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1.
Altern Ther Health Med ; 29(5): 242-254, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37052973

RESUMO

Context: The Da-yuan-yin (DYY) decoction is a classical prescription of traditional Chinese medicine that has antipyretic and anti-inflammatory effects. Network Pharmacology (NP) is an emerging discipline based on system-biology theory and biosystem network analysis that researchers can use to predict drug-action targets and mechanisms. Objective: The study intended to use NP evaluate the protective effects of the fifth eluting fraction of the supernatant of the DYY decoction (DYY-5) for mice induced with acute lung injury (ALI) using lipopolysaccharide (LPS) and to explore DYY-5's mechanisms. Design: The research team performed an animal study. Setting: The study took place at the College of Pharmaceutical Science at Soochow University in Suzhou, China. Animals: The animals were 42 male Balb/c mice, about 20 to 25 g in weight. Intervention: The research team: instilled 2 mg/kg of LPS intratracheally (i.t.) to induce ALI. The team divided the mice into seven groups of six mice: (1) a control group; (2) a negative control group-the DYY-5 group with mice treated only with a high dosage, 60 mg/kg, of DYY-5 to investigate the effects of DYY-5 on normal mice; (3) the positive control group, the LPS group, with induced ALI but no treatments; (4) the LPS+60 mg/kg-DYY-5 group with induced ALI treated with a high dosage of DYY-5; (5) the LPS+30 mg/kg-DYY-5 group with induced ALI treated with a medium dosage of DYY-5; (6) the LPS+15 mg/kg-DYY-5 group with induced ALI treated with a low dosage of DYY-5; and (7) a reference drug control group, the LPS+DXM group, with induced ALI treated with 5 mg/kg of dexamethasone (DXM). Outcome Measures: The research team: (1) determined the chemical components of DYY; (2) identified the anticomplementary activities of DYY-5; (3) took lung specimens, serum, and bronchoalveolar lavage fluid (BALF) from the mice for histopathological examination, Western blot, and biochemical analysis; (4) measured total protein concentrations and lung W/D ratios; (5) measured the expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) using quantitative real-time polymerase chain reaction (PCR); (6) measured the levels of pro-inflammatory and anti-inflammatory factors, the activity of myeloperoxidase (MPO) and superoxide dismutase (SOD), and the levels of complements, including complements 3 (C3), C3c, C5a, C5aR1, and C5b-9, using kits; (7) analyzed the levels of nuclear factor-kappa B (NF-κB) and IkB kinase (IKK) using Western blot; and (8) used network pharmacology (NP) to predict DYY-5's mechanisms and potential targets. Results: The study's results were consistent with the NP analysis, which reflected the multitarget and multipathway characteristics of DYY-5 in alleviating ALI. The LPS+30 mg/kg-DYY-5 group had significantly lower lung wet-to-dry (W/D) ratios and total protein concentrations in BALF than the LPS group did, with P < .01 and P < .0001, respectively as did the LPS+60 mg/kg-DYY-5 group (both P < .0001). The 60 mg/kg of DYY-5 compared to the LPS group: (1) regulated the levels tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and interleukin-1 beta (IL-1ß), with all P < .0001, anti-inflammatory factors-IL-4 (P < .05), IL-10 (P < .001), and IL-13 (P < .001); (2) increased the activity of SOD (P < .0001) and decreased the activity of MPO (P < .0001) and the expressions of iNOS and COX-2 mRNA (both P < .01); (3) blocked the activation of NF-κB and IKK; and (4) alleviated the pathological changes in the lung tissue, by reducing the depositions of C3c and decreasing the levels of C3, C5a and C5aR1 (all P < .0001), C5b-9 (P < .001) and C3c (P < .01) in serum. Conclusions: The protective effects of DYY-5 on ALI were related to antioxidation, anti-complementary activities, and regulation of inflammatory factors through the IKK/NF-κB signal pathway. DYY-5 may be useful as a potential therapeutic agent for treating ALI in clinics.


