Effects of 13-alkyl-substituted berberine alkaloids on the expression of COX-II, TNF-α, iNOS, and IL-12 production in LPS-stimulated macrophages
Introduction
Berberine, a main alkaloidal component of Coptidis Rhizoma and Phellodendri Cortex, has antibacterial activity (Hahn and Ciak, 1975), anti-inflammatory activity (Akhter et al., 1977), antitumor action (Makhey et al., 1995) and antimotility actions (Yamamoto et al., 1993). Extracts of berberine-containing plants have been used for many centuries in the treatment of diarrhea (Dutta et al., 1972), intestinal parasite infection, and ocular trachoma infections (Mohan et al., 1982). Recently we reported that berberine increases IL (interleukin)-12 p40 production by p38 mitogen-activated protein kinase in macrophages (Kang et al., 2002). IL-12 is a heterodimeric cytokine that is secreted by macrophages and other antigen-presenting cells and plays a critical role in determining the nature of immune response to exogenous or endogenous antigens. Because IL-12 is one of the central factors deciding the fate of the immune response concerning the polarity of Th1 vs. Th2 (in the presence of IL-12 shifts the balance towards a Th1 phenotype), this cytokine can be an ideal target for shaping the immune processes during autoimmune diseases (Hasko and Szabo, 1999). IL-12 deficiency has been associated with tumor growth, while this cytokine has been successfully administered in patient with cancer (Lotze et al., 1996). Recent evidence showed that administration of recombinant(r) IL-12 may be a key strategy in the treatment of Th2-dominated disease such as infectious diseases and allergic diseases Wynn et al., 1995, Cooper et al., 1997. We also suggested that IL-12-secreting fibroblast can effectively induce an antigen-specific Th1 response, which may be a therapeutic target in disorders caused by undesirable Th2-dominated responses (Kim et al., 2000). Since berberine has known to be a useful natural compound, we have synthesized many berberine analogs (Iwasa et al., 1998). Among them, we found that 13-alkyl-substituted berberines (Fig. 1) were shown to be better activity than berberine against certain bacteria species (Iwasa et al., 1998) and human cancer cell lines (Iwasa et al., 2001). However, pharmacological characterization on 13-alkyl derivatives of berberine has not been extensively performed. Therefore it is of great interest to investigate the effects of 13-alkylderivatives of berberine on the production of IL-12 as well as the expression of pro-inflammatory genes, such as iNOS, TNF-α and COX-II, which are considered to be important in the process of inflammation and related disorders. We provide evidence that 13-methylberberine (13-MB) and 13-ethylberberine (13-EB) increase production of IL-12 and inhibit the expression of iNOS at posttranscriptional level in macrophages activated with LPS. Furthermore, these alkaloids inhibited the production of PGE2 without affecting the expression of COX-II protein. Thus, 13-MB and 13-EB may be useful in certain type of cancers, inflammation and infectious disorders.
Section snippets
Materials
Lipopolysaccharide (E. Coli; serotype 0128:B12), sulphanilamide, N-(1- naphthyl) ethylenediamine, leupeptin, pepstatin A, phenylmethylsulfonylfluoride, dithiothreitol, berberine chloride, methyl iodide, and ethyl iodide were provided from Sigma (St. Louis, MO. USA). TNF-α, COX-II and iNOS antibodies were purchased from Transduction Laboratories (Lexington, KY, USA). Horseradish peroxidase labeled goat anti-rabbit IgG was purchased from Jackson ImmunoResearch Laboratories INC (West Grove, USA).
Effects on NO production
In cultured RAW 264.7 cells, nitrite time-dependently increased until 24 h (data not shown). The accumulated nitrite was 6 ± 0.8 μM in control media, which was increased to 64 ± 3.5 μM by LPS for 18 h. Co-treatment of 13-MB and 13-EB concentration-dependently decreased the nitrites, respectively (Fig. 2). The concentration of 50% inhibition of NO production (IC50) by 13-MB and 13-EB was 11.64 and 9.32 μM, respectively.
Effects on expression of TNF-α, COX-II and iNOS mRNA
To know whether 13-MB and 13-EB regulate pro-inflammatory gene expression,
Discussion
In this study, we demonstrated that 13-methylberberine (13-MB) and13-ethylberberine (13-EB), concentration-dependently, reduce the production of NO and the expression of iNOS protein in LPS-treated RAW cells. LPS and/or cytokines induce the expression of the iNOS isoform (Forstermann et al., 1993) in many cell types, including macrophages (Marletta et al., 1998). The concentration-dependent reduction of NO production by these compounds can be explained as the result of inhibition of iNOS
Acknowledgements
This work was supported by the Dongguk University Research Fund. The authors thank Young S. Ko, Gyeongsang National University, for technical assistance.
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