Multi-omics approaches reveal the therapeutic mechanism of Naoxintong capsule against ischemic stroke

ETHNOPHARMACOLOGICAL RELEVANCE: Ischemic stroke (IS) is a leading cause of long-term disability and mortality worldwide. The Chinese Pharmacopeia 2020 lists Naoxintong Capsule (NXT), a traditional Chinese medicine prescription, as having demonstrated substantial therapeutic efficacy for IS. AIM OF THE STUDY: Our study aimed to evaluate the mechanism by which NXT treats IS by integrating the microbiome, transcriptome, and metabolomics. MATERIALS AND METHODS: In a middle cerebral artery occlusion (MCAO) mouse model, the infarction rate, neurological scores, lipopolysaccharide (LPS) levels, inflammatory factor levels (IL-1β, IL-17A, and IL-6), and intestinal permeability proteins (ZO-1, MUC2, and MUC4) were measured to confirm the effect of NXT on the brain and colon. 16S rRNA sequencing, transcriptomics analysis, and targeted amino acid (AA) metabolism were employed to evaluate the mechanism by which NXT treats IS. Furthermore, the neuroprotective effects of specific AAs, identified through targeted AA metabolism, were assessed in PC12 cells following oxygen-glucose deprivation (OGD) injury. In addition, the TLR4/NF-κB pathway was evaluated by Western blot (WB). RESULTS: NXT administration substantially alleviated brain damage and colon injury by decreasing the infarction rate, neurological scores, LPS levels, and inflammatory factors, and increasing the expression of intestinal permeability protein. Transcriptomic analysis revealed that NXT regulated "inflammatory response," "Toll-like receptor signaling pathway,", and "NF-κB signaling pathway." Furthermore, WB confirmed that NXT inhibited the brain TLR4/NF-κB pathway. 16S rRNA sequencing indicated that NXT adjusted the intestinal microbiota composition and decreased the abundance of pathogenic bacteria, including Parasutterella_massiliensis and Ihubacter_excrementihominis. Targeted AA metabolism analysis demonstrated that NXT regulated the serum levels of serine, lysine, and proline in MCAO mice. Furthermore, serine, lysine, and proline inhibited the TLR4/NF-κB pathway to protect against OGD injury in PC12 cells. CONCLUSION: Our study indicates that NXT reduces the abundance of Parasutterella_massiliensis and Ihubacter_excrementihominis, while increasing the levels of serine, lysine, and proline. These changes are significantly associated with neuroinflammation. Furthermore, NXT alleviates IS-induced neuroinflammation by inhibiting the TLR4/NF-κB pathway. Importantly, our study provides novel insights into the mechanisms underlying NXT's therapeutic effects on IS.