低剂量Genistein诱导eNOS/Nrf2-ARE通路对缺血后神经元氧化应激损伤和空间学习记忆缺陷的保护作用
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摘要
目的：中风不仅是全球第二大主要死亡原因，而且已成为长期致残的首要病因之一。据统计每年新发中风病人约1600万，导致近600万人死亡。然而，目前尚未发现理想的治疗措施。缺血性脑损伤的病理过程极为复杂，其发病机制涉及脑组织能量代谢紊乱、兴奋性氨基酸毒性、自由基损伤、炎症反应等多环节。近些年来，氧化应激学说逐渐成为缺血性脑血管疾病的临床与基础研究的热点。
染料木黄酮（Genistein，GEN）是从植物中提取的大豆异黄酮类物质，大量研究报道GEN具有抗氧化作用。本研究在大鼠全脑缺血后5min尾静脉注射低剂量GEN，采用形态学、分子生物学、行为学等实验技术观察其对缺血后锥体神经元的保护作用和对大鼠空间学习记忆的影响，并探索其可能的信号转导机制。为探索缺血性脑中风的药物靶点和有效治疗措施提供重要的理论基础和实验依据。
方法：采用SD大鼠四动脉结扎全脑缺血模型，制备缺血再灌注5d的海马冠状冰冻切片，形态学（NeuN/Fluoro-Jade B、焦油紫染色、TUNEL染色等）实验技术观察海马CA1/3区神经元生存及损伤情况；制备缺血再灌注3d的海马冠状冰冻切片，采用免疫荧光双染或三染色技术观察激酶或蛋白的表达与亚细胞分布，观察海马CA1区神经元氧化应激损伤情况；制备海马CA1区总蛋白及胞浆、胞核组织蛋白匀浆液，采用westernblot技术进行激酶或蛋白如eNOS, p-eNOS, HSP90, Nrf2, HO-1的半定量分析，免疫共沉淀技术观察p-eNOS和HSP90的相互作用；生物素转换法观察Keap1的巯基亚硝基化（S-nitrosation）水平；TransAMTM试剂盒检测Nrf2的DNA结合活性；Morris Water Maze观察大鼠的空间学习记忆功能。
结果：1. GEN (1mg/kg)通过增加eNOS-HSP90的相互作用减轻缺血后海马CA1区神经元损伤
1) GEN显著降低全脑缺血诱导的迟发性神经元损伤。与sham组相比，溶剂对照组（缺血再灌注5d+溶剂对照）NeuN阳性细胞数显著减少，而TUNEL阳性细胞数显著增多；另外，缺血后给予GEN组海马CA1区NeuN染色阳性神经元数量较溶剂对照组显著增加，而凋亡样神经元明显减少；NOS抑制剂L-NAME可显著减弱GEN诱导的神经保护作用。
2) GEN显著增加大鼠海马CA1区神经元eNOS磷酸化水平及HSP90蛋白的表达。Western blot结果显示，与缺血再灌注相同时间点相比，GEN组再灌注30min、1d和3d时间点p-eNOS水平显著升高；HSP90的蛋白表达于再灌注的后期（1d、3d）GEN组较缺血再灌注（ischemia/reperfusion，I/R）组显著升高。
3) L-NAME显著抑制GEN诱导的p-eNOS水平和HSP90蛋白表达的升高，抑制了二者的相互结合。Western blot结果显示：与溶剂对照组相比（I/R3d+0.9%NaCl），缺血前30min给予1mg/kg L-NAME可显著降低GEN诱导的p-eNOS和HSP90水平的升高；共聚焦结果显示，与缺血再灌注3d组相比，GEN组p-eNOS免疫染色显著增强，主要分布在海马CA1区神经元的细胞浆，L-NAME显著废弃了此作用。与缺血再灌注组相比，GEN组p-eNOS和HSP90的免疫共结合显著升高，而L-NAME显著逆转了GEN的此作用，HSP90的蛋白表达在GEN组和L-NAME组并未发生显著变化。
4)于大鼠缺血后7-9d实施Morris水迷宫，观察GEN和L-NAME对大鼠寻找水下平台的时间的影响。实验结果显示：sham组和GEN处理组大鼠的潜伏时间随着训练时间的延长而缩短，而I/R组和L-NAME处理组无显著差异，且均较GEN组显著延长。移走水下平台后，I/R组和L-NAME组大鼠在平台原有象限探索的时间较GEN组和sham大鼠探索的时间显著缩短。结果表明GEN可改善大鼠缺血后空间学习记忆缺陷。2.1mg/kg GEN通过上调eNOS活性促进Keap1巯基亚硝基化，激活Nrf2-HO-1抗氧化信号通路，减轻大鼠缺血后氧化应激损伤
