OBJECTIVE  To rapidly identify the chemical constituents of Jiannao Tongluo capsule(JNTL) using UHPLC-Q-TOF-MS combined with molecular networking technology, and to further validate the potential mechanism of JNTL in treating cerebral infarction(CI) by integrating network pharmacology and in vitro experimental verification.

METHODS MS/MS spectral data of JNTL were acquired using both positive and negative ion modes mass spectrometry. A global natural products social molecular networking was constructed based on MS/MS fragmentation pattern similarity. Components of JNTL were rapidly identified by analyzing clusters of structurally similar molecules within the network, along with MS/MS data and relevant literature. Network pharmacology was applied to predict the core targets and potential mechanisms of JNTL in the treatment of CI, and visualization networks were generated. Finally, an in vitro oxygen-glucose deprivation/reoxygenation(OGD/R) model was established using HT22 mouse hippocampal neuronal cells for experimental validation.

RESULTS A total of 51 chemical constituents were identified in JNTL, including 11 organic acids, 7 flavonoids and their glycosides, 6 triterpenoid saponins, 2 iridoid glycosides, and 25 other compounds. Network pharmacology analysis revealed 105 overlapping targets between the component targets of JNTL and the disease targets of CI. Gene Ontology functional enrichment analysis identified 608 biological processes, 85 cellular components, and 159 molecular functions. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated 146 pathways(P<0.05). Protein-protein interaction network analysis identified core targets such as interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), and protein kinase B alpha(AKT1); molecular docking results indicated that astragaloside Ⅳ, cryptotanshinone and salvianolic acid B exhibited favorable binding activities with AKT1, TNF-α and IL-6. In vitro experimental results showed that JNTL-containing serum at concentrations up to 20% did not exhibit significant toxicity toward normal HT22 cells. In the OGD/R model, JNTL concentration-dependently increased cell viability and significantly alleviated neuronal injury(P<0.01). Furthermore, ELISA results demonstrated that JNTL significantly inhibited the release of the inflammatory factors TNF-α, IL-6, and IL-1β induced by OGD/R(P<0.05). Western blotting analysis indicated that JNTL activated the PI3K-Akt signaling pathway, as evidenced by significantly increased ratios of p-PI3K/PI3K and p-Akt/Akt, and effectively suppressed the abnormal accumulation of HIF-1α protein(P<0.01).

CONCLUSION For the first time, this study identified 51 chemical constituents from JNTL, thereby elucidating its chemical foundation. Furthermore, it demonstrate that JNTL may exert neuroprotective effects in the OGD/R model by activating the PI3K-Akt signaling pathway, inhibiting the release of inflammatory factors(TNF-α, IL-6, IL-1β), and downregulating HIF-1α protein expression. Additionally, it provides key scientific evidence for elucidating the pharmacodynamic material basis and molecular mechanisms underlying JNTL’s therapeutic effects on CI.