Anti-Inflammatory Effects of Idebenone Attenuate LPS-Induced Systemic Inflammatory Diseases by Suppressing NF-κB Activation

Abstract

Inflammation is a natural protective process through which the immune system responds to injury, infection, or irritation. However, hyperinflammation or long-term inflammatory responses can cause various inflammatory diseases. Although idebenone was initially developed for the treatment of cognitive impairment and dementia, it is currently used to treat various diseases. However, its anti-inflammatory effects and regulatory functions in inflammatory diseases are yet to be elucidated. Therefore, this study aimed to investigate the anti-inflammatory effects of idebenone in cecal ligation puncture-induced sepsis and lipopolysaccharide-induced systemic inflammation. Murine models of cecal ligation puncture-induced sepsis and lipopolysaccharide-induced systemic inflammation were generated, followed by treatment with various concentrations of idebenone. Additionally, lipopolysaccharide-stimulated macrophages were treated with idebenone to elucidate its anti-inflammatory effects at the cellular level. Idebenone treatment significantly improved survival rate, protected against tissue damage, and decreased the expression of inflammatory enzymes and cytokines in mice models of sepsis and systemic inflammation. Additionally, idebenone treatment suppressed inflammatory responses in macrophages, inhibited the NF-κB signaling pathway, reduced reactive oxygen species and lipid peroxidation, and normalized the activities of antioxidant enzyme. Idebenone possesses potential therapeutic application as a novel anti-inflammatory agent in systemic inflammatory diseases and sepsis.

Keywords: NF-κB; ROS; anti-inflammation; idebenone; inflammatory disease model.

Figure 1

Idebenone prevents inflammation-induced mortality and inflammatory responses and restores tissue damage in vivo. Idebenone decreased the mortality rate of mice with CLP-induced sepsis and LPS-induced inflammation. (A) Mice were subjected to either CLP or sham operation (n = 10/group) and then intraperitoneally injected with idebenone (10 mg/kg). Mortality in the CLP + phosphate-buffered saline (PBS; sham operation, white) and CLP + idebenone (black) groups (n = 10/group) was monitored daily for five days after surgery. (B) C57BL/6 mice were intraperitoneally injected with LPS (20 mg/kg), followed by treatment with idebenone (10 mg/kg). Mortality in the LPS-only (white box) and LPS + idebenone (black box) groups (n = 10/group) was monitored daily for 12 days after injection. Idebenone protects against inflammation-induced tissue damaged in mice with CLP-induced sepsis and LPS-induced inflammation. (C) iNOS expression in the lung, liver, kidney, and colon tissues of mice in the sham and CLP + idebenone groups was examined using immunohistochemical analysis. (D) HE staining was performed to examine histological changes in the lung, liver, kidney, and colon tissues of mice in sham and LPS + idebenone groups. All tissues were collected in each group at 24 h after CLP surgery and LPS injection, with or without idebenone administration.

Figure 2

Idebenone inhibits CLP- and LPS-induced inflammatory responses. Idebenone inhibits NO metabolites (NO), COX-2, and IL-1β levels in murine models of CLP- and LPS-induced inflammatory models. The secretion of NO metabolites after CLP (A) and LPS (E) were determined using NO assays. Protein expression of COX-2 and IL-1β in the (B) lung, (C) liver, and (D) kidney tissues of mice in the sham, CLP, and CLP + idebenone groups was analyzed using Western blotting. Protein expression of COX-2 and IL-1β in the (F) lung, (G) liver, and (H) kidney tissues of mice in the sham, LPS only, and LPS + idebenone groups was analyzed using Western blotting. All tissues were randomly collected from two mice per group at 24 h after CLP and LPS; β-actin was used as a loading control; the quantification of the blots described in (B–D,F–H) was performed using ImageJ software. Statistical analyses were performed using paired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, # p < 0.001.

Figure 3

Idebenone inhibits the inflammatory response in mouse models of CLP- and LPS-induced inflammation. Idebenone inhibits TNF-α, IL-1β, IL-6, and PGE₂ production in CLP- and LPS-induced inflammatory disease models. Serum levels of (A) TNF-α, (B) IL-1β, (C) IL-6, and (D) PGE₂ following treatment with or without idebenone after CLP were determined using the ELISA assay. Serum levels of (E) TNF-α, (F) IL-1β, (G) IL-6, and (H) PGE₂ following treatment with or without idebenone after the LPS injection were determined using the ELISA assay. n = 9/group; Graph represents the mean of three independent mouse serums levels of indicated mediators and cytokines. CLP-induced and LPS-induced mouse serum and tissue sample analyses were carried out by one-way ANOVA with Tukey’s Method using the GraphPad Prism 9 statistical program. Statistical significance was set at * p < 0.05, ** p < 0.01, and # p < 0.001.