1) GEN显著增加Keap1巯基的亚硝基化。采用生物素不可逆转换方法观察GEN对缺血后大鼠海马CA1区神经元Keap1亚硝基化水平的影响。结果显示：GEN显著升高缺血后3d Keap1的亚硝基化水平，缺血前给予L-NAME可降低此作用；而Keap1的总蛋白和β-actin水平没有显著变化。另外，共聚焦结果显示海马CA1区GEN组亚硝基化总蛋白的免疫染色较I/R组和L-NAME组显著增强，而且主要定位在神经元的细胞浆。
2) GEN增加了Nrf2的核转位。Western blot结果显示：在缺血再灌注组的后期（1d和3d）GEN显著增加了海马CA1区细胞核中Nrf2的蛋白水平，而其在细胞浆中的表达显著减少；激光共聚焦结果显示GEN组Nrf2和神经元核蛋白NeuN的免疫共定位较缺血再灌注组显著增强；L-NAME显著降低了GEN的此作用。
3) GEN显著增加海马CA1区Nrf2的DNA结合活性。与缺血再灌注3d组相比，GEN处理组Nrf2的DNA结合活性显著升高，而缺血前给予L-NAME显著废弃了GEN的此作用。
4) GEN显著增加海马CA1区Nrf2靶基因HO-1的蛋白表达。Westernblot分析显示：与缺血再灌注3d实验组相比，GEN显著增加了海马CA1区HO-1蛋白水平，而L-NAME降低了此作用。免疫荧光染色结果进一步证明，GEN增加了HO-1的荧光强度，其主要与细胞核荧光探针DAPI共定位，提示主要分布在CA1区神经元的细胞核中。
5) GEN降低脑缺血诱导的氧化应激损伤。采用免疫荧光染色技术观察GEN对脂质过氧化物最终产物4-HNE及DNA氧化损伤的特异性产物8-OHdG水平的影响。结果发现，sham组和GEN处理组海马CA1区4-HNE和8-OHdG免疫荧光的强度较弱，而缺血再灌注3d实验组和L-NAME处理组海马CA1区神经元4-HNE和8-OHdG的免疫染色显著增强。3. GEN可通过诱导海马CA3区Nrf2信号通路调控其神经保护作用
1) Keap1-tat小肽降低缺血后海马CA1区神经元损伤。焦油紫染色显示，与sham组相比缺血15min再灌注5d实验组和溶剂对照组海马CA1区神经元严重损伤，神经元密度降低；缺血前20min侧脑室注射Keap1-tat小肽（30，50，100μg）能显著增加海马CA1区生存的神经元数量，50μg剂量组生存的神经元数量最多，提示50μg为最佳剂量。
2) Keap1-tat可显著改善大鼠缺血后空间学习记忆功能。缺血15min再灌注7-9d实施Morris水迷宫试验。结果发现，与sham组相比溶剂对照组和缺血再灌注组大鼠寻找安全平台的潜伏期明显延长，而keap1-tat处理组(50μg)大鼠寻找安全平台的潜伏期较溶剂对照组显著降低；移走安全平台后，与sham组相比，90s内溶剂对照组和缺血再灌注组大鼠于平台所在象限探索的时间明显缩短，而keap1-tat小肽组大鼠在此象限停留时间较溶剂对照组显著延长。
3) Keap1-tat小肽未显著影响正常大鼠海马CA1区神经元的形态及大鼠的空间学习记忆功能。与sham组相比，脑室注射50μg keap1-tat小肽(sham+keap1-tat组)的正常大鼠海马CA1区神经元存活数量没有显著差异，而且两组大鼠的空间学习记忆功能无显著差异。
4) Keap1-tat小肽能显著增加细胞核中Nrf2水平。与再灌注同一时间点(I/R)组相比，侧脑室注射50μg keap1-tat小肽组海马CA1区神经元细胞核中Nrf2表达均显著升高。Sham组与sham+keap1-tat组海马CA1区神经元细胞核中Nrf2表达无显著差异。
