RESUMEN
OBJECTIVE: To investigate the mechanism of chrysin in regulating lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. METHODS: RAW264.7 cells were treated with different concentrations (0, 5, 10, 20, 40, 60, 80, 100, 150, and 200 µg/mL) of chrysin for 24 h, and the cell viability was measured using CCK-8. RAW264.7 cells were pre-treated with 10, 30, or 60 µg/mL chrysin for 2 h before stimulation with LPS for different times. The levels of TNF-α, IL-6 and MCP-1 were detected by ELISA, and Western blotting was used to detect the phosphorylation of JAK- 1, JAK-2, STAT-1 and STAT-3. The level of reactive oxygen species in RAW264.7 cells was detected by CM-H2DCFDA fluorescence probe. The effect of ROS on LPS-induced JAK-STATs signal and the inflammatory response of RAW264.7 cells was detected by ROS scavenger NAC. The transcription factors STAT-1 and STAT-3 nuclear translocation were observed by laser confocal microscopy. RESULTS: Chrysin below 60 µg/mL did not significantly affect the viability of RAW264.7 cells. At 10, 30, and 60 µg/mL, chrysin dose-dependently inhibited the expression of iNOS induced by LPS. Chrysin treatment also inhibited LPS-induced phosphorylation of JAK-STATs, nuclear translocation of STAT1 and STAT3, release of TNF-α, IL-6 and MCP-1, and the production of ROS in RAW264.7 cells; ROS acted as an upstream signal to mediate the activation of JAK-STATs signaling pathway. CONCLUSION: Chrysin blocks the activity of JAK-STATs mediated by ROS to inhibit LPS-induced inflammatory response in RAW264.7 cells.
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Antiinflamatorios/farmacología , Flavonoides/farmacología , Macrófagos/efectos de los fármacos , Transducción de Señal , Animales , Citocinas/metabolismo , Janus Quinasa 1/metabolismo , Janus Quinasa 2/metabolismo , Lipopolisacáridos , Macrófagos/metabolismo , Ratones , Óxido Nítrico Sintasa de Tipo II/metabolismo , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
OBJECTIVE: To investigate the effect of Nogo-66 receptor (NgR) silencing with specific small interfering RNA (siRNA) on brain injury repair in preterm rats with brain injury caused by intrauterine infection and related mechanism of action. METHODS: The pregnant Sprague-Dawley rats (with a gestational age of 15 days) were selected, and premature delivery was induced by RU486 or lipopolysaccharide (LPS). The preterm rats delivered by those treated with RU486 were selected as the control group. The preterm rats with brain injury caused by intrauterine infection induced by LPS were divided into model, empty vector, and NgR-siRNA groups, with 36 rats in each group. The rats in the control and model groups were given routine feeding only, and those in the empty vector and NgR-siRNA groups were given an injection of lentiviral empty vector or NgR-siRNA lentivirus via the lateral ventricle on postnatal day 1 (P1) and then fed routinely. On P3, P7, and P14, 8 rats in each group were randomly selected and sacrificed to harvest the brain tissue. RT-PCR was used to measure the mRNA expression of NgR. Western blot was used to to measure the protein expression of active RhoA. The immunofluorescence histochemistry was used to determine the degree of activation of microglial cells and the morphology of oligodendrocyte precursor cells (OPCs). Hematoxylin and eosin staining was used to observe the pathological changes in brain tissue. The behavioral score was evaluated on P30. RESULTS: On P3, the NgR-siRNA group had significantly lower mRNA expression of NgR and protein expression of active RhoA in brain tissue than the model and empty vector groups (P<0.05). In each group, the mRNA expression of NgR was positively correlated with the protein expression of active RhoA (P<0.05). The results of immunofluorescence histochemistry showed that on P3, the NgR-siRNA group had a significantly reduced fluorescence intensity of the microglial cells labeled with CD11b compared with the model and empty vector groups (P<0.05). The OPCs labeled with O4 antibody in the four groups were mainly presented with tripolar cell morphology. The results of pathological examination showed a normal structure of white matter with clear staining in the periventriclar area in the control group, a loose structure of white matter with disorganized fibers and softening lesions in the model and empty vector groups, and a loose structure of white matter with slightly disorganized fibers, slight gliocyte proliferation, and no significant necrotic lesions in the NgR-siRNA group. As for the behavioral score, compared with the model and empty vector groups, the NgR-siRNA group had a higher score in the suspension test, a longer total activity distance, and greater mean velocity and number of squares crossed, as well as a shorter time of slope test and a shorter time and distance of activity in the central area (P<0.05), while there were no significant differences in these parameters between the NgR-siRNA and control groups (P>0.05). CONCLUSIONS: NgR silencing with specific siRNA can effectively silence the expression of NgR in pertem rats with brain injury caused by interauterine infection and has a significant neuroprotective effect in brain injury repair.
