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1.
Gut ; 69(1): 146-157, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30723104

RESUMO

OBJECTIVE: We explored the hypothesis that TGR5, the bile acid (BA) G-protein-coupled receptor highly expressed in biliary epithelial cells, protects the liver against BA overload through the regulation of biliary epithelium permeability. DESIGN: Experiments were performed under basal and TGR5 agonist treatment. In vitro transepithelial electric resistance (TER) and FITC-dextran diffusion were measured in different cell lines. In vivo FITC-dextran was injected in the gallbladder (GB) lumen and traced in plasma. Tight junction proteins and TGR5-induced signalling were investigated in vitro and in vivo (wild-type [WT] and TGR5-KO livers and GB). WT and TGR5-KO mice were submitted to bile duct ligation or alpha-naphtylisothiocyanate intoxication under vehicle or TGR5 agonist treatment, and liver injury was studied. RESULTS: In vitro TGR5 stimulation increased TER and reduced paracellular permeability for dextran. In vivo dextran diffusion after GB injection was increased in TGR5-knock-out (KO) as compared with WT mice and decreased on TGR5 stimulation. In TGR5-KO bile ducts and GB, junctional adhesion molecule A (JAM-A) was hypophosphorylated and selectively downregulated among TJP analysed. TGR5 stimulation induced JAM-A phosphorylation and stabilisation both in vitro and in vivo, associated with protein kinase C-ζ activation. TGR5 agonist-induced TER increase as well as JAM-A protein stabilisation was dependent on JAM-A Ser285 phosphorylation. TGR5 agonist-treated mice were protected from cholestasis-induced liver injury, and this protection was significantly impaired in JAM-A-KO mice. CONCLUSION: The BA receptor TGR5 regulates biliary epithelial barrier function in vitro and in vivo through an impact on JAM-A expression and phosphorylation, thereby protecting liver parenchyma against bile leakage.


Assuntos
Sistema Biliar/fisiopatologia , Colestase Intra-Hepática/prevenção & controle , Receptores Acoplados a Proteínas-G/fisiologia , Animais , Bile/metabolismo , Ácidos e Sais Biliares/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Colestase Intra-Hepática/metabolismo , Impedância Elétrica , Epitélio/fisiopatologia , Ácidos Isonipecóticos/farmacologia , Ácidos Isonipecóticos/uso terapêutico , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oximas/farmacologia , Oximas/uso terapêutico , Permeabilidade , Fosforilação/fisiologia , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas-G/agonistas , Transdução de Sinais/fisiologia , Proteínas de Junções Íntimas/metabolismo
2.
Gut ; 69(1): 133-145, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31409605

RESUMO

OBJECTIVE: The Fragile X mental retardation (FMR) syndrome is a frequently inherited intellectual disability caused by decreased or absent expression of the FMR protein (FMRP). Lack of FMRP is associated with neuronal degradation and cognitive dysfunction but its role outside the central nervous system is insufficiently studied. Here, we identify a role of FMRP in liver disease. DESIGN: Mice lacking Fmr1 gene expression were used to study the role of FMRP during tumour necrosis factor (TNF)-induced liver damage in disease model systems. Liver damage and mechanistic studies were performed using real-time PCR, Western Blot, staining of tissue sections and clinical chemistry. RESULTS: Fmr1null mice exhibited increased liver damage during virus-mediated hepatitis following infection with the lymphocytic choriomeningitis virus. Exposure to TNF resulted in severe liver damage due to increased hepatocyte cell death. Consistently, we found increased caspase-8 and caspase-3 activation following TNF stimulation. Furthermore, we demonstrate FMRP to be critically important for regulating key molecules in TNF receptor 1 (TNFR1)-dependent apoptosis and necroptosis including CYLD, c-FLIPS and JNK, which contribute to prolonged RIPK1 expression. Accordingly, the RIPK1 inhibitor Necrostatin-1s could reduce liver cell death and alleviate liver damage in Fmr1null mice following TNF exposure. Consistently, FMRP-deficient mice developed increased pathology during acute cholestasis following bile duct ligation, which coincided with increased hepatic expression of RIPK1, RIPK3 and phosphorylation of MLKL. CONCLUSIONS: We show that FMRP plays a central role in the inhibition of TNF-mediated cell death during infection and liver disease.


Assuntos
Proteína do X Frágil de Retardo Mental/fisiologia , Hepatite Viral Animal/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/patologia , Linfócitos T CD8-Positivos/imunologia , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Morte Celular/fisiologia , Células Cultivadas , Colestase/imunologia , Colestase/metabolismo , Colestase/patologia , Proteína do X Frágil de Retardo Mental/metabolismo , Hepatite Viral Animal/patologia , Hepatite Viral Animal/prevenção & controle , Hepatócitos/patologia , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Vírus da Coriomeningite Linfocítica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia
3.
Gut ; 69(1): 177-186, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30954949

