Inhibition of inducible nitric oxide synthase by bis(helenalinyl)glutarate in RAW264.7 macrophages

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Abstract

Graphical abstract

Screening a phytochemical library for novel nitric oxide (NO) inhibitors, we identified bis(helalinyl)glutarate (BHG) as candidate compound. Indeed, BHG inhibited NO production and expression of inducible NO synthase. The glucocorticoid receptor and interleukin-1 and interleukin-10 signaling pathways were found as possible modes of action of BHG by means of mRNA microarray hybridization.

Nitric oxide (NO) plays a role in various physiological and pathophysiological conditions such as immunoregulatory and inflammatory processes. Hence, NO and its generating enzyme, inducible nitric oxide synthase (iNOS) may not only be of diagnostic and prognostic value, but may also serve as targets for novel therapeutic options. In the present investigation, we have screened a phytochemical library by correlating the IC50 values for 531 natural products of 60 cell lines with the microarray-based mRNA expression of 95 genes known to be involved in NO metabolism and signaling with the aim to identify candidate compounds as inhibitors for NO metabolism and signaling. We identified bis(helenalinyl)glutarate (BHG) as putative candidate compound. Indeed, BHG inhibited NO production (IC50 value: 0.90 ± 0.04 μM) and down-regulated iNOS protein expression (IC50 value: 1.12 ± 0.16 μM) of RAW264.7 mouse macrophages in the presence of lipopolysaccharide and interferon-γ. Performing XTT cytotoxicity assays, we found that BHG inhibited cell growth in a dose-dependent manner with an IC50 value of 5.6 μM. To gain insight into molecular pathways involved in NO inhibition and cytotoxicity, we performed microarray experiments which were exemplarily validated by real-time RT-PCR. A total of 227 genes (67 up- and 160 down-regulated) were obtained, which exhibited significant differences in mRNA regulation between BHG-treated and untreated RAW264.7 macrophages. Sixteen of 227 genes are known to be involved in NO-signaling. Pathway analyses revealed that further five and four down-regulated genes belong to the glucocorticoid receptor and interleukin-1 and interleukin-10 pathways, respectively. An interference of these two pathways and NO is known for inflammation and auto-immune diseases. The therapeutic potential of this compound has to be explored in the future.

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