Suppressive effects of nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression by Citrus reticulata extract in RAW 264.7 macrophage cells

Immature peels of Citrus reticulata extract (CR) are widely used as traditional herbal medicine in Korea. We studied its effects on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW 264.7 macrophage cells. NO production was assessed by nitrite assay and iNOS expression was identified by reverse transcription-polymerase chain reaction (RT-PCR), Real-time PCR and Western blot. The promoter activity of iNOS gene was also determined by luciferase reporter gene assay using 5′-flanking region of murine iNOS gene. Activation of nuclear factor kappa B (NF-κB) was determined by electrophoretic mobility shift assay (EMSA). CR (20, 50, and 100 μg/ml) significantly inhibited the lipopolysaccharide (LPS)-induced NO production (P < 0.01; 9.2 ± 1.5, 4.8 ± 0.6, and 3.3 ± 0.4 μM), iNOS protein (38.1 ± 3.8, 32.3 ± 5.8, and 36.8 ± 4.5%) and mRNA expression (34.2 ± 4.1, 13.1 ± 5.8, and 20.8 ± 1.2%) in RAW 264.7 macrophage cells. CR (20, 50, and 100 μg/ml) also reduced the iNOS promoter activity (68.7 ± 3.9, 50.6 ± 5.6, and 45.9 ± 3.9%) in piNOS-LUC-transfected cells. In addition, CR (20, 50, and 100 μg/ml) significantly inhibited the activity of NF-κB DNA binding activity in LPS-induced macrophage cells (P < 0.05; 51.8 ± 4.1, 32.7 ± 5.5, and 35.7 ± 2.9%).These results suggest that CR may suppress LPS-stimulated NO production by inhibiting of NF-κB.