Figure 4

Idebenone inhibits LPS-induced expression of pro-inflammatory mediators in RAW 264.7 cells. Idebenone inhibited LPS-induced increase in the levels of NO metabolites, TNF-α, IL-1β, IL-6, and PGE₂ in RAW 264.7 cells. (A) NO metabolite secretion was assessed using Griess reagent. (B) Western blotting was used to analyze the protein expression of inflammatory enzymes (iNOS, COX-2) and pro-inflammatory cytokines (IL-1β, TNF-α) in LPS-stimulated RAW 264.7 cells in the presence or absence of idebenone (1, 5, 10, 20 μM). Cells were pretreated with idebenone (1, 5, 10, 20 μM) for 2 h before LPS treatment (1 μg/mL). After 18 h, the cells were harvested for Western blotting, with β-actin as the loading control. The expression of (C) TNF-α, (D) IL-1β, (E) IL-6, and (F) PGE₂ in LPS-stimulated RAW 264.7 cells in the presence or absence of idebenone was determined using the ELISA assay. Graphs depict the mean of three independent experiments. Statistical analysis was performed using paired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, # p < 0.001.

Figure 5

Idebenone inhibits the LPS-induced activation of NF-κB in macrophages. (A) The phosphorylation levels of IκBα and (B) IKKβ in RAW 264.7 cells were examined using Western blotting analysis. The cells were pre-incubated with or without idebenone (20 μM) for 2 h and subsequently treated with 1 μg/mL of LPS for the indicated times. (C) Western blot analysis for p65 expression in the cytosol and nuclear fraction of RAW 264.7 cells treated with or without idebenone (20 μM), followed by stimulation with 1 μg/mL of LPS for 30 min; Lamin A and tubulin were used as nuclear and cytosolic fraction loading controls. (D) The nuclear translocation of the NF-κB p65 subunit in RAW 264.7 cells pretreated with or without 20 μM of idebenone for 2 h, followed by stimulation with 1 μg/mL of LPS for 30 min, was detected using immunofluorescence staining. Scale bar = 50 μm. (E) The phosphorylation levels of AKT, ERK, JNK, and p38 in macrophages were examined using Western blotting analysis. Macrophages were pre-incubated with or without idebenone (20 μM) for 2 h and subsequently stimulated with 1 μg/mL of LPS for the indicated times; β-actin was used as a loading control.

Figure 6

Idebenone suppresses ROS generation and lipid peroxidation in LPS-induced macrophage. (A) ROS production in DCFDA-stained RAW 264.7 cells was detected using flow cytometry. The cells were preincubated with or without 20 μM of idebenone for 2 h, followed by incubation with a medium containing 1 μg/mL of LPS for the indicated times. (B) The cells were preincubated with or without of idebenone (5, 10, 20, 40 μM) for 2 h, and then incubated with a medium containing 1 μg/mL of LPS for 2 h. (C) The cells were preincubated with 20 μM of idebenone and the ROS inhibitors NAC (5 mM), mito-TEMPO (50 μM), and DPI (10 μM) for 2 h, followed by incubation in a medium containing 1 μg/mL of LPS for 2 h. (D) MDA production in RAW 264.7 cells was measured using a lipid peroxidation assay kit. The cells were preincubated with idebenone (1, 5, 10, 20 μM) for 2 h, followed by incubation in a medium containing 1 μg/mL of LPS for 12 h. (E) RAW 264.7 cells were harvested, and the levels of SOD1 [Superoxide dismutase 1 (Cu-Zn)], SOD2 [Superoxide Dismutase 2 (MnSOD)], and catalase were measured using Western blotting. The cells were pretreated with 20 μM of idebenone for 2 h, followed by incubation in a medium containing 1 μg/mL of LPS for 12 h. Statistical analyses were performed using paired two-tailed Student’s t-test. ** p < 0.01, # p < 0.001.