Chlorophyllin suppression of lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells.
Chlorophyllin (CHL), a water-soluble derivative of chlorophyll, functions as an anticarcinogen and antioxidant. In the present study, we investigated the effect of CHL on nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Treatment with CHL inhibited nitric oxide production in the LPS-stimulated RAW 264. 7 cells in a dose-related manner. Competitive RT-PCR analysis, using a DNA competitor as an internal standard, demonstrated that the treatment with 1, 10, and 50 microM CHL decreased LPS-induced iNOS mRNA expression in a concentration-dependent manner. Since the expression of the iNOS gene is mainly regulated by NF-kappaB, we then examined the effects of CHL on the NF-kappaB DNA binding activity, using an electrophoretic mobility shift assay. CHL down-regulated the NF-kappaB DNA binding on its cognate recognition site at the concentrations just noted. Employing a transfection and reporter gene expression system with p(NF-kappaB)(3)-chloramphenicol acetyl transferase (CAT), the treatment of CHL produced a dose-dependent inhibition of CAT activity in RAW 264.7 cells. Furthermore, CHL partially restored LPS-decreased IkappaBalpha, an inhibitory protein against NF-kappaB activation, in the cytosolic extract from the LPS-treated cells determined by immunoblot analysis. CHL also protected the hydroxyl radical-induced cytotoxicity in RAW 264.7 cells, indicating its antioxidant effect. These results suggest that CHL suppresses the nitric oxide production and iNOS mRNA expression mediated by the inhibition of NF-kappaB activation, and its action mechanism may be based on its antioxidant effect.
Cho
K J
KJ
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusong-dong, Yusong-gu, Taejon, 305-701, Korea.
Han
S H
SH
Kim
B Y
BY
Hwang
S G
SG
Park
K K
KK
Yang
K H
KH
Chung
A S
AS
D000818:Animals; D016587:Antimutagenic Agents / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002735:Chlorophyllides / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004586:Electrophoresis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D017930:Genes, Reporter; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012486:Salmonella typhimurium; D014162:Transfection
D016587:Antimutagenic Agents; D002735:Chlorophyllides; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009573:Nitrites; D012333:RNA, Messenger; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D015500:Chloramphenicol O-Acetyltransferase; C007020:chlorophyllin