5) Keap1-tat具有与GEN相同的神经保护作用。侧脑室（CA3区）微量注射0.4μg/μl (5μl) KA，于第8d焦油紫染色发现海马CA3区神经元大量损伤，而CA1区仅有少量神经元损伤。与单纯KA(0.4μg/μl)处理组相比，KA+Keap1-tat组海马CA3区大量神经元形态正常，少见损伤的神经元，CA1区生存的神经元数量两组无显著差异。缺血10min再灌注5（dI/R）+KA组与单纯KA处理组相似，海马CA3区神经元严重损伤，而CA1区神经元损伤不显著。与I/R+KA处理组相比，缺血前侧脑室注射（CA3区）Keap1-tat小肽后再灌注5d海马CA1和CA3区神经元均无显著损伤；有趣的是，KA+I/R+GEN组可见海马CA3和CA1区神经元严重损伤。结果提示，0.4μg/μl (5μl) KA特异损伤海马CA3区神经元；Keap1-tat可有效降低缺血后CA1区神经元损伤；KA可阻抑GEN对缺血后海马CA1区神经元的保护作用。
6) Keap1-tat可诱导缺血后海马CA3区Nrf2的核转位，KA可降低此作用。免疫荧光双染色结果显示：缺血后再灌注1d时，CA3区给予Keap1-tat组Nrf2主要分布在神经元细胞核中，胞浆中分布较少；而KA+I/R+GEN处理组Nrf2除主要分布在胞浆外，在细胞核周围及核内也有较少分布。在缺血再灌注3d时，Keap1-tat组Nrf2几乎完全分布在细胞核中；KA+I/R+GEN组Nrf2几乎完全分布在细胞浆中。结果提示，在Keap1-tat处理组Nrf2可能随再灌注时间的延长发生了核转位，而KA阻断了此作用。结论：
缺血再灌注后给予低剂量GEN可通过上调eNOS-HSP90信号保护海马CA1区神经元，改善缺血后大鼠的空间学习和记忆功能；其分子机制可能是通过Keap1的亚硝基化诱导了Nrf2-HO-1抗氧化信号通路；海马CA3区Nrf2信号可能参与了GEN诱导神经保护作用的调控。我们的研究为临床探索治疗缺血性脑中风的药物研发提供了重要靶点，为临床防治中风的策略提供理论依据。
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 CA1region
1) 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-1
1) 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 GCI
1) 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.
染料木黄酮（Genistein，GEN）是从植物中提取的大豆异黄酮类物质，大量研究报道GEN具有抗氧化作用。本研究在大鼠全脑缺血后5min尾静脉注射低剂量GEN，采用形态学、分子生物学、行为学等实验技术观察其对缺血后锥体神经元的保护作用和对大鼠空间学习记忆的影响，并探索其可能的信号转导机制。为探索缺血性脑中风的药物靶点和有效治疗措施提供重要的理论基础和实验依据。
方法：采用SD大鼠四动脉结扎全脑缺血模型，制备缺血再灌注5d的海马冠状冰冻切片，形态学（NeuN/Fluoro-Jade B、焦油紫染色、TUNEL染色等）实验技术观察海马CA1/3区神经元生存及损伤情况；制备缺血再灌注3d的海马冠状冰冻切片，采用免疫荧光双染或三染色技术观察激酶或蛋白的表达与亚细胞分布，观察海马CA1区神经元氧化应激损伤情况；制备海马CA1区总蛋白及胞浆、胞核组织蛋白匀浆液，采用westernblot技术进行激酶或蛋白如eNOS, p-eNOS, HSP90, Nrf2, HO-1的半定量分析，免疫共沉淀技术观察p-eNOS和HSP90的相互作用；生物素转换法观察Keap1的巯基亚硝基化（S-nitrosation）水平；TransAMTM试剂盒检测Nrf2的DNA结合活性；Morris Water Maze观察大鼠的空间学习记忆功能。
结果：1. GEN (1mg/kg)通过增加eNOS-HSP90的相互作用减轻缺血后海马CA1区神经元损伤
1) GEN显著降低全脑缺血诱导的迟发性神经元损伤。与sham组相比，溶剂对照组（缺血再灌注5d+溶剂对照）NeuN阳性细胞数显著减少，而TUNEL阳性细胞数显著增多；另外，缺血后给予GEN组海马CA1区NeuN染色阳性神经元数量较溶剂对照组显著增加，而凋亡样神经元明显减少；NOS抑制剂L-NAME可显著减弱GEN诱导的神经保护作用。
