Elsevier

Biochemical Pharmacology

Volume 61, Issue 11, 1 June 2001, Pages 1417-1427
Biochemical Pharmacology

Inflammation and immunopharmacology
Wogonin, baicalin, and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expressions induced by nitric oxide synthase inhibitors and lipopolysaccharide1

https://doi.org/10.1016/S0006-2952(01)00594-9Get rights and content

Abstract

We previously reported that oroxylin A, a polyphenolic compound, was a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In the present study, three oroxylin A structurally related polyphenols isolated from the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, were examined for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. The results indicated that these three polyphenolic compounds inhibited LPS-induced NO production in a concentration-dependent manner without a notable cytotoxic effect on these cells. The decrease in NO production was in parallel with the inhibition by these polyphenolic compounds of LPS-induced iNOS gene expression. However, these three compounds did not directly affect iNOS enzyme activity. In addition, wogonin, but not baicalin or baicalein, inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity. Furthermore, N-nitro-l-arginine (NLA) and N-nitro-l-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS (but not COX-2) protein expression, which was inhibited by these three polyphenolic compounds. Wogonin, but not baicalin or baicalein, similarly inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cells. These results indicated that co-treatment with NOS inhibitors and polyphenolic compounds such as wogonin effectively blocks acute production of NO and, at the same time, inhibits expression of iNOS and COX-2 genes.

Introduction

Macrophages play an important role in the host defense mechanism against bacterial as well as viral infections [1], [2]. When activated by bacterial toxins such as LPS or lipoteicholic acid (LTA), macrophages inhibit the growth of a wide variety of tumor cells and invade microorganisms through releasing factors such as NO, cytokines, tumor necrosis factor-α, and eicosanoid mediators of the immune response [3]. NO has been shown to be a significant regulatory molecule in diverse physiological functions including vasodilation, neural communication, and host defense [4], [5]. Molecule cloning and sequencing analysis have revealed at least three types of NOS isoforms existing in cells [6], [7], [8]. NOS isozymes that appear in the vascular endothelium (eNOS) and central and peripheral neurons (nNOS) are constitutive (cNOS). Release of NO catalyzed by cNOS plays a role in maintaining active vasodilation through a Ca2+-dependent pathway. On the other hand, NOS in macrophages and hepatocytes is inducible, and its activation is Ca2+-independent. Following exposure to LPS or cytokines, iNOS can be induced in various cells such as macrophages, Kupffer cells, smooth muscle cells, and hepatocytes. iNOS activation catalyzes the formation of a large amount of NO, which plays a key role in the pathogenesis of a variety of diseases including septic shock [9], [10], [11]. Therefore, NO production by iNOS may reflect the degree of inflammation and provides a measure to assess the effect of drugs on the inflammatory process.

Cyclooxygenase is the enzyme catalyzing the conversion of arachidonic acid to prostaglandin H2, the precursor of a variety of biological active mediators such as PGE2, prostacyclin, and thromboxane A2[12], [13], [14]. Two forms of this enzyme have been identified: COX-1, a constitutive cyclooxygenase, and COX-2, an isoform induced in response to many stimulants and activated at the inflammatory site to give rise to pain, swelling, and stiffness [15], [16], [17]. Recent findings have suggested that COX-2 may play important roles in the pathogenesis of diseases such as colon carcinoma, Alzheimer’s disease, heart failure, and hypertension [18], [19], [20]. Therefore, there is an increasing interest in the usefulness of COX-2 inhibitors.

Medicinal plants have been used as traditional remedies for hundreds of years. Scutellaria baicalensis Georgi (Huang Qui) is one of the important medicinal herbs widely used for the treatment of various inflammatory diseases, hepatitis, tumors, and diarrhea in East Asian countries such as China, Korea, Taiwan, and Japan [21]. The plant has been reported to contain a large number of flavonoids, frequently found as glucosides and other constituents, including phenethyl alcohols, sterols, and essential oils and amino acids. In our previous study, oroxylin A (a polyphenolic compound) isolated from Huang Qui was found to be a potent inhibitor of LPS-induced NO and PGE2 productions by blocking iNOS and COX-2 gene activation [22]. In the present study, three oroxylin A structurally related polyphenolic compounds, i.e. baicalin, baicalein, and wogonin, were examined for their effects on LPS-induced iNOS and COX-2 gene expression. The results demonstrated that baicalin, baicalein, and wogonin significantly inhibited LPS-induced NO production and iNOS gene expression in a concentration-dependent manner, but did not inhibit iNOS enzyme activity. Furthermore, wogonin, but not baicalin or baicalein, inhibited LPS-induced PGE2 production and COX-2 gene expression. Similar results were obtained in NLA or L-NAME plus LPS-treated RAW 264.7 macrophages.

