Integrated bioinformatics and experimental verification to dissect the mechanisms and bioactive ingredients of Radix Rehmanniae in treating sclerosis

Multiple sclerosis (MS), as a primary cause of nontraumatic disability in young adults, has no effective treatment yet. Radix Rehmanniae (RR), a typical Traditional Chinese Medicine (TCM), is commonly used in MS patients as a most frequent herbal item in TCM formulas. Our recent study demonstrated that RR alleviated neurological deficits in an experimental MS model. However, direct evidence regarding the holistic mechanisms and bioactive components of RR for MS remains unclear. In this study, we employed an integrative strategy combining bioinformatics and experimental validation to profile the holistic mechanisms of RR, identify its bioactive components, and investigate their potential targets in MS. First, a network pharmacology approach was used to construct a "compound-target-pathway" network, indicating the action of RR on MS in a multicomponentmultitarget mode, and predicting Echinacoside and Acteoside as the primary bioactive ingredients. Bioinformatics analyses of transcriptomics and single-cell RNA sequencing based on GSE datasets indicated that oxidative stress and inflammatory/immune regulation in microglia might serve as crucial mechanisms of Echinacoside and Acteoside in MS pathology. Then, in vitro assays validated that Echinacoside and Acteoside possessed antiinflammatory and antioxidant properties by scavenging ONOO-and H2O2 directly, and suppressing microgliaderived ONOO-production through inhibition of NF-kappa B-mediated iNOS and NADPH oxidase. In addition, molecular docking showed strong affinities between Acteoside and inflammation-related targets TGF-beta and SMAD2. These findings provide the scientific evidence for clinical application of RR and bring novel insights into MS drug development.