RESUMO

OBJECTIVE: Increased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC). DESIGN: We investigated the functional contribution of fatty acid synthase (Fasn)-mediated de novo FA synthesis in a murine HCC model induced by loss of Pten and overexpression of c-Met (sgPten/c-Met) using liver-specific Fasn knockout mice. Expression arrays and lipidomic analysis were performed to characterise the global gene expression and lipid profiles, respectively, of sgPten/c-Met HCC from wild-type and Fasn knockout mice. Human HCC cell lines were used for in vitro studies. RESULTS: Ablation of Fasn significantly delayed sgPten/c-Met-driven hepatocarcinogenesis in mice. However, eventually, HCC emerged in Fasn knockout mice. Comparative genomic and lipidomic analyses revealed the upregulation of genes involved in cholesterol biosynthesis, as well as decreased triglyceride levels and increased cholesterol esters, in HCC from these mice. Mechanistically, loss of Fasn promoted nuclear localisation and activation of sterol regulatory element binding protein 2 (Srebp2), which triggered cholesterogenesis. Blocking cholesterol synthesis via the dominant negative form of Srebp2 (dnSrebp2) completely prevented sgPten/c-Met-driven hepatocarcinogenesis in Fasn knockout mice. Similarly, silencing of FASN resulted in increased SREBP2 activation and hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (HMGCR) expression in human HCC cell lines. Concomitant inhibition of FASN-mediated FA synthesis and HMGCR-driven cholesterol production was highly detrimental for HCC cell growth in culture. CONCLUSION: Our study uncovers a novel functional crosstalk between aberrant lipogenesis and cholesterol biosynthesis pathways in hepatocarcinogenesis, whose concomitant inhibition might represent a therapeutic option for HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Colesterol/biossíntese , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos/biossíntese , Neoplasias Hepáticas/metabolismo , Animais , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Ácido Graxo Sintase Tipo I/genética , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica , Genômica , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Transcriptoma
4.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(9): 769-775, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31750816

RESUMO

Objective To explore the functions and mechanisms of macrophages derived from PGRN gene knockout (PGRN-/- ) C57BL/6 mice in the invasion and migration of breast cancer cells. Methods Breast cancer cells were cultured in conditioned medium of macrophages derived from WT and PGRN-/- mice. TranswellTM assay and scratch assay were used to detect the invasion and migration ability of cancer cells. Western blot analysis was used to detect the expression of E-cadherin and N-cadherin in cancer cells. Cytokine array, real-time quantitative PCR and ELISA were performed to investigate the differences of cytokines secreted by macrophages derived from WT and PGRN-/- mice. Breast cancer cells were treated by the differentially expressed cytokine interleukin-6 (IL-6), and then the above methods were used to investigate its effect on cancer cells. Western blot analysis was used to verify the roles of NF-κB and JAK/STAT3 signaling pathways. Results The macrophages derived from PGRN-/- mice blocked NF-κB signaling pathway, reduced IL-6 secretion, and inhibited the invasion and migration of breast cancer cells. IL-6 activated JAK/STAT3 signaling pathway to promote the invasion and migration of breast cancer cells. Conclusion The macrophages derived from PGRN-/- mice can block the NF-κB and JAK/STAT3 signaling pathways, down-regulate IL-6 expression, and inhibit the invasion and migration of breast cancer cells.


Assuntos
Neoplasias da Mama/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interleucina-6/imunologia , Macrófagos/imunologia , Transdução de Sinais , Animais , Neoplasias da Mama/imunologia , Caderinas , Movimento Celular , Meios de Cultivo Condicionados , Granulinas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Invasividade Neoplásica , Fator de Transcrição STAT3/metabolismo , Células Tumorais Cultivadas
5.
Cell Physiol Biochem ; 53(4): 701-712, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31592599

