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-kappaB) was determined by electrophoretic mobility shift assay (EMSA). CR (20, 50, and 100 microg/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 microM), 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 microg/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 microg/ml) significantly inhibited the activity of NF-kappaB 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-kappaB.
Jung
Kyung Hee
KH
Department of Pharmacology, School of Medicine, Kyung Hee University, Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea.
Ha
Eunyoung
E
Kim
Mi Ja
MJ
Won
Hye-Jin
HJ
Zheng
Long Tai
LT
Kim
Hye Kyung
HK
Hong
Seung Jae
SJ
Chung
Joo Ho
JH
Yim
Sung-Vin
SV
D000818:Animals; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D002957:Citrus / Q000737:chemistry*; D024202:Electrophoretic Mobility Shift Assay; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D008262:Macrophage Activation / Q000187:drug effects*; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000276:immunology / Q000378:metabolism; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000276:immunology; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D010936:Plant Extracts / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection
D008070:Lipopolysaccharides; D016328:NF-kappa B; D010936:Plant Extracts; D009569:Nitric Oxide; D008156:Luciferases; D052247:Nitric Oxide Synthase Type II