Multi-omics profiling and functional characterization of Salvia miltiorrhiza-derived nanovesicles: A novel therapeutic approach for atherosclerosis via cross-kingdom nanovesicle regulation

Research on nanovesicles from plants, particularly medicinal varieties, is less developed than studies on those derived from animals. Nonetheless, focused investigations on Salvia miltiorrhiza-derived nanovesicles (SDNV) remain limited. This study aimed to extract dried (SDNV-D) and fresh (SDNV-F) Salvia miltiorrhiza-derived nanovesicles from the decoction and roots of Salvia miltiorrhiza cultivated for one year. The extraction utilized ultra-high-speed differential and sucrose concentration gradient centrifugation. The miRNA of the SDNVs was sequenced using the Illumina SE50 platform, and bioinformatics tools were employed to predict the target genes of the miRNA. Metabolites were extracted via the methanol precipitation method, while isopropanol and acetonitrile precipitation techniques were used for lipids. A total protein extraction kit was used for proteins. Sequencing and analyzing metabolites, lipids, and proteins in SDNVs were conducted using liquid chromatography-tandem mass spectrometry. The functions of SDNV-D were examined through molecular cytology experiments and animal studies. SDNV-D was tagged with Dir and administered via the caudal vein, allowing it to reach various tissues and organs and exert biological effects. SDNV-D injections significantly reduced blood lipids and inflammatory factors in experiments involving model mice with atherosclerosis, helping alleviate and prevent arterial plaque formation. This effect was mediated through the TLR4/NLRP3 inflammatory pathway, which aligned with the anticipated target genes of the miRNAs. To clarify the cellular targets and molecular mechanism of SDNV-D, we conducted in vitro experiments using mouse aortic vascular smooth muscle cells (MOVAS). PKH26 fluorescent labeling confirmed that SDNV-D is actively internalized by MOVAS cells and localized in the cytoplasm. Functional assays showed that SDNV-D significantly inhibits ox-LDL-induced abnormal proliferation of MOVAS cells, reduces the secretion of pro-inflammatory factors, and downregulates key indicators in the TLR4/NLRP3 pathway. Overall, this comprehensive study highlights the potential for plant-derived miRNAs to influence atherosclerosis by regulating gene expression across kingdoms in mammals, providing confidence in the research's depth and hope for advancements in cardiovascular treatments. • First multi-omics atlas of Salvia miltiorrhiza nanovesicles (SDNVs). • SDNV-D targets TLR4/NLRP3 to halt atherosclerosis via cross-kingdom nanovesicle. • Dir tracking reveals multi-organ biodistribution of SDNVs in vivo. • Plant nanovesicles: Dual role as therapeutics and drug carriers. • Optimized scalable SDNV extraction ensures biosafety and stability.