Assuntos
Lesão Pulmonar Aguda , NF-kappa B , Masculino , Camundongos , Animais , NF-kappa B/genética , NF-kappa B/metabolismo , Lipopolissacarídeos , Ciclo-Oxigenase 2/efeitos adversos , Complexo de Ataque à Membrana do Sistema Complemento/efeitos adversos , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Camundongos Endogâmicos BALB C , RNA Mensageiro , Superóxido Dismutase
4.
Pharm Biol ; 61(1): 228-240, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36655330

RESUMO

CONTEXT: Da-Yuan-Yin is a Chinese traditional prescription. OBJECTIVE: This study explores the therapeutic effects of the Da-Yuan-Yin decoction polyphenol fraction (DYY-4) on acute lung injury (ALI) in mice induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: The mice (n = 10) were orally administrated with DYY-4 (15, 30, and 60 mg/kg) or DXM (5 mg/kg), half an hour after LPS (2 mg/kg) instilled intratracheally. The protein content and the levels of inflammatory factors, the levels of complements, the mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the level of myeloperoxidase (MPO) and superoxide dismutase (SOD), the expression of the IkB kinase (IKK) and nuclear factor-kappa B (NF-κB), the lung wet-to-dry weight (W/D) ratio and lung tissue were evaluated, 24 h after LPS challenge. Network pharmacology predicted potential targets. RESULTS: DYY-4 (30, 60 mg/kg, p < 0.01, p < 0.01) decreased the lung W/D ratio, total protein concentration, the levels of C3, C3c and C5a, the levels of TNF-α, IL-6, and IL-1ß, while increased the levels of IL-4 and IL-10. DYY-4 (60 mg/kg) decreased the levels of C5aR1, C5b-9 and COX-2 mRNA (p < 0.05), the levels of MPO and iNOS mRNA, the activation of the IKK/NF-κB pathway (p < 0.01), and increased the levels of IL-13 and SOD (p < 0.01). DYY-4 (60 mg/kg) relieved the lung tissue pathological changes and reduced the C3c deposition. DISCUSSION AND CONCLUSIONS: Network pharmacology combined with animal experiments revealed the targets of DYY-4 alleviating ALI.


Assuntos
Lesão Pulmonar Aguda , NF-kappa B , Camundongos , Animais , NF-kappa B/metabolismo , Lipopolissacarídeos/toxicidade , Polifenóis/efeitos adversos , Ciclo-Oxigenase 2/genética , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/prevenção & controle , Pulmão , Superóxido Dismutase , RNA Mensageiro
5.
J Proteome Res ; 21(8): 2063-2070, 2022 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-35820187

RESUMO

Kinases play important roles in cell signaling, and adenosine monophosphate (AMP) is known to modulate cellular energy homeostasis through AMP-activated protein kinase (AMPK). Here, we explored novel AMP-binding kinases by employing a desthiobiotin-conjugated AMP acyl-phosphate probe to enrich efficiently AMP-binding proteins. Together with a parallel-reaction monitoring-based targeted proteomic approach, we uncovered 195 candidate AMP-binding kinases. We also enriched desthiobiotin-labeled peptides from adenine nucleotide-binding sites of kinases and analyzed them using LC-MS/MS in the multiple-reaction monitoring mode, which resulted in the identification of 44 peptides derived from 43 kinases displaying comparable or better binding affinities toward AMP relative to adenosine triphosphate (ATP). Moreover, our proteomic data revealed a potential involvement of AMP in the MAPK pathway through binding directly to the relevant kinases, especially MEK2 and MEK3. Together, we revealed the AMP-binding capacities of a large number of kinases, and our work built a strong foundation for understanding how AMP functions as a second messenger to modulate cell signaling.


Assuntos
Proteoma , Proteômica , Proteínas Quinases Ativadas por AMP/metabolismo , Monofosfato de Adenosina , Trifosfato de Adenosina/metabolismo , Cromatografia Líquida , Peptídeos , Proteoma/genética , Espectrometria de Massas em Tandem
6.
Toxicol Lett ; 352: 61-69, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34624459

