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通讯作者:

马涛,E-mail:tmadoc@njmu.edu.cn

中图分类号:R363.2

文献标识码:A

文章编号:1007-4368(2023)04-468-07

DOI:10.7655/NYDXBNS20230404

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目录contents

    摘要

    目的:检测洛伐他汀(lovastatin,LOV)对N-甲基-D-天门冬氨酸(N-methyl-D-aspartate,NMDA)诱导的兴奋性毒性的神经保护作用并探讨LOV调节NMDA受体功能在神经保护中的潜在机制。方法:培养的大鼠原代神经元细胞分未处理组、 LOV组、NMDA组、LOV+NMDA组、谷氨酸(glutamate,Glu)组及Glu+APV(一种特异性NMDA受体拮抗剂)组。免疫荧光染色检测神经元形态,TUNEL分析检测神经元凋亡,免疫印迹测定蛋白水平,生物素化法检测细胞表面受体。结果:①与NMDA组或Glu组少数幸存微管相关蛋白(microtubule-associated protein 2,MAP-2)阳性神经元相比,LOV+NMDA组和Glu+APV组MAP- 2阳性神经元的数量明显增多,神经元树突的数目和长度均明显增加(P < 0.001);②与NMDA组或Glu组TUNEL阳性细胞显著增多相比,LOV+NMDA组或Glu+APV组TUNEL阳性细胞显著减少(P < 0.001);③与未处理组相比,NMDA组NMDA受体蛋白(N-methyl-D-aspartate receptor,NR2B)表达显著减少(P < 0.001),而LOV预处理后增加NR2B蛋白表达(P < 0.05);④生物素化法检测细胞表面受体显示,NMDA 处理导致细胞表面大部分NR2B 丢失(P < 0.001),LOV 预处理能显著减少NMDA 诱导的细胞表面 NR2B 丢失(P < 0.05)。进一步研究显示,NMDA 处理后,NR2B 在酪氨酸(tyrosine,Tyr)1472 位点的磷酸化下降 (P < 0.05),LOV预处理显著恢复Tyr1472位点的磷酸化(P < 0.05)。结论:LOV能减轻NMDA诱导的兴奋性毒性损害,这一作用可能与其影响NMDA受体亚单位NR2B的细胞内吞和/或胞内降解,进而调节NR2B表面分布有关。

    Abstract

    Objective:To explore the neuroprotective effect of lovastatin(LOV)on N - methyl - D - aspartate(NMDA)induced excitotoxicity and the potential mechanism of LOV in regulating the function of NMDA receptors in neuroprotection. Methods:The primary cultured rat neurons were divided into the vehicle group,LOV group,NMDA group,LOV + NMDA group,glutamate(Glu) group and Glu + APV(a specific NMDA receptor antagonist)group. Neuronal morphology was detected by immunofluorescence staining,neuronal apoptosis was detected by TUNEL analysis,protein levels were detected by Western blotting,cell surface receptors were detected by biotinylation. Results:①Compared with the few surviving microtubule-associated protein 2(MAP-2)positive neurons in the NMDA group or the Glu group,the number of MAP -2 immunopositive neurons in the LOV+NMDA group and the Glu+APV group was significantly increased,as well as the number and length of neuronal dendrites were significantly increased(P < 0.001). ②Compared with the significantly increased TUNEL-positive cells in the NMDA group or the Glu group,the TUNEL-positive cells in the LOV +NMDA group or the Glu +APV group were significantly decreased(P < 0.001). ③Compared with the vehicle group,the expression of N - methyl - D - aspartate receptor(NR2B)in the NMDA group was significantly decreased(P < 0.001),while LOV pretreatment could increase the expression of NR2B when compared with the NMDA group(P < 0.05). ④The cell surface receptor biotinylation assay showed that NMDA treatment resulted in the loss of the most NR2B on the cell surface(P < 0.001),while LOV pretreatment could significantly reduce the NMDA-induced loss of NR2B(P < 0.05). Further studies showed that phosphorylation of NR2B at tyrosine(Tyr)1472 was decreased after NMDA treatment(P < 0.05),while pretreatment with LOV significantly restored the phosphorylation of Tyr1472(P < 0.05). Conclusion:LOV may significantly attenuate the excitotoxicity induced by NMDA,and its neuroprotective effect is probably related to the selective regulation of NR2B surface expression by affecting the intracellular endocytosis and/or intracellular degradation of NR2B.

