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1.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(7): 577-582, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31537240

RESUMO

Objective To investigate the roles of Th1 cytokines tumor necrosis factor α (TNF-α), interferon gamma (IFN-γ) and multifunctional T cells in nucleotides binding oligomer domain 2 knockout (NOD2-/-) mice infected with Mycobacterium tuberculosis (MTB) H37Ra. Methods Mouse models of pulmonary infection were established by tracheal instillation of MTB strain H37Ra into NOD2-/- mice and C57BL/6 mice (n=10 each group). Lung tissues were removed and stained by HE staining and pathological scores were evaluated 4 weeks after infection. The levels of TNF-α and IFN-γ in the lung homogenates were detected by ELISA, and the ratio of multifunctional CD4+ T and CD8+ T cells in the spleen were examined by flow cytometry. Results MTB infection promoted lung inflammation of NOD2-/- mice. The levels of TNF-α and IFN-γ in the lung tissues of NOD2-/- mice increased. Compared with normal saline group, TNF-α+, IFN-γ+ cells and TNF-α+IFN-γ+ cells in CD4+/CD8+T cells significantly increased in NOD2-/- mice and C57BL/6 mice after the infection. TNF-α+CD4+T cells, IFN-γ+CD4+T cells and IFN-γ+CD8+T cells in MTB-infected NOD2-/- mice were significantly higher than those in MTB-infected C57BL/6 mice. Conclusion H37Ra can induce Th1 immune response in NOD2-/- mice.


Assuntos
Mycobacterium tuberculosis , Células Th1/imunologia , Tuberculose/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Interferon gama/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Adaptadora de Sinalização NOD2/genética , Baço/citologia , Baço/imunologia , Fator de Necrose Tumoral alfa/imunologia
2.
Nihon Yakurigaku Zasshi ; 154(3): 92-96, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31527366

RESUMO

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel that responds to mechanical, thermal, and chemical stimuli in addition to various endogenous ligands, such as arachidonic acid metabolites. The present study aimed to elucidate the expression of TRPV4 in the gastrointestinal tract and the pathogenic roles of TRPV4 in dextran sulphate sodium (DSS)-induced colitis. TRPV4-immunoreactivity was detected in epithelial-like cells of the mouse tongue, esophagus, stomach, ileum, and colon; TRPV4 expression in the tongue was higher than other gastrointestinal tracts. TRPV4 colocalized with a type IV cell marker sonic hedgehog in circumvallate papillae. These findings suggest that TRPV4 contributes to sour taste sensing by regulating type III taste cell differentiation in mice. DSS-induced colitis was significantly attenuated in TRPV4-knockout (TRPV4KO) mice when compared to wild-type mice. DSS treatment upregulated TRPV4 expression in vascular endothelia of colonic mucosa and submucosa. DSS treatment increased vascular permeability, which was abolished in TRPV4KO mice. The activation of TRPV4 decreased VE-cadherin expression in mouse aortic endothelial cells exposed to TNF-α. These findings indicate that the upregulation of TRPV4 in vascular endothelial cells contributes to the progression of colonic inflammation via the activation of vascular permeability. Thus, TRPV4 is an attractive target for the treatment of inflammatory bowel diseases.


Assuntos
Colite/fisiopatologia , Células Endoteliais/fisiologia , Trato Gastrointestinal/fisiologia , Trato Gastrointestinal/fisiopatologia , Canais de Cátion TRPV/fisiologia , Animais , Colite/induzido quimicamente , Sulfato de Dextrana , Camundongos , Camundongos Knockout , Língua/fisiologia
3.
Adv Exp Med Biol ; 1155: 155-162, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468394

