Scutellarin combined with paeoniflorin for protects against permanent cerebral ischemia in rats through the activation of sonic hedgehog pathway

Abstract

Objective To investigate the protective effect and mechanism of scutellarin combined with paeoniflorin after permanent cerebral ischemia in rats. Methods Forty-eight adult male SD rats were randomly divided into four groups: sham-operation, cerebral ischemia, scutellarin+ paeoniflorin, and cyclopamine (n=12 in each group). A model of permanent middle cerebral artery occlusion was induced by suture method. The intraperitoneal injection of cyclopamine 6 mg/kg, a specific inhibitor of sonic hedgehog (SHH) pathway, at 15 min before ischemia in the cyclopamine group, while other groups were intraperitoneally injected an equal volume of saline. At 0 hour and 3 hours after ischemia, the scutellarin+ paeoniflorin group and cyclopamine group were intraperitoneally injected scutellarin (20 mg/kg) and paeoniflorin (30 mg/kg), while other groups were intraperitoneally injected an equal volume of saline. Neurological deficit scores were performed at 24 hours after ischemia, and then the rats were decapitated. The cerebral infarct volume was measured by using 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Real-time fluorescent quantitative polymerase chain reaction and Western blotting were used respectively to detect the expression levels of SHH, Patched-1, Gli-1 mRNAs and proteins in the ischemic cortex. Results The neurological deficit scores in the cerebral ischemia group, scutellarin+ paeoniflorin group, and cyclopamine group were 3.33±0.52, 1.50±0.55, and 3.67±0.52, respectively. The neurological deficit score in the scutellarin+ paeoniflorin group was significantly lower than that in the cerebral ischemia group (P<0.05), and the neurological deficit score in the cyclopamine group was significantly higher than that in the scutellarin+ paeoniflorin group (P<0.05). The infarct volume percentage in the cerebral ischemia group, scutellarin+ paeoniflorin group, and cyclopamine group were 31.77%±1.19%, 22.94%±2.65%, and 35.53%±0.20%, respectively. The infarct volume in the scutellarin+ paeoniflorin group was significantly less than that in the cerebral ischemia group (P<0.05), and the infarct volume in the cyclopamine group was significantly larger than that of the scutellarin+ paeoniflorin group (P<0.05). The expression levels of SHH, Patched-1, Gli-1 mRNAs and proteins in the cerebral ischemia group, scutellarin+ paeoniflorin group, and cyclopamine group were significantly higher than those in the sham-operation group (all P<0.05). The expression levels of SHH, Patched-1, Gli-1 mRNAs and proteins in the scutellarin+ paeoniflorin group were significantly higher than those in the in the cerebral ischemia group (all P<0.05), and the expression levels of Gli-1 mRNA and protein in the cyclopamine group were significantly lower than those in the scutellarin+ paeoniflorin group (all P<0.05). Conclusions The scutellarin combined with paeoniflorin has certain protective effect on focal cerebral ischemia injury in rats. Its mechanism is associated with the activation of SHH signaling pathway. Key words: Brain Ischemia; Flavonoids; Paeoniflorin; Hedgehog Proteins; Signal Transduction; Neuroprotective Agents; Disease Models, Animal; Rats; Scutellarin