressive characteristics, which could significantly alleviate the severity of CIA based on their stable suppressive ability in an inflammatory microenvironment and further influence the function of other downstream cell subtypes. Human CD8+Tregs might be a therapeutic strategy for rheumatoid arthritis.


Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Imunoterapia/métodos , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Antígenos CD/metabolismo , Artrite Experimental/terapia , Artrite Reumatoide/terapia , Antígenos CD8/metabolismo , Células Cultivadas , Colágeno/imunologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunoglobulina G/metabolismo , Cadeias alfa de Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Tolerância a Antígenos Próprios , Sirolimo/farmacologia , Linfócitos T Reguladores/transplante , Fator de Crescimento Transformador beta/metabolismo
13.
Pharm Res ; 36(5): 70, 2019 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-30888509

RESUMO

PURPOSE: To prepare an oligo(lactic acid)8-rapamycin prodrug (o(LA)8-RAP)-loaded poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA) micelle for injection and characterize its compatibility and performance versus a RAP-loaded PEG-b-PLA micelle for injection in vitro and in vivo. METHODS: Monodisperse o(LA)8 was coupled on RAP at the C-40 via DCC/DMAP chemistry, and conversion of o(LA)8-RAP prodrug into RAP was characterized in vitro. Physicochemical properties of o(LA)8-RAP- and RAP-loaded PEG-b-PLA micelles and their antitumor efficacies in a syngeneic 4 T1 breast tumor model were compared. RESULTS: Synthesis of o(LA)8-RAP prodrug was confirmed by 1H NMR and mass spectroscopy. The o(LA)8-RAP prodrug underwent conversion in PBS and rat plasma by backbiting and esterase-mediated cleavage, respectively. O(LA)8-RAP-loaded PEG-b-PLA micelles increased water solubility of RAP equivalent to 3.3 mg/ml with no signs of precipitation. Further, o(LA)8-RAP was released more slowly than RAP from PEG-b-PLA micelles. With added physical stability, o(LA)8-RAP-loaded PEG-b-PLA micelles significantly inhibited tumor growth relative to RAP-loaded PEG-b-PLA micelles in 4 T1 breast tumor-bearing mice without signs of acute toxicity. CONCLUSIONS: An o(LA)8-RAP-loaded PEG-b-PLA micelle for injection is more stable than a RAP-loaded PEG-b-PLA micelle for injection, and o(LA)8-RAP converts into RAP rapidly in rat plasma (t1/2 = 1 h), resulting in antitumor efficacy in a syngeneic 4 T1 breast tumor model.


Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos/química , Lactatos/química , Polietilenoglicóis/química , Pró-Fármacos/administração & dosagem , Sirolimo/administração & dosagem , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/toxicidade , Liberação Controlada de Fármacos , Feminino , Lactatos/toxicidade , Ácido Láctico/química , Camundongos , Micelas , Polietilenoglicóis/toxicidade , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Pró-Fármacos/toxicidade , Ratos , Transdução de Sinais , Sirolimo/química , Sirolimo/farmacologia , Sirolimo/toxicidade , Solubilidade , Serina-Treonina Quinases TOR/metabolismo
14.
Nat Commun ; 10(1): 651, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30783116

RESUMO

Ageing constitutes the most important risk factor for all major chronic ailments, including malignant, cardiovascular and neurodegenerative diseases. However, behavioural and pharmacological interventions with feasible potential to promote health upon ageing remain rare. Here we report the identification of the flavonoid 4,4'-dimethoxychalcone (DMC) as a natural compound with anti-ageing properties. External DMC administration extends the lifespan of yeast, worms and flies, decelerates senescence of human cell cultures, and protects mice from prolonged myocardial ischaemia. Concomitantly, DMC induces autophagy, which is essential for its cytoprotective effects from yeast to mice. This pro-autophagic response induces a conserved systemic change in metabolism, operates independently of TORC1 signalling and depends on specific GATA transcription factors. Notably, we identify DMC in the plant Angelica keiskei koidzumi, to which longevity- and health-promoting effects are ascribed in Asian traditional medicine. In summary, we have identified and mechanistically characterised the conserved longevity-promoting effects of a natural anti-ageing drug.