RESUMO

Fragile X syndrome is an X-linked dominant disorder and the most common cause of inherited mental retardation. It is caused by trinucleotide repeat expansion in the fragile X mental retardation 1 gene (FMR1) at the Xq27.3. The expansion blocks expression of the gene product, Fragile X Mental Retardation Protein (FMRP). The syndrome includes mild to moderate mental retardation and behavioral manifestations such as tactile defensiveness, gaze avoidance, repetitive motor mannerisms, perseverative (repetitive) speech, hyperarousal and it frequently includes seizures. This behavioral phenotype overlaps significantly with autism spectrum disorder. The knockout mice lack normal Fmr1 protein and show macro-orchidism, learning deficits, and hyperactivity. Consequently, this knockout mouse may serve as a valuable tool in the elucidation of the physiological role of FMR1 and the mechanisms involved in macroorchidism, abnormal behavior, abnormalities comparable to those of human fragile X patients. In this study we evaluated the effects of taurine on the testicular physiology to better understand the cellular mechanisms underlying macro-orchidism. We found that there was a significant decrease in the number of Leydig cells in the testis of fragile X mouse. Furthermore, the expression of somatostatin was drastically decreased and differential expression pattern of CDK5 in fragile X mouse testis. In the control testis, CDK is expressed in primary and secondary spermatids whereas in the Fmr1 ko mice CDK 5 is expressed mainly in spermatogonia. Taurine supplementation led to an increase in CDK5 expression in both controls and Ko mice. CDKs (Cyclin-dependent kinases) are a group of serine/threonine protein kinases activated by binding to a regulatory subunit cyclin. Over 20 functionally diverse proteins involved in cytoskeleton dynamics, cell adhesion, transport, and membrane trafficking act as CDK5 substrates elucidating the molecular mechanisms of CDK5 function. CDK5 phosphorylates a diverse list of substrates, implicating it in the regulation of a range of cellular processes. CDK5 is expressed in Leydig cells, Sertoli cells, spermatogonia and peritubular cells indicating a role in spermatogenesis. In this study we examined the expression levels of CDK5 and how it is affected by taurine supplementation in the testes and found that taurine plays an important role in testicular physiology and corrected some of the pathophysiology observed in the fragile x mouse testis.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Taurina/farmacologia , Testículo/fisiopatologia , Animais , Suplementos Nutricionais , Modelos Animais de Doenças , Síndrome do Cromossomo X Frágil/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Expansão das Repetições de Trinucleotídeos
4.
Adv Exp Med Biol ; 1155: 523-529, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468428

RESUMO

Mammalian tissues, especially the heart, contain high concentrations of taurine, a beta-amino acid that possesses a variety of physiological functions. While it is well known that taurine reacts with several metabolites, such as bile acids and fatty acids, taurine-conjugated metabolites in the heart have not been specifically studied. Recently, we performed Liquid chromatography-mass spectrometry- (LC-MS-) based metabolome analysis, comparing metabolome profiles of hearts from taurine transporter knockout (TauTKO) mice and wild-type mice to identify differences in taurine-conjugated metabolite content of the two phenotypes. Comparison of the metabolite profiles revealed taurine-containing dipeptides, such as glutamyltaurine, which are present in wild-type but not in TauTKO hearts. These data suggest that taurine functions not only as a free osmolyte but also as a conjugated metabolite within the heart.


Assuntos
Coração , Metaboloma , Miocárdio/metabolismo , Taurina/metabolismo , Animais , Cromatografia Líquida , Camundongos , Camundongos Knockout , Espectrometria de Massas em Tandem
5.
Adv Exp Med Biol ; 1155: 543-553, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468430

RESUMO

Taurine transporter (TauT) has been identified as a target gene of p53 tumor suppressor. TauT is also found to be overexpressed in variety type of human cancers, such as leukemia. This study showed that expression of TauT was upregulated by c-Myc and c-Jun oncogenes. To explore whether blocking of TauT inhibits tumor development, the RNA interference (RNAi) and immune targeting approaches were tested in tumor cells in vitro and in p53 mutant mice in vivo. Knockdown of TauT expression by RNAi resulted in cell cycle G2 arrest and suppressed human breast cancer MCF-7 cells proliferation determined by colonies production and cell migration assays. Knockdown of TauT also rendered MCF-7 cells more susceptible to chemotherapeutic drug-induced apoptosis. An antibody specifically against TauT blocked taurine uptake and induced cell cycle G2 arrest leading to cell death of variety type of tumor cells without affecting the viability of normal mammalian cells. TauT peptide vaccination significantly increased median lifespan (1.5-fold) of the p53 null mice and rescued p53+/- mice by extending the median lifespan from 315 days to 621 days. Furthermore, single dose treatment of tumor-bearing (thymic lymphoma) p53 null mice with TauT peptide reduced tumor size by about 50% and significantly prolonged survival of these mice from average 7 days (after observing the thymic lymphoma) to 21 days. This finding demonstrates that a novel TauT peptide vaccine can delay, inhibit, and/or treat p53 mutation related spontaneous tumorigenesis in vivo. Therefore, TauT peptide may be used as a universal cancer vaccine to prevent and/or treat patients with p53 mutation-mediated cancers.