RESUMO

Mitomycin treatment induces pulmonary toxicity, and alveolar epithelial cell senescence is crucial in the pathogenesis of the latter. However, the mechanism by which mitomycin induces alveolar epithelial cell senescence has yet to be elucidated. In this work, different doses (37.5-300 nM) of mitomycin induced the senescence of human alveolar type II-like epithelial cells and enhanced the phosphorylation of GSK3ß (S9). The GSK3ß (S9A) mutant reversed the senescence of mitomycin-treated alveolar epithelial cells. Pharmacological inhibition and gene deletion of Akt1, a kinase that regulates the phosphorylation of GSK3ß (S9), suppressed mitomycin-induced alveolar epithelial cell senescence. The knockdown of p53, a downstream effector of GSK3ß and an important regulator of cell senescence, repressed mitomycin-induced alveolar epithelial cell senescence. Treatment with baicalein weakened the phosphorylation of GSK3ß (S9) and alleviated the senescence of alveolar epithelial cells brought about by mitomycin treatment. GSK3ß (S9) phosphorylation appears to be the first signal involved in the mitomycin-induced senescence of alveolar epithelial cells and may present a potential target for attenuating mitomycin-induced pulmonary toxicity.


Assuntos
Alquilantes/toxicidade , Regulação para Baixo/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Mitomicina/toxicidade , Alvéolos Pulmonares/efeitos dos fármacos , Células A549 , Senescência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Flavanonas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Imidazóis/farmacologia , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Alvéolos Pulmonares/citologia , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
7.
Bioorg Med Chem Lett ; 50: 128319, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34403728

RESUMO

Tigliane esters show many biological activities, including anti-HIV-1 activity. Our aim in this study was to establish structure-anti-HIV activity relationships for four series of tigliane-type diterpenoids. We synthesized and evaluated 29 new phorbol ester derivatives for anti-HIV activity and for cytotoxicity against human tumor cell lines. Among them, three derivatives, two phorbol-13-monoesters (5d and 5e) and a phorbol-12,13-diester (6a), showed significant anti-HIV activity. We found that better anti-HIV activity was often associated with a shorter acyl ester at C-13. Particularly, compounds with a phenyl ring in the ester side chain exhibited excellent anti-HIV activity and had good safety indexes. Due to its significant anti-HIV potency with a high selectivity index, phorbol-12,13-dicinnamoate (6a) was chosen as the potential candidate for further preclinical trials.


Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , HIV-1/fisiologia , Ésteres de Forbol/química , Ésteres de Forbol/farmacologia , Replicação Viral/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
8.
Protein J ; 39(5): 411-421, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33009960

RESUMO

Interleukin enhancer-binding factor 2 (ILF2) forms a heterodimer with interleukin enhancer-binding factor 3 (ILF3) via double-stranded RNA-binding motif and zinc finger associated domain and thus regulates gene expression and cancer cell growth. However, how ILF2 is degraded in cells remains elusive. In this work, using stable isotope labeling by amino acids in cell culture (SILAC) quantitative proteomics, we find that ILF2 is downregulated in cells expressing cereblon (CRBN). Using affinity purification and immunoblotting analysis, we demonstrate that CRBN interacts with ILF2 and functions as a substrate receptor of the cullin-4 RING E3 ligase complex. Biochemical experiments disclose that CRBN expression reduces ILF2 protein level and this reduction is diminished when the proteasome is inhibited. Upon protein synthesis inhibition, the degradation of ILF2 is enhanced by CRBN. Moreover, CRBN promotes the ubiquitination of ILF2 and thus results in the ubiquitin-mediated proteasomal degradation. Analyses of previously identified post-translational modification sites and the crystal structure of ILF2 discover the potential ubiquitination sites on ILF2. Through mutagenesis and biochemical experiments, we further reveal that the K45R mutation completely abolishes the effect of CRBN on ILF2, suggesting that this is the key residue responsible for its ubiquitination. Taken together, we identify an E3 ligase that regulates ILF2 and uncover a molecular pathway for its degradation. This work might be helpful to elucidate the molecular mechanism by which CRBN regulates diverse cellular functions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína do Fator Nuclear 45/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal/genética , Células HEK293 , Humanos , Proteína do Fator Nuclear 45/genética , Complexo de Endopeptidases do Proteassoma/genética , Ubiquitina-Proteína Ligases/genética
9.
J Proteome Res ; 17(4): 1509-1520, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29533670