RESUMO

BACKGROUND/AIMS: Cholinergic signalling mediated by the activation of muscarinic and nicotinic receptors has been described in the literature as a classic and important signalling pathway in the regulation of the inflammatory response. Recent research has investigated the role of acetylcholine, the physiological agonist of these receptors, in the control of energy homeostasis at the central level. Studies have shown that mice that do not express acetylcholine in brain regions regulating energy homeostasis present with excessive weight gain and hyperphagia. However, it has not yet been well-described in the literature which cholinergic receptor subunits are involved in this response; moreover, the signalling pathways responsible for the observed effects are not fully delineated. The hypothalamus is the regulating centre of energy homeostasis, and the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR) is highly expressed in this region. When active, α7nAChR recruits proteins such as JAK2/STAT3 to mediate its signalling; the same intracellular components are required by leptin, an anorexigenic hormone. The aim of the present study was to evaluate the role of the hypothalamic α7nAChR in the control of energy homeostasis. METHODS: The work was performed on Swiss male mice. Initially, using immunofluorescent staining on brain sections, the presence of α7nAChR in hypothalamic cells regulating energy homeostasis was evaluated. Animals were submitted to stereotaxis in the lateral ventricle and intracerebroventricular stimulation (ICV) was used for the administration of an agonist (PNU) or antagonist (α-bungarotoxin) of α7nAChR. Metabolic parameters were evaluated and the expression of neuropeptides was evaluated in the hypothalamus by real-time PCR and western blot. The expression of hypothalamic neuropeptides was evaluated in mice treated with siRNA or inhibitors of JAK2/STAT3 (AG490 and STATTIC) proteins. We also evaluated food intake in α7nAChR knockout animals (α7KO). Additionally, in mouse hypothalamic cell culture (the mypHoA-POMC/GFP lineage), we evaluated the expression of neuropeptides and pSTAT3 after stimulation with PNU. RESULTS: Our results indicate co-localisation of α7nAChR with α-MSH, AgRP and NPY in hypothalamic cells. Pharmacological activation of α7nAChR reduced food intake and increased hypothalamic POMC expression and decreased NPY and AgRP mRNA levels and the protein content of pAMPK. Inhibition of α7nAChR with an antagonist increased the mRNA content of NPY and AgRP. Inhibition of α7nAChR with siRNA led to the suppression of POMC expression and an increase in AgRP mRNA levels. α7KO mice showed no changes in food intake. Inhibition of proteins involved in the JAK2/STAT3 signalling pathway reversed the effects observed after PNU stimulation. POMC-GFP cells, when treated with PNU, showed increased POMC expression and nuclear translocation of pSTAT3. CONCLUSION: Thus, selective activation of α7nAChR is able to modulate important markers of the response to food intake, suggesting that α7nAChR activation can suppress the expression of orexigenic markers and favour the expression of anorexics using the intracellular JAK2/STAT3 machinery.


Assuntos
Proteína Relacionada com Agouti/metabolismo , Janus Quinase 2/metabolismo , Pró-Opiomelanocortina/metabolismo , Fator de Transcrição STAT3/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Proteína Relacionada com Agouti/genética , Animais , Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Bungarotoxinas/farmacologia , Linhagem Celular , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Pró-Opiomelanocortina/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Receptor Nicotínico de Acetilcolina alfa7/genética
6.
Life Sci ; 237: 116930, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31610190

RESUMO

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by symptoms that include social communication impairments, interaction deficits, and repetitive and stereotyped behaviors. Recent studies have suggested that imbalanced cytokine levels are associated with impaired behavioral outcomes in individuals with ASD. VGX-1027 is a potent immunomodulatory compound that has shown promise for the treatment of several neuroinflammatory disorders. Here, we studied the effects of VGX-1027 on BTBR T+ Itpr3tf/J (BTBR) mice, an animal model of autism. BTBR mice exhibit most of the core behavioral features of ASD, such as reduced sociability and increased repetitive behaviors. In this study, we investigated the effects of VGX-1027 on self-grooming, marble burying and sociability tests using BTBR mice. We further examined its effect on IL-1ß, IL-6, IL-10, TNF-α, IFN-γ, and NF-κB p65 production in splenic CD4+ cells and on IL-1ß, IL-6, IL-10, TNF-α, IFN-γ, COX-2, and iNOS (NOS2) protein and mRNA expression in brain tissues. The administration of VGX-1027 was found to attenuate self-grooming and marble burying behaviors, and enhance social interactions in BTBR mice. Additionally, VGX-1027 treatment resulted in a substantial decrease in IL-1ß, IL-6, TNF-α, IFN-γ, and NF-κB p65 production, but increased IL-10 production in CD4+ T cells. Moreover, this agent was also found to significantly decrease IL-1ß, IL-6, TNF-α, IFN-γ, COX-2, and NOS2 levels and increase IL-10 expression at the protein and mRNA level in brain tissues. Based on results using BTBR mice, our data provide the first evidence that VGX-1027 could potentially be used for the amelioration of autism-like symptoms.


Assuntos
Acetatos/farmacologia , Transtorno Autístico/prevenção & controle , Comportamento Animal/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Mediadores da Inflamação/metabolismo , Inflamação/prevenção & controle , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Oxazóis/farmacologia , Animais , Transtorno Autístico/etiologia , Transtorno Autístico/metabolismo , Transtorno Autístico/patologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais
7.
Life Sci ; 237: 116915, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31610207

RESUMO

AIMS: The objective of the study was to determine whether ß-caryophyllene (BCP) exerts a neuroprotective effect in cerebral ischemia-reperfusion (I/R) injury by inhibiting microglial activation and modulating their polarization via the TLR4 pathway. MAIN METHODS: Wild-type (WT) and TLR4 knockout (KO) C57BL/6J mice were subjected to cerebral I/R injury and neurologic dysfunction, cerebral infarct volume, brain edema, microglia activation and polarization, and TLR4 expression were determined. In vitro, primary microglia were stimulated with LPS and IFN-γ or IL-4 to induce polarization of microglia toward M1 or M2 phenotypes. KEY FINDINGS: BCP reduced cerebral infarct volume, brain edema, and neurologic deficits in WT mice after I/R. The optimal dose of BCP, 72 mg/kg body weight, inhibited microglial activation and reduced the secretion of proinflammatory cytokines interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6 by microglia of WT mice. BCP inhibited the level of TLR4 in WT mice, and partially reduced neurologic deficits, infarct volume, and brain edema in TLR4 KO mice. Importantly, BCP reduced the number of activated M1-type microglia and increased the number of M2-type microglia in the ipsilateral cortex of both WT and TLR4 KO mice. In vitro, BCP decreased the secretion of proinflammatory cytokines induced by LPS plus IFN-γ, downregulated the level of TLR4 protein, and polarized microglia towards the M2 phenotype. SIGNIFICANCES: The decrease in TLR4 activity mediated, at least in part, the anti-inflammatory effects of BCP and its ability to shift microglia polarization from the M1 to M2 phenotype.