Assuntos
Vacinas Anticâncer , Imunoterapia , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Interferência de RNA , Proteína Supressora de Tumor p53/genética , Animais , Anticorpos Monoclonais/farmacologia , Pontos de Checagem do Ciclo Celular , Técnicas de Silenciamento de Genes , Genes jun , Genes myc , Humanos , Células MCF-7 , Camundongos , Camundongos Knockout , Terapia de Alvo Molecular , Mutação , Taurina , Vacinas de Subunidades
6.
Adv Exp Med Biol ; 1155: 905-921, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468456

RESUMO

Taurine is abundant in various tissues including the brain, muscle, heart, spleen, liver and kidney with various physiological functions. Since taurine is produced by cysteine sulfinic acid decarboxylase (CSAD) in the liver and kidney, taurine-deficient mice without CSAD have been investigated for abnormal physiological functions such as retinal development, immune, pancreatic and liver function. In this study, the behavioral effects and abnormal brain development caused by low taurine in the developing brain were examined. In neonatal brains of homozygous CSAD knockout mice (HO), taurine was reduced by 85%, compared to wild-type mice (WT). Taurine was reduced by 35% in the brains of 2 month-old HO, compared to WT. Anxiety, motor coordination and autistic-like behaviors were evaluated at 2 months of age using five behavioral tests: elevated plus maze, open field, social approach, marble burying and accelerating rotarod. Mice were tested from 3 groups including WT, HO and HO with oral treatment of 0.2% taurine in the drinking water (HOT). HOT were born from HO dams treated with taurine from before pregnancy and were continuously treated with taurine in the drinking water after weaning. The taurine levels in the brain and plasma of HOT were restored to WT at 2 months of age. Taurine-deficiency did not lead to changes in autistic-like behaviors as the HO were not significantly different from WT in marble burying and social approach. However, taurine-deficiency increased anxiety-like behavior in HO in the elevated plus maze and open field, compared to WT. Taurine treatment significantly restored the HOT to WT levels of anxiety-like behavior in the elevated plus maze. However, changes in exploratory activity in the open field were not improved with taurine treatment. There was a slight difference in motor ability as the WT mice stayed on the accelerating rotarod longer that the HO and HOT, but the difference was significant in the HOT during the first trial only, compared to WT.These data support hypothesis that taurine is essential for the emotional development of the brain. First, taurine is remarkably low in the neonatal brain of HO, compared to the adult brain of HO. Second, taurine treatment in HO partially improves anxiety-like behavior to WT.


Assuntos
Ansiolíticos/farmacologia , Ansiedade/tratamento farmacológico , Taurina/farmacologia , Animais , Comportamento Animal , Camundongos , Camundongos Knockout
7.
Adv Exp Med Biol ; 1155: 977-985, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468461

RESUMO

Taurine (2-aminoethanesulfonic acid) is a sulfur-containing amino acid. It is one of the most abundant free amino acids in many excitable tissues, including the brain, skeletal and cardiac muscles. Physiological actions of taurine are widespread and include regulation of plasma glucose levels, bile acid conjugation, detoxification, membrane stabilization, blood pressure regulation, osmoregulation, neurotransmission, and modulation of mitochondria function and cellular calcium levels. Taurine plays an important role in modulating glutamate and GABA neurotransmission and prevents excitotoxicity in vitro primarily through modulation of intracellular calcium homeostasis. Taurine supplementation prevents age-dependent decline of cognitive functions. Because of the wide spread actions of taurine, its levels are highly regulated through enzymatic biosynthesis or dietary intake. Furthermore, depletion of endogenous or dietary supplementation of exogenous taurine have been shown to induce wide spread actions on multiple organs. Cysteine sulfonic acid decarboxylase (CSAD) was first identified in the liver and is thought to be the rate-limiting enzyme in taurine biosynthesis. CSAD mRNA is expressed in the brain in astrocytes. Homozygous knockout mice lacking CSAD (CSAD-KO) have very reduced taurine content and show severe functional histopathology in the visual system, skeletal system, heart, pancreas and brain. Conversely, dietary supplementation of taurine results in significant health benefits acting through the same organ systems. Fluctuation of taurine bioavailability lead to changes in the expression levels of taurine transporters in neuronal plasma membranes, endothelial cells forming the blood-brain barrier and proximal cells of the kidneys. Suggesting a highly regulated mechanism for maintaining taurine homeostasis and organ systems function. Here we show how alterations in taurine levels directly affect the function of one organ system and through functional interaction and compensatory adaptation; these effects extend to another organ systems with focus on the nervous system.


