Abstract

Cross-kingdom regulation by microRNAs (miRNAs) has been widely observed across species. Plant-derived miRNAs can be absorbed through the mammalian gastrointestinal tract, subsequently enter the bloodstream, and reach various target tissues. However, the specific composition, types, and functions of miRNAs in traditional Chinese medicinal herbs, such as Forsythia suspensa, remain unexplored. In this study, we identify 225 conserved miRNAs and 121 novel miRNAs in F. suspensa fruit by high-throughput sequencing. Target gene prediction of abundant Forsythia-derived miRNAs reveals that multiple downstream targets of fsu-miR2916-p5 and fsu-miR6478 are components of the human PI3K/AKT pathway, suggesting this pathway may serve as a key regulatory axis co- modulated by Forsythia-derived miRNAs. Overexpression of fsu-miR2916-p5 and fsu-miR6478 in human colorectal epithelial cells DLD1 suppresses the expression of AKT1 and AKT3, as well as the levels of phosphorylated AKT (p-AKT), as demonstrated by western blot analysis. Furthermore, RNA sequencing reveals downregulation of pro- inflammatory genes. RT-qPCR confirms the presence of Forsythia-derived miRNAs in the mouse colon after oral administration of F. suspensa extract, and confocal microscopy analysis validates their accumulation in colonic cells following gavage of synthetic Forsythia-derived miRNAs. Moreover, uptake of synthetic fsu-miR2916-p5 and fsu- miR6478 in DLD1 cells attenuates LPS-induced mitochondrial reactive oxygen species (ROS) production and reduces the expression of inflammation-associated molecules, including ICAM1, TGFBR1, and IL1R1, as assessed by immunofluorescence and western blot analysis. These findings identify miRNAs in F. suspensa and elucidate their regulatory influence on human gene expression and anti-inflammatory activity, thus offering a novel perspective on miRNA-mediated, plant-based therapeutic strategies for colitis.