Assuntos
Isquemia Encefálica/prevenção & controle , Macrófagos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Sesquiterpenos/farmacologia , Acidente Vascular Cerebral/prevenção & controle , Receptor 4 Toll-Like/fisiologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/imunologia , Microglia/metabolismo , Microglia/patologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia
8.
Nat Cell Biol ; 21(10): 1248-1260, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31576060

RESUMO

While nuclear lamina abnormalities are hallmarks of human diseases, their interplay with epigenetic regulators and precise epigenetic landscape remain poorly understood. Here, we show that loss of the lysine acetyltransferase MOF or its associated NSL-complex members KANSL2 or KANSL3 leads to a stochastic accumulation of nuclear abnormalities with genomic instability patterns including chromothripsis. SILAC-based MOF and KANSL2 acetylomes identified lamin A/C as an acetylation target of MOF. HDAC inhibition or acetylation-mimicking lamin A derivatives rescue nuclear abnormalities observed in MOF-deficient cells. Mechanistically, loss of lamin A/C acetylation resulted in its increased solubility, defective phosphorylation dynamics and impaired nuclear mechanostability. We found that nuclear abnormalities include EZH2-dependent histone H3 Lys 27 trimethylation and loss of nascent transcription. We term this altered epigenetic landscape "heterochromatin enrichment in nuclear abnormalities" (HENA). Collectively, the NSL-complex-dependent lamin A/C acetylation provides a mechanism that maintains nuclear architecture and genome integrity.


Assuntos
Núcleo Celular/ultraestrutura , Histona Acetiltransferases/metabolismo , Lamina Tipo A/metabolismo , Proteínas Nucleares/metabolismo , Acetilação , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Epigênese Genética , Fibroblastos , Heterocromatina , Histona Acetiltransferases/genética , Histonas/genética , Histonas/metabolismo , Lamina Tipo A/química , Lamina Tipo A/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética
9.
J Agric Food Chem ; 67(46): 12752-12760, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31642668

RESUMO

Atherosclerosis, the major risk of cardiovascular events, is a chronic vascular inflammatory disease. Pterostilbene is a naturally occurring dimethylated analogue of resveratrol and has recently been demonstrated to be beneficial against cardiovascular diseases. However, the underlying mechanisms of pterostilbene on atherosclerosis remain elusive. Experimental atherosclerosis was induced by a high-fat diet (HFD) in apolipoprotein E knockout (ApoE-/-) mice. Pterostilbene was administered intragastrically for 16 weeks. We found that pterostilbene significantly attenuated thoracic and abdominal atherosclerotic plaque formation in HFD-fed ApoE-/-mice, accompanied by modulated lipid profiles and reduced production of proinflammatory cytokines (including IL-6, IFN-γ, and TNF-α). In addition, pterostilbene restored vascular redox balance in thoracic and abdominal aorta, evidenced by enhanced catalase (CAT) expression and activities, and decreased malondialdehyde and H2O2 production. Notably, pterostilbene specifically induced CAT expression and activities in the vascular smooth muscle cells (VSMCs) of thoracic and abdominal aorta. In vitro, pterostilbene markedly promoted the expression and activity of CAT and decreased ox-low-density lipoprotein (LDL)-mediated VSMC proliferation and intracellular H2O2 production, which was abolished by CAT siRNA knockdown or inhibition. Pterostilbene-induced CAT expression was associated with inhibition of Akt, PRAS40, and GSK-3ß signaling activation and upregulation of PTEN. Our data clearly demonstrated that pterostilbene exerted an antiatherosclerotic effect by inducing CAT and modulating the VSMC function.


Assuntos
Aterosclerose/tratamento farmacológico , Catalase/metabolismo , Músculo Liso Vascular/enzimologia , Estilbenos/administração & dosagem , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Catalase/genética , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lipoproteínas LDL/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Oxirredução
10.
Cancer Immunol Immunother ; 68(11): 1769-1778, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31616964

RESUMO

Various immune cells are recruited in the tumor microenvironment. It is well established that cellular immune responses, such as cytotoxic or suppressive activities, play an important role in regulating tumor growth and metastasis. However, the contribution of humoral immune responses against tumors is poorly understood. Fc receptors constitute critical elements for the up- or downregulation of immune responses through immune complexes. Here, we examined the potential role of the inhibitory Fc receptor, Fcγ receptor IIB (FcγRIIB), in tumor immunity using a mouse model. Our findings indicated that tumor-specific antibodies are induced in tumor-bearing mice and control tumor immunity. FcγRIIB deletion significantly improved both cellular and humoral immunity against tumors and delayed tumor growth. These findings indicated that spontaneous antibodies against tumors create a suppressive tumor microenvironment through FcγRIIB signaling, thus suggesting an attractive therapeutic target for cancer immunotherapy.


