Inhibition of lipopolysaccharide-induced nitric oxide production by flavonoids in RAW264.7 macrophages involves heme oxygenase-1.
The role of heme oxygenase-1 (HO-1) played in the inhibitory mechanism of flavonoids in lipopolysaccharide (LPS)-induced responses remained unresolved. In the present study, flavonoids, including 3-OH flavone, baicalein, kaempferol, and quercetin, induced HO-1 gene expression at the protein and mRNA levels in the presence or absence of LPS in RAW264.7 macrophages. This effect was associated with suppression of LPS-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) protein expression. Hemin induced HO-1 protein expression and this was associated with the suppression of LPS-induced NO production and iNOS protein expression in a dose-dependent manner. In addition, an increase in bilirubin production was found in flavonoid- and hemin-treated cells. Hemin, at the doses of 10, 20, and 50 microM, dose-dependently stimulated the flavonoid (50 microM)-induced HO-1 protein expression, and enhanced their inhibitory effects on LPS-induced NO production and iNOS protein expression. Pretreatment of the HO-1 inhibitor, tin protoporphyrin (10 microM), attenuated the inhibitory activities of the indicated flavonoids on LPS-induced NO production. Morphologic analysis showed that 3-OH flavone, baicalein, kaempferol, quercetin, hemin, and tin protoporphyrin did not cause any change in cell viability in the presence or absence of LPS. In contrast, only 3-OH flavone showed a significant inhibition of cell growth using the MTT assay. Transfection of an HO-1 vector in macrophages (HO-1/RAW264.7) resulted in a 3-fold increase in HO-1 protein compared with that the parental RAW264.7 cells. NO production mediated by LPS in HO-1 over-expressed RAW264.7 cells (HO-1/RAW264.7) was significant less than that in parental RAW264.7 cells. 3-OH Flavone, baicalein, kaempferol, and quercetin showed a more significant inhibition on LPS-induced NO production in HO-1/RAW264.7 cells than in parental RAW264.7 cells. These results provide evidence on the role of HO-1 in the inhibition of LPS-induced NO production by flavonoids. A combination of HO-1 inducers (i.e. hemin) and flavonoids might be an effective strategy for the suppression of LPS-induced NO production.
Lin
Hui-Yi
HY
Graduate Institute of Pharmaceutical Sciences, School of Pharmacy, Taipei Medical University, Taipei, Taiwan ROC.
Juan
Shu-Hui
SH
Shen
Shing-Chuan
SC
Hsu
Feng-Lin
FL
Chen
Yen-Chou
YC
D000818:Animals; D002460:Cell Line; D004347:Drug Interactions; D004790:Enzyme Induction / Q000187:drug effects; D044950:Flavanones; D047309:Flavones; D005419:Flavonoids / Q000737:chemistry / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis* / Q000235:genetics; D051547:Heme Oxygenase-1; D006427:Hemin / Q000494:pharmacology; D044949:Kaempferols / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D008565:Membrane Proteins; D008665:Metalloporphyrins / Q000494:pharmacology; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D011524:Protoporphyrins / Q000494:pharmacology; D011794:Quercetin / Q000494:pharmacology
D044950:Flavanones; D047309:Flavones; D005419:Flavonoids; D044949:Kaempferols; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D008665:Metalloporphyrins; D011524:Protoporphyrins; D009569:Nitric Oxide; C006680:baicalein; C006552:kaempferol; D006427:Hemin; D011794:Quercetin; C032628:tin protoporphyrin IX; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse; C043562:flavone