RESUMO

Protein post-translational modification by ubiquitin-fold modifier 1, UFM1, regulates many biological processes such as response to endoplasmic reticulum stress and regulation of tumor progression. A recent study has indicated that the UFM1-binding and PCI domain-containing protein 1 (UFBP1) is required for the conjugation of UFM1 to a substrate. However, other biological functions of UFBP1 have not been explored. Here, we use immunoprecipitation and label-free quantitative proteomics to identify UFBP1-interacting proteins in a mammalian cell line. About 80 potential interacting proteins are obtained from MS analyses of three biological replicates. Bioinformatics analyses of these proteins suggest that UFBP1 may participate in the regulation of protein folding, stability, and trafficking. Biochemical experiments discover that UFBP1 expression downregulates the protein level and reduces the stability of several of its interacting proteins, while UFBP1 knockdown increases their protein levels. Protein synthesis inhibition and proteasomal inhibition experiments reveal that UFBP1 promotes their ubiquitination and degradation. Experiments using a model UFBP1-interacting protein ANT3 demonstrate that UFBP1 enhances the interaction between ANT3 and its E3 ligase and thus promotes its ubiquitination and degradation. Our work elucidates a novel molecular mechanism by which UFBP1 regulates protein ubiquitination and degradation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Processamento de Proteína Pós-Traducional , Proteólise , Proteômica , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Translocador 3 do Nucleotídeo Adenina/metabolismo , Animais , Linhagem Celular , Regulação para Baixo , Células HEK293 , Humanos , Mamíferos , Ligação Proteica , Ubiquitina-Proteína Ligases/metabolismo
10.
J Asthma ; 55(2): 111-118, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28399677

RESUMO

OBJECTIVE: This study aimed to explore the value of elevated serum levels of tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and eosinophil cationic protein (ECP) in the diagnosis of bronchial asthma (BA). METHODS: A total of 170 patients with BA (case group, 85 patients in acute attack and 85 patients in clinical remission) and 150 healthy individuals (control group) were enrolled in this study. Enzyme-linked immunosorbent assay and receiver operating characteristic (ROC) curves were calculated for the contents and diagnostic values of serum TNF-α, IL-8, and ECP in BA. RESULTS: Compared with the control group, patients in acute attack and clinical remission had higher TNF-α, IL-8, and ECP levels (p < 0.05). The serum level of TNF-α was positively correlated with IL-8 and ECP (p < 0.05). ROC curves showed that the diagnostic threshold value of IL-8 was 13.53 ng/ml, its area under the curve (AUC) was 0.87, its specificity was 99.3%, and its sensitivity was 57.6%. The diagnostic threshold value of TNF-α was 1.29 ng/ml with AUC being 0.94, specificity was 89.3%, and sensitivity was 83.5%. ECP showed 7.22 ng/ml diagnostic threshold value (AUC = 0.88, specificity = 74.0%, sensitivity = 86.5%). The FEV1/pre(%) and FEV1/FVC were negatively correlated and the Z5/pre(%) and resonance frequency (Fres) were positively correlated with the serum levels of TNF-α, IL-8, and ECP in patients in acute attack and in clinical remission (all p < 0.05). CONCLUSION: Our findings reveal that elevated serum levels of TNF-α, IL-8, and ECP can be involved in the development and progression of BA.


Assuntos
Asma/sangue , Proteína Catiônica de Eosinófilo/sangue , Interleucina-8/sangue , Fator de Necrose Tumoral alfa/sangue , Adulto , Asma/etiologia , Asma/fisiopatologia , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Pessoa de Meia-Idade , Curva ROC , Capacidade Vital
11.
J Proteomics ; 148: 94-104, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27432471

