A Prenylated Xanthone, Cudratricusxanthone A, Isolated from Cudrania tricuspidata Inhibits Lipopolysaccharide-Induced Neuroinflammation through Inhibition of NF-κB and p38 MAPK Pathways in BV2 Microglia

Abstract

Cudrania tricuspidata Bureau (Moraceae) is an important source of traditional Korean and Chinese medicines used to treat neuritis and inflammation. Cudratricusxanthone A (1), a prenylated xanthone, isolated from C. tricuspidata, has a variety of biological and therapeutic activities. The goal of this study was to examine the effects of compound 1 on neuroinflammation and characterize its mechanism of action in lipopolysaccharide (LPS)-stimulated BV2 microglia. Cudratricusxanthone A (1) suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 enzymes and decreased the production of iNOS-derived nitric oxide and COX-2-derived prostaglandin E2 in LPS-stimulated mouse BV2 microglia. The compound also decreased tumor necrosis factor-α, interleukin (IL)-1β, and IL-12 production; inhibited the phosphorylation and degradation of IκB-α; and blocked the nuclear translocation of p50 and p65 in mouse BV2 microglia induced by LPS. Cudratricusxanthone A (1) had inhibitory effects on nuclear factor kappa B DNA-binding activity. Additionally, it inhibited the p38 mitogen-activated protein kinase signaling pathway. Our data suggests that cudratricusxanthone A (1) may be a useful therapeutic agent in the treatment of neurodegenerative diseases caused by neuroinflammation.

Keywords: Cudrania tricuspidata; cudratricusxanthone A; microglia; mitogen-activated protein kinase; neuroinflammation; nuclear factor-kappa B.

Figure 1

Chemical structures of compounds 1–9.

Figure 2

The effects of compounds 1–9 on NO production in BV2 microglia stimulated with LPS. The cells were pretreated for 3 h with the indicated concentrations of compounds 1–9 and stimulated for 24 h with LPS (1 μg/mL). The LPS treatment was performed in the presence of compound. The concentrations of nitrite were determined as described in the Experimental Section. The data show the means ± SDs of three experiments. * p < 0.05 compared with the group treated with LPS. “+” is treated, “−“ is not treated.

Figure 3

The effects of cudratricusxanthone A (1) on TNF-α (A); IL-1β (B); IL-12 (C); and IL-6 (D) mRNA expression in LPS-stimulated BV2 cells. Cells were pretreated for 3 h with the indicated concentrations of cudratricusxanthone A (1) and then stimulated for 12 h with LPS (1 μg/mL). The LPS treatment was performed in the presence of compound. The concentrations of TNF-α (A), IL-1β (B); IL-12 (C); and IL-6 (D) were determined as described in the Experimental Section. RNA quantification was performed as described in the Experimental Section, and representative blots of three independent experiments are shown. The data represent the means ± SDs of three experiments. * p < 0.05 compared with the group treated with LPS. “+” is treated, “−“ is not treated.

Figure 4

The effects of cudratricusxanthone A (1) on the PGE₂ production (A) and protein expression of iNOS and COX-2 (B) in BV2 microglia stimulated with LPS. Cells were pretreated for 3 h with the indicated concentrations of cudratricusxanthone A (1) and then stimulated for 24 h with LPS (1 μg/mL). The LPS treatment was performed in the presence of compound. The concentrations of iNOS and COX-2 (B) were determined as described in the Experimental Section. Western blot analyses were performed as described in the Experimental Section, and representative blots of three independent experiments are shown. The band intensity was quantified by densitometry and normalized to β-actin, and the values are presented at the bottom of the each band. Relative data represent the means ± SDs of three experiments. * p < 0.05 compared with the LPS-treated group. “+” is treated, “−“ is not treated.

Figure 5

The effects of cudratricusxanthone A (1) on IκB-α phosphorylation and degradation (A); NF-κB translocation (B,C); NF-κB localization as determined by immunofluorescence analysis (D); and NF-κB DNA binding activity (E) in BV2 microglia. Cells were pretreated for 3 h with the indicated concentrations of cudratricusxanthone A (1), and stimulated for 1 h with LPS (1 μg/mL). The LPS treatment was performed in the presence of compound. Western blot analyses of IκB-α and p-IκB-α in the cytoplasm (A) and NF-κB in the cytoplasm (B) and nucleus (C) and immunofluorescence analyses (E) were carried out as described in the Experimental Section. The band intensity was quantified by densitometry and normalized to β-actin and PCNA, and the values are presented at the bottom of the each band. Relative data represent the means ± SDs of three experiments. * p < 0.05 compared with the LPS-treated group. “+” is treated, “−“ is not treated.

Figure 6

Effects of cudratricusxanthone A (1) on ERK, JNK, and p38 MAPK phosphorylation and protein expression. Cells were pretreated for 3 h with the indicated concentrations of cudratricusxanthone A (1) and stimulated for 1 h with LPS (1 μg/mL). The LPS treatment was performed in the presence of compound. The levels of (A) phosphorylated ERK (p-ERK); (B) phosphorylated JNK (p-JNK); and (C) phosphorylated p38 MAPK (p-p38 MAPK) were determined by western blotting. Representative blots from three independent experiments with similar results and densitometric evaluations are shown. Band intensity was quantified by densitometry and normalized to β-actin, and the values are presented at the bottom of each band. “+” is treated, “−“ is not treated.