Bulk and single-cell transcriptomics reveal neuroprotective molecular mechanism of Rosa roxburghii polysaccharides in cerebral ischemia-reperfusion injury

Rosa roxburghii polysaccharides (RP) have anti-inflammatory and neuroprotective properties, but their therapeutic mechanisms in cerebral ischemia-reperfusion injury (CIRI) remain undefined. Our goal was to clarify functional mechanisms of RP alleviating neuropathological damage caused by CIRI. Male C57BL/6 mice (n = 18 per group) were used for in vivo experiments to evaluate neuroprotective effects. Middle cerebral artery occlusion (MCAO) was employed to construct CIRI model, and administered RP at doses of 200, 400, and 800 mg/kg. Bulk RNA sequencing combined with network pharmacology analyses was utilized to pinpoint potential RP target genes. Bulk RNA-seq and scRNA-seq analyses were conducted on independent sets of brain samples. PPI networks, along with TF and miRNA regulatory networks, were developed for identification. Single-cell RNA sequencing (scRNA-seq) was conducted to describe cell populations, intercellular communication, and pseudotime trajectories in CIRI. Quantitative PCR (qPCR) was performed to validate gene expression. RP significantly alleviated neuronal damage, reduced neuroinflammation, and inhibited neuronal apoptosis in CIRI mice. Twelve candidate target genes were identified, and among them GFAP, HMOX1, LGALS3, and TLR2 were recognized as hub genes based on PPI network analysis and multiple centrality algorithms. These genes were expressed predominantly in immune-related cell types, including microglia, neutrophils, astrocytes, and neural progenitor cells. Microglia showed extensive interactions with other immune cells. Trajectory analysis revealed that M2 microglia were located closer to the root, while M1 microglia were near the terminal branch. HMOX1, LGALS3, and TLR2 were highly expressed in both M1 and M2 microglial subtypes. All four hub genes were upregulated in CIRI and significantly downregulated after RP treatment. RP exerts neuroprotective effects in CIRI by modulating key inflammatory targets and immune cell responses. These findings highlight RP's therapeutic potential in ischemic stroke in preclinical mouse models and warrant further investigation into its active components and clinical application.