穗花杉双黄酮通过抑制细胞焦亡减轻小鼠急性肺损伤

doi:10.12122/j.issn.1673-4254.2025.04.03

摘要/Abstract

摘要：

目的探究穗花杉双黄酮（AF）通过抑制NLRP3/ASC/Caspase-1轴介导的细胞焦亡减轻急性肺损伤（ALI）的机制。方法体内实验中，将60只BALB/c雌性小鼠随机分为对照组（Control组）、模型组（LPS组）、AF低剂量治疗组（LPS+AF-LD组）、AF中剂量治疗组（LPS+AF-MD组）、AF高剂量治疗组（LPS+AF-HD组），12只/组。灌胃给药0.5 h后气管滴注LPS造模，6 h后取材。体外细胞实验中，将RAW264.7细胞分为对照组（Control组）、模型组（LPS+ATP组）、AF治疗组（LPS+ATP+AF组）、焦亡抑制剂组（LPS+ATP+AC组）、联合处理组（LPS+ATP+AF+AC组），药物预处理2 h后进行造模、指标检测。HE染色法观察肺组织的病理改变，同时检测肺肿胀程度、BALF中蛋白水平、肺组织MPO水平。采用CCK-8法检测AF的细胞增殖毒性及对细胞活力的影响。ELISA法检测IL-1β、IL-6、IL-18、TNF-α水平，通过免疫组化、免疫荧光、免疫印迹等方法检测NLRP3、ASC、Cleaved Caspase-1、GSDMD N等蛋白水平。结果在体内实验中，相较于LPS组，AF药物治疗能改善小鼠肺组织的病理损伤状态，改善肺肿胀程度，降低BALF中蛋白水平、肺组织MPO水平（P<0.01）。AF药物治疗抑制了NLRP3/ASC/Caspase-1轴的高表达（P<0.01），对于细胞焦亡的核心基因GSDMD N起到抑制效应（P<0.01），减少了细胞焦亡导致的IL-1β、IL-6、IL-18、TNF-α等炎性因子释放（P<0.01）。在细胞实验中，通过细胞增殖毒性实验确定了AF的安全浓度，AF能够减少由于LPS+ATP刺激导致的细胞死亡（P<0.01），抑制NLRP3/ASC/Caspase-1轴的高表达，降低GSDMD N及炎症因子的表达水平（P<0.01）。焦亡抑制剂显示出与AF药物治疗类似的效果，而联合处理组的效果更加明显。结论 AF可能通过抑制NLRP3/ASC/Caspase-1轴介导的细胞焦亡发挥减轻ALI的效应。

关键词: 穗花杉双黄酮, 急性肺损伤, 细胞焦亡, NLRP3/ASC/ Caspase-1轴

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

孙亚磊, 罗萌, 郭长胜, 高静, 苏凯奇, 陈立典, 冯晓东. 穗花杉双黄酮通过抑制细胞焦亡减轻小鼠急性肺损伤[J]. 南方医科大学学报, 2025, 45(4): 692-701.

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.

图/表 7

图1 AF对肺组织病理改变及炎性反应的影响

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.

表1 引物序列

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

图2 AF对炎症因子水平的影响

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.

图3 AF对NLRP3、ASC水平的影响

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.

图4 AF对NLRP3/ASC/ Caspase-1轴及细胞焦亡水平的影响

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.

图5 AF对细胞增殖毒性、细胞活性及炎性反应的影响

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.

图6 AF对细胞NLRP3/ASC/ Caspase-1轴及细胞焦亡水平的影响

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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