The Protective Role Induced By Low Genistein From Oxidative Stress Injury And Spatial Learning And Memory Deficits Via ENOS/Nrf2-ARE Signaling Following Global Cerebral Ischemia

Objects：Stroke is the second leading cause of mortality and the thirdleading cause of disability worldwide. Approximately16million first-everstrokes occur each year, leading to nearly6million deaths. Nevertheless,currently very few therapeutic options are available. Stroke with complexmechanisms is well known to involve in energy metabolism disorder,excitatory amino acids toxicity, free radicals and inflammation. Considerableevidence implicates oxidative stress in the pathophysiology of ischemic strokehas now become a major of focus of both clinical and basic science research.The current study examined the potential beneficial effect and underlyingmechanisms of post-treatment with the naturally occurring isoflavonicphytoestrogen, genistein (GEN), which has been implicated to attenuateoxidative stress. Genistein (1mg/kg) was administered iv5-min afterreperfusion in rats subjected to global cerebral ischemia (GCI), andfurthermore using morphology, molecular biology and behavior methodsobserved the neuro-protective role, special learning and memory function ofGEN-treatment, as well as the possible mechanisms. Our current study willprovide a new insight for exploring drug targets, therapeutic strategy ofischemic stroke.Methods: Global cerebral ischemia (GCI) is induced by four-vesselocclusion (4-VO) in SD rats. Coronal frozen sections of5d reperfusion werestaining with NeuN/Fluoro-Jade B, Cresyl Violet staining or TUNEL methodto investigate neuronal survival/death of hippocampal CA1/3region; Coronalfrozen sections of3d reperfusion were used to detect the proteins/kinasesexpression and oxidative stress injury in the hippocampal CA1region;Homogenate (total protein, cytoplasm or nucleus) from hippocampal CA1was for western blot analysis for eNOS, p-eNOS, HSP90, Nrf2, HO-1and forco-immunoprecipitation for HSP90with p-eNOS. Biotin switch technique wasused to investigate S-nitrosation level of Keap1and Nrf2DNA bindingactivity was detected using TransAMTM kit. Morris Water Maze was used toobserve spatial learning and memory function.Results:11mg/kg GEN decreases neuronal damage through increasing interaction ofeNOS-HSP90following GCI in hippocampal CA1region1) GEN strongly protects the hippocampal CA1region from GCI-induceddelayed neuronal cell death. NeuN and TUNEL staining results showed thatGCI (ischemia10min followed by5d reperfusion) induced a significant loss ofhippocampal CA1region neurons, as indicated by a marked decrease ofNeuN-positive cells compared with sham group. In addition, the number ofTUNEL-positive cell was markedly increased in the hippocampal CA1regionfollowing GCI, compared with sham. More important, GEN post-treatmentstrongly attenuated the delayed neuronal cell death in the hippocampal CA1region, as evidenced by a decreased number of TUNEL-positive cells andincreased number of NeuN-positive cells compared with the vehicle-controlgroup. L-NAME, an inhibitor of eNOS markedly abolished theneuro-protective role induced by GEN-treatment.2) GEN induces a significant increase in phospho-eNOS levels and HSP90protein expression in the hippocampal CA1region following GCI. Westernblot analysis revealed that GEN-treatment significantly increased p-eNOSlevels at30min,1d and3d reperfusion, as compared with the I/R group. Inaddition, the protein expression of HSP90was enhanced by GEN at late phaseof ischemia/reperfusion (1d,3d). The levels of total eNOS and β-actin werenot significantly changed between groups.3) L-NAME reverses the GEN-induced elevation the levels of e-NOS,HSP90, as well as the interaction between the two proteins. Western blotresults showed that pre-treatment with L-NAME (1mg/kg)30min prior toischemia reversed the GEN-induced elevation of p-eNOS as compared with the vehicle group (I/R3d+0.9%NaCl). Furthermore, confocal result showedthe representative double immunohistochemistry results for NeuN andp-eNOS in the CA1region at reperfusion day3. The results mirror theWestern blot results observed with a robust increase of p-eNOSimmunofluorescence observed in the GEN group, which was abolished byL-NAME pre-treatment. Note that the p-eNOS immunostaining colocalizedwell