Assuntos
Sistemas Neurossecretores/fisiologia , Taurina/farmacologia , Animais , Barreira Hematoencefálica , Membrana Celular , Células Endoteliais , Homeostase , Rim , Camundongos , Camundongos Knockout
9.
Gene ; 717: 143998, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31381951

RESUMO

Eid1 is a member of the EID protein family, which regulates differentiation, transcription and acetyltransferase activity. Accumulating evidence suggests that Eid1 is relevant to neurological disorder, but the main function of Eid1 is still unclear, especially in the brain. To better understand this issue, we generated Eid1-knockout (Eid1-KO) mice and profiled its gene expression changes in the brain by RNA sequencing. This study identified 2531 genes differentially expressed in Eid1-KO mice compared with the wild-type, then qRT-PCR verification demonstrated that the transcriptomic data are reliable. By protein-protein interaction cluster analysis, 'regulation of cell proliferation' were unexpectedly discovered as important Eid1 functions. We then isolated neural progenitor cells (NPCs) and showed that the number of neurospheres and the proliferation rate of Eid1-KO NPCs were obviously lower than that in the control group, furthermore, CCK-8 and immunofluorescence assay clearly demonstrated that the Eid1-KO NPCs showed significantly less cell proliferation than the control group. To the best of our knowledge, this is the first comprehensive report of the Eid1-KO transcriptome of mice brain. Our analysis and experimental data provide a foundation for further studies on understanding function of Eid1 in the brain.


Assuntos
Encéfalo/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/fisiologia , Proliferação de Células/genética , Feminino , Perfilação da Expressão Gênica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Gravidez , Mapas de Interação de Proteínas , Análise de Sequência de RNA
10.
Cell Physiol Biochem ; 53(3): 439-452, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31436397

RESUMO

BACKGROUND/AIMS: Among the assisted reproductive techniques, the in vitro maturation of oocytes (IVM) is less developed than other techniques, but its implementation would entail a qualitative advance. This technique consists in the extraction of immature oocytes from antral ovarian follicles with the patient under low hormone stimulation or without hormone to mature exogenously in culture media supplemented with different molecules to promote maturation. In this sense, we are interested in the role that cannabinoids could have as IVM promoters because cannabinoid's molecular pathway is similar to the one by which oocyte's meiosis resumption is activated. With the intention of advancing in the possible use of cannabinoids as supplements for the media for in vitro maturation of oocytes, we intend to deepen the study of the function of the phytocannabinoid Δ-9-tetrahydrocannabinol (THC) in the IVM process. METHODS: By immunocytochemistry, we detected the location pattern of cannabinoid receptor type 1 (CB1) and type 2 (CB2) during oocyte maturation in presence or absence of THC, as well as, the staining pattern of p-AKT and p-ERK. We used a genetic/ pharmacological approach generating knockout oocytes for CB1 and/or CB2 and they were incubated with THC during the oocyte maturation to visualize the physiological effects of THC, observing the rate of blastocyst achieved by oocyte. RESULTS: This study confirms that the incubation of oocytes with THC during IVM accelerated some events of that process like the phosphorylation pattern of ERK and AKT and was able to increase the blastocyst rate in response to IVF. Moreover, it seems that both CB1 and CB2 are necessary to maintain a healthy oocyte maturation. CONCLUSION: Our data suggest that THC may be useful IVM supplements in clinic as is more feasible and reliable than any synthetic cannabinoid.


