Multi-omics analysis of Nanovesicles derived from Salvia miltiorrhiza under different treatment conditions and functional evaluation of SDNV-D
Research on plant-derived nanovesicles, especially those from medicinal plants, is relatively limited compared to the extensive studies of animal-derived nanovesicles. One such medicinal plant, Salvia miltiorrhiza, is widely used to treat cardiovascular diseases in various forms worldwide. However, studies explicitly focusing on Salvia miltiorrhiza-derived nanovesicles (SDNV) remain scarce. This study aimed to extract dried (SDNV-D) and fresh (SDNV-F) Salvia miltiorrhiza-derived nanovesicles from decoction and fresh roots grown for one year. The comprehensive extraction process involved ultra-high-speed differential and sucrose concentration gradient centrifugation. The morphology and particle size of the SDNVs were analyzed using transmission electron microscopy and nanoparticle tracking technology. The Illumina SE50 platform was used to sequence the SDNVs' miRNA and predict the miRNA target genes through bioinformatics techniques. The metabolites were extracted using the methanol precipitation method. For lipids, isopropanol and acetonitrile precipitation techniques were used. For protein, the plant total protein extraction kit was used. The metabolites, lipids, and proteins in SDNVs were sequenced and analyzed by liquid chromatography-mass spectrometry. Functions of specific miRNAs were evaluated through molecular cytology experiments and animal studies. The results indicated that SDNVs possess ideal particle sizes, well-defined morphological structures, and negative surface charge. These natural nanovesicles are rich in miRNAs, metabolites, and proteins and demonstrate biosafety. SDNV-D was labeled with Dir and injected via the caudal vein; it reached multiple tissues and organs to exert its effects. In experiments with atherosclerosis model mice, injection of SDNV-D effectively reduced blood lipids and inflammatory factors, helping to mitigate and prevent arterial plaque formation. This effect operated through the TLR4/NLRP3 inflammatory pathway, aligning with the predicted target genes of miRNAs. Overall, this comprehensive study provides valuable insights into the potential of plant-derived miRNAs to intervene in atherosclerosis through cross-kingdom regulation of gene expression in mammals, offering reassurance in the thoroughness of the research.