with NeuN, suggesting the neuronal induction of p-eNOS followinggenistein treatment in the hippocampal CA1region. Additionally, GENmarkedly increased the interaction between p-NOS and HSP90compared withI/R3d group, which was reversed by L-NAME-treatment. While the HSP90protein level was no change between the two groups (GEN group andL-NAME group).4) Effect of L-NAME on genistein-induced improvement in spatial learningand memory. Morris water maze that was carried out during7-9d afterischemia results illustrated the effects of genistein and L-NAME treatment onlatency time in finding the hidden platform during latency trials. Latency timein sham-operated and genistein groups became progressively shorter in aday-dependent manner, while there was no significant difference in ischemicvehicle control and L-NAME treated groups that showed a longer timesearching for the hidden platmorm compared to GEN group. In probe trailscharacterized by the removal of the hidden platform, I/R and L-NAME treatedrats spent less time in the goal quadrant, which previously contained theplatform compared to GEN-treated group and sham group, displayedimproved learned bias, as evidenced by spending more time in the goalquadrant.2.1mg/kg GEN enhanced S-nitrosylation of Keap1via up-regulating eNOSactivation, which induced the anti-oxidative stress signaling pathway ofNrf2-HO-11) Genistein induces S-nitrosylation of Keap1in hippocampal CA1region.S-nitrosylation of Keap1was assayed in CA1protein samples at3daysreperfusion using the biotin-switching method. The result showed that GEN markedly induced S-nitrosylation of Keap1as compared with the I/R group,while pre-treatment with L-NAME markedly attenuated this effect. TotalKeap1and β-actin proteins were not significantly changed between groups.Additionally, GEN induced a significant increase of S-nitrosylated proteinimmunostaining in CA1pyramidal neurons that was localized primarily in thecytosol. L-NAME treatment markedly attenuated the effect, implicating a rolefor eNOS in regulating CA1protein nitrosylation.2) GEN enhances nuclear accumulation of Nrf2in hippocampal CA1neurons. Western blot analysis revealed that GEN significantly elevated Nrf2levels in the nucleus, with a corresponding decrease in the cytoplasm, at1dand3d reperfusion, suggesting that genistein treatment induced nucleartranslocation of Nrf2at these later time points. Interestingly, L-NAMEpre-treatment markedly attenuated the nuclear translocation of Nrf2at3-daysreperfusion. Furthermore, the Western blot results were confirmed by confocalanalysis of double immunohistochemistry for Nrf2and NeuN, showing higherneuronal Nrf2immunoreactivity in the genistein group, as compared to I/Rgroup and L-NAME-treated groups at3d reperfusion.3) GEN enhances DNA binding activity of Nrf2in hippocampal CA1region.DNA binding activity of Nrf2was significantly increased by GEN, ascompared with the ischemic control group (reperfusion3d), an effect that wasreversed by pretreatment with L-NAME.4) GEN up-regulates HO-1protein expression in hippocampal CA1region.Western blot analysis showed that HO-1levels were significantly increased byGEN, as compared with control groups at3d reperfusion, and L-NAMEattenuated the increase. In agreement with the results, confocalimmunochemistry analysis showed that genistein significantly enhanced thelevels of HO-1at3d reperfusion, with co-localization mainly with DAPI (anuclear fluorescent probe), indicating that HO-1mainly localized in thenucleus.5) GEN attenuates oxidative damage in hippocampal CA1neuronsfollowing GCI. The effects of GEN on oxidative stress were observed using immunostaining analysis at3d reperfusion for4-HNE (marker for lipidperoxidation) and8-OHdG (marker for oxidative DNA damage). The resultsshowed that sham and GEN groups presented very weak straining for4-HNEand8-OHdG in hippcampal CA1region. However, robust increases inimmunostaining intensity for4-HNE and8-OHdG were observed in bothischemic control and L-NAME pre-treated groups.3. Nrf2signaling in CA3region contributes the neuro-protective role inducedby1mg/kg GEN on CA1neurons following GCI1) Keap1-tat peptide exerts a robust neuroprotective role against GCI.Histology of hippocampal CA1neurons at5d reperfusion after GCI revealedthat vehicle-treated and ischemia/reperfusion