Fig 2: The schematic presentation elucidating proposed mechanisms that icariside II protects neurons against cerebral I/R‐induced injury. Cerebral I/R induces excessive autophagy, resulting in neuronal death. ICS II, a PDE 5 inhibitor, reduces autophagic influx after OGD/R insults via mediating the PKG/GSK‐3β/autophagy axis

Fig 3: Icariside II (ICS II) attenuates oxygen–glucose deprivation and reoxygenation (OGD/R)‐induced cytotoxicity and inhibited PDE 5. (a) Primary cortical neurons were treated with different concentrations of icariside II and cell viability was determined by MTT (n = 5). Neuronal cells were treated with or without icariside II for 24 hr after OGD/R. (b) Cell viability was measured using the MTT assay (n = 5). (c) Cytotoxicity was detected using the intracellular lactate dehydrogenase (LDH) release assay (n = 5). (d) The morphology of neuronal cells was observed using reverse‐phase microscope. (e) PDE 5A expression. (f) Quantitation of PDE 5A (n = 5). (g) PDE 5 activity. (h) cGMP level (n = 5). (i) PKG activity (n = 5). The docking results of icariside II with the PDE 5 complex. (j) The whole view of the icariside II dimer and PDE 5 displaying the molecular binding pocket. (k) A close‐up amplification of the molecule binding pocket from the side. (l) Crystal structure of icariside II (blue) displaying PDE 5 (yellow and pink) bound to the docking pocket. Hydrogen bonds were shown as green dotted lines. Data were expressed as mean ± SEM of five independent experiments. *P < .05 versus control group; # P < .05 versus OGD/R group

Fig 4: Icariside II (CS II) suppresses oxygen–glucose deprivation and reoxygenation (OGD/R)‐induced neuronal injury by mediating PKG/GSK‐3β/autophagy axis. (a) The binding energy has been surfaced and key residues of icariside II with GSK‐3β were displayed using in silico molecular docking. (A) The substrate binding surface. (B) The substrate binding sites. (C) The substrate binding pocket. (D) Amino acid residues. Neurons were treated with or without icariside II and GSK‐3β inhibitor SB216763 after OGD/R for 24 hr. (b) Cell viability was detected using the MTT assay. (c) Cell toxicity was detected using the intracellular lactate dehydrogenase (LDH) release assay. (d) PD5 activity (n = 5). (e) cGMP level (n = 5). (f) PKG activity (n = 5). (g) Representative Western blots were shown for PDE 5A, LC3B, and Beclin 1 expression. (h) Quantitation of PDE 5A expression, LC3‐II/LC3‐I ratio, and Beclin 1 expression (n = 3). Neurons were treated with or without icariside II and PKG inhibitor KT5823 after OGD/R for 24 hr. (i) Cell viability was detected using the MTT assay. (j) Cell toxicity was detected using the LDH release assay. (k) PD5 activity (n = 5). (l) cGMP level (n = 5). (m) PKG activity (n = 5). (n) Representative Western blots were shown for PDE 5A expression and levels of p‐ser9‐GSK‐3β and p‐tyr216‐GSK‐3β. (o) Quantitation of PDE 5A expression and levels of p‐ser9‐GSK‐3β and p‐tyr216‐GSK‐3β. The data are presented as the mean ± SEM. *P < .05 versus control group; # P < .05 versus OGD/R group; ▲ P < .05 versus OGD/R + ICS II 25 μM group

Fig 5: The interaction between PKG and GSK‐3β. (a) The interaction between PKG and GSK‐3β was displayed using ZDOCK. (A) The substrate binding sites. (B) The substrate binding surface. (C) PKG bound to GSK‐3β. (D) Interactions of PKG with active site amino acids and Pi of GSK‐3β. The PKG protein was shown in red colour solid ribbon, while GSK‐3β protein was in green. (b) The binding affinity of PKG with GSK‐3β was evaluated by a surface plasmon resonance assay. Various concentrations of PKG were mixed with GSK‐3β, and binding was detected