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Lesiones Encefálicas/terapia , Receptor Nogo 1/genética , ARN Interferente Pequeño/genética , Animales , Animales Recién Nacidos , Femenino , Silenciador del Gen , Infecciones/complicaciones , Lentivirus/genética , Masculino , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To investigate the molecular mechanism by which salidroside protects PC12 cells from H2O2-induced apoptosis. METHODS: PC12 cells cultured in DMEM supplemented with 10% horse serum and 5% fetal bovine serum were pretreated with different doses of salidroside for 2 h and then stimulated with H2O2 for different lengths of time. The expression levels of PARP and caspase 3 and the phosphorylation of p38, ERK and JNK were determined with Western blotting. The cell nuclear morphology was observed after DAPI staining. The production of ROS was detected using a ROS detection kit, and the levels of gp91phox and p47phox in the membrane and cytoplasm were detected by membrane-cytoplasm separation experiment; the binding between gp91phox and p47phox was assayed by coimmunoprecipitation experiment. RESULTS: Salidroside dose-dependently suppressed cell apoptosis, lowered phosphorylation levels of p38, ERK and JNK, inhibited the production of ROS, reduced the binding between gp91phox and p47phox, and inhibited the activity of NOX2 in PC12 cells exposed to H2O2. CONCLUSION: Salidroside protects PC12 cells from H2O2-induced apoptosis at least partly by suppressing NOX2-ROS-MAPKs signaling pathway.
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Apoptosis , Glucósidos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Fenoles/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Caspasa 3/metabolismo , Peróxido de Hidrógeno , NADPH Oxidasa 2 , Fármacos Neuroprotectores/farmacología , Células PC12 , Fosforilación , RatasRESUMEN
The diagnostic value of membrane glycolipid biochemistry index, the lipid-bound sialic acid (LSA) and total sialic acid (TSA) in cerebrospinal fluid (CSF) was evaluated in 30 intracranial and 65 gastrointestinal tumors. The plasma LSA, TSA and red cell membrane sialic acid (R-SA) in were determined according to the method of Sevenmerhulm. Our results showed that the levels of LSA and TSA in CSF of intracranial tumor patients was higher than that of normal group(p<0.01). The concentration of TSA and LSA in patients with malignant glioma was higher than that of benign meningioma patients(P<0.01). No significance was found between intracranial halmatoma patients and normal control group for levels of membrane glycolipids (p>0.05). Results also found that the plasma LSA, TSA and R-SA of gastric carcinoma were significantly higher than those of control group (p<0.05); while no significant difference was found in the plasma LSA, TSA and R-SA levels between chronic gastritis, gastrohelcoma and normal control group (p>0.05). Plasma LSA, TSA and R-SA levels of gastric carcinoma patient were significantly higher than those of chronic gastritis patients and gastrohelcoma patients(p<0.05). It was also found that plasma LSA, TSA and R-SA contents were significantly higher in large intestine carcinoma patients than in benign in stestine tumor patients (p<0.05) while no significant difference was found between intestine benign tumor and normal control group (p>0.05). The levels of LSA, TSA and R-SA were obviously higher in the patients with metastasis than in the ones without (p<0.05.) The membrane glycolipid biochemistry index LSA and TSA in CSF are sensive markers for diagnosing intracranial tumors. For gastrointestinal malignant tumors the plasma LSA TSA and red blood cell membrane SA may be considered as auxiliary indicators for diagnosis. They can be used for distinguishing benign from malignant tumors.
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Neoplasias Encefálicas/patología , Membrana Eritrocítica/metabolismo , Gangliósidos/metabolismo , Neoplasias Gastrointestinales/patología , Glucolípidos/metabolismo , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/líquido cefalorraquídeo , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/diagnóstico , Gangliósidos/sangre , Gangliósidos/líquido cefalorraquídeo , Gastritis/patología , Neoplasias Gastrointestinales/diagnóstico , Glioma/diagnóstico , Glioma/patología , Glucolípidos/sangre , Glucolípidos/líquido cefalorraquídeo , Humanos , Meningioma/diagnóstico , Meningioma/patología , Ácido N-Acetilneuramínico/sangre , Ácido N-Acetilneuramínico/líquido cefalorraquídeo , Ácido N-Acetilneuramínico/metabolismoRESUMEN
To study the effect of sodium aescinate in inducing human breast cancer MCF-7 cells apoptosis and its possible mechanism. MTT assay was used to detect the inhibitory effect of sodium aescinate on the proliferation of MCF-7 cells. The morphological changes were observed under inverted microscope. DAPI nuclear staining was used to detect the changes in cell nucleus. Annexin V-FITC/PI flow cytometry was adopted to test the apoptosis rate. Changes in apoptosis-related proteins (PARP, cleaved caspase-8 and pro-caspase-3), cell survival-associated signal molecules (AKT and ERK) and their common upstream kinase SRC was detected by Western blotting. The result showed that after different concentrations of sodium aescinate were used to treat breast cancer MCF-7 cells, they inhibited the proliferation of MCF-7 cells in a dose-dependent manner, induced cell apoptosis (typical morphological changes in nucleus, significant increase in cell apoptosis rate). The expressions of cleaved PARP and caspase-8 increased, while the expression of pro-caspase-3 decreased, which further verified sodium aescinate's effect in inducing cell apoptosis. Sodium aescinate significantly inhibited the phosphorylation of cell survival-related signal molecules (AKT, ERK) and down-regulate the activation of their common up-stream kinase SRC. The findings indicated that sodium aescinate can block signals transiting to downstream molecules AKT, ERK, inhibit the proliferation of breast cancer cell MCF-7 cell apoptosis and induced cell apoptosis by suppressing the activation of SRC.