Assuntos
Envelhecimento/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Flavonoides/farmacologia , Longevidade/efeitos dos fármacos , Envelhecimento/fisiologia , Angelica/química , Animais , Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Transporte de Cátions/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Drosophila melanogaster/efeitos dos fármacos , Flavonoides/administração & dosagem , Fatores de Transcrição GATA/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Longevidade/fisiologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Medicina Tradicional do Leste Asiático , Camundongos , Camundongos Endogâmicos C57BL , Isquemia Miocárdica/tratamento farmacológico , Extratos Vegetais/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Sirolimo/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética
15.
Phytomedicine ; 57: 203-214, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30785016

RESUMO

BACKGROUND: Huangkui capsule (HKC), an anti-inflammatory Chinese modern patent medicine, has been now widely applied to the clinical therapy of diabetic nephropathy (DN). However, the overall therapeutic mechanisms in vivo are still unclear. Renal tubular epithelial-to-mesenchymal transition (EMT) is one of the major pathogenesis of renal interstitial fibrosis in DN. Recently, the physiological roles of NLRP3 inflammasome activation and toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signaling are closely linked to EMT. But, it remains elusive whether HKC regulates renal tubular EMT in vivo through targeting NLRP3 inflammasome activation and TLR4/NF-κB signaling in the kidneys. PURPOSE: This study thereby aimed to clarify the therapeutic effects of HKC on renal tubular EMT in DN and its underlying mechanisms in vivo, compared to rapamycin (RAP). METHODS: Thirty-two rats were randomly divided into 4 groups: the Sham group, the Vehicle group, the HKC group and the RAP group. The early DN rat models were induced by unilateral nephrectomy combined with intraperitoneal injection of streptozotocin, and administered with HKC suspension or RAP suspension or vehicle after modeling for 4 weeks. Changes in the incipient renal lesions-related parameters in urine and blood were analyzed, respectively. Renal interstitial tissues were isolated for histomorphometry, immunohistochemistry and Western blotting at sacrifice. RESULTS: For the early DN rat models, HKC at the suitable dose of 2 g/kg/day ameliorated the general condition and biochemical parameters partially including kidney weight (KW), urinary albumin (UAlb), serum creatinine (Scr) and serum albumin (Alb), attenuated renal tubular EMT significantly and inhibited the activation of NLRP3 inflammasome in the kidneys obviously, which was superior to RAP generally. In addition to these, HKC also suppressed TLR4/NF-κB signaling in the kidneys of the DN model rats accurately, which was different from RAP specifically. CONCLUSION: The results of this study further indicated that HKC, different from RAP, can alleviate renal tubular EMT in the DN model rats, likely by inhibiting NLRP3 inflammasome activation and TLR4/NF-κB signaling in the kidneys. Our findings thus provide the more accurate information in vivo about a clinical value of HKC, a traditional anti-inflammatory phytomedicine, in the treatment of the early DN patients.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Fibrose/tratamento farmacológico , Fibrose/patologia , Inflamassomos/antagonistas & inibidores , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , NF-kappa B/metabolismo , Nefrectomia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia
16.
Mol Pain ; 15: 1744806919830240, 2019 Jan-Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30717619

RESUMO

BACKGROUND: Pain is one of the most common and distressing symptoms suffered by patients with progression of cancer; however, the mechanisms responsible for hyperalgesia are not well understood. Since the midbrain periaqueductal gray is an important component of the descending inhibitory pathway controlling on central pain transmission, in this study, we examined the role for pro-inflammatory cytokines of the periaqueductal gray in regulating mechanical and thermal hyperalgesia evoked by bone cancer via phosphatidylinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signals. METHODS: Breast sarcocarcinoma Walker 256 cells were implanted into the tibia bone cavity of rats to induce mechanical and thermal hyperalgesia. Western blot analysis and ELISA were used to examine PI3K/protein kinase B (Akt)/mTOR and pro-inflammatory cytokine receptors and the levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α). RESULTS: Protein expression levels of p-PI3K/p-Akt/p-mTOR were amplified in the periaqueductal gray of bone cancer rats, and blocking PI3K-mTOR pathways in the periaqueductal gray attenuated hyperalgesia responses. In addition, IL-1ß, IL-6, and TNF-α were elevated in the periaqueductal gray of bone cancer rats, and expression of their respective receptors (namely, IL-1R, IL-6R, and tumor necrosis factor receptor (TNFR) subtype TNFR1) was upregulated. Inhibition of IL-1R, IL-6R, and TNFR1 alleviated mechanical and thermal hyperalgesia in bone cancer rats, accompanied with downregulated PI3K-mTOR. CONCLUSIONS: Our data suggest that upregulation of pro-inflammatory cytokine signal in the periaqueductal gray of cancer rats amplifies PI3K-mTOR signal in this brain region and alters the descending pathways in regulating pain transmission, and this thereby contributes to the development of bone cancer-induced pain.