Assuntos
Anticorpos/imunologia , Carcinoma Pulmonar de Lewis/terapia , Imunoterapia , Receptores de IgG/fisiologia , Timoma/terapia , Neoplasias do Timo/terapia , Microambiente Tumoral/imunologia , Animais , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de IgG/imunologia , Timoma/imunologia , Timoma/metabolismo , Timoma/patologia , Neoplasias do Timo/imunologia , Neoplasias do Timo/metabolismo , Neoplasias do Timo/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Hypertension ; 74(5): 1181-1191, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31564162

RESUMO

Brain renin angiotensin system within the paraventricular nucleus plays a critical role in balancing excitatory and inhibitory inputs to modulate sympathetic output and blood pressure regulation. We previously identified ACE2 and ADAM17 as a compensatory enzyme and a sheddase, respectively, involved in brain renin angiotensin system regulation. Here, we investigated the opposing contribution of ACE2 and ADAM17 to hypothalamic presympathetic activity and ultimately neurogenic hypertension. New mouse models were generated where ACE2 and ADAM17 were selectively knocked down from all neurons (AC-N) or Sim1 neurons (SAT), respectively. Neuronal ACE2 deletion revealed a reduction of inhibitory inputs to AC-N presympathetic neurons relevant to blood pressure regulation. Primary neuron cultures confirmed ACE2 expression on GABAergic neurons synapsing onto excitatory neurons within the hypothalamus but not on glutamatergic neurons. ADAM17 expression was shown to colocalize with angiotensin-II type 1 receptors on Sim1 neurons, and the pressor relevance of this neuronal population was demonstrated by photoactivation. Selective knockdown of ADAM17 was associated with a reduction of FosB gene expression, increased vagal tone, and prevented the acute pressor response to centrally administered angiotensin-II. Chronically, SAT mice exhibited a blunted blood pressure elevation and preserved ACE2 activity during development of salt-sensitive hypertension. Bicuculline injection in those models confirmed the supporting role of ACE2 on GABAergic tone to the paraventricular nucleus. Together, our study demonstrates the contrasting impact of ACE2 and ADAM17 on neuronal excitability of presympathetic neurons within the paraventricular nucleus and the consequences of this mutual regulation in the context of neurogenic hypertension.


Assuntos
Proteína ADAM17/metabolismo , Angiotensina II/farmacologia , Hipertensão/fisiopatologia , Núcleo Hipotalâmico Paraventricular/metabolismo , Peptidil Dipeptidase A/metabolismo , Sistema Renina-Angiotensina/genética , Animais , Sistema Nervoso Autônomo/efeitos dos fármacos , Sistema Nervoso Autônomo/fisiopatologia , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/fisiologia , Hipertensão/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Optogenética/métodos , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/patologia , Distribuição Aleatória , Sistema Renina-Angiotensina/efeitos dos fármacos
13.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(3): 298-304, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31631593

RESUMO

Objective: To determine the effect of Huntingtin-associated protein 1 ( Hap1) on fibroblast proliferation. Methods: Hap1 knockout ( Hap1 -/-) primary fibroblasts were isolated and cultured in vitro. The proliferation of Hap1 -/- fibroblasts was detected by EdU proliferation assay and cell flow assay. Transcriptome sequencing of the wild-type and Hap1 -/- fibroblasts was screened for proliferation-related genes. Real-time quantitative PCR (qPCR) was performed to verify changes in expressions of related genes. Skin repair was examined in Hap1 knockdown mice with skin wounds. The proliferation of fibroblasts during wound repair was detected by PCNA immunohistochemical staining. Results: Hap1 -/- fibroblasts were successfully cultured. Compared with WT, EdU-positive fibroblasts decreased in Hap1 -/-,with less cells entering the S phase. Transcriptome sequencing of primary fibroblasts identified genes of Cdc25C, E2f7, E2f8 and Ccl5. qPCR confirmed that Hap1 knockout increased E2f7 expression. Hap1 +/- mice had larger skin lesions, slower healing and lower positive density of fibroblast proliferation than those of wild type mice. Conclusion: Hap1 may positively regulate fibroblast proliferation by inhibiting the expression of cell cycle negative regulator E2f7.Its deletion inhibits fibroblasts entering the S phase, thereby reducing cell proliferation and affecting wound repair.


