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