  • 兴奋性毒性的特征是Ca2+ 依赖的神经元细胞在过量谷氨酸(glutamate,Glu)刺激下死亡,有证据提示这与脑缺血和神经退行性疾病有关[1-3]。突触Glu 过度释放会激活突触后Glu受体,导致Ca2+ 依赖性蛋白酶钙蛋白酶过度活化,促进关键细胞骨架和膜蛋白的降解[4]。尽管几乎所有Glu受体亚型都参与介导兴奋性毒性信号转导,但普遍认为 N⁃甲基⁃D⁃天冬氨酸(N⁃methyl⁃D⁃aspartate,NMDA)受体由于其高钙渗透性而发挥主要作用[3]。其中NR1为最基本的功能单位,NR2为重要的调节亚单位。含NR2A 的 NMDA 受体通常与细胞存活有关,而 NR2B 型突触外受体与细胞死亡信号有关[5]。有研究报道,兴奋毒性谷氨酸能神经元中 NR2B 表达和调控受钙蛋白酶(calpain)和细胞周期素依赖性激酶 5(cyclin dependent kinase5,CDK5)[6-7] 调节。他汀类药物是最为常用的降胆固醇药物,越来越多的研究支持他汀对卒中事件和神经退行性疾病的保护作用[8-11]。我们先前研究表明,洛伐他汀(lovastatin,LOV)能抑制 calpain 和 CDK5 的过度激活而发挥神经保护作用[12],然而,这其中的确切机制仍需进一步研究。本研究在培养的大鼠原代神经元中检测 LOV 对 NMDA 诱导的兴奋性毒性的保护作用,并探讨 LOV在兴奋性毒性信号转导中的潜在保护机制。

  • 1 材料和方法

  • 1.1 材料

  • LOV、Glu、NMDA、APV(特异性NMDA受体拮抗剂)、环己酰胺(cyclohexamide,CHX)购自美国Sigma 公司,蛋白酶抑制剂(cocktail of protease inhibitors) 和原位细胞死亡检测试剂盒TUNEL 购自瑞士Roche 公司,Sulfo ⁃NHS ⁃LC⁃ biotin[sulfosuccinimydyl⁃ 6 ⁃ (biotinamido)⁃hexanoate]和抗生蛋白链菌素(strep⁃ tavidin)购自美国 Pierce 公司,抗微管相关蛋白 (microtubule⁃associated protein 2,MAP⁃2)和 4′,6′⁃ diamidino⁃2⁃phenylindole(DAPI)购自美国 Chemicon 公司。 Alexa⁃594 结合的羊抗鼠 IgG 购自美国Invitrogen 公司。

  • 1.2 方法

  • 1.2.1 大鼠皮质神经元的培养及处理

  • 参照先前研究[13],从孕 17 d 的 Sprague⁃Dawley 大鼠胚胎中分离出皮质神经元。细胞培养 2 周待 NMDA受体发育成熟后使用。将发育成熟的原代神经元细胞分未处理(vehicle)组、LOV 组、NMDA 组、LOV+NMDA 组、Glu 组及 Glu+APV 组。未处理组不做特殊处理;LOV 组用 LOV 500 nmol/L 预处理大鼠皮质神经元 3 d;NMDA 组和 Glu 组分别应用 100 μmol/L NMDA和100 μmol/L Glu处理培养的神经元 15 min;NMDA+LOV 组在 NMDA 处理之前,用LOV 500 nmol/L预处理大鼠皮质神经元3 d,以观测LOV对这些应激是否有保护作用;Glu+APV组在 Glu 处理神经元之前 30 min,应用 NMDA 受体特异性的拮抗剂100 μmol/L APV预处理。

  • 1.2.2 免疫荧光染色

  • 各组经过处理的标本用 4%多聚甲醛固定 20 min 后,加入含有 0.2%Triton X⁃100/PBS 5 min, PBS清洗后,滴加血清封闭液,室温封闭1 h。使用鼠抗MAP⁃2(1∶500)4℃孵育过夜。Alexa⁃594结合的羊抗鼠 IgG(1∶500)室温孵育 1 h 后 DAPI 复染。在荧光显微镜下观察,并对每个MAP⁃2阳性神经元突起的数量及突起的长度进行定量分析。

  • 1.2.3 TUNEL分析

  • 参照原位细胞死亡检测试剂盒的方法行TUNEL 染色,随后细胞用 DAPI(1 μg/mL)复染 5 min。荧光封片剂封片后,于荧光显微镜下观察,并计数 TUNEL 阳性细胞,计算 TUNEL 阳性细胞率。对每组3~4次独立实验的6~8个盖玻片观察计数。

  • 1.2.4 蛋白免疫印迹(Western blot)

