A Newly Synthesized Flavone from Luteolin Escapes from COMT-Catalyzed Methylation and Inhibits Lipopolysaccharide-Induced Inflammation in RAW264.7 Macrophages via JNK, p38 and NF-κB Signaling Pathways

Abstract

Luteolin is a common dietary flavone possessing potent anti-inflammatory activities. However, when administrated in vivo, luteolin becomes methylated by catechol-O-methyltransferases (COMT) owing to the catechol ring in the chemical structure, which largely diminishes its anti-inflammatory effect. In this study, we made a modification on luteolin, named LUA, which was generated by the chemical reaction between luteolin and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Without a catechol ring in the chemical structure, this new flavone could escape from the COMT-catalyzed methylation, thus affording the potential to exert its functions in the original form when administrated in the organism. Moreover, an LPS-stimulated RAW cell model was applied to detect the anti-inflammatory properties. LUA showed much more superior inhibitory effect on LPS-induced production of NO than diosmetin (a major methylated form of luteolin) and significantly suppressed upregulation of iNOS and COX-2 in macrophages. LUA treatment dramatically reduced LPS-stimulated reactive oxygen species (ROS) and mRNA levels of pro-inflammatory mediators such as IL-1β, IL-6, IL-8 and IFN-β. Furthermore, LUA significantly reduced the phosphorylation of JNK and p38 without affecting that of ERK. LUA also inhibited the activation of NF-κB through suppression of p65 phosphorylation and nuclear translocation.

Keywords: 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH); Luteolin; catechol-O-methyltransferases (COMT); inflammation.

Fig. 1. Synthetic pathway and chemical structure of LUA.

LUA: LUAAPH-1.

Fig. 2. Effect of LUA on cell viability in RAW267.4 cells.

The cells were treated with 0.2% DMSO or various concentrations (1-50 μM) of LUA for 24 h. Cell viability was assessed using the WST-1 assay. Each value was presented as mean ± SD (n = 10). ****p < 0.0001 vs. 0.2% DMSO-treated cells.

Fig. 3. LUA inhibited LPS-induced production of NO in Raw264.7 cells via the downregulation of iNOS expression.

(A) Cells were incubated with various doses (1-20 μmol/l) of LUA in the absence or presence of LPS. The media were collected after 24 h and assayed for NO production (n = 13). (B) Cells were incubated with luteolin, diosmetin and LUA at the concentration of 15 uM in the absence or presence of LPS. The media were collected after 24 h and assayed for NO (n = 4). (C and D) Cells were treated with LUA at the concentration of 15 uM in the absence or presence of LPS. (C) After incubation for 6h, total RNA was isolated and RT-PCR was conducted for iNOS mRNA level (n = 3). (D) After incubation for 24 h, cell lysates were subjected to Western blot analysis with an iNOS or β-actin-specific antibody (n = 5). The results were displayed as means ± SD. Data were presented as percentage of control group. ####p < 0.0001 vs. Ctrl group; ***p < 0.001, ****p < 0.0001 vs. LPS group. Ctrl: control; LUT: luteolin; DIO: diosmetin; LUA: LUAAPH-1.

Fig. 4. Effects of LUA on LPS-induced COX-2 mRNA (A) and protein expression (B) in RAW264.7 cells.

Cells were stimulated with or without LPS in the presence or absence of LUA (15 μM). (A) The COX-2 mRNA expression level was detected by RT-PCR after treatment for 6h (n = 4). (B) The COX-2 protein expression level was measured by Western blot after stimulation for 24 h (n = 4). Data were presented as percentage of control group. The results were displayed as means ± SD. ####p < 0.0001 vs. Ctrl group; ***p < 0.001 vs. LPS group. Ctrl: control; LUA: LUAAPH-1.

Fig. 5. Effects of LUA on LPS-induced pro-inflammatory cytokines in RAW264.7 cells.

Cells were treated with LPS in the absence or presence of LUA (15 μM). (A-D) Following stimulation with LPS for 6 h, the IL-1β (A) (n = 3), IL-6 (B) (n = 6), IL-8 (C) (n = 5), IFN-β (D) (n = 4) mRNA expression levels were determined by RT-PCR. (E) Following incubation for 24 h, the protein quantity of IL-1β was measured by Western blot (n = 4). Data were presented as percentage of control group. The results were displayed as means ± SD. ####p < 0.0001 vs. Ctrl group; *p < 0.05, **p < 0.01, ****p < 0.0001 vs. LPS group. Ctrl: control; LUA: LUAAPH-1.

Fig. 6. Intracellular ROS levels in RAW264.7 cells.

After incubating with LPS in the presence or absence of LUA (15 μM) for 24 h, the cells were exposed to DCFH-DA for 30 min at 37°C. The fluorescence was measured at 485 nm (excitation) and 535 nm (emission). Data were presented as percentage of control group (n = 6). The results were displayed as means ± SD. ####p < 0.0001 vs. Ctrl group; ****p < 0.0001 vs. LPS group. Ctrl: control; LUA: LUAAPH-1.

Fig. 7. Effects of LUA on LPS-induced MAPK signaling pathways in RAW264.7 cells.

Cells were incubated with LPS for 30 min in the absence or presence of LUA (15 μM). Cells lysates were subjected to Western blot for analysis of p-JNK (A) (n = 4), p-ERK (B) (n = 5)and p-P38 (C) (n = 4). The relative abundance of the phosphorylated form to its total protein was quantified. Data were presented as percentage of control group. The results were displayed as means ± SD. ####p < 0.0001 vs. Ctrl group; ***p < 0.001, ****p < 0.0001 vs. LPS group. Ctrl: control; LUA: LUAAPH-1.

Fig. 8. Effects of LUA on LPS-induced NF-κB signaling pathway in RAW264.7 cells.

(A) Cells were incubated with LPS for 30 min in the absence or presence of LUA (15 μM). Cells lysates were subjected to Western blot for analysis of p-p65 (n = 4). The relative abundance of p-p65 to t-p65 was quantified. (B) Cells were incubated with LPS for 1 h in the absence or presence of LUA (15 μM). Nuclear translocation of p65 was visualized by immunofluorescence analysis with a confocal laser scanning microscope. Data were presented as percentage of control group. The results were displayed as means ± SD. ####p < 0.0001 vs. Ctrl group; ****p < 0.0001 vs. LPS group. Ctrl: control; LUA: LUAAPH-1.

Fig. 9. A schematic model that proposes the synthesis procedure, advantages as well as the potential contribution of LUA in anti-inflammatory signaling pathways in LPS-activated RAW264.7 macrophages.

LUT: luteolin; LUA: LUAAPH-1; AAPH: 2,2'-azobis(2-amidinopropane) dihydrochloride; DIO: diosmetin; CHR: chrysoeriol; COMT: catechol-O-methyltransferases; LPS: lipopolysaccharide; IKK: inhibitor-κB kinase; IκBα: inhibitor kappa B alpha; MKKs: MAPK kinases; ROS: reactive oxygen species.