Amentoflavone attenuates abdominal aortic aneurysm progression by suppressing vascular smooth muscle cell phenotypic switching via targeting NF-κB-MMP2/9 axis

To investigate the role and mechanism of amentoflavone (AMF) in abdominal aortic aneurysm (AAA) pathogenesis, providing novel therapeutic candidates and theoretical foundations.

① Animal studies: AAA models were induced by angiotensin Ⅱ (Ang Ⅱ) in 6 to 8-week-old male ApoE^-/- mice randomized (n=10/group) into saline+DMSO, saline+AMF, Ang Ⅱ+DMSO (1000 ng·kg^-1·min^-1), and Ang Ⅱ+AMF (10, 20, 50 mg/kg) groups. AMF or DMSO (100 μL) was intraperitoneally administered every 48 h from day 2 for 28 d; 20 mg/kg AMF (optimal dose by AAA incidence) was selected for subsequent experiments. Alternatively, AAA was induced by periaortic elastase incubation in C57 BL/6J mice randomized (n=6) into sham+DMSO, sham+AMF (heat-inactivated elastase), elastase+DMSO, and elastase+AMF groups for 14 d with identical AMF/DMSO protocols. Survival was monitored; AAA dilation was assessed by ultrasonography; elastic fiber degradation was evaluated via HE and VVG staining; MMP2/MMP9 expression was quantified by RT-qPCR/Western blotting. ② Network pharmacology: AAA-related targets (top 2000 genes from Genecards) and AMF targets (predicted via Galaxy WEB/SuperPred/SwissTargetPrediction) were intersected; protein-protein interaction (PPI) networks were constructed using STRING and visualized in Cytoscape. ③ Cellular experiments: Primary rat aortic vascular smooth muscle cells (RAVSMCs) were treated with: DMSO+CTL, DMSO+PDGFBB (25 ng/mL), AMF+CTL (20 μmol/L), or AMF+PDGFBB. Calponin, αSMA, MMP2, MMP9, IκBα, and phospho-IκBα levels were assessed; NF-κB activation was evaluated via p65 nuclear translocation; proliferation (CCK-8) and migration (scratch/Transwell) were measured. Pathway rescue: TNF-α (20 ng/mL) was co-administered with AMF+PDGFBB to assess target reversibility.

① AMF significantly improved the survival rate of Ang Ⅱ-induced AAA model mice (P<0.05), decreased the incidence of AAA, reduced the degree of abdominal aortic dilation (P<0.01), attenuated elastic fiber fragmentation and degradation in the aortic media (P<0.001), and inhibited the expression of MMP2 (P<0.01) and MMP9 (P<0.05) in AAA tissues. AMF also markedly reduced the extent of abdominal aortic dilation (P<0.001), mitigated medial elastic fiber disruption (P<0.001), and suppressed MMP2 (P<0.001) and MMP9 (P<0.05) expression in the elastase-induced AAA model. ② PPI network analysis revealed that MMP2 and MMP9 occupied a core position within the therapeutic target network. ③ RT-qPCR analysis showed that AMF treatment (20 μmol/L) significantly reversed the PDGF BB (25 ng/mL)-induced increases in MMP2 (P<0.05) and MMP9 (P<0.01) mRNA levels and the decreases in Calponin (P<0.001) and αSMA (P<0.05) mRNA levels in RAVSMCs. Western blotting demonstrated that AMF attenuated the PDGF BBinduced upregulation of MMP2 (P<0.001) and MMP9 (P<0.001) proteins and the downregulation of Calponin (P<0.01) and αSMA (P<0.001) proteins. The CCK-8 assay indicated that AMF markedly suppressed PDGFBB-induced excessive proliferation (P<0.001), and wound healing and Transwell assays showed that AMF significantly inhibited PDGFBB-induced cell migration (P<0.001). TRRUST transcription factor network analysis identified RELA (p65) as the top-ranked transcription factor. Western blotting revealed that AMF substantially decreased the phosphorylation level of IκBα (P<0.001), and immunofluorescence staining demonstrated that AMF effectively reduced the nuclear translocation of p65, thereby inhibiting the NF-κB signaling pathway. In the pathway rescue experiment, RT-qPCR showed that TNF-α (20 ng/mL) treatment counteracted the AMF-induced decreases in MMP2 (P=0.087) and MMP9 (P<0.05) mRNA levels and the increases in calponin (P<0.05) and αSMA (P<0.05) mRNA levels. Western blotting confirmed that TNF-α reversed the AMF-mediated reduction in MMP2 (P<0.001) and MMP9 (P<0.001) protein expression and the elevation of Calponin (P<0.05) and αSMA (P=0.063) protein levels.

AMF attenuates AAA progression by targeting the NF-κB-MMP2/9 axis, demonstrating its potential as a novel AAA therapeutic agent.