Assuntos
Proliferação de Células , Fibroblastos/citologia , Proteínas do Tecido Nervoso/genética , Cicatrização , Animais , Células Cultivadas , Técnicas de Inativação de Genes , Camundongos , Camundongos Knockout , Pele/patologia
14.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(3): 334-338, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31631599

RESUMO

Objective: To study the mechanism of renal injury in Lepr db/ db mice with the leptin receptor homozygous deficiency. Methods: Ten male of 28-week-old Lepr db/+ mice with leptin receptor heterozygous deficiency were selected as control group and ten male Lepr db/ db mice with leptin receptor homozygous deficiency were used in this study. After fasting for 8 hours, the body mass, fasting blood glucose (FBG) and glycosylated hemoglobulin (HbA1c) of the mice were measured. Blood of the mice was obtained from femoral artery before euthanasia. Serum creatinine (CRE), blood urea nitrogen (BUN), superoxide dismutase (SOD), glutathione (GSH) and malonaldehyde (MDA) were detected by corresponding kits, and serum interleukin-1ß (IL-1ß), monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) were measured using enzyme-linked immunosorbent assay (ELISA) method. The kidney was taken for pathological observation. The expression levels of nuclear factor E2-related factor 2 (Nrf2) and nuclear factor kappa B (NF-κB) in renal were analyzed by Western blot. The mitochondria of renal was isolated by the corresponding kit. Meanwhile, the expression level of lipoic acid synthase (LIAS) in renal mitochondria was measured by Western blot. Results: The body mass, FPG, HbA1c, CRE and BUN levels of the Lepr db/ db mice were significantly increased in comparison with the Lepr db/+ mice ( P<0.05). Compared with the Lepr db/+ mice, the Lepr db/ db mice renal exhibited glomerular hypertrophy, thickened basement membrane and capillary wall, the mesangial matrix expansion and mesangial cell hyperplasia. Compared with the Lepr db/+ mice, the serum level of GSH in the Lepr db/ db mice was decreased significantly ( P<0.05). The levels of MDA and concentrations of MCP-1, IL-1ß and TNF-α in serum of the Lepr db/ db mice were higher than those of the Lepr db/+ mice ( P<0.05). Compared with the Lepr db/+ mice, the expression of LIAS and Nrf2 protein in the Lepr db/ db mice renal were decreased ( P<0.05), while the expression of NF-κB protein was increased ( P<0.05). Conclusion: LIAS, Nrf2 and NF-κB might play significant roles through regulation of oxidative stress and inflammation in the renal injury of Lepr db/ db mice.


Assuntos
Rim/patologia , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Receptores para Leptina/genética , Sulfurtransferases/metabolismo , Animais , Masculino , Camundongos , Camundongos Knockout , Estresse Oxidativo
15.
Zhonghua Shao Shang Za Zhi ; 35(10): 740-745, 2019 Oct 20.
Artigo em Chinês | MEDLINE | ID: mdl-31658545

RESUMO

Objective: To construct and identify a mouse model with conditional knockout (cKO) of p75 neurotrophin receptor (p75NTR-cKO) gene in epidermis cells by Cre-loxP system. Methods: Five p75NTR(flox/flox) transgenic C57BL/6J mice (aged 6-8 weeks, male and female unlimited, the age and sex of mice used for reproduction were the same below) and five keratin 14 promotor-driven (KRT14-) Cre(+ /-) transgenic C57BL/6J mice were bred and hybridized via Cre-loxP system. Five p75NTR(flox/+) ·KRT14-Cre(+ /-) mice selected from the first generation of mice were mated with five p75NTR(flox/flox) mice to obtain the second generation hybrids. After the second generation mice were born 20-25 days, the parts of the mice tail were cut off to identify the genotype by polymerase chain reaction method. Four p75NTR gene complete cKO mice (6 weeks old) and 4 wild-type mice (6 weeks old) were selected and sacrificed respectively. The abdominal skin tissue and brain tissue were excised to observe the expression of p75NTR in the two tissue of two types of mice by immunohistochemical staining. The abdominal skin tissue of two types of mice was obtained to observe the histomorphological changes by hematoxylin and eosin staining. Results: (1) Twenty second generation mice were bred. The genotype of 4 mice was p75NTR(flox/flox)·KRT14-Cre(+ /-)(p75NTR(-/-)), i. e. p75NTR gene complete cKO mice; the genotype of 5 mice was p75NTR(flox/+) ·KRT14-Cre(+ /-), i. e. p75NTR gene partial cKO mice; the genotype of 5 mice was p75NTR(flox/flox)·KRT14-Cre(-/-), and that of 6 mice was p75NTR(flox/+) ·KRT14-Cre(-/-), all of which were wild-type mice. (2) The expression of p75NTR was negative in skin epidermis tissue of p75NTR gene complete cKO mice, while numerous p75NTR positive expression was observed in skin epidermis tissue of wild-type mice. Abundant p75NTR positive expression was observed in brain tissue of both wild-type mice and p75NTR gene complete cKO mice. (3) There was no abnormal growth of skin epidermis tissue in both wild-type mice and p75NTR gene complete cKO mice, with intact hair follicle structure. Conclusions: Applying Cre-loxP system can successfully construct a p75NTR-cKO mice model in epidermis cells without obvious changes in skin histomorphology.


