Synthetic Fluororutaecarpine Inhibits Inflammatory Stimuli and Activates Endothelial Transient Receptor Potential Vanilloid-Type 1

Abstract

The natural product, rutaecarpine (RUT), is the main effective component of Evodia rutaecarpa which is a widely used traditional Chinese medicine. It has vasodilation, anticoagulation, and anti-inflammatory activities. However, further therapeutic applications are limited by its cytotoxicity. Thus, a derivative of RUT, 10-fluoro-2-methoxyrutaecarpine (F-RUT), was designed and synthesized that showed no cytotoxicity toward RAW264.7 macrophages at 20 μM. In an anti-inflammation experiment, it inhibited the production of nitric oxide (NO) and tumor necrosis factor (TNF)-α in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages; cyclooxygenase (COX)-2 and inducible NO synthase (iNOS) induced by LPS were also downregulated. After 24 h of treatment, F-RUT significantly inhibited cell migration and invasion of ovarian A2780 cells. Furthermore, F-RUT promoted expressions of transient receptor potential vanilloid type 1 (TRPV1) and endothelial (e)NOS in human aortic endothelial cells, and predominantly reduced the inflammation in ovalbumin/alum-challenged mice. These results suggest that the novel synthetic F-RUT exerts activities against inflammation and vasodilation, while displaying less toxicity than its lead compound.

Keywords: TRPV-1; eNOS; fluoro-rutaecarpine; iNOS; inflammation.

Scheme 1

Synthesis of 10-fluoro-2-methoxyrutaecarpine (F-RUT) and 10-fluoro-2,3-dimethoxyrutaecarpine.

Figure 1

Effects of 10-fluoro-2-methoxyrutaecarpine (F-RUT) on nitric oxide (NO) and tumor necrosis factor (TNF)-α release by lipopolysaccharide (LPS)-treated (40 ng/mL) RAW264.7 macrophages. (a) NO levels were detected in culture medium using the Griess reaction; (b) TNF-α release in cell supernatants was detected using a mouse TNF-α Quantikine kit; (c) Cell viability upon F-RUT and rutaecarpine (RUT) treatment for 24 h in an MTT assay. Values are expressed as the mean ± SE. * p < 0.05, ** p < 0.01.

Figure 1

Effects of 10-fluoro-2-methoxyrutaecarpine (F-RUT) on nitric oxide (NO) and tumor necrosis factor (TNF)-α release by lipopolysaccharide (LPS)-treated (40 ng/mL) RAW264.7 macrophages. (a) NO levels were detected in culture medium using the Griess reaction; (b) TNF-α release in cell supernatants was detected using a mouse TNF-α Quantikine kit; (c) Cell viability upon F-RUT and rutaecarpine (RUT) treatment for 24 h in an MTT assay. Values are expressed as the mean ± SE. * p < 0.05, ** p < 0.01.

Figure 2

Effect of 10-fluoro-2-methoxyrutaecarpine (F-RUT) on inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions by lipopolysaccharide (LPS)-treated RAW264.7 macrophages (a), and luciferase reporter plasmid-transfected macrophages (b). Cells were transfected with 2.5 μg of the pGL4.32 [luc2P/NF-κB-RE/Hygro] reporter plasmid, then treated with different concentrations of F-RUT and LPS (40 ng/mL) for 24 h. Levels of luciferase activity were determined as described in Materials and Methods. Values are expressed as the mean ± SE of triplicate tests. * p < 0.05, ** p < 0.01 versus LPS treatment.

Figure 3

Effects of 10-fluoro-2-methoxyrutaecarpine (F-RUT) on migration and invasion. Cell migration (a) and cell invasion (b) were detected following F-RUT treatment for 0–24 h, and photographed with a microscope (upper panel). The statistical analysis is shown in the lower panel. (* p < 0.05, ** p < 0.01).

Figure 4

Effects of 10-fluoro-2-methoxyrutaecarpine (F-RUT) on transient receptor potential vanilloid-type 1 (TRPV-1) expression and endothelial nitric oxide synthase (eNOS) phosphorylation in human aortic endothelial cells (HAECs). The densitometric ratio is indicated.

Figure 5

Effects of 10-fluoro-2-methoxyrutaecarpine (F-RUT) on ovalbumin (OVA)-challenged mice. Data are representative of three to five mice per group. Scale bar is 100 µm.