Neuroprotection of rhubarb extract against cerebral ischaemia-reperfusion injury via the gut-brain axis pathway

Background: The gut -brain axis (GBA) plays a central role in cerebral ischaemia-reperfusion injury (CIRI). Rhubarb, known for its purgative properties, has demonstrated protective effects against CIRI. However, it remains unclear whether this protective effect is achieved through the regulation of the GBA. Aim: This study aims to investigate the mechanism by which rhubarb extract improves CIRI by modulating the GBA pathway. Methods: We identified the active components of rhubarb extract using LC-MS/MS. The model of middle cerebral artery occlusion (MCAO) was established to evaluate the effect of rhubarb extract. We conducted 16S rDNA sequencing and untargeted metabolomics to analyze intestinal contents. Additionally, we employed HE staining, TUNEL staining, western blot, and ELISA to assess intestinal barrier integrity. We measured the levels of inflammatory cytokines in serum via ELISA. We also examined blood -brain barrier (BBB) integrity using Evans blue (EB) penetration, transmission electron microscopy (TEM), western blot, and ELISA. Neurological function scores and TTC staining were utilized to evaluate neurological outcomes. Results: We identified twenty-six active components in rhubarb. Rhubarb extract enhanced alpha-diversity, reduced the abundance of Enterobacteriaceae, and partially rectified metabolic disorders in CIRI rats. It also ameliorated pathological changes, increased the expressions of ZO-1, Occludin, and Claudin 1 in the colon, and reduced levels of LPS and D -lac in serum. Furthermore, it lowered the levels of IL -18, IL -6, IL -10, IL -17, and TNF-alpha in serum. Rhubarb extract mitigated BBB dysfunction, as evidenced by reduced EB penetration and improved hippocampal microstructure. It upregulated the expressions of ZO-1, Occludin, Claudin 1, while downregulating the expressions of TLR4, MyD88, and NF-kappa B. Similarly, rhubarb extract decreased the levels of IL -18, IL -6, and TNF-alpha in the hippocampus. Ultimately, it reduced neurological function scores and cerebral infarct volume. Conclusion: Rhubarb effectively treats CIRI, potentially by inhibiting harmful bacteria, correcting metabolic disorders, repairing intestinal barrier function, alleviating BBB dysfunction, and ultimately improving neurological outcomes.