Buyang huanwu decoction inhibits the activation of the RhoA/Rock2 signaling pathway through the phenylalanine metabolism pathway, thereby reducing neuronal apoptosis following cerebral ischemia-reperfusion injury.

Ethnopharmacological relevance: Buyang Huanwu Decoction (BYHWD) exerts its anti-cerebral ischemia effects through multiple pathways and targets, although its specific mechanisms remain unclear.

Aim of the study: Ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS) metabolomics and other methods were employed to investigate the role of BYHWD in inhibiting neuronal apoptosis following cerebral ischemia-reperfusion by modulating the RhoA/Rock2 pathway.

Methods: A rat model of exhaustion swimming combined with middle cerebral artery occlusion (ES + I/R) was established to evaluate the intervention effects of Buyang Huanwu Decoction on cerebral ischemia-reperfusion. This was assessed using neurological function scores, Qi deficiency and blood stasis syndrome scores, HE staining, Nissl staining and TT staining. Differential metabolites and metabolic pathways associated with cerebral ischemia-reperfusion were identified using UPLC-QTOF-MS metabolomics, with key differential metabolites validated through ELISA. Molecular docking techniques were employed to predict interactions between the key differential metabolite, hippuric acid, and its primary downstream pathways. Finally, the levels of neurocellular apoptosis, as well as key molecules in the RhoA/Rock2 signaling pathway and the mitochondrial apoptosis pathway, were measured.

Results: The results indicated that the primary differential metabolites associated with BYHWD's protective effects against ischemia-reperfusion injury were hippuric acid, lysophosphatidic acid, and lysophosphatidylethanolamine, with the main metabolic pathway being phenylalanine metabolism. Molecular docking studies demonstrated that malonic acid exhibited a strong affinity for proteins related to the RhoA/Rock2 signaling pathway and the mitochondrial apoptosis pathway.Furthermore, we found that BYHWD treatment significantly decreased the apoptosis rate of cells following cerebral ischemia-reperfusion and inhibited the expression of key molecules in both the RhoA/Rock2 signaling pathway and the mitochondrial apoptosis pathway in brain tissue.

Conclusion: BYHWD ameliorated brain tissue injury after cerebral ischemia/reperfusion in rats with qi deficiency and blood stasis. The underlying mechanism may involve BYHWD's inhibition of the RhoA/Rock2 signaling pathway activation through modulation of the phenylalanine metabolism pathway, thereby reducing neuronal apoptosis mediated by the mitochondrial apoptosis pathway.