Assuntos
Dor do Câncer/complicações , Citocinas/metabolismo , Encefalite/etiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Substância Cinzenta Periaquedutal/metabolismo , Transdução de Sinais/fisiologia , Animais , Neoplasias Ósseas/complicações , Neoplasias Ósseas/secundário , Dor do Câncer/etiologia , Carcinoma 256 de Walker/patologia , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Hiperalgesia/etiologia , Imunossupressores/farmacologia , Masculino , Morfolinas/farmacologia , Medição da Dor , Substância Cinzenta Periaquedutal/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Gastroenterology ; 156(6): 1775-1787, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30710527

RESUMO

BACKGROUND & AIMS: Crohn's disease (CD) is characterized by an imbalance of effector and regulatory T cells in the intestinal mucosa. The efficacy of anti-adhesion therapies led us to investigate whether impaired trafficking of T-regulatory (Treg) cells contributes to the pathogenesis of CD. We also investigated whether proper function could be restored to Treg cells by ex vivo expansion in the presence of factors that activate their regulatory activities. METHODS: We measured levels of the integrin α4ß7 on Treg cells isolated from peripheral blood or lamina propria of patients with CD and healthy individuals (controls). Treg cells were expanded ex vivo and incubated with rapamycin with or without agonists of the retinoic acid receptor-α (RARA), and their gene expression profiles were analyzed. We also studied the cells in cytokine challenge, suppression, and flow chamber assays and in SCID mice with human intestinal xenografts. RESULTS: We found that Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. The pathway that regulates the expression of integrin subunit α is induced by retinoic acid (RA). Treg cells from patients with CD incubated with rapamycin and an agonist of RARA (RAR568) expressed high levels of integrin α4ß7, as well as CD62L and FOXP3, compared with cells incubated with rapamycin or rapamycin and all-trans retinoic acid. These Treg cells had increased suppressive activities in assays and migrated under conditions of shear flow; they did not produce inflammatory cytokines, and RAR568 had no effect on cell stability or lineage commitment. Fluorescently labeled Treg cells incubated with RAR568 were significantly more likely to traffic to intestinal xenografts than Treg cells expanded in control medium. CONCLUSIONS: Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. Incubation of patients' ex vivo expanded Treg cells with rapamycin and an RARA agonist induced expression of α4ß7 and had suppressive and migratory activities in culture and in intestinal xenografts in mice. These cells might be developed for treatment of CD. ClinicalTrials.gov, Number: NCT03185000.


Assuntos
Doença de Crohn/imunologia , Integrinas/metabolismo , Receptor alfa de Ácido Retinoico/agonistas , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Adulto , Animais , Antineoplásicos/farmacologia , Estudos de Casos e Controles , Técnicas de Cultura de Células , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Imunossupressores/farmacologia , Integrinas/genética , Mucosa Intestinal/imunologia , Mucosa Intestinal/transplante , Selectina L/metabolismo , Ativação Linfocitária , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Compostos Orgânicos/farmacologia , Sirolimo/farmacologia , Linfócitos T Reguladores/imunologia , Transcriptoma/efeitos dos fármacos , Tretinoína/farmacologia
18.
Carbohydr Polym ; 210: 364-371, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30732772

RESUMO

Many pathogenic bacteria can invade phagocytic and non-phagocytic cells and colonize inside, which protects them from the attack by host immune system and antibiotics. A novel amphiphilic molecule was synthesized through conjugation of streptomycin and decylamine to hyaluronan. Rapamycin was encapsulted by the spontaneous self-assembly of hyaluronan-based amphiphilic molecules. The newly formed micelles not only facilitated the entry of drugs into host cells in part via CD44 phagocytic receptor, released streptomycin in the acidic compartment, but also could activate autophagy. The micelles elicited an efficient killing capacity against intracellular bacteria through the promotion of streptomycin uptake and rapamycin-initiated activation of autophagy. This strategy might highlight an acid-sensitive hyaluronan-based drug delivery system for effective treatment of intracellular infections.


