Idebenone attenuates cerebral ischemia-reperfusion injury in rats by inhibiting neuronal apoptosis through regulating the GSK-3β/β-catenin pathway

Ischemic stroke is one of the major neurological diseases leading to death and long-term disability. Although reperfusion can restore blood flowing ischemic brain tissue, it can further induce oxidative stress, blood-brain barrier disruption, and neuronal apoptosis; therefore, identifying safe and effective neuroprotective agents is of great clinical significance. This study investigated whether idebenone attenuates cerebral ischemia-reperfusion injury in rats by inhibiting neuronal apoptosis through the glycogen synthase kinase-3 beta (GSK-3β)/β-catenin signaling pathway.

A middle cerebral artery occlusion/reperfusion (MCAO/R) model was established in 87 adult male SD rats (body weight 250 to 300 g) by inserting a suture into the internal carotid artery via the left common carotid artery to occlude the left middle cerebral artery for 2 h, followed by suture withdrawal and 50 h of reperfusion. ① Pharmacodynamic evaluation experiment: Rats were randomly divided into sham operation (Sham) group, ischemia/reperfusion (I/R) group, and I/R+idebenone (IDB) group, with 21 rats per group. Neurological function was assessed using the Modified Neurological Severity Score (mNSS); brain water content was measured by the wet-dry weight method and brain index was calculated; cerebral infarct area was detected by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and 7.0 T magnetic resonance imaging (MRI); blood-brain barrier permeability was assessed by Evans Blue (EB) extravasation staining; neuronal morphology was observed by HE staining; neuronal apoptosis was detected by TUNEL; and the expression of Bcl-2, Bax, and cytochrome c (Cyt c) was detected by Western blotting. ② LRP5/6 co-receptor inhibition experiment: Molecular docking was used to predict the binding of IDB to Frizzled-5. Rats were randomly divided into Sham group, I/R group, I/R+IDB group, and I/R+IDB+Dickkopf-1 (DKK-1) group, with 3 rats per group, and Western blotting was used to detect the expression of total GSK-3β, phosphorylated GSK-3β (Ser9), total β-catenin, and phosphorylated β-catenin (Ser33/Ser37/Thr41). ③ GSK-3β inhibition experiment: Using the highly selective GSK-3β inhibitor TDZD-8, rats were randomly divided into Sham group, I/R group, I/R+IDB group, and I/R+IDB+TDZD-8 group, with 3 rats per group, and Western blotting was used to detect the expression of total GSK-3β, phosphorylated GSK-3β (Ser9), total β-catenin, and phosphorylated β-catenin (Ser33/Ser37/Thr41).

① Pharmacodynamic results showed that compared with the Sham group, the I/R group had significantly increased cerebral EB extravasation, neuronal morphological damage, and percentage of TUNEL-positive cells(P<0.01), accompanied by a decreased Bcl-2/Bax ratio and elevated cytoplasmic Cyt c (P<0.001), indicating that the MCAO/R model caused significant neurological and tissue damage. Compared with the I/R group, the I/R+IDB group showed a decreased mNSS score (P<0.001), reduced brain water content (P<0.001) and brain index (P<0.01), improved MRI-related infarct indicators (P<0.05), smaller corrected cerebral infarct area by TTC (P<0.01), reduced EB extravasation (P<0.001), increased proportion of surviving neurons (P<0.001), and fewer TUNEL-positive cells (P<0.05); Bcl-2 expression and the Bcl-2/Bax ratio were increased, Bax expression was decreased (P<0.05), mitochondrial Cyt c was increased (P<0.05), and cytoplasmic Cyt c was decreased (P<0.01). ② LRP5/6 co-receptor inhibition experiment: Molecular docking confirmed that idebenone had potential binding ability to the Frizzled-5 receptor, with a binding energy of -5.6 kcal/mol. Compared with the I/R group, the I/R+IDB group showed increased phosphorylated GSK-3β (Ser9) and decreased phosphorylated β-catenin (Ser33/Ser37/Thr41) (all P<0.05). After combined intervention with DKK-1, these changes persisted, with no statistically significant difference compared with the I/R+IDB group. ③ GSK-3β inhibition experiment revealed that compared with the I/R group, I/R+IDB alone upregulated GSK-3β phosphorylation (P<0.01) and decreased intracellular phosphorylated β-catenin expression (P<0.05), and TDZD-8 intervention showed a similar regulatory trend to IDB; there was no statistically significant difference in the phosphorylation levels of the above proteins between the I/R+IDB+TDZD-8 group and the I/R+IDB group, and no obvious additive effect was observed.

Idebenone can alleviate neurological deficits, brain edema, cerebral infarction, blood-brain barrier disruption, and neuronal apoptosis after cerebral ischemia-reperfusion in rats, and its effects may be related to inhibiting GSK-3β activity and stabilizing β-catenin signaling; however, whether it directly acts on Frizzled-5 and the dependence on upstream receptors require further verification.