Objectives: Ischemic stroke is a severe neurological disorder with high morbidity, mortality, and disability rates, posing a substantial burden on patients, families, and healthcare systems. Soy isoflavone (SI), a naturally occurring phytoestrogen, has demonstrated promising neuroprotective effects. This study aimed to evaluate the anti-stroke efficacy of SI and elucidate its underlying mechanisms through integrated phytochemical profiling, network pharmacology, and both in vitro and in vivo experimental validation. Methods: Active constituents of SI were extracted via reflux and identified using liquid chromatography–mass spectrometry (LC-MS). Network pharmacology was employed to predict therapeutic targets and signaling pathways. The neuroprotective effects of SI were first assessed in PC12 cells subjected to oxygen–glucose deprivation/reoxygenation (OGD/R) injury in vitro. For in vivo evaluation, transient cerebral ischemia–reperfusion injury was induced using the bilateral common carotid artery occlusion (BCCAO) model in adult male ICR rats (27.3 ± 1.8 g; 6–8 weeks old), obtained from the Shanghai Experimental Animal Center, Chinese Academy of Sciences. Forty-eight rats were randomly assigned into four groups (n = 12): sham, model (BCCAO), SI-treated (100 mg/kg, oral gavage for 5 days), and edaravone (EDA)-treated (10 mg/kg, i.p., positive control). All procedures were approved by the Institutional Animal Care and Use Committee of Changchun Normal University (Approval No. 2024003, 13 March 2024) and conducted in accordance with the NIH guidelines and ARRIVE 2.0 reporting standards. Results: In vitro, SI significantly enhanced PC12 cell viability from 57.23 ± 2.88% to 80.76 ± 4.43% following OGD/R. It also reduced intracellular Ca²⁺ by 58.42%, lactate dehydrogenase (LDH) release by 37.67%, caspase-3 activity by 55.05%, and reactive oxygen species (ROS) levels by 74.13% (p < 0.05). A flow cytometry analysis revealed that OGD/R increased the apoptosis rate from 5.34% (control) to 30.85% (model group), which was significantly attenuated by SI treatment, especially in the 560 µg/mL group (20.00%), followed by the 140 and 280 µg/mL groups. In vivo, SI improved neurological scores from 8.3 ± 1.09 to 6.8 ± 1.68, reduced cerebral infarction volume by 18.49%, and alleviated brain edema by 10.42% (p < 0.05). SI also decreased malondialdehyde (MDA) and LDH levels by 31.15% and 39.46%, respectively, while increasing the activity of antioxidant enzymes: superoxide dismutase (SOD) by 11.70%, catalase (CAT) by 26.09%, and glutathione peroxidase (GSH-px) by 27.55% (p < 0.01). Scratch assay results showed that SI restored the impaired migratory ability of the OGD/R-treated PC12 cells, further supporting its role in cellular repair. A Western blot analysis demonstrated the upregulation of nuclear factor erythroid 2–related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NAD(P)H:quinone oxidoreductase 1 (NQO1) and the downregulation of Kelch-like, ECH-associated protein 1 (Keap1) in the cerebral ischemia–reperfusion model. Conclusions: These findings indicate that soy isoflavone confers significant neuroprotective effects against cerebral ischemia–reperfusion injury by enhancing endogenous antioxidant defense mechanisms, reducing oxidative stress, inhibiting apoptosis, and promoting cell migration. The protective effects are likely mediated through the activation of the Nrf2/Keap1 signaling pathway, supporting the therapeutic potential of SI in ischemic stroke treatment.

The flowers of Pueraria lobate (Puerariae Flos) have served as a traditional Chinese medicinal and food herbage plant for many years. Tectoridin is one of the most active metabolites extracted from flowers of Pueraria lobate and has a variety of beneficial activities, including antioxidative, hypoglycemic, and anti-inflammatory activities. Nevertheless, the functions and potential mechanisms underlying tectoridin in cerebral ischemia/reperfusion injury have not been well interpreted; thus, a network analysis strategy was performed to systematically evaluate its pharmacological mechanisms, which were further validated in rats with cerebral ischemia. Network analysis predicted that tectoridin could attenuate brain damage after stroke by modulating signaling pathways associated with redox, inflammation, and autophagy. The experimental results demonstrated an improvement in neurological function in rats treated with tectoridin, along with a significant reduction in cerebral infarction volume. The neuroprotective benefits of tectoridin stem, in part, from its antioxidant capabilities, which include the upregulation of Nrf2/HO-1 protein expression, reduction of the TLR4/MYD88/NF-κB inflammatory pathway, and inhibition of the PI3K/Akt/mTOR pathway, contributing to its anti-apoptotic effects. This investigation offers a thorough examination of the pathways and targets linked to the therapeutic effects of tectoridin on ischemic stroke, highlighting its anti-inflammatory, antioxidative, and anti-apoptotic mechanisms. These findings serve as a valuable reference for the development and exploration of effective anti-ischemic stroke medications.

Paeoniflorin (PF), a monomeric compound extracted from the dry roots of Paeonia lactiflora, has been widely used in the treatment of nervous system diseases, marking it as a critical formula in Parkinson’s disease (PD). However, the action of PF against PD and its molecular mechanism are still unclear. In this study, tandem mass tags quantitative proteomics was performed to systematically clarify the underlying mechanism of action of PF against PD and to confirm it using in vivo and in vitro studies. The results showed that PF notably enhanced the viability of PC12 cells and mitigated MPP⁺-induced mitochondrial dysfunction, oxidative stress, and apoptosis. Tandem mass tag-based quantitative proteome analysis revealed the identification of 6405 proteins, of which 92 were downregulated and 190 were upregulated. Among them, the levels of PI3K, AKT, CREB, and BDNF in the MPP⁺-induced PC12 cell and MPTP mice were considerably lower than in the control group, indicating the role of the BDNF/CREB pathway in the pathogenesis of neuroprotection. The related DEP (PI3K, AKT, CREB, and BDNF) expression levels were verified by Western blot. PF effectively restored the altered expression of the four DEPs induced by MPP⁺ and MPTP. Summarily, PF exerted remarkable neuroprotective effects in MPP⁺-induced PC12 cell and MPTP-induced mice. Further, our research may provide proteomics insights that contribute to the further exploration of PF as a potential treatment for PD.