Fisetin administration improves LPS-induced acute otitis media in mouse in vivo

Abstract

Acute otitis media is one of the most common infectious diseases worldwide in spite of the widespread vaccination. The present study was conducted to explore the effects of fisetin on mouse acute otitis media models. The animal models were established by lipopolysaccharide (LPS) injection into the middle ear of mice via the tympanic membrane. Fisetin was administered to mice for ten days through intragastric administration immediate after LPS application. Hematoxylin and eosin (H&E) staining was performed and the pro-inflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6 and VEGF, were measured through enzyme-linked immunosorbent assay (ELISA) method and RT-qPCR analysis. Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway was detected by immunoblotting assays. Reactive oxygen species (ROS) generated levels were determined through assessment of anti-oxidants, and TXNIP/MAPKs signaling pathways were explored to reveal the possible molecular mechanism for acute otitis media progression and the function of fisetin. Fisetin reduced mucosal thickness caused by LPS. In fisetin-treated animals, pro-inflammatory cytokine release was downregulated accompanied with TLR4/NF-κB inactivation. ROS production was significantly decreased in comparison to the LPS-treated group. The TXNIP/MAPKs signaling pathway was inactivated for fisetin treatment in LPS-induced mice with acute otitis media. The above results indicated that fisetin improved acute otitis media through inflammation and ROS suppression via inactivating TLR4/NF-κB and TXNIP/MAPKs signaling pathways.

Figure 1

Fisetin ameliorates the middle ear injury of mice after lipopolysaccharide (LPS) exposure. (A) H&E staining analysis of the middle ear sections obtained from mice under different conditions (scale bar, 500 μm). (B) The representative images of ME histophathology in LPS-treated mice with or without fisetin administration exhibited by H&E staining (scale bar, 100 μm) (A). (C) The quantification of mucosa thickness is shown. Data are expressed as the mean ± SEM (n=10). ^*p<0.05, ^**p<0.01 and ^***p<0.001 vs. the control (Con) group; ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. the LPS group.

Figure 2

Fisetin reduced pro-inflammatory cytokine release in lipopolysaccharide (LPS)-induced mice with acute otitis media. The pro-inflammatory cytokines in serum of mice were calculated through enzyme-linked immunosorbent assay (ELISA) methods, including (A) interleukin-1β (IL-1β), (B) tumor necrosis factor-α (TNF-α), (C) IL-6 and (D) VEGF. The pro-inflammatory cytokines in the middle ear effusions of mice were assessed via ELISA kits, including (E) IL-1β, (F) TNF-α, (G) IL-6 and (H) VEGF. The quantification is displayed. Data are expressed as the mean ± SEM (n=10). ^*p<0.05, ^**p<0.01 and ^***p<0.001 vs. the control (Con) group; ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. the LPS group.

Figure 3

Fisetin ameliorates lipopolysaccharide (LPS)-induced inflammation in the middle ear of mice through Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway. Western blot analysis was conducted to explore (A) TLR4 and (B) MyD88 protein expression levels. (C) TLR4 and (D) MyD88 mRNA levels were measured via RT-qPCR assays, and the relative fold quantification is exhibited. The immunoblotting analysis was performed to determine (E) p-IKKα, (F) p-IκBα and (G) p-NF-κB protein levels in the middle ear of mice. Data are expressed as the mean ± SEM (n=10). ^*p<0.05, ^**p<0.01 and ^***p<0.001 vs. the control (Con) group; ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. the LPS group.

Figure 4

Fisetin inhibits apoptosis in the middle ear of mice treated with lipopolysaccharides (LPS). (A) Flow cytometry was used to examine apoptosis in lipopolysaccharide (LPS)-treated mice with acute otitis media after fisetin administration at different concentrations. (B) The percentage of apoptotic cells following flow cytometry analysis was shown. (C) The representative images of cleaved caspase-3, cleaved PARP, Bax, Bad, Bcl-2, and Bcl-xL were shown through western blot analysis. The quantification of (D) cleaved caspase-3, (E) cleaved PARP, (F) Bax, (G) Bad, (H) Bcl-2, and (I) Bcl-xL is exhibited. RT-qPCR assays were used to determine (J) Bax, (K) Bad, (L) Bcl-2 and (M) Bcl-xL gene levels. Data are expressed as the mean ± SEM (n=10). ^*p<0.05, ^**p<0.01 and ^***p<0.001 vs. the control (Con) group; ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. the LPS group.

Figure 5

Fisetin upregulates anti-oxidants levels in lipopolysaccharide (LPS)-exposed mice with acute otitis media. (A) SOD activity, and (B) MDA levels in serum were measured. (C) SOD activity and (D) MDA levels in the middle ear tissues were analyzed. Western blot analysis was included to detect SOD1, SOD2, HO-1 and Nrf2 protein expression levels in the middle ear tissue of mice with LPS induction in the presence or absence of fisetin at different doses. (E) Representative images of western blot analysis are displayed. (F) SOD1, SOD2, HO-1 and Nrf2 protein levels were quantified following western blot results. Data are expressed as the mean ± SEM (n=10). ^*p<0.05, ^**p<0.01 and ^***p<0.001 vs. the control (Con) group; ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. the LPS group.

Figure 6

Fisetin-improves acute otitis media triggered by lipopolysaccharides (LPS) is associated with TXNIP and MAPKs signaling pathways. Western blot assays were conducted to assess (A) TXNIP, (B) NLRP3, (C) ASC, (D) cleaved caspase-1, (E) phosphorylated ERK1/2 and (F) phosphorylated p38 levels in the middle ear of mice after LPS treatment. Data are expressed as the mean ± SEM (n=10). ^*p<0.05, ^**p<0.01 and ^***p<0.001 vs. the control (Con) group; ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. the LPS group.

Figure 7

Fisetin ameliorates the acute otitis media in the middle ear of mice through TXNIP/NLRP3 suppression. The immunofluorescent analysis was used to analyze (A) TXNIP and (B) NLRP3 levels in the middle ear sections with the specific antibodies. The quantification of influorescent intensity is dispalyed. (C) TXNIP and (D) NLRP3 mRNA levels were calculated through RT-qPCR assays. Data are expressed as the mean ± SEM (n=10). ^*p<0.05, ^**p<0.01 and ^***p<0.001 vs. the control (Con) group; ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. the lipopolysaccharides (LPS) group.

Figure 8

Fisetin ameliorates the acute otitis media in the middle ear of lipopolysaccharides (LPS)-treated mice by inhibiting MAPK signaling pathway. (A) p-p38 and (B) p-ERK1/2 levels were determined via the immunofluorescent analysis. The quantification of phosphorylated p38 and ERK1/2 is shown. Data are expressed as the mean ± SEM (n=10). ^*p<0.05, ^**p<0.01 and ^***p<0.001 vs. the control (Con) group; ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. the LPS group.