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.