Skimmianine Pretreatment Attenuates Cerebellar Neuroinflammation and Myelin Injury Following Experimental Cerebral Ischemia-Reperfusion

Objective: Cerebral ischemia/reperfusion (I/R) injury triggers oxidative stress, neuroinflammation, neuronal degeneration, and white matter damage not only in directly affected cerebral regions but also in remote brain areas such as the cerebellum. Skimmianine, a naturally occurring furoquinoline alkaloid, has been reported to possess antioxidant and anti-inflammatory properties. This study investigated the protective effects of skimmianine pretreatment against secondary cerebellar injury following experimental cerebral I/R.

Materials and methods: Thirty-two female Wistar rats were randomly assigned to sham, Skimmianine, I/R, and I/R + Skimmianine groups (n = 8/group). Cerebral I/R was induced by transient middle cerebral artery occlusion for 60 min followed by 23 h reperfusion. Skimmianine (40 mg/kg/day, intraperitoneally) was administered for 14 days before ischemia induction. Oxidative stress markers, neuroinflammatory mediators, histopathological alterations, behavioral outcomes, and ultrastructural changes were evaluated. In addition, network pharmacology and molecular docking analyses were performed to explore potential molecular mechanisms.

Results: Cerebral I/R significantly decreased TAS levels compared with sham (0.89 ± 0.15 vs. 1.52 ± 0.18 mmol Trolox Eq/L) and increased TOS (15.60 ± 3.03 vs. 6.80 ± 1.41 µmol H₂O₂ Eq/L), OSI (17.48 ± 0.50 vs. 4.43 ± 0.47), TNF-α (68.4 ± 10.2 vs. 18.6 ± 4.4 pg/mL), Iba1 (41.3 ± 9.7 vs. 11.7 ± 1.6 pg/mL), and GFAP levels (334.5 ± 12.5 vs. 87.7 ± 9.5 ng/mL; all p < 0.001). I/R also impaired motor performance, as shown by increased beam crossing time (11.7 ± 2.2 vs. 4.8 ± 0.7 s) and grid foot fault rate (18.6 ± 4.0% vs. 3.4 ± 1.1%). Skimmianine pretreatment significantly improved these alterations, increasing TAS to 1.29 ± 0.20 mmol Trolox Eq/L and reducing TOS, OSI, TNF-α, Iba1, and GFAP levels to 9.20 ± 2.04, 7.07 ± 0.47, 34.9 ± 7.4, 24.2 ± 6.9, and 237.0 ± 7.9, respectively, compared with the untreated I/R group. Histopathological scores for Purkinje cell loss, edema, vascular congestion, and TNF-α expression were also significantly reduced by skimmianine. Quantitative TEM analysis showed that I/R reduced myelin thickness (0.29 ± 0.05 vs. 0.53 ± 0.07 µm), increased G-ratio values (0.75 ± 0.05 vs. 0.63 ± 0.04), and increased vacuolized fibers (24.70 ± 4.20% vs. 3.20 ± 1.10%), whereas skimmianine partially restored myelin thickness (0.42 ± 0.07 µm), reduced the G-ratio (0.68 ± 0.05), and decreased vacuolized fibers (11.20 ± 2.80%; p < 0.05 vs. I/R). Molecular docking demonstrated favorable binding between skimmianine and TNF-α, with a predicted binding energy of -6.953 kcal/mol.

Conclusions: These findings indicate that skimmianine exerts neuroprotective effects against secondary cerebellar injury following cerebral I/R through coordinated modulation of oxidative stress, systemic neuroinflammatory responses, astroglial injury-associated pathways, and inflammation-related mechanisms.