Assuntos
Células Epidérmicas , Folículo Piloso/crescimento & desenvolvimento , Receptor de Fator de Crescimento Neural , Animais , Feminino , Folículo Piloso/metabolismo , Integrases , Queratina-14 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
16.
Cell Physiol Biochem ; 53(5): 774-793, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31647207

RESUMO

BACKGROUND/AIMS: Deregulation of the complex interaction among host genetics, gut microbiota and environmental factors on one hand and aberrant immune responses on the other hand, are known to be associated with the development of inflammatory bowel disease. Recent studies provided strong evidence that autophagy plays a key role in the etiology of Crohn's disease (CD). Probiotics may exhibit many therapeutic properties, including anti-inflammatory abilities. While successful results have been obtained in ulcerative colitis patients, probiotics remain inefficient in CD for unknown reason. It remains therefore important to better understand their molecular mechanisms of action. METHODS: The activation of autophagy was examined by stimulating bone marrow-derived dendritic cells by the bacteria, followed by confocal microscopy and western blot analysis. The impact of blocking in vitro autophagy was performed in peripheral blood mononuclear cells using 3-methyl adenine or bafilomycin followed by cytokine secretion measurement by ELISA. The role of autophagy in the anti-inflammatory capacities of the bacterial strains was evaluated in vivo using an acute trinitrobenzene sulfonic acid-induced murine model of colitis. The impact of BMDC was evaluated by adoptive transfer, notably using bone marrow cells derived from autophagy-related 16-like 1-deficient mice. RESULTS: We showed that selected lactobacilli and bifidobacteria are able to induce autophagy activation in BMDCs. Blocking in vitro autophagy abolished the capacity of the strains to induce the release of the anti-inflammatory cytokine interleukin-10, while it exacerbated the secretion of the pro-inflammatory cytokine interleukin-1ß. We confirmed in the TNBS-induced mouse model of colitis that autophagy is involved in the protective capacity of these selected strains, and showed that dendritic cells are involved in this process. CONCLUSION: We propose autophagy as a novel mechanism involved in the regulatory capacities of probiotics.


Assuntos
Autofagia , Bifidobacterium/fisiologia , Lactobacillus/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Proteínas Relacionadas à Autofagia , Células da Medula Óssea/citologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Colite/induzido quimicamente , Colite/microbiologia , Colite/patologia , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Feminino , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Macrolídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout
17.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(8): 689-694, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31638565

RESUMO

Objective To study the effect of deletion of Toll-like receptor 2 (TLR2) gene on insulin resistance and polarization of macrophages in mice. Methods The wild-type (WT) and TLR2 knockout (TLR2-/-) C57BL/6 male mice, aged 28 days, were selected, with 12 mice in each group. The genotype of each mouse was identified by PCR. After mice were fed with basic diet for 3 months, the glucose tolerance test (GTT) and insulin resistance test (ITT) were performed. The mononuclear cells isolated from peripheral blood were stimulated with GM-CSF/IFN-γ and M-CSF/IL-4/IL-13, respectively, to induce differentiation to M1-like and M2-like macrophages. The CD11b, F4/80, CD11c, CD206 and early growth response 2 (EGR2) were detected by flow cytometry to determine the phenotype of macrophages. The levels of TNF-α, IL-6 and IL-10 in the culture supernatant of macrophages were detected using ELISA. Results The result of PCR identification was consistent with the genotype of mice. Compared to WT mice, TLR2-/- mice exhibited the significantly improved glycemic control at 30 min during GTT and the significantly increased insulin sensitivity at 15 minutes during ITT. The flow cytometry showed that M1 markers decreased and M2 macrophages increased in the TLR2-/- mice. ELISA showed that the levels of IL-6 and TNF-α significantly decreased in the culture supernatant of M1 macrophages, while the level of IL-10 significantly increased in the culture supernatant of M2 macrophages in TLR2-/- mice compared with WT mice. Conclusion TLR2 signal has an effect on the polarization of macrophages and makes macrophages tend to switch to M1 phenotype. A higher number of pro-inflammatory factors secreted by M1 macrophages contribute to a low-grade inflammation state in the body, which leads to a decrease in glucose tolerance and insulin sensitivity.


Assuntos
Resistência à Insulina , Macrófagos , Receptor 2 Toll-Like , Animais , Resistência à Insulina/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 2 Toll-Like/genética
18.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(8): 714-720, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31638569

RESUMO

Objective To investigate the effect of progranulin (PGRN) on osteoporosis in ovariectomized mice. Methods PGRN-knockout (PGRN-/-) and wild-type mice were ovariectomized to induce postmenopausal osteoporosis models. Next, the bone tissues in all mice were scanned by Micro-CT and three-dimensional reconstruction was performed to detect the micro-structure, followed by trabecula data analysis. The morphology and osteoclasts in the bone tissues of PGRN-/- and wild-type mice were observed by HE staining and TRAP staining, respectively. The expression of receptor activator for nuclear factor-κB ligand (RANKL), tumor necrosis factor α (TNF-α) and P65 were detected by immunohistochemistry. The expression of TRAP mRNA in the mice was measured using fluorescence quantitative PCR and the protein expression of MMP9, MMP14, P65 was detected by Western blot analysis. Results Bone mineral density (BMD), bone volume fraction (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) in the PGRN-/- group were significantly higher than those in the wild-type group, while the trabecular separation (Tb.S) in the PGRN-/- group was in the contrary. The degree of osteoporosis was less severe and number of osteoclasts in the PGRN-/- mice were reduced, likewise, RANKL, TNF-α, MMP9, MMP14 and P65 as well as TRAP mRNA were down-regulated in the PGRN-/- group compared with the wild-type group. Conclusion PGRN aggravates the postmenopausal osteoporosis in ovariectomized mice.


