Scutellarin Alleviates Neuronal Apoptosis in Ischemic Stroke via Activation of the PI3K/AKT Signaling Pathway

Abstract

Among all stroke types, ischemic stroke (IS) occurs most frequently, resulting in neuronal death and tissue injury within both the central infarct region and surrounding areas. This study explored the neuroprotective mechanisms of scutellarin, a flavonoid compound, through an integrated strategy that merged in silico analyses (including network pharmacology and molecular docking simulations) with both in vitro and in vivo experimental verification. We identified 1887 IS-related targets and 129 scutellarin targets, with 23 overlapping targets. PPI network analysis revealed five core targets, and molecular docking demonstrated strong binding affinities between scutellarin and these targets. Bioinformatic analyses, including GO functional annotation and KEGG pathway mapping, indicated that the PI3K/AKT cascade represents the primary signaling mechanism. An in vitro experimental system was developed using PC12 cells under oxygen-glucose deprivation conditions to investigate how scutellarin regulates neuronal cell death via the PI3K/AKT pathway. Western blot quantification demonstrated that treatment with scutellarin enhanced the expression of p-PI3K, p-AKT, and Bcl-2 proteins, while simultaneously reducing levels of apoptotic markers Bax and cleaved caspase-3. Furthermore, pharmacological intervention with the selective PI3K inhibitor LY294002 attenuated these molecular alterations, resulting in diminished expression of p-PI3K, p-AKT, and Bcl-2, accompanied by elevated levels of Bax and cleaved caspase-3. In a rat model of middle cerebral artery occlusion, scutellarin administration demonstrated comparable neuroprotective effects, maintaining neuronal survival and modulating apoptotic protein expression via PI3K/AKT pathway activation. Collectively, this study demonstrates the therapeutic potential of scutellarin in cerebral ischemia through PI3K/AKT pathway modulation, suggesting its possible application in treating ischemic disorders.

Keywords: PI3K/AKT pathway; ischemic stroke; molecular docking; network pharmacology; neuronal apoptosis; scutellarin.

Figure 1

Differential expression analysis between MCAO and sham samples: (A) Distribution of gene expression in the samples. (B) Distance heatmap showing clustering relationships among the samples. (C,D) Volcano plot and heatmap illustrating differentially expressed genes.

Figure 2

Screening of intersection gene targets: (A) Target intersection analysis between scutellarin and cerebral ischemia using Venn diagram. (B) STRING-based protein–protein interaction network construction. (C) Topological visualization of interaction networks. (D) Hierarchical representation of five key molecular targets based on degree centrality.

Figure 3

Functional annotation analysis: (A) Enriched biological processes visualization. (B) Cellular component distribution analysis. (C) Molecular function enrichment patterns. (D) KEGG pathway enrichment landscape.

Figure 4

Molecular docking visualization of scutellarin with core targets: (A) Visualization of scutellarin docking with the CASP1 molecule. (B) Visualization of scutellarin docking with the CASP3 molecule. (C) Visualization of scutellarin docking with the CCL2 molecule. (D) Visualization of scutellarin docking with the EGFR molecule. (E) Visualization of scutellarin docking with the PTGS2 molecule.

Figure 5

Scutellarin alleviates neuronal apoptosis in the ischemic cortex of MCAO rats at three days: Bar = 50 μm, n = 3. (A) TUNEL immunofluorescence images; (B) Quantitative analysis. **** p < 0.001 vs. Sham group; ## p < 0.01 vs. MCAO group.

Figure 6

Effects of scutellarin on Nissl staining in the ischemic cortex of MCAO rats (×100).

Figure 7

PI3K and AKT phosphorylation analysis in MCAO rat ischemic cortex following scutellarin treatment at day 3: (A,C) Representative Western blots and immunofluorescence micrographs; (B,D) Statistical analyses. Green fluorescence indicates NeuN-positive neurons, blue represents DAPI-labeled nuclei, and red shows Cy3-tagged proteins. Statistical significance: ** p < 0.01 vs. Sham; # p < 0.05, ## p < 0.01 vs. MCAO; Scale bar: 50 μm, n = 5.

Figure 8

Apoptotic protein expression analysis in MCAO rat ischemic cortex after 3-day scutellarin treatment: (A,C) Western blot bands and immunofluorescence microscopy; (B,D) Quantitative data analysis. Neurons (NeuN, green), nuclei (DAPI, blue), target proteins (Cy3, red). Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Sham; # p < 0.05, ## p < 0.01 vs. MCAO; Scale = 50 μm, n = 5.

Figure 9

Effect of scutellarin on the mRNA expression of pathway-enriched genes in PC12 cells. Statistical significance: * p < 0.05, ** p < 0.01 vs. Control group; # p < 0.05, ## p < 0.01 vs. OGD group; n = 5.

Figure 10

Impact of PI3K inhibition on scutellarin-mediated protein regulation in PC12 cells: (A,C) Western blot bands; (B,D) Quantitative data analysis. Statistical significance: * p < 0.05, ** p < 0.01 vs. Control; # p < 0.05, ## p < 0.01 vs. OGD; ^▲ p < 0.05, ^▲▲ p < 0.01 vs. OGD+S; n = 5.