RESUMEN
OBJECTIVE: 4-Hydroxynonenal (4-HNE) can increase the synthesis of interleukin-8 (IL-8) in bronchial epithelium cells (16HBE). This study was to explore the role of ginkgolide B in inhibiting the synthesis of IL-8 induced by 4-HNE in 16HBE. METHODS: The experiments were divided into 3 groups: a group treated with 4-HNE (10 micromol/L), a group treated with ginkgolide B (100 micromol/L) + 4-HNE (10 micromol/L), and a control group. IL-8 and IL-8 mRNA were measured after 4-HNE (or serum-free medium) stimulation for 0.5, 2, 4, 8, 12 hours. The phosphorylation of ERK1/2, JNK, p38MAPK and the combining activity of AP-1 after 4-HNE stimulation for 0.5, 2, 4, 8, 12 hours were all examined. IL-8 and the combining activity of AP-1 were measured after the 16HBE were pre-incubated with 50 micromol/L PD98059 (MEK1 inhibitor) for 2 hours before 4-HNE stimulation. The combining activity of AP-1 in 10 micromol/L 4-HNE, 100 micromol/L ginkgolide B + 10 micromol/L 4-HNE, and the control groups were all measured by EMSA. RESULTS: The level of IL-8 in 10 micromol/L 4-HNE, 100 micromol/L ginkgolide B + 10 micromol/L 4-HNE, the control groups after 4-HNE stimulating for 4 h were (98.3 +/- 4.2), (88.2 +/- 5.3), (65.3 +/- 6. 2) and (116.5 +/- 5.6), (102.8 +/- 4.7), (63.7 +/- 6.6) microg/L for 12 h. The level of IL-8 and IL-8 mRNA after 4-HNE stimulation in the ginkgolide B + 4-HNE group were lower than those in the 4-HNE group while higher than those in the control groups. The level of phosphorylation of ERK1 in the 4-HNE group at 0.5, 2, 4, 8, 12 hours were higher than those in the control groups (t = 2.83 - 14.03, P < 0.05). The AP-1 combining activity in the 4-HNE group, the ginkgolide B + 4-HNE group, PD98059 + 4-HNE group, and the control group were significantly different (F = 21.49 - 194.16, P < 0.01). The expression of IL-8 and the AP-1 combining activity in groups of pre-incubated with PD98059 2 hours before 4-HNE stimulation were lower than that without PD98059. The combining activity of AP-1 in the ginkgolide B + 4-HNE group was decreased as compared to the 4-HNE groups. CONCLUSION: 4-HNE increased the expression of Interleukin-8 in bronchial epithelium cells, via increasing the transcription activities of AP-1 by ERK1 cell signal transduction pathways. Ginkgolide B inhibited synthesis of IL-8 by blocking ERK1-AP1 transduction pathways.
Asunto(s)
Aldehídos/efectos adversos , Células Epiteliales/efectos de los fármacos , Ginkgólidos/farmacología , Interleucina-8/metabolismo , Lactonas/farmacología , Células Cultivadas , Antagonismo de Drogas , Células Epiteliales/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas c-jun/metabolismoRESUMEN
OBJECTIVE: To study the effect of benzo(a)pyrene [B(a)P] on the expression of gamma-glutamate-cysteine ligase (gamma-GCS) in rat alveolar epithelium cells (CCL-149 cell line). METHODS: Rat alveolar cells of the line CCL-149 were cultured and exposed to B(a)P of the concentrations of 0, 100, 500, and 5000 microg/L respectively for 24, 36, 48, and 72 hours respectively. Then the A values was measured to observe the influence of B(a)P on the growth of the CCL-149 cells. Another CCL-149 cells were exposed to B(a)P of the concentration of 200 microg/L for 6 h, then the nuclear protein was extracted. Electrophoretic mobility shift assays (EMSA) and antibody supershift assay were used to observe the specific binding of aryl hydrocarbon receptor nuclear translocator (ARNT) to the E-box element. CCL-149 cells were cultured to 95% confluent and transfected with GCLC-luc and GCLC-delE-box-luc for 6 h, exposed to B(a)P of different concentrations (2, 20, and 200 microg/L) for 2, 6, 12, or 24 h, then the cells were harvested and the luciferase activity was measured. Cells treated with DMSO were used as negative control group. RESULTS: B(a)P of the concentrations over 5000 microg/L significantly influenced the growth of the CCL-149 cells (all P < 0.01). Treated with B(a)P induced binding of AHR/ARNT to E-box element was significantly increased by treatment of B(a)P. Treatment of B(a)P of different concentrations at different time points did not significantly influence the luciferase activity (all P > 0.05). CONCLUSION: B(a)P induces the binding of ARNT to the E-box element on the gamma-GCS gene, but this interaction between E-box and ARNT seems not to have effect on the gene expression of gamma-GCS.
Asunto(s)
Benzo(a)pireno/farmacología , Células Epiteliales/efectos de los fármacos , Glutamato-Cisteína Ligasa/metabolismo , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Elementos E-Box/genética , Células Epiteliales/metabolismo , Glutamato-Cisteína Ligasa/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Alveolos Pulmonares/citología , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Factores de Tiempo , TransfecciónRESUMEN
OBJECTIVE: To study the effects of allitridum on the antioxidative capability of rat lung epithelial L2 cells (CCL-149 cell line) by observing the changes of glutathione (GSH), malondialdehyde (MDA) content, and the expressing level of gamma-glutamyl-cysteine synthetase (gamma-GCS) protein. METHODS: The cell viability of CCL-149 cells treated with allitridum was detected by MTT assay. CCL-149 cells were incubated with 0, 0.1, 1.0, 5.0 microg/ml of allitridum for 6, 12, 24, 48 h. After treatment, GSH content and MDA formation were measured by spectrophotometric assay, and the gamma-GCS protein was semi-quantified by Western blot. RESULTS: Allitridum (0.1, 1.0, 5.0 microg/ml) showed no side effects on cell growth (P > 0.05). Treatment with allitridum increased GSH levels and decreased MDA formation in CCL-149, the most significant time being incubation at 24 h. The GSH content were (13.34 +/- 0.62), (27.67 +/- 2.39), (29.54 +/- 0.71), (30.25 +/- 1.05) mg/g prot, and the MDA content were (1.25 +/- 0.08), (0.90 +/- 0.06), (0.84 +/- 0.06), (0.81 +/- 0.02) nmol/mg prot when CCL-149 cells were treatment with 0, 0.1, 1.0, 5.0 microg/ml of allitridum for 24 h, respectively. The effect showed no dose dependent effects (P > 0.05). After 6, 12, 24 h incubation, the expression of gamma-GCS protein was increased as compared to the control cells (t = 2.82 - 9.92, P < 0.05). CONCLUSION: Allitidum may increase the antioxidative ability of CCL-149 cells by increasing the expression of gamma-GCS protein and the content of GSH.