Assuntos
Osteoporose Pós-Menopausa , Progranulinas , Animais , Densidade Óssea/genética , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Knockout , Osteoporose Pós-Menopausa/genética , Osteoporose Pós-Menopausa/fisiopatologia , Ovariectomia , Progranulinas/genética , Progranulinas/metabolismo
19.
Gynecol Oncol ; 155(2): 349-358, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31477281

RESUMO

OBJECTIVE: Platinum compounds have been widely used as a primary treatment for many types of cancer. However, resistance is the major cause of therapeutic failure for patients with metastatic or recurrent disease, thus highlighting the need to identify novel factors driving resistance to Platinum compounds. Metadherin (MTDH, also known as AEG-1 and LYRIC), located in a frequently amplified region of chromosome 8, has been consistently associated with resistance to chemotherapeutic agents, though the precise mechanisms remain incompletely defined. METHODS: The mRNA of FANCD2 and FANCI was pulled down by RNA-binding protein immunoprecipitation. Pristimerin-loaded nanoparticles were prepared using the nanoprecipitation method. Immunocompromised mice bearing patient-derived xenograft tumors were treated with pristimerin-loaded nanoparticles, cisplatin and a combination of the two. RESULTS: MTDH, through its recently discovered role as an RNA binding protein, regulates expression of FANCD2 and FANCI, two components of the Fanconi anemia complementation group (FA) that play critical roles in interstrand crosslink damage induced by platinum compounds. Pristimerin, a quinonemethide triterpenoid extract from members of the Celastraceae family used to treat inflammation in traditional Chinese medicine, significantly decreased MTDH, FANCD2 and FANCI levels in cancer cells, thereby restoring sensitivity to platinum-based chemotherapy. Using a patient-derived xenograft model of endometrial cancer, we discovered that treatment with pristimerin in a novel nanoparticle formulation markedly inhibited tumor growth when combined with cisplatin. CONCLUSIONS: MTDH is involved in post-transcriptional regulation of FANCD2 and FANCI. Pristimerin can increase sensitivity to platinum-based agents in tumors with MTDH overexpression by inhibiting the FA pathway.


Assuntos
Antineoplásicos/farmacologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/antagonistas & inibidores , Proteínas de Grupos de Complementação da Anemia de Fanconi/antagonistas & inibidores , Proteínas de Membrana/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Cisplatino/farmacologia , Cistadenocarcinoma Seroso/tratamento farmacológico , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Masculino , Camundongos Knockout , Nanopartículas , Neoplasias Uterinas/tratamento farmacológico
20.
Genes Dev ; 33(19-20): 1381-1396, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31488579

RESUMO

Short telomere syndromes manifest as familial idiopathic pulmonary fibrosis; they are the most common premature aging disorders. We used genome-wide linkage to identify heterozygous loss of function of ZCCHC8, a zinc-knuckle containing protein, as a cause of autosomal dominant pulmonary fibrosis. ZCCHC8 associated with TR and was required for telomerase function. In ZCCHC8 knockout cells and in mutation carriers, genomically extended telomerase RNA (TR) accumulated at the expense of mature TR, consistent with a role for ZCCHC8 in mediating TR 3' end targeting to the nuclear RNA exosome. We generated Zcchc8-null mice and found that heterozygotes, similar to human mutation carriers, had TR insufficiency but an otherwise preserved transcriptome. In contrast, Zcchc8-/- mice developed progressive and fatal neurodevelopmental pathology with features of a ciliopathy. The Zcchc8-/- brain transcriptome was highly dysregulated, showing accumulation and 3' end misprocessing of other low-abundance RNAs, including those encoding cilia components as well as the intronless replication-dependent histones. Our data identify a novel cause of human short telomere syndromes-familial pulmonary fibrosis and uncover nuclear exosome targeting as an essential 3' end maturation mechanism that vertebrate TR shares with replication-dependent histones.


Assuntos
Proteínas de Transporte/genética , Fibrose Pulmonar Idiopática/genética , Mutação com Perda de Função , Proteínas Nucleares/genética , RNA/metabolismo , Telomerase/metabolismo , Animais , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Linhagem Celular , Cílios/genética , Feminino , Ligação Genética , Células HCT116 , Humanos , Fibrose Pulmonar Idiopática/enzimologia , Fibrose Pulmonar Idiopática/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Transtornos do Neurodesenvolvimento/genética , Linhagem , Processamento Pós-Transcricional do RNA/genética , Encurtamento do Telômero/genética
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