Inhibitory Effects of Paeonia Suffruticosa on the Expression of Inducible Nitric Oxide Synthase Gene in RAW264.7 Macrophages / 대한비뇨기과학회지

PURPOSE: The aim of the present study was to examine the effect of the root cortex of Paeonia suffruticosa, an anti-inflammatory folk medicine, on nitric oxide (NO) synthesis and the induction of inducible NO synthase (iNOS) gene expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. MATERIALS AND METHODS: We studied the effects of the RAW264.7 macrophage cell line of the extract from the root cortex of P. suffruticosa and NO with western blot and northern blot assays. RESULTS: We found that treatment of RAW264.7 macrophages with the ethylacetate fraction of the ethanolic extract of the root cortex of P. suffruticosa inhibits LPS-induced synthesis of NO in vitro. This effect is dose-dependent and appears to involve the suppression of iNOS gene expression, thereby reducing excessive NO synthesis in LPS-stimulated RAW264.7 macrophages. CONCLUSIONS: As NO is a pro-inflammatory molecule and iNOS inhibitors may have therapeutic potential, our findings may explain the anti-inflammatory property of the root cortex of P. suffruticosa. This root has been used in oriental folk medicine for the treatment of various inflammatory diseases with relatively little knowledge regarding its mechanism of anti-inflammatory action.

Screening of promoter DNA-binding protein of iNOS gene by phage display technique from human liver cDNA library / 解放军医学杂志

Objective To screen promoter DNA-binding protein of inducible nitric oxide synthase gene by using phage display technique from human liver cDNA library, and to study the expression and regulation mechanism of iNOS gene. Methods The sequence of iNOS promoter was identified in GenBank by bioinformatics based on the open reading frame(ORF) of iNOS gene and amplified from HepG2 genome by polymerase chain reaction (PCR). The amplified product was subcloned into pCAT3-Basic reporter vector, named as pCAT3-iNOSp. The HepG2 cell line was then transfected with pCAT3-Basic to serve as negative control, and pCAT3-promoter which contains the promoter region of CMV served as the positive control subject, and pCAT3-iNOSp served as the test subject, respectively. The choloraphenical acetyltransferase(CAT)activity was determined by enzyme linked immunosorbent assay(ELISA) kit. The T7 Select human liver cDNA library was biopanned and positive clones were selected. After screening, positive plaque was performed to amplify and PCR products were sequenced. Results The expression of CAT in transfection of pCAT3-PS1TP1p was 4.2 times as higher as pCAT3-Basic plasmid. Sequence analysis was performed in 12 positive plaque, which were the iNOSp binding protein. Conclusion The iNOS gene promoter identified in this study has shown to have transcription activity,and iNOS promoter DNA-binding proteins havs been screened. The results will be useful for further study of the expression and regulation mechanism of iNOS in liver cell.