RESUMO

UNLABELLED: A Disintegrin and Metalloproteinase 12 (ADAM12) is expressed significantly higher in multiple tumors than in normal tissues and has been used as a prognostic marker for the evaluation of cancer progression. Although several ADAM12 substrates have been identified biochemically and its proteolytic function has been explored, the upstream regulators and the interacting proteins have not been systematically investigated. Here, we use immunoprecipitation and mass spectrometry (MS)-based quantitative proteomic approaches to identify 28 interacting partners for the long form of ADAM12 (ADAM12-L) in HeLa cells. Proteins that regulate cell proliferation, invasion, and epithelial to mesenchymal transition are among the identified ADAM12-interacting proteins. Further biochemical experiments discover that the protein level and the stability of ADAM12 are upregulated by one of its interacting proteins, myoferlin. In addition, myoferlin also increases the proteolytic activity of ADAM12, leading to the reduction of an ADAM12 substrate, E-cadherin. This result implies that ADAM12 and its interacting proteins might converge to certain signaling pathways in the regulation of cancer cell progression. The information obtained here might be useful in the development of new strategies for modulating cell proliferation and invasion involved in the regulation between ADAM12 and its interacting partners. MS data are available via ProteomeXchange with identifier PXD003560. BIOLOGICAL SIGNIFICANCE: Regulation of the proliferation and invasion of cancer cells is important in cancer treatment. ADAM12 has been found to play important roles in regulating these processes and identification of its interacting partners will improve our understanding of its biological functions and provide basis for functional modulation. Through mass spectrometry-based quantitative proteomic approaches, we identify the interacting partners for ADAM12 in a human cancer cell line and find many proteins that are involved in the proliferation and invasion of cancer cells. A novel regulator, myoferlin, of ADAM12 is discovered and this protein increases ADAM12 expression level, stability, and its enzymatic activity, leading to the reduction of its substrate, E-cadherin, which plays important roles in the regulation of cell adhesion and tumor metastasis. This result provides a connection for two highly expressed proteins in cancer cells and may shed light on the regulation of their biological functions in cancer progression.


Assuntos
Proteína ADAM12/metabolismo , Caderinas/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Musculares/fisiologia , Mapas de Interação de Proteínas , Proteômica/métodos , Proteína ADAM12/análise , Caderinas/análise , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ligação ao Cálcio/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/isolamento & purificação , Proteínas Musculares/análise , Proteínas Musculares/isolamento & purificação , Invasividade Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia
12.
FASEB J ; 29(12): 4829-39, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26231201

RESUMO

The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains and that thalidomide blocks the formation of CRBN-ubiquitin conjugates. Furthermore, we show that ubiquitinated CRBN is targeted for proteasomal degradation. Treatment of human myeloma cell lines such as MM1.S, OPM2, and U266 with thalidomide (100 µM) and its structural analog lenalidomide (10 µM) results in stabilization of CRBN and elevation of CRBN protein levels. This in turn leads to the reduced level of CRBN target proteins and enhances the sensitivity of human multiple myeloma cells to IMiDs. Our results reveal a novel mechanism by which thalidomide and its analogs modulate the CRBN function in cells. Through inhibition of CRBN ubiquitination, thalidomide and its analogs allow CRBN to accumulate, leading to the increased cullin-4 RING E3 ligase-mediated degradation of target proteins.


Assuntos
Mieloma Múltiplo/metabolismo , Peptídeo Hidrolases/metabolismo , Talidomida/análogos & derivados , Talidomida/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Células HEK293 , Humanos , Lenalidomida , Mieloma Múltiplo/patologia , Peptídeo Hidrolases/genética
13.
PLoS One ; 10(7): e0132480, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26176961

RESUMO

The purpose of this study was to establish a method for monitoring the neural differentiation of stem cells using ferritin transgene expression, under the control of a neural-differentiation-inducible promoter, and magnetic resonance imaging (MRI). Human adipose tissue-derived mesenchymal stem cells (hADMSCs) were transduced with a lentivirus containing the human ferritin heavy chain 1 (FTH1) gene coupled to one of three neural cell-specific promoters: human synapsin 1 promoter (SYN1p, for neurons), human glial fibrillary acidic protein promoter (GFAPp, for astrocytes), and human myelin basic protein promoter (MBPp, for oligodendrocytes). Three groups of neural-differentiation-inducible ferritin-expressing (NDIFE) hADMSCs were established: SYN1p-FTH1, GFAPp-FTH1, and MBPp-FTH1. The proliferation rate of the NDIFE hADMSCs was evaluated using a Cell Counting Kit-8 assay. Ferritin expression was assessed with western blotting and immunofluorescent staining before and after the induction of differentiation in NDIFE hADMSCs. The intracellular iron content was measured with Prussian blue iron staining and inductively coupled plasma mass spectrometry. R2 relaxation rates were measured with MRI in vitro. The proliferation rates of control and NDIFE hADMSCs did not differ significantly (P > 0.05). SYN1p-FTH1, GFAPp-FTH1, and MBPp-FTH1 hADMSCs expressed specific markers of neurons, astrocytes, and oligodendrocytes, respectively, after neural differentiation. Neural differentiation increased ferritin expression twofold, the intracellular iron content threefold, and the R2 relaxation rate two- to threefold in NDIFE hADMSCs, resulting in notable hypointensity in T2-weighted images (P < 0.05). These results were cross-validated. Thus, a link between neural differentiation and MRI signals (R2 relaxation rate) was established in hADMSCs. The use of MRI and neural-differentiation-inducible ferritin expression is a viable method for monitoring the neural differentiation of hADMSCs.


