The Mechanism Of Icariside ? In Suppressing Oxygen-glucose Deprivation/reperfusion-induced Primary Neuronal Death

Objective:To investigate the effect of ICS ? against primary neuronal damage induced by oxygen-glucose deprivation and re-oxygenation(OGD/R)and explore its potential mechanism.Methods:The stimulant brain ischemic/reperfusion model was induced by OGD/R in rat primary neurons.Sildenafil(SIL)was used as positive control.Primary hippocampal neurons were randomly divided into seven groups:control group,control+ICS ? 50 ?M group,OGD/R group,OGD/R+ICS ? 12.5 ?M group,OGD/R+ICS ? 25 ?M group,OGD/R+ICS ? 50 ?M group and OGD/R+SIL 20 ?M group.Primary cortical neurons were randomly divided into eight groups:control group,control+ICS ? 25 ?M group,OGD/R group,OGD/R+ICS ? 6.25 ?M group,OGD/R+ICS ? 12.5 ?M group,OGD/R+ICS ? 25 ?M group,OGD/R+3-methyladenine(3-MA)1 mM group and OGD/R+rapamycin 20 ?M group.Effects of the ICS ? on OGD/R-induced primary hippocampal and cortical neuronal injury were examined by MTT assay,lactate dehydrogenase(LDH)release,TUNEL staining and double staining of AnnexinV-FITC/PI,respectively.Autophagic flux was examined by adenovirus with mRFP-GFP-LC3.Expressions of memory-related,apoptotic and autophagic signaling pathways proteins were measured using Western blot analysis.The direct interaction between ICS ? and PDE5 was further evaluated by molecular docking experiment.Results:(1)ICS ?(12.5 ?M,25 ?M,50 ?M)markedly abrogated OGD/R-induced hippocampal neuronal death as suggested by the increase in cell viability and reduce in cellular LDH release.Furthermore,ICS II effectively restored the cyclic guanosine monophosphate(cGMP)level and the activity of its downstream target protein kinase G(PKG),increased brain-derived neurotrophic factor(BDNF),tyrosine protein kinase B(TrkB)and cAMP response element-binding protein(CREB)expressions,and suppressed the ratio of Bax/Bcl-2 and activated caspase-3.Mechanistically,those favorable effects of ICS ? were attributed to its activation of the PKG/TrkB/BDNF via increasing BDNF expression,evidenced by that the inhibition effects of ICS ?were almost abrogated by Rp-8-Br-cGMPS(PKG inhibitor)or ANA-12(TrkB inhibitor).ICS ? also decreased both protein expression and activity of PDE5.Notably,ICS ? effectively bound and inhibited PDE5 as demonstrated by relatively high binding score.(2)ICS ?(6.25 ?M,12.5 ?M,25 ?M)markedly abrogated OGD/R-induced primary cortical neuronal death as suggested by the increase in cell viability and decrease in cellular LDH release.Moreover,autophagy was activated as evidenced by triggering autophagic influx and increasing autophagy-related gene(ATG)expressions after stimulation of OGD/R.However,ICS ? significantly inhibited excessive autophagy as evidenced by repressing autophagic flux,combined with decreasing ATG expressions including microtubule-associated protein 1 light chain 3(LC3 ?),Beclin 1,ATG5 and ATG7.ICS ?,a PDE5 inhibitor,not only restored cGMP/PKG pathway,but also inactivated GSK-3? through mediating its phosphorylation.Moreover,siGSK-3?obviously promoted the beneficial effect of ICS ?.Conclusion:ICS ?,a PDE5 inhibitor,reduces OGD/R-induced apoptosis in primary hippocampal neurons,and autophagy in primary cortical neurons under the laboratory conditions,and its underlying mechanism may be due to the regulation of BDNF/TrkB/CREB and PKG/GSK-3? signaling pathway.