Protective Effects Of17β-estradiol Conjugate On Cerebral Ischemia-reperfusion-induced Neurocyte Injury And The Molecular Mechanisms

OBJECTIVE: To demonstrate the effect of E2-BSA on cerebral ischemia reperfusioninjury and possible mechanism using cerebral ischemia reperfusion (CIR) model inSprague-Dewley rat.METHODS: Through establishing the cerebral ischemia reperfusion(CIR)model inSprague-Dewley rat, hematoxylin-eosin（HE）staining were conducted to test the differentanti-ischemic neuronal injury effects of different concentrations of E2-BSA, and screen thebest pre-protective concentration of E2-BSA; The rats divided into7groups, shamoperation group (SH), ischemic reperfusion model group (IRI), and differentconcentrations E2-BSA treatment groups (5,10,15μM E2-BSA), intranuclear estrogenreceptor (ER) inhibitor ICI182780treatment group (ICI) and PI3K inhibitor LY294002treatment group. Then DNA ladder, Real-time quantitative PCR (RT-PCR) and westernblot were conducted to determine the relationship between the anti-apoptosis effect ofE2-BSA and extranuclear ER in injured nerve cells. Then we investigated the relationshipbetween the protective effect of E2-BSA and PI3K/Akt signaling pathway, and determinedthe activity of Caspase3, the downstream molecule of PI3K/Akt signaling pathway.RESULTS: Our data demonstrated that E2-BSA had a neuroprotective role in cerebralischemia-reperfusion-induced neurocyte injury and10μM E2-BSA was the bestpre-protective concentration compared with other concentrations,. Western blot showedthat cerebral ischemia-reperfusion inhibited the activity of Akt and E2-BSA attenuated thedecrease in Akt activation by cerebral ischemia-reperfusion. PI3K inhibitor LY294002 prevented E2-BSA to upregulate the activity of Akt. DNA ladder showed that cerebralischemia-reperfusion induced neurocyte apoptosis and10μM E2-BSA inhibitedischemia-reperfusion-induced neurocyte apoptosis. This protective effect of E2-BSA wasreversered by co-treatment with PI3K inhibitor LY294002, but not by co-treatment withintranuclear ER inhibitor ICI182780. RT-PCR and western blot showed that cerebralischemia-reperfusion upregulated caspase-3expression and10μM E2-BSA inhibitedischemia-reperfusion-induced overexpression of caspase-3. This inhibitory effect ofE2-BSA on caspase-3expression was reversered by co-treatment with PI3K inhibitorLY294002, but not by co-treatment with intranuclear ER inhibitor ICI182780.CONCLUSION: E2-BSA treatment plays neuroprotective roles in cerebralischemia-reperfusion-induced neurocyte injury in SD mouse. The extranuclear rapidsignaling pathway, but not nuclear ER, was involved in the neuroprotective effect ofE2-BSA. The neuroprotective of E2-BSA is by inhibition of downstream caspase3activities via activating PI3K/Akt signaling pathway.