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Biomed Chromatogr ; 34(3): e4767, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31785600


Disorders of certain branched-chain amino acids may be associated with the occurrence and development of non-alcoholic fatty liver disease. Measurement of related branched-chain amino acid levels could provide a reference for the clinical and scientific research of the non-alcoholic fatty liver disease. An established HPLC-FLD method was used to quantify aspartic acid, glutamate, glutamine, glycine, taurine, tyrosine, 4-amino butanoic acid, tryptophan, methionine, valine, phenylalanine, isoleucine and leucine in mouse brain tissue. Brain tissue samples mixed with internal standard (3-aminobutyric acid) were processed, then derivatized with 2-O-phthaldialdehyde, and finally separated on an ODS2 column through gradient elution at a flow rate of 1.0 ml·min-1 . The excitation and emission wavelengths were set at 340 and 455 nm, respectively. The mobile phase A was 100% methanol and the mobile phase B consisted of 30 mmol·L-1 sodium acetate (pH 6.8). The injection volume was 20 µl and the single run time was 45 min. Several parameters, accuracy, precision, and stability, were verified and the results showed the established method had good sensitivity and resolution for all of the 13 compounds and internal standard in mouse brain.

Curr Med Sci ; 39(4): 526-533, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31346986


Activation of macrophages is a key event for the pathogenesis of various inflammatory diseases. Notch signaling pathway recently has been found to be a critical pathway in the activation of proinflammatory macrophages. Salidroside (Sal), one of main bioactive components in Rhodiola crenulata (Hook. F. et Thoms) H. ohba, reportedly possesses anti-inflammatory activity and ameliorates inflammation in alcohol-induced hepatic injury. However, whether Sal regulates the activation of proinflammatory macrophages through Notch signaling pathway remains unknown. The present study investigated the effects of Sal on macrophage activation and its possible mechanisms by using both alcohol and lipopolysaccharide (LPS) to mimic the microenvironment of alcoholic liver. Detection of THP-1-derived macrophages exhibited that Sal could significantly decrease the expression of tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1ß) and IL-6 in the macrophages at both mRNA and protein levels. Furthermore, Sal significantly suppressed NF-κB activation via Notch-Hes signaling pathway in a dose-dependent manner. Moreover, in the microenvironment of alcoholic liver, the expression of Notch-dependent pyruvate dehydrogenase phosphatase 1 (PDP1) was elevated, and that of M1 gene expression [inducible NO synthase (NOS2)] was up-regulated. These changes could all be effectively ameliorated by Sal. The aforementioned findings demonstrated that Sal could inhibit LPS-ethanol-induced activation of proinflammatory macrophages via Notch signaling pathway.

Glucosídeos/farmacologia , Inflamação/tratamento farmacológico , Fígado/efeitos dos fármacos , Fenóis/farmacologia , Rhodiola/química , Citocinas/genética , Etanol/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Interleucina-1beta/genética , Interleucina-6/genética , Lipopolissacarídeos/toxicidade , Fígado/lesões , Fígado/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , NF-kappa B/genética , Proteína Fosfatase 2C/genética , RNA Mensageiro/genética , Receptores Notch/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética
Sheng Li Xue Bao ; 59(2): 197-203, 2007 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-17437043


To investigate the roles of glycogen synthase kinase 3beta (GSK3beta) and adenomatous polyposis coli (APC) protein in wound repair of airway epithelial cells (AECs), we established a wound model of airway epithelium in vitro. Then the following tests were undertaken: (1) Western blot was used to detect the levels of total GSK3beta and phosphorylated GSK3beta in human bronchial epithelial (16HBE) cells; (2) The localizations of APC protein was observed by using immunofluorescence technique; (3) Immunoprecipitation was used to investigate the relationship between APC protein and GSK3beta during the repair of 16HBE cells. The results were as follows: (1) The level of phosphorylated GSK3beta increased 0.5 h after scratching (P<0.05), reached a maximum at 6 h (P<0.05), and maintained until 12 h, while the total level of GSK3beta remained constant; (2) Results of immunofluorescence study showed that APC protein clustered with tubulin in the region of the migrating leading cells 6 h after scratching, which was dissimilar with that in the cells 0 h after scratching; (3) GSK3beta and APC protein were immunoprecipitated and analysed on SDS-PAGE. We found that GSK3beta and APC protein were precipitated, indicating that the two proteins existed in a complex. After scratching, dissociation of the two proteins occurred. Taken together, we conclude that scratching caused a decrease in phosphorylation of GSK3beta, and that reduced phosphorylation of GSK3beta promoted APC protein to bind to the plus ends of microtubules and stabilize the growing ends. These observations suggest that APC protein and GSK3beta may synergistically play an important role in the repair of airway epithelium.