Assuntos
Blastocisto/efeitos dos fármacos , Dronabinol/farmacologia , Oócitos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Blastocisto/citologia , Blastocisto/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fertilização In Vitro , Técnicas de Maturação in Vitro de Oócitos , Meiose/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Oócitos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo
11.
Life Sci ; 234: 116755, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31415769

RESUMO

AIMS: Vitamin D and its receptor, vitamin D receptor (VDR), have renoprotection effect against diabetic nephropathy (DN). But the exact mechanism has not been fully elucidated. Epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP) epoxygenase-derived metabolites of arachidonic acid, protecting against diabetes and DN. Herein, we hypothesized that activation of VDR attenuated high glucose-induced cellular injury in renal tubular epithelial cells partially through up-regulating CYP2J5 expression. MAIN METHODS: Streptozotocin (STZ) was injected to induce diabetic in wild type and Vdr-/- mice. The effects of VDR knockout and an activator of VDR, paricalcitol, on the renal injury were detected. In vitro, a murine kidney proximal tubule epithelial cell line BU.MPT induced by high glucose were treated with or without paricalcitol (30 mM) for 12 h or 24 h. KEY FINDINGS: The expression of CYP2J5 was significantly decreased both in wild type and Vdr-/- diabetic mice induced by STZ. The STZ-induced kidney architecture damage and apoptosis rate in Vdr-/- mice were more severe. In vitro, high glucose treatment strongly reduced the CYP2J5 expression and the synthesis of 14,15-EET in BU.MPT cells. Supplement of 14,15-EET significantly reduced the lactate dehydrogenase (LDH) release induced by high glucose in BU.MPT cells. Furthermore, treatment with paricalcitol attenuated cellular injury and restored the expression of CYP2J5 reduced by high glucose in BU.MPT cells. SIGNIFICANCE: We conclude that activation of VDR attenuates high glucose-induced cellular injury partially dependent on CYP2J5 in murine renal tubule epithelial cells and paricalcitol may represent a potential therapy for DN.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/etiologia , Ergocalciferóis/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Receptores de Calcitriol/agonistas , Animais , Linhagem Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Ergocalciferóis/uso terapêutico , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Knockout , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo
12.
Life Sci ; 234: 116793, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31465735

RESUMO

INTRODUCTION: Environmental factors have a key role in the control of gut microbiota and obesity. TLR2 knockout (TLR2-/-) mice in some housing conditions are protected from diet-induced insulin resistance. However, in our housing conditions these animals are not protected from diet-induced insulin-resistance. AIM: The aim of the present study was to investigate the influence of our animal housing conditions on the gut microbiota, glucose tolerance and insulin sensitivity in TLR2-/- mice. MATERIAL AND METHODS: The microbiota was investigated by metagenomics, associated with hyperinsulinemic euglycemic clamp and GTT associated with insulin signaling through immunoblotting. RESULTS: The results showed that TLR2-/- mice in our housing conditions presented a phenotype of metabolic syndrome characterized by insulin resistance, glucose intolerance and increase in body weight. This phenotype was associated with differences in microbiota in TLR2-/- mice that showed a decrease in the Proteobacteria and Bacteroidetes phyla and an increase in the Firmicutesphylum, associated with and in increase in the Oscillospira and Ruminococcus genera. Furthermore there is also an increase in circulating LPS and subclinical inflammation in TLR2-/-. The molecular mechanism that account for insulin resistance was an activation of TLR4, associated with ER stress and JNK activation. The phenotype and metabolic behavior was reversed by antibiotic treatment and reproduced in WT mice by microbiota transplantation. CONCLUSIONS: Our data show, for the first time, that the intestinal microbiota can induce insulin resistance and obesity in an animal model that is genetically protected from these processes.


Assuntos
Microbioma Gastrointestinal , Resistência à Insulina , Insulina/metabolismo , Receptor 2 Toll-Like/genética , Animais , Estresse do Retículo Endoplasmático , Deleção de Genes , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Intolerância à Glucose/microbiologia , Abrigo para Animais , Resistência à Insulina/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 2 Toll-Like/metabolismo
13.
Toxicol Lett ; 315: 23-30, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31442584