Assuntos
Ferritinas/metabolismo , Células-Tronco Mesenquimais/fisiologia , Neurogênese , Células Cultivadas , Ferritinas/genética , Expressão Gênica , Humanos , Imageamento por Ressonância Magnética , Regiões Promotoras Genéticas , Ativação Transcricional
14.
Neurourol Urodyn ; 33(1): 147-52, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23495084

RESUMO

AIMS: We investigated the mechanisms of diabetic bladder dysfunction (BD) through analysis of the roles of L- and T-type voltage-gated calcium channels (VGCCs), with the ultimate goal of identifying potential drug targets for diabetic BD. METHODS: Bladder function of db/db (type 2 diabetes) and wild type (Wt) mice was evaluated by behavioral tests and in vivo cystometry. Contractile responses of bladder strips to carbachol were measured with or without pre-treatment with nifedipine (a L-type VGCC blocker) or mibefradil (a T-type VGCC blocker). Furthermore, the effects of mibefradil and nifedipine on the proliferation of human bladder smooth muscle cells (BSMCs) were studied. RESULTS: db/db mice had significantly increased voiding frequency, bladder weight, bladder compliance and capacity, and heightened contractile response to carbachol, compared to Wt mice. Nifedipine, but not mibefradil, dramatically suppressed bladder tissue contraction in Wt mice. Whereas nifedipine nearly completely inhibited bladder contraction in db/db mice, mibefradil "normalized" the heightened bladder contractility of db/db mice to the level of Wt mice. In culture, mibefradil, but not nifedipine, inhibited the proliferation of human BSMCs. CONCLUSION: Our results indicate that while L-type VGCCs play a major role in the contraction of both diabetic and non-diabetic bladders, T-type VGCCs are involved in the contraction of diabetic bladders and mediate BSMC proliferation. This study provides support for further investigations on the effect of blockade of T-type VGCC or combined blockade of both types of VGCCs in the treatment of diabetic BD.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo T/metabolismo , Diabetes Mellitus Tipo 2/complicações , Contração Muscular , Doenças da Bexiga Urinária/etiologia , Bexiga Urinária/metabolismo , Acetilcolina/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo T/efeitos dos fármacos , Canais de Cálcio Tipo T/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Masculino , Mibefradil/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Nifedipino/farmacologia , RNA Mensageiro/metabolismo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/patologia , Bexiga Urinária/fisiopatologia , Doenças da Bexiga Urinária/metabolismo , Doenças da Bexiga Urinária/patologia , Doenças da Bexiga Urinária/fisiopatologia
15.
Asian Pac J Cancer Prev ; 14(10): 5805-10, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24289581

RESUMO

THis study was conducted to analyze the molecular mechanisms responsible for anti-proliferation effects of glaucocalyxin A in cultured MCF-7 and Hs578T breast cancer cells. The concentration that reduced cell viability to 50% (IC50) after 72 h treatment was derived and potential molecular mechanisms of anti-proliferation using the IC50 were investigated as changes in cell cycle arrest and apoptosis. Gene and protein expression changes related to apoptosis were investigated by semi-quantitative RT-PCR and western blotting, respectively. Involvement of phosphorylated mitogen-activated protein kinases and JNK signaling in regulation of these molecules was characterized by western blotting. Cell viability decreased in a concentration-dependent manner and the IC50 was determined as 1 µM in MCF-7 and 4 µM in Hs578T cell. Subsequently, we demonstrated that the GLA-induced MCF-7 and Hst578T cell death was due to cell cycle arrest at the G2/M transition and was associated with activation of the c-jun N-terminal kinase (JNK) pathway. We conclude that GLA has the potential to inhibit the proliferation of human breast cancer cells through the JNK pathway and suggest its application forthe effective therapy for patients with breast cancer.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Diterpenos do Tipo Caurano/farmacologia , Proteína Ligante Fas/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Transdução de Sinais/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Células MCF-7 , Proteínas Quinases Ativadas por Mitógeno/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Transdução de Sinais/genética
16.
Tumour Biol ; 34(6): 3619-25, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23832539