Proteína da Polipose Adenomatosa do Colo/fisiologia , Brônquios/lesões , Células Epiteliais/patologia , Quinase 3 da Glicogênio Sintase/fisiologia , Cicatrização/fisiologia , Brônquios/citologia , Linhagem Celular , Células Epiteliais/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Fosforilação
Sheng Li Xue Bao ; 59(2): 204-9, 2007 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-17437044


The effect of glycogen synthase kinase 3beta (GSK3beta) has been repeatedly implicated in cell proliferation, but studies on the effect of GSK3beta in different cell lines with different stimuli have drawn different conclusions. To investigate the direct effect of GSK3beta on cell growth in human lung adenocarcinoma cell line A549, we changed its activity by transient transfection with two kinds of GSK3beta mutant plasmids, constitutively active form S9A-GSK3beta and dominant negative form KM-GSK3beta. Twenty-four hours later, cell counting, flow cytometry and Western blot detection were made respectively. The results showed that enhancing GSK3beta activity caused a decrease in cell number, as well as a higher percentage of cells at G(1) phase. Further, the expression of cyclin D1 was down-regulated by GSK3beta. Taken together, our observations suggest that GSK3beta may induce G(1) cell cycle arrest in a cyclin D1-dependent fashion and therefore possibly plays a growth-inhibitory role in A549 cells.

Adenocarcinoma/patologia , Pontos de Checagem do Ciclo Celular , Ciclina D1/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Neoplasias Pulmonares/patologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Glicogênio Sintase Quinase 3 beta , Humanos , Transfecção
Food Chem Toxicol ; 44(9): 1590-6, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16750592


Epidemiological evidence suggests that cigarette smoke induces squamous metaplasia in human tracheobronchial epithelium that can progress to lung squamous carcinoma. But it is not well understood how tracheobronchial epithelial cells transduce the signals that mediate cigarette smoke-induced squamous differentiation or squamous metaplasia. In the present study, we found that in vitro cigarette smoke components notably inhibited glycogen synthase kinase 3 (GSK3) and induced the expression of involucrin, a marker of squamous differentiation. The inactivation of GSK3 by two highly selective inhibitors, lithium and SB216763, also significantly enhanced involucrin expression in cultured porcine tracheobronchial epithelial cells (PTBECs). Moreover, we demonstrated that cigarette smoke components significantly promoted activator protein-1 (AP-1) binding activities to the upstream regulatory region of involucrin gene, and similar results were observed by further studies through using GSK3 inhibitors to imitate the effects of cigarette smoke components. Taken together, we conclude that GSK3 is involved in involucrin expression induced by cigarette smoke in PTBEC probably via negatively regulating AP-1 activity, implying a possible mechanism responsible for squamous differentiation induced by cigarette smoke.

Transformação Celular Neoplásica/induzido quimicamente , Quinase 3 da Glicogênio Sintase/metabolismo , Mucosa Respiratória/enzimologia , Fumaça/efeitos adversos , Animais , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/patologia , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Indóis/farmacologia , Lítio/farmacologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Maleimidas/farmacologia , Metaplasia/induzido quimicamente , Metaplasia/enzimologia , Metaplasia/patologia , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/patologia , Precursores de Proteínas/metabolismo , Mucosa Respiratória/patologia , Transdução de Sinais/efeitos dos fármacos , Suínos , Fator de Transcrição AP-1/biossíntese , Fator de Transcrição AP-1/genética