Cytisine Exerts A Significant Neuroprotection Against Focal Cerebral Ischemia-induced Impairment In Mice By Regulating Extrasynaptic NR2B-ERK/CREB Signal Pathway

Objectives In the present study, we investigated whether CYT(Cytisine) would exert an neuroprotection to the brain against the focal cerebral ischemia injury in mice and elucidated the reveal potential mechanismMethods 1. To obseve the protective effects of CYT on focal cerebral ischemia in mice ① ICR mice were successfully subjected to middle cerebral artery suture-occlusion(MCAO)by using intraluminal suture. ② The method of Bederson was used to test the Neurological deficit scores in mice. ③ Volume of brain infarction was measured by 2% 2,3,5-triphen-yltetrazolium chloride(TTC) staining. ④ The area of cerebral infarction and the volume of cerebral infarction were assessed with the method of TTC staining in brain tissues. ⑤ The dry/wet weight method was used to evaluate the brain water content in brain tissues. ⑥ Histopathological impairment was estimated by performing hematoxylin and eosin(HE) staining in ischemic brain tissues. ⑦ The apoptosis rate of neurons in ischemic cortex and hippocampus were detected by Terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL) staining in ischemic brain tissues. ⑧ Ultrastructural changes was evaluated by Electron Microscopy. 2. To explore the possible protective mechanism of CYT on cerebral ischemia and reperfusion injury in mice. ① Antioxidant enzyme activity and content of malondialdehyde(MDA)were assessed by the method of spectrophotometry in brain tissues. ② Expression of NR2 B,p-ERK/t-ERK and p-CREB/t CREB were determined by Immunofluorescence andWestern blot assay in brain tissues, respectively. ③ Reverse transcriptase–polymerase chain reaction(RT-PCR)was used to quantify the m RNA level,of NR2 B, ERK and CREB in ischemic brain tissues.Results 1. Neuroprotection of CYT on focal cerebral ischemia injury in mice. Compared to the model group: ① CYT(0.5, 1 mg/kg) and Nimodipine(6 mg/kg) reduced the neurological deficits scores(P<0.01). ② CYT(0.5, 1 mg/kg) and Nimodipine(6 mg/kg)reduced the volume of cerebral infarction(P<0.01). ③ CYT(1 mg/kg) and Nimodipine(6 mg/kg)reduced the brain water content(P<0.05). ④ CYT(0.5, 1 mg/kg) and Nimodipine(6 mg/kg) ameliorated histopathological morphological lesion dramaticly. ⑤ CYT(0.25, 0.5 and 1 mg/kg) and Nimodipine(6 mg/kg) alleviated neuronal apoptosis ischemic cortex and hippocampus(P<0.05,P<0.01). ⑥ CYT(1 mg/kg) and Nimodipine(6 mg/kg)observationally resisted organelles impairment and ultrastructural changes. 2. Possible protective mechanism of CYT on cerebral ischemic injury in mice. Compared to the model group: ① CYT(0.5, 1 mg/kg) and Nimodipine(6 mg/kg) increased the activity of SOD, CAT and reduced the content of MDA(P<0.05,P<0.01). CYT(0.25, 0.5, 1 mg/kg)and Nimodipine(6 mg/kg) enhanced GSH-Px(P<0.05,P<0.01).② CYT(1 mg/kg) and Nimodipine(6 mg/kg) suppressed the NR2 B immunoreactivity of NR2 B, decreased the m RNA and protein expression of NR2B(P<0.01). ③ CYT(1 mg/kg) and Nimodipine(6 mg/kg) enhanced the immunoreactivity of p-ERK(P<0.05,P<0.01), facilitated the the expression of the m RNA and protein expression of ERK(P<0.01). ④ CYT(1 mg/kg) and Nimodipine(6 mg/kg) strengthened the immunoreactivity of p-CREB(P<0.01), increased the protein(P<0.01)and m RNA(P>0.05)expression of CREB.Conclusions The results indicate that CYT has significantly protective effects against the focal cerebral ischemia-induced injury in mice and may confer neuroprotection bymodifying the extrasynaptic NR2B-ERK/CREB cascade signal pathway.