Section snippets

Cells

RAW 264.7, a mouse macrophage cell line, was obtained from the American Type Culture Collection. Cells were cultured in RPMI-1640 medium supplemented with 2 mM glutamine, antibiotics (100 U/mL of penicillin and 100 U/mL of streptomycin), and 10% heat-inactivated fetal bovine serum (GIBCO/BRL) and maintained at 37° in a humidified incubator containing 5% CO2.

Agents

Three structurally related polyphenolic compounds (baicalin, baicalein, and wogonin) were isolated from the Chinese herbal plant Huang Qui.

Inhibition of LPS-induced NO production by baicalin, baicalein, and wogonin in RAW 264.7 macrophages

The chemical structures of baicalin, baicalein, and wogonin are shown in Fig. 1. These polyphenolic compounds are flavonoids. The extraction and isolation of each compound from the Chinese herb Huang Qui (S. baicalensis) was described in Materials and Methods, and the purity of each compound was more than 99.5%. The effects of baicalin, baicalein, and wogonin on LPS-induced NO production in RAW 264.7 macrophages were investigated by measuring the accumulated nitrite, as estimated by the Griess

Discussion

NO has been recognized to be an important mediator of cellular communication in several preparations (in addition to endothelial cells) such as macrophages, neutrophils, smooth muscle, autonomic nervous system, and central nervous system [28], [29], [30], [31], [32], [33]. Several studies have demonstrated that induction of iNOS produces a large amount of NO during endotoxemia and under inflammatory conditions. Therefore, drugs that inhibit iNOS expression and/or enzyme activity resulting in

Acknowledgements

This study was supported by grants from the National Science Council (NSC 89-2314-B-038-035), National Institutes of Health (HL 27763), Southern Illinois University (CRC/EAM), and the Juridical Person of Yen’s Foundation.

References (51)

  • G.A. Joly et al.

    Potent inhibition of inducible nitric oxide synthase by geldanamycin, a tyrosine kinase inhibitor, in endothelial, smooth muscle cells and in rat aorta

    FEBS Lett

    (1997)
  • G.K. Lopes et al.

    Polyphenol tannic acid inhibits hydroxyl radical formation from Fenton reaction by complexing ferrous ions

    Biochim Biophys Acta

    (1999)
  • P.S. Ray et al.

    The red wine antioxidant resveratrol protects isolated rat heart from ischemia reperfusion injury

    Free Radic Biol Med

    (1999)
  • F. Yang et al.

    Green tea polyphenols block endotoxin-induced tumor necrosis factor-production and lethality in a murine model

    J Nutr

    (1998)
  • J.K. Lin et al.

    Cancer chemoprevention by tea polyphenols through mitotic signal transduction blockade

    Biochem Pharmacol

    (1999)
  • Y. Surh

    Molecular mechanisms of chemopreventive effects of selected dietary and medicinal phenolic substances

    Mutat Res

    (1999)
  • H.K. Kim et al.

    Effects of naturally occurring flavonoids on nitric oxide production in the macrophage cell lines RAW 264.7 and their structure–activity relationships

    Biochem Pharmacol

    (1999)
  • R.M. Palmer et al.

    Vascular endothelial cells synthesize nitric oxide from l-arginine

    Nature

    (1988)
  • C.J. Lowenstein et al.

    Nitric oxide inhibits viral replication in murine myocarditis

    J Clin Invest

    (1996)
  • J.B. Hibbs et al.

    Macrophage cytotoxicityrole of l-arginine deiminase and imino nitrogen oxidation to nitrite

    Science

    (1987)
  • S. Moncada et al.

    Nitric oxidephysiology, pathophysiology and pharmacology

    Pharmacol Rev

    (1992)
  • M.A. Marletta et al.

    Macrophage oxidation of l-arginine to nitrite and nitratenitric oxide is an intermediate

    Biochemistry

    (1988)
  • D.L. Duval et al.

    Characterization of hepatic nitric oxide synthaseidentification as the cytokine-inducible form primarily regulated by oxidants

    Mol Pharmacol

    (1996)
  • C. Nathan

    Nitric oxide as a secretory product of mammalian cells

    FASEB J

    (1992)
  • M. Ueno et al.

    Endotoxin decreases the contractile responses of the porcine basilar artery to vasoactive substances

    J Cereb Blood Flow Metab

    (1993)
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    Abbreviations: NO, nitric oxide; iNOS, inducible nitricoxide synthase; COX-2, cyclooxygenase-2; PGE2,prostaglandin E2; MTT,3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, LPS,lipopolysaccharide; NLA, N-nitro-l-arginine; andL-NAME, N-nitro-l-arginine methyl ester.

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