RESUMO

Ulcerative colitis2 (UC) is an inflammatory bowel disease3 (IBD) that causes long-lasting inflammation and ulcers in the human digestive tract. The repair function of TLR4 in the intestinal epithelium is still unknown. Here, wild-type4 (WT) mice, TLR4-knockout mice5 (KO; TLR4-/-) and commensal-depleted mice were used as dextran sulfate sodium6 (DSS)-induced or radiation-induced colitis and injury models to explore the role of TLR4 signaling in intestinal injury. Exogenous lipopolysaccharide7 (LPS) promoted DSS-induced inflammatory cytokines and aggravated intestinal damage. TLR4 deficiency and commensal bacterial depletion inhibited the toxic effects of LPS, but these mice were more susceptible to DSS-induced and radiation-induced intestinal damage. Compared with WT mice, neither DSS nor radiation promoted production of more inflammatory cytokines in the guts of TLR4-KO and commensal-depleted mice. Introducing the cytokine repair factors, PGE2 and GM-CSF, increased the cytokine levels in the guts of DSS-induced colitis mice. We hypothesized that TLR4 and its ligands repaired the epithelium after DSS-induced and radiation-induced intestinal damage by upregulating PGE2 and GM-CSF. Transwell migration assays suggested that LPS, IL6, TNF, PGE2 and GM-CSF promoted intestinal cell migration, and cell viability analysis suggested that these factors protected against radiation-induced intestinal damage. Our data underscore the importance of the balancing role of TLR4 in intestinal injury and repair.


Assuntos
Linhagem Celular/efeitos da radiação , Colite/induzido quimicamente , Colite/fisiopatologia , Sulfato de Dextrana/toxicidade , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos da radiação , Receptor 4 Toll-Like/efeitos da radiação , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação
14.
Sheng Li Xue Bao ; 71(4): 588-596, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31440756

RESUMO

The aim of the study was to establish Ace2 (angiotensin-converting enzyme 2) knockout mouse model with CRISPR/Cas9 gene targeting technology. A vector targeting Ace2 gene knockout was constructed with the primers of single-guide RNA (gRNA), and then transcribed gRNA/Cas9 mRNA was micro-injected into the mouse zygote. The deletion of exons 3 to 18 of Ace2 gene in mice was detected and identified by PCR and gene sequencing. The Ace2 gene knock-out mice were bred and copulated. Ace2 protein and mRNA expression were detected by Western blot and qRT-PCR in F3 progeny knock-out male mice. The gRNA expression vector was successfully constructed and transcribed in vitro, and active gRNA and Cas9 mRNA were injected directly into zygote. The deletion of exons 3 to 18 of Ace2 gene in six positive founder mice as the F0 generation were confirmed by PCR and gene sequencing. Six founder mice were mated with wild-type mice, then achieved F1 generation were mated and produced F2 generation. The female positive mouse of F2 was selected to mate with wild-type mice and produce Ace2-/Y mice of F3 generation. Ace2 mRNA and protein were not detected in tissues of these Ace2-/Y mice. In conclusion, a mouse model with Ace2 deficiency has been successfully established with CRISPR/Cas9 technique, which shall lay a foundation for future investigation of Ace2.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Camundongos Knockout , RNA Guia/genética , Animais , Feminino , Marcação de Genes , Masculino , Camundongos
15.
Nat Commun ; 10(1): 2921, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266943

RESUMO

Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signaling cascades, including the JAK-STAT pathway. Our understanding of how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. Here, we use an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and IFN-γ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA.


Assuntos
Regulação da Expressão Gênica , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Fator Gênico 3 Estimulado por Interferon/metabolismo , Interferons/metabolismo , Fator de Transcrição STAT2/metabolismo , Animais , Feminino , Humanos , Fator Gênico 3 Estimulado por Interferon/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Células RAW 264.7 , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/genética , Transcrição Genética
16.
Nat Commun ; 10(1): 2914, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266968

RESUMO

The deubiquitylase OTUD3 plays a suppressive role in breast tumorigenesis through stabilizing PTEN protein, but its role in lung cancer remains unclear. Here, we demonstrate that in vivo deletion of OTUD3 indeed promotes breast cancer development in mice, but by contrast, it slows down KrasG12D-driven lung adenocarcinoma (ADC) initiation and progression and markedly increases survival in mice. Moreover, OTUD3 is highly expressed in human lung cancer tissues and its higher expression correlates with poorer survival of patients. Further mechanistic studies reveal that OTUD3 interacts with, deubiquitylates and stabilizes the glucose-regulated protein GRP78. Knockdown of OTUD3 results in a decrease in the level of GRP78 protein, suppression of cell growth and migration, and tumorigenesis in lung cancer. Collectively, our results reveal a previously unappreciated pro-oncogenic role of OTUD3 in lung cancer and indicate that deubiquitylases could elicit tumor-suppressing or tumor-promoting activities in a cell- and tissue-dependent context.