Assuntos
Ácido Hialurônico/química , Espaço Intracelular/microbiologia , Micelas , Sirolimo/química , Sirolimo/farmacologia , Estreptomicina/química , Estreptomicina/farmacologia , Animais , Autofagia/efeitos dos fármacos , Cápsulas , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Sinergismo Farmacológico , Interações Hidrofóbicas e Hidrofílicas , Espaço Intracelular/efeitos dos fármacos , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Células RAW 264.7 , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/fisiologia
19.
Nat Commun ; 10(1): 714, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755615

RESUMO

Glucose homeostasis is partly controlled by the energy sensor mechanistic target of rapamycin (mTOR) in the muscle and liver. However, whether mTOR in the small intestine affects glucose homeostasis in vivo remains unknown. Here, we first report that delivery of rapamycin or an adenovirus encoding the dominant negative acting mTOR-mutated protein into the upper small intestine is sufficient to inhibit small intestinal mTOR signaling and lower glucose production in rodents with high fat diet-induced insulin resistance. Second, we found that molecular activation of small intestinal mTOR blunts the glucose-lowering effect of the oral anti-diabetic agent metformin, while inhibiting small intestinal mTOR alone lowers plasma glucose levels by inhibiting glucose production in rodents with diabetes as well. Thus, these findings illustrate that inhibiting upper small intestinal mTOR is sufficient and necessary to lower glucose production and enhance glucose homeostasis, and thereby unveil a previously unappreciated glucose-lowering effect of small intestinal mTOR.


Assuntos
Glicemia/metabolismo , Glucose/biossíntese , Intestino Delgado/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Adenoviridae/genética , Animais , Dieta Hiperlipídica , Homeostase , Resistência à Insulina , Intestino Delgado/efeitos dos fármacos , Masculino , Metformina/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
20.
PLoS Genet ; 15(2): e1007957, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30742617

RESUMO

Mucormycosis-an emergent, deadly fungal infection-is difficult to treat, in part because the causative species demonstrate broad clinical antifungal resistance. However, the mechanisms underlying drug resistance in these infections remain poorly understood. Our previous work demonstrated that one major agent of mucormycosis, Mucor circinelloides, can develop resistance to the antifungal agents FK506 and rapamycin through a novel, transient RNA interference-dependent mechanism known as epimutation. Epimutations silence the drug target gene and are selected by drug exposure; the target gene is re-expressed and sensitivity is restored following passage without drug. This silencing process involves generation of small RNA (sRNA) against the target gene via core RNAi pathway proteins. To further elucidate the role of epimutation in the broad antifungal resistance of Mucor, epimutants were isolated that confer resistance to another antifungal agent, 5-fluoroorotic acid (5-FOA). We identified epimutant strains that exhibit resistance to 5-FOA without mutations in PyrF or PyrG, enzymes which convert 5-FOA into the active toxic form. Using sRNA hybridization as well as sRNA library analysis, we demonstrate that these epimutants harbor sRNA against either pyrF or pyrG, and further show that this sRNA is lost after reversion to drug sensitivity. We conclude that epimutation is a mechanism capable of targeting multiple genes, enabling Mucor to develop resistance to a variety of antifungal agents. Elucidation of the role of RNAi in epimutation affords a fuller understanding of mucormycosis. Furthermore, it improves our understanding of fungal pathogenesis and adaptation to stresses, including the evolution of drug resistance.


Assuntos
Farmacorresistência Fúngica Múltipla/genética , Mucor/efeitos dos fármacos , Mucor/patogenicidade , Antifúngicos/farmacologia , Epigênese Genética , Genes Fúngicos , Humanos , Mucor/genética , Mucormicose/tratamento farmacológico , Mucormicose/microbiologia , Mutação , Orotato Fosforribosiltransferase/genética , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Orotidina-5'-Fosfato Descarboxilase/genética , Interferência de RNA , RNA Fúngico/genética , Sirolimo/farmacologia , Tacrolimo/farmacologia
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