Effects of antioxidant and NF-κB on the induction of iNOS gene in rat pulmonary microvascular endothelial cells in vitro / 中国病理生理杂志

ABSTRACT

AIM：To investigate the role of NF-κB in the activation of inducible nitric oxide synthase (iNOS) gene by tumor necrosis factor alpha (TNF α) and lipopolysaccharide (LPS) in endothelial cells and effect of antioxidant on the induction of iNOS. METHODS：Rat pulmonary microvascular endothelial cell (RPMEC) was cultured and the cells were identified with antiendothelial cell antibody CD31 using immunohistochemistry(ABC). The concentration of nitrite in the culture media was determined based on Griess reaction. iNOS mRNA was analyzed using RT-PCR and Northern blot. NF-κB in cell nuclei was detected with electrophoresis mobility shift assay (EMSA). RESULTS：A marked production of nitrite in RPMECs was found after 24 hours treatment with TNF α(105 U/L) and LPS (1 mg/L) (P＜0.01). The level of iNOS mRNA increased significantly after adding TNF α(105 U/L) and LPS (1 mg/L) to the cell media for 2 hours (P＜0.05). Pretreatment with cycloheximide (CHX, 10 mg/L) or antioxidant, PDTC (0.1 mmol/L) or NAC (20 mmol/L) significantly decreased nitrite production and iNOS mRNA expression induced by TNF α(105 U/L) and LPS (1 mg/L) (P＜0.05). Furthermore, there was a dose-effect relationship between PDTC/NAC and inhibitory effect. TNF α (105 U/L) and LPS (1 mg/L) triggered the activation and translocation of NF-κB. This effect was blocked by adding PDTC (0.1 mmol/L) or NAC (20 mmol/L) to the cell media for 1.5 hours.CONCLUSION：1.TNFα and LPS may induce iNOS gene expression at transcriptional or posttranscriptional level. The upregulation of iNOS depends on new protein synthesis. 2. The induction of iNOS gene expression by TNFα and LPS is dependent on the activation of NF-κB. 3. Antioxidants may inhibit the induction of iNOS gene through the inhibition of NF-κB activation.