animals that underwent15-minGCI displayed a significant neuronal loss as compared to sham controls.Keap1-tat (30,50,100μg) administered by icv20min prior to GCI couldsignificantly increased the number of survival neuron in the hippocampal CA1region compared to vehicle-treated groups. While among the three differentdoses,50μg keap1-tat peptide exerted the best effect on neuro-protectionagainst GCI insult.2) Keap-1-tat peptide enhances cognitive outcome following GCI. Morriswater maze experiment showed that vehicle-treated and ischemia/reperfusionanimals that underwent GCI showed significant longer latencies in finding thesubmerged platform on days7-9after stroke as compared to sham controls. Incontrast, pre-treated with keap1-tat (50μg) had significantly decreasedlatencies to find the submerged platform on day7-9as compared to thevehicle group. Furthermore, when the platform was removed on day9,vehicle-treated and ischemia/reperfusion animals spent significantly less timewithin90s in the quadrant where the submerged platform was located ascompared to sham animals. In contrast, keap1-tat (50μg)-treated rats spentsignificantly greater amount of time in the quadrant where the submergedplatform was located as compared to vehicle group.3) Keap1-tat peptide had no significant effects on either histology or speciallearning and memory function of sham group rats. Results in Morris water maze showed that50μg keap1-tat peptide in nonischemic control animals (icvfor5d) markedly unchanged histology of hippocampal CA1neuron and alsounaffected congnitive outcome at7-9d compared with non-ischemic controlsham.4) Keap1-tat peptide increased the level of Nrf2in nucleus fraction. Inkeap1-tat pre-treatment groups, Nrf2levels in nucleus fraction markedlyincreased at3h,6h,12h,1d,3d compared with the same time-point animalsthat without keap1-tat administration. Between sham contral animals andsham+keap1-tat groups showed no significant change in Nrf2expression innucleus fraction of hippocampal CA1region.5) Similar to GEN, Keap1-tat protected CA1neurons from ischemic insult.0.4μg KA was injected into CA3region by icv, and Cresyl Violet stainingshowed that CA3neuronal loss was observed, while a majority of CA1neurons are intact at8d after treatment with KA. KA with Keap1-tat (injectinto CA3by icv at72h after KA-treatment) had no significant change in thesurvival neuronal number of CA1region, while it markedly decreased CA3neuronal loss compared with KA alone treatment. In addition, treatment withKA72h prior to ischemia resulted in a large of CA3neuronal loss, while CA1neurons were almost survival. Compared with I/R+KA group, administratedthe rats with Keap1-tat (CA3region by icv)20min prior to ischemia perfectlyprotected CA3neurons with intact CA1neuronal histology from GCI insult.Intriguingly, KA significantly abolished the protective role of GEN on CA1neurons following GCI, as evidence by a large number of neurons not only inCA1also CA3regions were lost.6) Keap1-tat induced nucleus translocation of Nrf2following GCI and KAabolished the function. Confocal results showed that at1d reperfusion, intreatment with Keap1-tat by icv to CA3region group Nrf2mainly distributedin CA3neuronal nucleus with a little in the cytoplam, while in KA+I/R+GENgroup Nrf2mainly presented in cytoplasm with a little in perinuclear andnuclear. At3d reperfusion, in Keap1-tat-treatment group Nrf2almostpresented in neuronal nuclear of CA3region, while in KA+I/R+GEN group Nrf2practically distributed in the cytoplasm. The results indicated that Nrf2might nucleus translocated from cytoplasm depending on the time ofreperfusion and the KA lesion leading initially to loss of CA3hippocampalneurons blocked Nrf2nucleus translocation induced by GEN.Conclusion:The current study adds to a growing literature on beneficial effects ofphytoestrogen compounds, and provides important insights into theantioxidant and neuroprotective mechanisms of GEN. Specifically, low doseGEN was demonstrated to exert profound neuroprotection, antioxidant andcognitive function preservation effects in GCI via enhanced eNOS activationand (NO)-mediated thiol modification of Keap1, with subsequentup-regulation of the Nrf2/HO-1antioxidative signaling pathway, which mayinvolve a regulation of Nrf2signaling of CA3on CA1neuronal survivalfollowing GCI.