Multi-Method Investigation of the Role of the PI3K/Akt Pathway in Sinomenine-Mediated Neuroprotection Against Acute Ischemic Stroke.

BACKGROUND: Effective neuroprotective therapies for acute ischemic stroke (AIS) remain limited due to the complex interplay between neuroinflammation and apoptosis. Sinomenine (Sino), a bioactive alkaloid derived from Sinomenium acutum, exhibits anti-inflammatory and anti-apoptotic activities; however, its molecular targets and mechanisms in AIS remain unclear. This study aimed to identify potential targets and key pathways of Sino and validate its neuroprotective effects in AIS.

METHODS: A combined approach integrating network pharmacology, Mendelian randomization (MR), molecular docking, and in vivo validation was adopted. Potential targets of Sino and ischemic stroke were identified using public databases. Overlapping targets were analyzed through protein-protein interaction network construction and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Sprague-Dawley rats were randomly assigned to four groups, and a middle cerebral artery occlusion/reperfusion (MCAO/R) model was established (n = 12 per group): Sham, MCAO/R, Sino (20 mg/kg), and Sino +LY294002 (LY, 10 mg/kg). Sino and Sino + LY were administered intraperitoneally within 6 h after surgery and once daily thereafter for three days. Sham and MCAO groups were given the same amount of physiological saline undergoing the same procedures. Sino was administered intraperitoneally within 6 h after surgery and once daily thereafter for three days. Neurological deficits, infarct volume, neuronal injury, apoptosis, activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway, and inflammatory responses were assessed using behavioral tests, 2,3,5-Triphenyltetrazolium chloride (TTC)/Nissl/Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) staining, Western blotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA).

RESULTS: Twelve overlapping targets between Sino and ischemic stroke were identified, with Akt1 recognized as a central hub. Enrichment analysis highlighted the PI3K/Akt pathway as a critical signaling axis, while MR analysis indicated a nominal association between Akt1 and ischemic stroke. Molecular docking predicted stable binding between Sino and Akt1. In MCAO/R rats, Sino significantly improved neurological function, reduced infarct volumes, attenuated neuronal apoptosis, and increased neuronal survival. Mechanistically, Sino increased the p-PI3K/PI3K and p-Akt/Akt ratios, upregulated Bcl-2 expression, and decreased the expression of Bax, cleaved caspase-3, ionized calcium-binding adapter molecule 1 (Iba1), inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). These beneficial effects were notably attenuated by LY.

CONCLUSION: This study establishes PI3K/Akt as a functionally necessary mediator of Sino's neuroprotection against cerebral ischemia/reperfusion injury. The incomplete LY reversal indicates multi-target activity, supporting Sino's development as an adjunctive therapeutic candidate for ischemic stroke.