8-Methoxybicolosin C from Lespedeza bicolor Attenuates Inflammation and Oxidative Stress via Nrf2/HO-1 and NF-κB/MAPK Pathways in Lipopolysaccharide-Induced Mouse Kupffer Cells

Abstract

Lespedeza bicolor (L. bicolor) is known for its anti-inflammatory, antioxidant, and anticancer properties, making it a common choice in traditional medicine practices. Researchers in several recent studies have focused on isolating individual phytochemicals from this plant through chromatography analysis to explore their therapeutic potential. In our previous work, we identified 8-methoxybicolosin C (8-MC) as a novel flavonoid derivative, isolated and purified from the roots of L. bicolor, which exhibited inhibitory effects on cell proliferation. In this study, we further investigated the biological activities of 8-MC by examining its antioxidant and anti-inflammatory effects in LPS-induced mouse Kupffer cells. The results showed that 8-MC suppresses the expression of inflammation-related mediators, including inducible nitric oxide synthase (iNOS), nitric oxide (NO), and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β, in a dose-dependent manner. Additionally, 8-MC improves the GSH/GSSG balance by increasing glutathione (GSH) levels and decreasing oxidized glutathione (GSSG) levels. Interestingly, 8-MC was found to bind Keap1, preventing roteasomal degradation, and promoting the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), thereby increasing the expression of antioxidant-related proteins such as heme oxygenase-1 (HO-1). Moreover, 8-MC suppressed the activation of inflammatory signaling pathways, including c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinases (MAPKs), while also inhibiting the nuclear translocation of nuclear factor kappa B (NF-κB), effectively reducing inflammatory responses. These findings collectively demonstrated that 8-MC possesses potent anti-inflammatory and antioxidant activities through the regulation of NF-κB, MAPK, and Nrf2/HO-1 signaling pathways. Consequently, 8-MC shows potential as a valuable therapeutic agent for managing various inflammatory disorders.

Keywords: 8-Methoxybicolosin C; MAPKs; NF-κB; Nrf2/HO-1 pathway; anti-inflammation; antioxidant.

Fig. 1. Effect of 8-MC on cell viability and GSH/GSSG ratio.

ImKCs were treated with 8-MC at the indicated concentrations for 24 h. (A) The results of cell viability were assessed and presented as a percentage in comparison to the untreated group. The cells were pre-treated with 8-MC at the indicated concentrations for 2 h and then induced with lipopolysaccharide (LPS, 0.5 μg/ml) for 24 h. The levels of reduced glutathione (GSH) (B), oxidized glutathione (GSSG) (C), and the GSH/GSSG ratio (D) were calculated based on these measurements. Each result represents the average value ± SEM from three individual replicates. ##p < 0.01 in comparison with the untreated group. *p < 0.05, **p < 0.01 in comparison with the LPS-treated group. ns, not significant. ImKCs, immortalized Kupffer cells; 8-MC, 8-methoxybicolosin C.

Fig. 2. The impact of 8-MC on iNOS-driven nitric oxide (NO) production in LPS-induced ImKCs.

ImKCs were pre-treated with 8-MC at the indicated concentrations for 2 h and then induced with LPS (0.5 μg/ml) for 24 h. (A) The levels of NO production were assessed by utilizing Griess reagents. (B) Protein expression levels were assessed via western blot analysis (WB) and normalized using β-actin. Each result represents the average value ± SEM from three individual replicates. #p < 0.05, ##p < 0.01 in comparison with the untreated group. *p < 0.05, *p < 0.01 in comparison with the LPS-treated group.

Fig. 3. Pro-inflammatory cytokine inhibition effect of 8-MC in LPS-induced ImKCs.

ImKCs were pre-treated with 8-MC at the indicated concentrations for 2 h and then induced with LPS (0.5 μg/ml) for 24 h. (A-C) The secretion of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β in the cell supernatant was assessed through ELISA analysis. Each result represents the average value ± SEM from three individual replicates. ##p < 0.01 in comparison with the untreated group. **p < 0.01 in comparison with the LPS-treated group. ns, not significant.

Fig. 4. Comparative molecular docking analysis of 8-MC and PC with Keap1.

(A–C) Binding mode of 8-MC with Keap1: (A) 3D binding complex showing hydrogen bonds formed at Arg483, (B) 2D interaction map highlighting hydrogen bonds and hydrophobic contacts, and (C) Venn diagram showing overlapping binding residues with Nrf2. (D–F) Binding mode of the positive control (PC) with Keap1: (D) 3D complex visualization, (E) detailed interaction map, and (F) Venn diagram displaying the overlap of binding sites between PC and Nrf2 on Keap1. These results suggest that both 8-MC and PC may competitively occupy the Nrf2-binding interface on Keap1.

Fig. 5. Upregulation of HO-1 and Nrf2 expression by 8-MC in LPS-induced ImKCs.

ImKCs were pre-treated with 8-MC at the indicated concentrations for 2 h and then induced with LPS (0.5 μg/ml) for 4 h (B and C) or 24 h (A). (A and B) Protein expression levels were assessed via the WB and normalized using β- actin. Each result represents the average value ± SEM from three individual replicates. (C) A representative image captured via immunofluorescence depicts the movement of Nrf2 (red) into the DAPI-stained nucleus (blue). The image scale is set to 10 μm. #p < 0.05 in comparison with the untreated group. ns, not significant. *p < 0.05 in comparison with the LPS-treated group.

Fig. 6. ImKCs were pre-treated with 8-MC (10 μM) for 2 h, with or without Sn protoporphyrin (SnPP, 10 μM), and then induced with LPS (0.5 μg/ml) for 24 h.

(A) The levels of NO production were assessed by utilizing Griess reagents. (B) The secretion of pro-inflammatory cytokines in the cell supernatant was assessed through ELISA analysis. Each result represents the average value ± SEM from three individual replicates. ###p < 0.001 in comparison with the untreated group. ***p < 0.001 in comparison with the LPS-treated group.

Fig. 7. Inhibition of IκB degradation and NF-κB translocation by 8-MC in LPS-induced ImKCs.

ImKCs were pre-treated with 8-MC (2.5 and 10 μM) for 2 h and then induced with LPS (0.5 μg/ml) for 10 min. (A) Protein expression levels were assessed via the WB and normalized using β-actin. Each result represents the average value ± SEM from three individual replicates. (B) A representative image captured via immunofluorescence depicts the movement of NF-κB (red) into the DAPIstained nucleus (blue). The image scale is set to 10 μm. #p < 0.05 in comparison with the untreated group. *p < 0.05 in comparison with the LPS-treated group.

Fig. 8. Inhibition of MAPK phosphorylation by 8-MC in LPS-induced ImKCs.

ImKCs were pre-treated with 8-MC at the indicated concentrations for 2 h and then induced with LPS (0.5 μg/ml) for 10 min. Protein expression levels were assessed via the WB and normalized using β-actin. The phosphorylation levels of each protein were individually adjusted by normalizing them with the total levels of p38, JNK, and ERK. Each result represents the average value ± SEM from three individual replicates. #p < 0.05 in comparison with the untreated group. *p < 0.05 in comparison with the LPS-treated group. ns, not significant.