Amentoflavone alleviates acute lung injury in mice by inhibiting cell pyroptosis

doi:10.12122/j.issn.1673-4254.2025.04.03

Abstract

Abstract:

Objective To investigate the effect of amentoflavone (AF) for alleviating lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and inhibiting NLRP3/ASC/Caspase-1 axis-mediated pyroptosis. Methods Female BALB/c mice were randomly divided into control group, LPS group, and AF treatment groups at low, moderate and high doses (n=12). ALI models were established by tracheal LPS instillation, and in AF treatment groups, AF was administered by gavage 30 min before LPS instillation. Six hours after LPS instillation, the mice were euthanized for examining lung tissue histopathological changes, protein levels in BALF, and MPO levels in the lung tissue. In the in vitro experiment, RAW264.7 cells were pretreated with AF, AC (a pyroptosis inhibitor), or their combination for 2 h before stimulation with LPS and ATP. The changes in cell proliferation and viability were detected using CCK-8 assay, and IL-1β, IL-6, IL-18, and TNF-α levels were determined with ELISA. Immunohistochemistry, immunofluorescence assay, and immunoblotting were used to detect the protein levels of NLRP3, ASC, cleaved caspase-1, and GSDMD N in rat lung tissues and the treated cells. Results In mice with LPS exposure, AF treatment significantly improved lung pathologies and edema, reduced protein levels in BALF and pulmonary MPO level, inhibited the high expression of NLRP3/ASC/Aspase-1 axis, reduced the expression of GSDMD N, and lowered the release of IL-1β, IL-6, IL-18, and TNF‑α. In RAW264.7 cells with LPS and ATP stimulation, AF pretreatment effectively reduced cell death, inhibited activation of the NLRP3/ASC/Aspase-1 axis, and reduced GSDMD N expression and the inflammatory factors. The pyroptosis inhibitor showed a similar effect to AF, and their combination produced more pronounced effects in RAW264.7 cells. Conclusion Amentoflavone can alleviate ALI in mice possibly by inhibiting NLRP3/ASC/Caspase-1 axis-mediated cell pyroptosis.

Key words: amentoflavone, acute lung injury, pyroptosis, NLRP3/ASC/Caspase-1 axis

Yalei SUN, Meng LUO, Changsheng GUO, Jing GAO, Kaiqi SU, Lidian CHEN, Xiaodong FENG. Amentoflavone alleviates acute lung injury in mice by inhibiting cell pyroptosis[J]. Journal of Southern Medical University, 2025, 45(4): 692-701.

Figures/Tables 7

Fig.1 Pathological changes and inflammatory response in the lung tissue of the mice in different groups (Mean±SD). A: Protocols for treatment of the mice. B: HE stainin of mouse lung tissue sections. C: Mouse lung histopathological scores (n=4). D: Wet/Dry ratio of mouse lung tissue (n=4). E: Protein levels in BALF (n=6). F: MPO levels in mouse lung tissue (n=6). ##P<0.01 vs Control group. **P<0.01 vs LPS group.

Tab.1 Primer sequences for RT-qPCR

Gene	Forward primer	Reverse primer
IL-1β	TCG CAG CAG CAC ATC AAC AAG AG	AGG TCC ACG GGA AAG ACA CAG G
IL-6	CTC CCA ACA GAC CTG TCT ATA C	CCA TTG CAC AAC TCT TTT CTC A
IL-18	CAAAGTGCCAGTGAACCCCAGAC	ACAGAGAGGGTCACAGCCAGTC
TNF-ɑ	CAC GCT CTT CTG TCT ACT GAA CTT C	CTT GGT GGT TTG TGA GTG TGA GG
GAPDH	AGG TCG GTG TGA ACG GAT TTG	GGG GTC GTT GAT GGC AAC A

Fig. 2 Levels of inflammatory cytokines in the lung tissue of the mice in different groups (Mean±SD, n=6). A-D: Serum levels of IL-1β, IL-6, IL-18 and TNF‑α. E-H: Levels of IL-1β, IL-6, IL-18 and TNF‑α in BALF. I-J: Immunohistochemical results and quantitative analysis of IL-1β in lung tissue (scale bar=100 μm). ##P<0.01 vs Control group. **P<0.01 vs LPS group.

Fig.3 NLRP3 and ASC levels in the lung tissue of the mice in different groups (Mean±SD, n=6). A: NLRP3 immunofluorescence results. B, C: Quantitative analysis of immunofluorescence results. D: ASC immunofluorescence results. ##P<0.01 vs Control group. **P<0.01 vs LPS group.

Fig.4 Activation of the NLRP3/ASC/Caspase-1 axis and pyroptosis level of in RAW264.7 cells with different treatments (Mean±SD, n=3). A: Western blotting of the proteins. B-E: quantitative analysis of the protein levels. ##P<0.01 vs Control group. **P<0.01 vs LPS group.

Fig.5 Proliferative toxicity, cell viability and inflammatory response in RAW264.7 cells with different treatments. A: Viability of the cells treated with different concentrations of AF (n=3). B: Viability of RAW264.7 cells with different treatments (n=3). C-F: Levels of inflammatory cytokines IL-1β, IL-6, IL-18, and TNF-α in the cells (n=6). G-J: mRNA levels of inflammatory cytokines IL-1β, IL-6, IL-18, and TNF‑α in the cells (n=6). ##P<0.01 vs Control group. **P<0.01 vs LPS+ATP group. &&P<0.01 vs LPS+ATP+AC and LPS+ATP+AF group. &P<0.05 vs LPS+ATP+AC and LPS+ATP+AF group.

Fig.6 Activation of the NLRP3/ASC/Caspase-1 axis and pyroptosis of RAW264.7 cells with different treatments (Mean±SD, n=3). A: Western blotting of the proteins. B-E: quantitative analysis of the protein levels. ##P<0.01 vs Control group; *P<0.05, **P<0.01 vs LPS+ATP group; &P<0.05, &&P<0.01 vs LPS+ATP+AC and LPS+ATP+AF groups.

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