Assuntos
Proteínas de Choque Térmico/metabolismo , Neoplasias Pulmonares/enzimologia , Proteases Específicas de Ubiquitina/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Proteínas de Choque Térmico/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteases Específicas de Ubiquitina/genética
17.
J Cancer Res Clin Oncol ; 145(9): 2241-2250, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31342168

RESUMO

PURPOSE: The tripartite motif (TRIM)16 acts as a tumour suppressor in both squamous cell carcinoma (SCC) and melanoma. TRIM16 is known to be secreted by keratinocytes, but no studies have been reported yet to assess the relationship between TRIM16 keratinocyte expression and melanoma development. METHODS: To study the role of TRIM16 in skin cancer development, we developed a keratinocyte TRIM16-specific knockout mouse model, and used the classical two-stage skin carcinogenesis challenge method, to assess the loss of keratinocyte TRIM16 on both papilloma, SCC and melanoma development in the skin after topical carcinogen treatment. RESULTS: Heterozygous, but not homozygous, TRIM16 knockout mice exhibited an accelerated development of skin papillomas and melanomas, larger melanoma lesions and an increased potential for lymph node metastasis. CONCLUSION: This study provides the first evidence that keratinocyte loss of the putative melanoma tumour suppressor protein, TRIM16, enhances melanomagenesis. Our data also suggest that TRIM16 expression in keratinocytes is involved in cross talk between keratinocytes and melanocytes, and has a role in melanoma tumorigenesis.


Assuntos
Proteínas de Transporte/genética , Queratinócitos/metabolismo , Perda de Heterozigosidade/fisiologia , Linfonodos/metabolismo , Melanócitos/metabolismo , Melanoma/genética , Neoplasias Cutâneas/genética , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas de Transporte/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Regulação Neoplásica da Expressão Gênica , Queratinócitos/patologia , Linfonodos/patologia , Metástase Linfática , Melanócitos/patologia , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Knockout , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
18.
Nat Commun ; 10(1): 2943, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270333

RESUMO

Mutations exclusively in equilibrative nucleoside transporter 3 (ENT3), the only intracellular nucleoside transporter within the solute carrier 29 (SLC29) gene family, cause an expanding spectrum of human genetic disorders (e.g., H syndrome, PHID syndrome, and SHML/RDD syndrome). Here, we identify adult stem cell deficits that drive ENT3-related abnormalities in mice. ENT3 deficiency alters hematopoietic and mesenchymal stem cell fates; the former leads to stem cell exhaustion, and the latter leads to breaches of mesodermal tissue integrity. The molecular pathogenesis stems from the loss of lysosomal adenosine transport, which impedes autophagy-regulated stem cell differentiation programs via misregulation of the AMPK-mTOR-ULK axis. Furthermore, mass spectrometry-based metabolomics and bioenergetics studies identify defects in fatty acid utilization, and alterations in mitochondrial bioenergetics can additionally propel stem cell deficits. Genetic, pharmacologic and stem cell interventions ameliorate ENT3-disease pathologies and extend the lifespan of ENT3-deficient mice. These findings delineate a primary pathogenic basis for the development of ENT3 spectrum disorders and offer critical mechanistic insights into treating human ENT3-related disorders.


Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Transporte de Nucleosídeos/metabolismo , Adenosina/metabolismo , Adenilato Quinase/metabolismo , Células-Tronco Adultas/ultraestrutura , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Autofagia , Transporte Biológico , Diferenciação Celular , Autorrenovação Celular , Metabolismo Energético , Ácidos Graxos/metabolismo , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fenótipo , Ribonucleotídeos/farmacologia , Transdução de Sinais , Análise de Sobrevida , Serina-Treonina Quinases TOR/metabolismo
19.
J Agric Food Chem ; 67(32): 8794-8809, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31345023