RESUMO

Peroxisome proliferator-activated receptor δ gene (PPARδ) is correlated with carcinogenesis of colorectal cancer, but the regulation of its gene transcription remains unclear. We herein report that AP1 binds the promoter and regulates PPARδ gene expression. With a luciferase reporter system, we identified a functional promoter region of 30 bp of PPARδ gene by deletion and electrophoretic mobility shift assays (EMSA). Using site-directed mutagenesis and decoy analyses, we demonstrated that AP1 bound the functional transcriptional factor binding site in a region extending from -176 to -73 of the PPARδ promoter, which was confirmed using EMSA and supershift assays. Consequently, inhibition of the AP1 binding site led to decreased PPARδ mRNA. Our study demonstrated that AP1 is the transcriptional factor that contributes to PPARδ expression in LoVo cells.


Assuntos
Regulação da Expressão Gênica , PPAR delta/genética , Regiões Promotoras Genéticas/genética , Fator de Transcrição AP-1/metabolismo , Antracenos/farmacologia , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Luciferases/genética , Luciferases/metabolismo , Mutagênese Sítio-Dirigida , PPAR delta/metabolismo , Ligação Proteica/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-1/antagonistas & inibidores , Transfecção
17.
Int J Biol Macromol ; 58: 349-53, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23603082

RESUMO

In our study, a water-soluble polysaccharide (CCPa-1) was successfully purified from the fruiting bodies of Coprinus comatus by DEAE-cellulose and Sepharose CL-6B column chromatography. The molecular weight was evaluated to be 53.6 kDa as determined by high performance size exclusion chromatography (HPSEC). Sugar composition analysis revealed that CCPa-1 consisted primarily of galactose, glucose and arabinose in a molar ratio of 6.6:1.2:2.2. CCPa-1 could not only inhibit the growth of sarcoma 180 (S180) tumor transplanted in mice, but also increase the relative spleen/thymus indexes and body weight of tumor bearing mice. Moreover, Con A- or LPS-induced splenocyte proliferation was also enhanced after CCPa-1 administration in tumor-bearing mice. Furthermore, CCPa-1 significantly enhanced the Con A- or LPS-induced splenocyte proliferation and increased the production of TNF-α and IL-2. All the data demonstrated that CCPa-1 had a potential application as natural antitumor agent with immunomodulatory activity.


Assuntos
Antineoplásicos/farmacologia , Coprinus/química , Polissacarídeos Fúngicos/farmacologia , Fatores Imunológicos/farmacologia , Animais , Antineoplásicos/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Polissacarídeos Fúngicos/isolamento & purificação , Fatores Imunológicos/isolamento & purificação , Interleucina-2/sangue , Masculino , Camundongos , Camundongos Endogâmicos ICR , Sarcoma Experimental/tratamento farmacológico , Sarcoma Experimental/imunologia , Sarcoma Experimental/patologia , Baço/efeitos dos fármacos , Baço/patologia , Fator de Necrose Tumoral alfa/sangue
18.
BJU Int ; 110(3): 413-9, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22115428

RESUMO

OBJECTIVE: • To examine functional and molecular changes of the bladders from elastin-haploinsufficient mice (Eln(+/-) ) at baseline as well as in response to partial bladder outlet obstruction (pBOO). MATERIALS AND METHODS: • Female Eln(+/-) and wild type (Wt) mice (3-4 months old) were studied. • The bladder elastin content was quantified by measuring desmosine. • Mice were divided into two groups to undergo surgery to create pBOO or to undergo sham surgery. Three days after surgery, bladder function was evaluated by in vivo cystometry, and the contractile response of bladder strips exposed to electrical field stimulation (EFS) and carbachol was examined by ex vivo myography. RESULTS: • The Eln(+/-) -sham mice had a 33.6% decrease in bladder elastin compared with Wt-sham mice. • Cystometry showed significantly decreased bladder compliance and capacity in Eln(+/-) -sham vs Wt-sham mice; pBOO increased bladder compliance and capacity to a greater extent in Eln(+/-) mice compared with Wt mice. • Bladder strips from Eln(+/-) -sham mice showed a significantly heightened contractile response to both EFS and carbachol compared with Wt-sham mice. • A significantly increased contractile response to carbachol was detected in Wt-pBOO vs Wt-sham but not between Eln(+/-) -pBOO and Eln(+/-) -sham mice. CONCLUSION: • The results that elastin-deficient mice had decreased bladder compliance and capacity and increased bladder contractility; and that Wt-pBOO mice showed an enhanced contractile response to carbachol, but Eln(+/-) -pBOO mice did not, suggest that elastin is critical for normal bladder function and is involved in bladder response to pBOO.


