Jiajian Shuyu Pills ameliorates cerebral ischemia-reperfusion injury by regulation hippo signaling and the lipid metabolism-ferroptosis axis.

Ischemic stroke is a life-threatening cerebrovascular disease characterized by focal injury to the central nervous system. Jiajian Shuyu Pills (JJSYP), a modified traditional Chinese medicine formulation derived from Shuyu Pills, consist of multiple herbs, including Rhizoma Dioscoreae, Polygonum multiflorum Thunb, Rehmannia glutinosa Libosch, Codonopsis pilosula (Franch.) Nannf, Atractylodes macrocephala Koidz, Poria cocos (Schw.) Wolf, Paeonia lactiflora Pall, Angelica sinensis (Oliv.) Diels, Ligusticum chuanxiong Hort, Eucommia ulmoides Oliv, Polygala tenuifolia Willd, Acorus tatarinowii Schott, Lycium barbarum L, and Schisandra chinensis (Turcz.) Baill. JJSYP show therapeutic potential for ischemic stroke; however, their bioactive components and molecular mechanisms remain insufficiently defined. This study aimed to evaluate the therapeutic efficacy of JJSYP against cerebral ischemia-reperfusion injury (CIRI) and to elucidate its underlying molecular mechanisms through comprehensive multi-omics integration, thereby providing a scientific basis for the clinical application of JJSYP and the development of novel therapeutic strategies for CIRI. A systematic, multi-step experimental strategy was employed. The protective effects of JJSYP against CIRI-induced neurological deficits were evaluated in a transient middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model, in which mice underwent 1 h of middle cerebral artery occlusion followed by 24 h of reperfusion. And proteomic analysis was performed to identify differentially expressed proteins and predict the signaling pathways involved in the anti-CIRI effects of JJSYP. Then, the bioactive components of JJSYP were identified through chemical profiling combined with network pharmacology. Untargeted metabolomics was used to characterize changes in metabolic profiles, and a "component-target-metabolite-pathway" network was constructed to clarify their potential associations. Finally, molecular biological experiments and lipidomic analysis were conducted to validate the anti-CIRI mechanisms of JJSYP. In vivo experiments showed that JJSYP significantly alleviated cerebral tissue injury and improved neurological function in CIRI mice. Proteomic analysis indicated that JJSYP may mitigate CIRI primarily by regulating the Hippo signaling pathway, which is closely associated with cell survival, proliferation, and apoptosis. Integrated network pharmacology and metabolomics analyses identified six core JJSYP components that potentially modulate seven key targets and regulate six critical CIRI-related metabolic pathways. Validation experiments further confirmed that JJSYP modulated Hippo signaling-related proteins, including p-YAP/YAP, SOX2, and YWHAZ, and improved lipid peroxidation- and ferroptosis-related markers, such as 4-HNE, ACSL4, and PLA2G2A, suggesting that JJSYP may exert anti-CIRI effects by regulating Hippo signaling and the lipid metabolism-ferroptosis axis. This is the first study to systematically investigate the potential anti-CIRI mechanisms of JJSYP through multi-omics analysis. The findings preliminarily suggest that JJSYP alleviates CIRI by modulating the Hippo signaling pathway and the lipid metabolism-ferroptosis axis. This study provides preclinical scientific evidence for the therapeutic effects of JJSYP and offers a feasible strategy for elucidating the mechanisms of traditional Chinese medicine formulas, thereby facilitating their modernization and internationalization.