2) GEN显著增加大鼠海马CA1区神经元eNOS磷酸化水平及HSP90蛋白的表达。Western blot结果显示，与缺血再灌注相同时间点相比，GEN组再灌注30min、1d和3d时间点p-eNOS水平显著升高；HSP90的蛋白表达于再灌注的后期（1d、3d）GEN组较缺血再灌注（ischemia/reperfusion，I/R）组显著升高。
3) L-NAME显著抑制GEN诱导的p-eNOS水平和HSP90蛋白表达的升高，抑制了二者的相互结合。Western blot结果显示：与溶剂对照组相比（I/R3d+0.9%NaCl），缺血前30min给予1mg/kg L-NAME可显著降低GEN诱导的p-eNOS和HSP90水平的升高；共聚焦结果显示，与缺血再灌注3d组相比，GEN组p-eNOS免疫染色显著增强，主要分布在海马CA1区神经元的细胞浆，L-NAME显著废弃了此作用。与缺血再灌注组相比，GEN组p-eNOS和HSP90的免疫共结合显著升高，而L-NAME显著逆转了GEN的此作用，HSP90的蛋白表达在GEN组和L-NAME组并未发生显著变化。
4)于大鼠缺血后7-9d实施Morris水迷宫，观察GEN和L-NAME对大鼠寻找水下平台的时间的影响。实验结果显示：sham组和GEN处理组大鼠的潜伏时间随着训练时间的延长而缩短，而I/R组和L-NAME处理组无显著差异，且均较GEN组显著延长。移走水下平台后，I/R组和L-NAME组大鼠在平台原有象限探索的时间较GEN组和sham大鼠探索的时间显著缩短。结果表明GEN可改善大鼠缺血后空间学习记忆缺陷。2.1mg/kg GEN通过上调eNOS活性促进Keap1巯基亚硝基化，激活Nrf2-HO-1抗氧化信号通路，减轻大鼠缺血后氧化应激损伤
1) GEN显著增加Keap1巯基的亚硝基化。采用生物素不可逆转换方法观察GEN对缺血后大鼠海马CA1区神经元Keap1亚硝基化水平的影响。结果显示：GEN显著升高缺血后3d Keap1的亚硝基化水平，缺血前给予L-NAME可降低此作用；而Keap1的总蛋白和β-actin水平没有显著变化。另外，共聚焦结果显示海马CA1区GEN组亚硝基化总蛋白的免疫染色较I/R组和L-NAME组显著增强，而且主要定位在神经元的细胞浆。
2) GEN增加了Nrf2的核转位。Western blot结果显示：在缺血再灌注组的后期（1d和3d）GEN显著增加了海马CA1区细胞核中Nrf2的蛋白水平，而其在细胞浆中的表达显著减少；激光共聚焦结果显示GEN组Nrf2和神经元核蛋白NeuN的免疫共定位较缺血再灌注组显著增强；L-NAME显著降低了GEN的此作用。
3) GEN显著增加海马CA1区Nrf2的DNA结合活性。与缺血再灌注3d组相比，GEN处理组Nrf2的DNA结合活性显著升高，而缺血前给予L-NAME显著废弃了GEN的此作用。
4) GEN显著增加海马CA1区Nrf2靶基因HO-1的蛋白表达。Westernblot分析显示：与缺血再灌注3d实验组相比，GEN显著增加了海马CA1区HO-1蛋白水平，而L-NAME降低了此作用。免疫荧光染色结果进一步证明，GEN增加了HO-1的荧光强度，其主要与细胞核荧光探针DAPI共定位，提示主要分布在CA1区神经元的细胞核中。
5) GEN降低脑缺血诱导的氧化应激损伤。采用免疫荧光染色技术观察GEN对脂质过氧化物最终产物4-HNE及DNA氧化损伤的特异性产物8-OHdG水平的影响。结果发现，sham组和GEN处理组海马CA1区4-HNE和8-OHdG免疫荧光的强度较弱，而缺血再灌注3d实验组和L-NAME处理组海马CA1区神经元4-HNE和8-OHdG的免疫染色显著增强。3. GEN可通过诱导海马CA3区Nrf2信号通路调控其神经保护作用
1) Keap1-tat小肽降低缺血后海马CA1区神经元损伤。焦油紫染色显示，与sham组相比缺血15min再灌注5d实验组和溶剂对照组海马CA1区神经元严重损伤，神经元密度降低；缺血前20min侧脑室注射Keap1-tat小肽（30，50，100μg）能显著增加海马CA1区生存的神经元数量，50μg剂量组生存的神经元数量最多，提示50μg为最佳剂量。