Assuntos
Elastina/deficiência , Obstrução do Colo da Bexiga Urinária/fisiopatologia , Bexiga Urinária/fisiopatologia , Animais , Colágeno Tipo III/metabolismo , Desmosina/metabolismo , Estimulação Elétrica , Feminino , Hidroxiprolina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular/fisiologia , Tamanho do Órgão , RNA Mensageiro/metabolismo , Tropoelastina/metabolismo
19.
Fa Yi Xue Za Zhi ; 27(6): 405-8, 412, 2011 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-22393586

RESUMO

OBJECTIVE: To explore the effect of ketamine on adrenal pheochromocytoma (PC12) cell proliferation inhibition and induction of apoptosis and its mechanism. METHODS: PC12 cells of rats were models for dopaminergic neuron. PC12 cells were cultured with ketamine at concentrations of 0.9, 1.2, 1.5, 1.8 and 2.1 mmol/L, respectively. The cell viability was measured by MTT method after incubation at 12, 24, 48 and 72h. Hoechst stain was used to observe the morphological changes of apoptosis. PC12 cells cultured after 48 h with different concentrations of ketamine were selected to detect apoptotic rate using flow cytometry and detect the expression of bax and bcl-2 proteins using Western blotting. RESULTS: For different concentrations of ketamine, vitality of PC12 cells significantly decreased with increase of the incubation time. Apoptosis was obviously observed using Hoechst staining. Flow cytometry showed that apoptosis rates significantly increased with increasing ketamine concentrations. CONCLUSION: Ketamine can inhibit the proliferation of PC12 cell by inducing apoptosis of the PC12 cell in a concentrations-dependent manner. The underlying mechanism may be related to promoting the expression of bax and inhibiting the expression of bcl-2 in the cells.


Assuntos
Anestésicos Dissociativos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ketamina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Western Blotting , Relação Dose-Resposta a Droga , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Ketamina/administração & dosagem , Células PC12 , Ratos , Fatores de Tempo , Proteína X Associada a bcl-2/metabolismo
20.
Fa Yi Xue Za Zhi ; 25(5): 348-51, 358, 2009 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-20000043

RESUMO

OBJECTIVE: To explore the correlation between signs similar to schizophrenia in mice after ketamine administration and the expressions of NRG1 and ErbB4 mRNA in order to explain the possible pathogenesis of schizophrenia. METHODS: Fifty KM mice were randomly divided into 5 groups which were administered intraperitoneally with saline, clozapine and different dosages ketamine. The ketamine groups were administered intraperitoneally with low dosage (25 mg/kg), middle dosage (50 mg/kg) and high dosage (100 mg/kg) one time every day for 7 days. After administration of 100 mg/kg ketamine for 7 days, the clozapine group was introgastrically administered 20 mg/kg with clozapine one time every day for 7 days. The pathological changes of hippocampus neurons were observed by HE stain. The expressions of the NRG1 and ErbB4 mRNA in hippocampus were detected by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: In the group with high dosage of ketamine, the levels of NRG1 and ErbB4 mRNA were significantly lower than that of the group with saline. CONCLUSION: Ketamine may induce signs similar to schizophrenia in KM mice. The mechanism may be involved in the reduction of NRG1 and ErbB4 mRNA expression.


Assuntos
Receptores ErbB/metabolismo , Hipocampo/metabolismo , Ketamina/efeitos adversos , Neuregulina-1/metabolismo , Esquizofrenia/genética , Animais , Clozapina/uso terapêutico , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Receptores ErbB/genética , Hipocampo/patologia , Masculino , Camundongos , Neuregulina-1/genética , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Receptor ErbB-4 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esquizofrenia/induzido quimicamente
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