| Objective To investigate the neuroprotective effects of Angong Niuhuang Pill (ANP) on cerebral ischemia-reperfusion injury (CIRI) in rats and explore the potential underlying mechanisms. Methods Network pharmacology was employed to identify the active components of ANP and stroke-related targets and pathways, followed by GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses. A CIRI model was induced via middle cerebral artery occlusion/reperfusion (MCAO/R) method. Forty-eight healthy male Sprague-Dawley (SD) rats were randomly divided into four groups (n=12 per group): sham operation group, model group, ANP treatment group, and Protein Kinase C Epsilon (PKCε) inhibitor group. Behavioral performance was evaluated by the Morris water maze test, neurological function was assessed using the modified Garcia score, and cerebral infarct volume was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Intracellular Ca2? concentration was detected by flow cytometry, pathological changes were observed by hematoxylin-eosin staining, and the expression of PKCε and sulfonylurea receptor 1 (SUR1) was analyzed via immunohistochemistry. Western blotting was performed to detect the protein levels of PKCε, SUR1, inward-rectifier potassium channel subfamily J member 11 (Kir6.2), caspase-3, and caspase-9. HT22 cells were divided into control, model, ANP, and PKCε inhibitor groups. Except for the control group, an oxygen–glucose deprivation/reoxygenation (OGD/R) model was established in vitro. Calcium concentration, reactive oxygen species (ROS) levels, apoptosis, and protein expression were assessed by flow cytometry and Western blot to investigate the involvement of the PKCε/mitochondrial ATP-sensitive potassium (mitoK_ATP) pathway. Results Target intersection analysis between ischemic stroke and drug-related targets identified 212 overlapping targets, mainly associated with apoptosis and mitochondrial regulation. GO and KEGG analyses suggested that ANP exerts neuroprotective effects by modulating intrinsic and neuronal apoptosis, with the PKCε/mitoK_ATP pathway potentially serving as a key mechanism. In vivo, ANP treatment improved cognitive performance in cerebral ischemia–reperfusion injury (CIRI) rats, as evidenced by increased time spent in the target quadrant [(17.59 ± 0.81) vs. (10.60 ± 2.05) s, P < 0.001] and distance traveled [(299.12 ± 21.88) vs. (165.84 ± 36.86) cm, P < 0.001] in the Morris water maze, elevated neurological scores [(15.40 ± 1.52) vs. (12.00 ± 1.00), P < 0.05], reduced infarct size [(16.82 ± 2.78)% vs. (49.43 ± 2.91)%, P < 0.001], decreased brain water content [(72.54 ± 1.75)% vs. (83.58 ± 1.65)%, P < 0.001], and alleviated histopathological damage, accompanied by reduced intracellular Ca2? levels [(3,351.67 ± 456.22) vs. (21,107.00 ± 4,698.18) RFU, P < 0.001]. Meanwhile, ANP upregulated PKCε expression [(0.77 ± 0.08) vs (0.51 ± 0.04), P < 0.01] and downregulated Kir6.2 [(0.56 ± 0.03) vs (0.98 ± 0.01), P < 0.05], SUR1 [(0.35 ± 0.08) vs (0.70 ± 0.10), P < 0.05], caspase-3 [(0.67 ± 0.17) vs (1.18 ± 0.20), P < 0.05], and caspase-9 [(0.70 ± 0.07) vs (1.01 ± 0.11), P < 0.05] expression. In vitro, PKCε inhibition significantly increased calcium [(14,213.33 ± 283.38) vs. (7,699.00 ± 401.98) RFU, P < 0.05], ROS [(39,943.00 ± 1,297.52) vs. (19,495.67 ± 714.18) RFU, P < 0.05], and apoptosis rate [(24.94 ± 2.51)% vs. (17.92 ± 1.46)%, P < 0.05] compared with the ANP group. PKCε inhibitor treatment reduced PKCε protein [(0.43 ± 0.03) vs. (0.56 ± 0.07), P < 0.05] and increased SUR1 [(0.70 ± 0.07) vs. (0.42 ± 0.12), P < 0.05], Kir6.2 [(1.10 ± 0.16) vs. (0.76 ± 0.08), P < 0.05], caspase-3 [(0.56 ± 0.02) vs. (0.46 ± 0.05), P < 0.05], and caspase-9 [(0.59 ± 0.04) vs. (0.44 ± 0.03), P < 0.05] expression. Conclusion ANP may exert a neuroprotective effect against CIRI by activating the PKCε/mitoKATP pathway, thereby reducing calcium overload and inhibiting the caspase-dependent apoptotic cascade. |