2) Keap1-tat可显著改善大鼠缺血后空间学习记忆功能。缺血15min再灌注7-9d实施Morris水迷宫试验。结果发现，与sham组相比溶剂对照组和缺血再灌注组大鼠寻找安全平台的潜伏期明显延长，而keap1-tat处理组(50μg)大鼠寻找安全平台的潜伏期较溶剂对照组显著降低；移走安全平台后，与sham组相比，90s内溶剂对照组和缺血再灌注组大鼠于平台所在象限探索的时间明显缩短，而keap1-tat小肽组大鼠在此象限停留时间较溶剂对照组显著延长。
3) Keap1-tat小肽未显著影响正常大鼠海马CA1区神经元的形态及大鼠的空间学习记忆功能。与sham组相比，脑室注射50μg keap1-tat小肽(sham+keap1-tat组)的正常大鼠海马CA1区神经元存活数量没有显著差异，而且两组大鼠的空间学习记忆功能无显著差异。
4) Keap1-tat小肽能显著增加细胞核中Nrf2水平。与再灌注同一时间点(I/R)组相比，侧脑室注射50μg keap1-tat小肽组海马CA1区神经元细胞核中Nrf2表达均显著升高。Sham组与sham+keap1-tat组海马CA1区神经元细胞核中Nrf2表达无显著差异。
5) Keap1-tat具有与GEN相同的神经保护作用。侧脑室（CA3区）微量注射0.4μg/μl (5μl) KA，于第8d焦油紫染色发现海马CA3区神经元大量损伤，而CA1区仅有少量神经元损伤。与单纯KA(0.4μg/μl)处理组相比，KA+Keap1-tat组海马CA3区大量神经元形态正常，少见损伤的神经元，CA1区生存的神经元数量两组无显著差异。缺血10min再灌注5（dI/R）+KA组与单纯KA处理组相似，海马CA3区神经元严重损伤，而CA1区神经元损伤不显著。与I/R+KA处理组相比，缺血前侧脑室注射（CA3区）Keap1-tat小肽后再灌注5d海马CA1和CA3区神经元均无显著损伤；有趣的是，KA+I/R+GEN组可见海马CA3和CA1区神经元严重损伤。结果提示，0.4μg/μl (5μl) KA特异损伤海马CA3区神经元；Keap1-tat可有效降低缺血后CA1区神经元损伤；KA可阻抑GEN对缺血后海马CA1区神经元的保护作用。
6) Keap1-tat可诱导缺血后海马CA3区Nrf2的核转位，KA可降低此作用。免疫荧光双染色结果显示：缺血后再灌注1d时，CA3区给予Keap1-tat组Nrf2主要分布在神经元细胞核中，胞浆中分布较少；而KA+I/R+GEN处理组Nrf2除主要分布在胞浆外，在细胞核周围及核内也有较少分布。在缺血再灌注3d时，Keap1-tat组Nrf2几乎完全分布在细胞核中；KA+I/R+GEN组Nrf2几乎完全分布在细胞浆中。结果提示，在Keap1-tat处理组Nrf2可能随再灌注时间的延长发生了核转位，而KA阻断了此作用。结论：
缺血再灌注后给予低剂量GEN可通过上调eNOS-HSP90信号保护海马CA1区神经元，改善缺血后大鼠的空间学习和记忆功能；其分子机制可能是通过Keap1的亚硝基化诱导了Nrf2-HO-1抗氧化信号通路；海马CA3区Nrf2信号可能参与了GEN诱导神经保护作用的调控。我们的研究为临床探索治疗缺血性脑中风的药物研发提供了重要靶点，为临床防治中风的策略提供理论依据。
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 CA1region
1) 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-1
1) 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 GCI
1) 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.
引文
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