Protective Effect And Potential Mechanism Of Icariside Ⅱ On Cerebral Ischemia-reperfusion Injury In Mice

Objective:To investigate the neuroprotective effects of icarisideⅡ（ICS Ⅱ）on cerebral ischemia-reperfusion injury（CIRI）and explore its possible mechanism.Methods:The CIRI mice model was established by the middle cerebral artery occlusion（MCAO）method,and the cerebral blood flow（CBF）during the surgical procedure was monitored using laser speckle flowmeter.The mice were randomly divided into seven groups:sham operation group（sham）,sham+ICS Ⅱ-high dose group（sham+ICS Ⅱ 20 mg/kg）,model group（model）,model+ICS Ⅱ-low,-medium and-high dose groups（model+ICS Ⅱ5 mg/kg,ICS Ⅱ 10 mg/kg,ICS Ⅱ 20 mg/kg）and positive drug group（model+edaravone4.5 mg/kg）.Male Nrf2 knockout(Nrf2^-/-)and homologous wild type(Nrf2^+/+)mice were randomly divided into four groups:sham group（sham）,sham+ICS Ⅱ-high dose group（sham+ICS Ⅱ 20 mg/kg）,model group（model）,model+ICS Ⅱ-high dose group（model+ICS Ⅱ 20 mg/kg）.Drug treatment groups were pre-treatement with different doses（5,10,20mg/kg）of ICS Ⅱ or edaravone（4.5 mg/kg）for 7 days.The sham group and model group were given the volume-matched normal saline.Three days after CIRI,neurological functional scores of mice were measured by improved Longa 5 method.The cerebral infarction volume of mice was measured by TTC staining.The presence of ICS Ⅱ in mice brain tissue was determined using liquid chromatography-tandem mass spectrometry,and the molecular docking technology was used to evaluate the binding capacity between ICS Ⅱ and Nrf2.The ROS and MDA levels,and the SOD activity were evaluated using ELISA assay.Results:The results indicated that neurological function scores and cerebral infarction volume of mice were significantly increased in model group those of sham group,the levels of ROS and MDA were augmented,and the activity of SOD was reduced in model group those of sham group;whereas,neurological function scores and cerebral infarction volume of mice were significantly reduced in model group those of sham group,the levels of ROS and MDA were reduced,and the activity of SOD was augmented in model group those of model group.Moreover,ICS Ⅱ not only promoted CBF recovery of mice,but also passed through the blood brain barrier.Of note,the binding energy between ICS Ⅱ and Nrf2 was-7.06 kcal/mol,which suggested that ICS Ⅱ directly bound with Nrf2,and the binding sites including Val608,Val370,Val369,Arg326 Thr609.On this basis,Nrf2^-/-mice were used to further explore the role of Nrf2 in the protective effects of ICS Ⅱ after CIRI.The results revealed that neurological function scores and cerebral infarction volume of model group in Nrf2^-/-mice were significantly lowered than those of sham group in Nrf2^+/+mice;the levels of ROS and MDA were enhanced,and the activity of SOD was reduced;whereas,the protective effects of ICS Ⅱ on CIRI in mice were partially abolished by Nrf2 gene deletion.Conclusion:ICS Ⅱ exerts neuroprotective effects on CIRI,at least partly,via regulation of Nrf2/antioxidative signaling pathway.Whereas,its detailed mechanism deserves further study.