Unraveling ancient wisdom: The molecular mechanism of Gujin Luyan Xuming decoction in combating cerebral ischemia-reperfusion injury.

Background: Cerebral infarction significantly impairs the quality of life of affected patients, emphasizing the need for research into novel targeted therapeutic agents. For nearly 2000 years, Gujin Luyan Xuming decoction (GJLYXMD) has been a classical treatment for this condition; however, the precise molecular mechanism remains unclear.

Purpose: This study aimed to examine the mechanism of GJLYXMD in middle cerebral artery occlusion/reperfusion (MCAO/R) rats via the integration of multi-omics approaches, network pharmacology, and molecular dynamics simulation.

Methods: The efficacy of GJLYXMD in treating cerebral ischemia-reperfusion injury (CIRI) was initially assessed in vivo. The identification of active ingredients of GJLYXMD absorbed in the brain was conducted using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Subsequently, 16S rDNA sequencing, fecal metabolomics, ischemic lateral cerebral cortex metabolomics, proteomics, and network pharmacology were employed to elucidate potential mechanisms of GJLYXMD in the treatment of CIRI. The therapeutic feasibility of GJLYXMD for CIRI was ultimately supported by molecular docking, molecular dynamics simulation, and correlation analysis.

Results: Pharmacodynamic assessment indicated that GJLYXMD could ameliorate cerebral ischemia injury, with the most pronounced effect observed in the GJLYXMD-H group. A total of 14 active ingredients absorbed in the brain were identified by UPLC-MS/MS analysis. Analysis of 16S rDNA and fecal metabolites indicated that GJLYXMD-H could modulate intestinal microbiota and metabolites, restore the intestinal barrier, and reduce inflammatory cytokine levels. 235 differential metabolites were identified in the cerebral cortex between the Model and GJLYXMD-H groups. The differential proteins between these groups were enriched in the P53 and necroptosis signaling pathways, further validated by network pharmacology and animal studies. Molecular docking and molecular dynamics simulation studies indicated that the primary component of GJLYXMD can bind to the P53 protein.

Conclusion: This study identifies, for the first time, 14 active components absorbed in the brain of GJLYXMD. Besides, GJLYXMD can alter the composition of intestinal flora and fecal metabolic pathways, and inhibit P53-mediated necroptosis, consequently improving CIRI. These findings illustrate considerable translational potential of GJLYXMD in the management of CIRI and warrant further research and development.