Si-Miao-Yong-An Decoction ameliorates deep vein thrombosis by suppressing NAT10-mediated ac4C modification of HMOX1...

Deep vein thrombosis (DVT) is a common and clinically significant vascular disorder characterized by abnormal thrombus formation in the peripheral venous system. Si-Miao-Yong-An Decoction (SMYAD), a classic traditional Chinese medicine formula with heat-clearing and detoxifying properties, has been widely used in clinical treatment of DVT, yet its precise molecular mechanisms remain unclear.

To demonstrate that SMYAD alleviates DVT by suppressing vascular endothelial ferroptosis via regulation of N-acetyltransferase 10 (NAT10) mediated N4-acetylcytidine (ac4C) modification.

The therapeutic efficacy of SMYAD was evaluated by Murine Doppler Ultrasound, hematoxylin and eosin staining, as well as analyses of ferroptosis indicators and inflammatory factors. To elucidate the regulatory mechanism of SMYAD on NAT10, we performed ultra-high performance liquid chromatography-tandem mass spectrometry, network pharmacology analysis, cellular thermal shift assay, molecular docking and surface plasmon resonance analysis. Furthermore, various assays were performed to evaluate ferroptosis-related parameters, including cellular iron content, lipid reactive oxygen species levels, ac4C dot blotting, qRT-PCR, and western blotting.

After treatment with SMYAD, ac4C modification and NAT10 mRNA and protein levels in vascular tissues were significantly reduced. Containing serum of SMYAD could inhibit the mRNA and protein levels of NAT10, thereby reducing the mRNA and protein levels of Heme oxygenase 1 (HMOX1), and effectively alleviate ferroptosis. The research further confirmed that endothelial-specific knockout of NAT10 mimicked the protective effects of SMYAD. In addition, we identified kaempferol and calycosin as the bioactive constituents of SMYAD, which bind synergistically to NAT10 and downregulate ac4C modification of HMOX1.

This finding reveals a novel mechanism by which SMYAD exerts anti-thrombotic effects, offering a scientific rationale for its clinical application and demonstrating its therapeutic potential in targeting NAT10-regulated ac4C modification of HMOX1 against DVT.