RESUMO

Oxidative stress may play a critical role in the progression of liver disorders. Increasing interest has been given to the associations among diet, oxidative stress, gut-liver axis, and nonalcoholic fatty liver disease. Here, we investigated the effects of processed meat proteins on biomarkers of lipid homeostasis, hepatic metabolism, antioxidant functions, and gut microbiota composition in glutaredoxin1 deficient (Glrx1-/-) mice. The wild-type (WT) and Glrx1-/- mice were fed a soy protein diet (SPD), a dry-cured pork protein diet (DPD), a braised pork protein diet (BPD), and a cooked pork protein diet (CPD) at a dose of 20% of protein for 3 months. Serum and hepatic total cholesterol, serum endotoxin, hepatic liver droplet %, and antioxidant capacity were significantly increased in the CPD fed WT mice. In addition, CPD fed Glrx1-/- mice significantly increased total cholesterol, triacylglycerol, and pro-inflammatory cytokines which are accompanied by higher steatosis scores, intrahepatic lipid accumulation, and altered gene expression associated with lipid metabolism. Furthermore, hepatic gene expression of Nrf2/keap1 signaling pathway and its downstream signaling targets were determined using RT-qPCR. Glrx1 deficiency increased Nrf2 activity and expression of its target genes (GPx, catalase, SOD1, G6pd, and Bbc3), which was exacerbated by intake of CPD. Metagenomic analyses revealed that Glrx1-/- mice fed meat protein diets had higher abundances of Mucispirillum, Oscillibacter, and Mollicutes but lower abundances of Bacteroidales S24-7 group_norank, Blautia, and Anaerotruncus than their wild-type counterparts. In summary, Glrx1 deficiency induced an increase in serum biomarkers for lipid homeostasis, gut microbiota imbalance, and upregulation of Nrf2/Keap1 and antioxidant defense genes, which was aggravated by cooked meat protein diet.


Assuntos
Glutarredoxinas/genética , Inflamação/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lipogênese , Fígado/metabolismo , Produtos da Carne/efeitos adversos , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Colesterol/sangue , Citocinas/metabolismo , Feminino , Microbioma Gastrointestinal , Glutarredoxinas/deficiência , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/microbiologia , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Carne Vermelha , Transdução de Sinais , Triglicerídeos/sangue
20.
Toxicol Lett ; 313: 188-195, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31284022

RESUMO

Brucine is one of the main bioactive and toxic constituents of the herb drug Semen Strychni. Here we aimed to determine dosing time-dependent hepatotoxicity of brucine, and to investigate the role of metabolism in generation of brucine chronotoxicity. Brucine was administered to wild-type or Npas2-/- (a clock disrupted model) mice at different circadian time points for toxicity and pharmacokinetic characterization. The hepatotoxicity was evaluated by plasma alanine aminotransferase and aspartate aminotransferase measurements and histopathological analysis. The role of Cyp3a11 in brucine metabolism was determined by chemical inhibition assays and Cyp3a11-overexpressing HEK293 cells. Hepatic circadian Cyp3a11 mRNA and protein levels were determined by qPCR and Western blotting, respectively. The toxicity of brucine was more severe in the light phase [Zeitgeber time (ZT) 2 and ZT8] than in the dark phase (ZT14 and ZT20). Chemical inhibition and substrate metabolism assays suggested Cyp3a11 as a significant contributor to brucine metabolism. The Cyp3a11 mRNA, protein and activity in the livers of wild-type mice displayed significant circadian fluctuations. Npas2 ablation markedly down-regulated Cyp3a11 mRNA, protein and activity, and abrogated their circadian rhythms. The circadian time differences in brucine pharmacokinetics and liver distribution were lost in Npas2-/- mice, so were the time differences in brucine hepatotoxicity. In conclusion, chronotoxicity of brucine was determined by circadian variations in Cyp3a11 metabolism. The findings have implications in improving brucine (and possibly Semen Strychni) efficacy via dosing time optimization.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Ritmo Circadiano , Citocromo P-450 CYP3A/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Proteínas de Membrana/metabolismo , Fotoperíodo , Estricnina/análogos & derivados , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Ritmo Circadiano/genética , Cronoterapia Farmacológica , Células HEK293 , Humanos , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Estricnina/administração & dosagem , Estricnina/metabolismo , Estricnina/farmacocinética , Estricnina/toxicidade
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