  • 按照文献所述制备蛋白及进行蛋白免疫印迹检测[14]。大鼠皮质神经元经过处理后,弃培养液,冷PBS 洗两遍,加入预冷的细胞裂解液(10 mmol/L Tris,pH 7.4,150 mmol/L NaCl,5 mmol/L EDTA, 5 mmol/L EGTA,50 mmol/L NaF,1 mmol/L Na3VO4,5 mmol/L DTT,1% NP⁃40 及蛋白酶抑制剂)作用 20 min,离心取上清蛋白。蛋白浓度的测定采用 Lowry 法。取等量样品(20 μg),以4%~20% SDS⁃聚丙烯酰胺预制凝胶进行电泳分离蛋白。电泳结束后将蛋白转至 PVDF 膜(300 mA,90 min),用含 5% 脱脂奶粉的PBST 封闭60 min,加入一抗 4℃过夜。一抗具体为:β⁃actin(1∶5 000,Sigma 公司,美国), NR2B(1∶400)、NR1(1∶1 000)、GluR2(1∶1 000)、 phosphor ⁃ NR2B(Tyr1472,1∶500)、phosphor ⁃ NR2B (Tyr1336)(1∶1 000,Millipore 公司,美国)。一抗孵育后,PBST洗涤,辣根过氧化物酶标记的羊抗兔或鼠IgG二抗(1∶5 000 稀释)孵育1 h,PBST洗涤,此后按ECL显色试剂盒说明书操作,曝光后在激光光密度图像扫描系统进行光密度分析。以β⁃actin 为内参照,实验重复3 次以上。

  • 1.2.5 生物素化法检测细胞表面受体

  • 生物素化法检测细胞表面受体参照 Hawasli 等[15]的研究方法。培养的大鼠皮质神经元用冷 PBS 洗2遍后,加入预冷的含0.5 mg/mL Sulfo⁃NHS⁃ LC ⁃biotin 的 PBS,4℃孵育 20 min。冷 PBS 洗 3 遍后,加入细胞裂解液(含蛋白酶抑制剂、0.1% SDS、 1% Triton X⁃100 的 PBS)作用 30 min,用细胞刮刮下细胞,冰上放置 30 min,4℃、14 000 r/min 离心 10 min,取上清蛋白。蛋白浓度的测定采用 Lowry 法。取等量样品(500 μg),加入 streptavidin 4℃ 孵育过夜。免疫沉淀物溶于1×上样缓冲液中,通过蛋白免疫印迹法测定其蛋白水平。

  • 应用蛋白合成抑制剂环己酰胺抑制NR2B的合成,以观察 NR2B 的降解情况。在 100 μmol/L 环己酰胺加入培养液后不同时间点(0、30、60 min),收集各组细胞,蛋白定量后Western blot检测NR2B,动态观察LOV对NR2B稳定性的影响。

  • 1.3 统计学方法

  • 数据分析使用 SPSS 18.0 统计软件进行,数据使用均数 ± 标准差(x- ± s)表示,采用 Student’s t 检验对数据进行统计比较,P <0.05 为差异有统计学意义。

  • 2 结果

  • 2.1 LOV对神经元兴奋毒性的保护作用

  • 本课题组之前的研究显示,LOV在低浓度(0.5~1.0 μmol/L)下对 NMDA 具有最佳的神经保护作用[12-14]。本研究用0.5 μmol/L LOV对原代培养的神经元进行预处理 3 d,以研究 LOV 的兴奋毒性保护作用和潜在的机制。MAP⁃2 是一种神经元特异性细胞骨架蛋白,对 MAP⁃2 的免疫荧光染色显示 (图1A~C),与未处理组相比,NMDA 组或 Glu 组 MAP⁃2阳性神经元显著减少,少数幸存神经元的染色多集中在胞体,树突的数目和长度均明显减少。与 NMDA 组相比,在 NMDA 处理前加用 LOV 预处理,其MAP⁃2免疫阳性神经元的数量明显增多,且神经元树突的数目和长度均明显增加(P <0.001)。与Glu组相比,在Glu处理前使用NMDA受体特异性拮抗剂APV预处理,其MAP⁃2免疫阳性神经元的数量明显增多,神经元树突的数目和长度均明显增加 (P <0.001)。TUNEL 染色的结果进一步支持 LOV 的神经保护作用。TUNEL 染色显示(图2A~B),未处理组凋亡细胞很少,NMDA 组或 Glu 组 TUNEL 阳性细胞显著增多,在 NMDA 或 Glu 处理前加用 LOV 或 APV 预处理,能显著减少 TUNEL 阳性细胞(P <0.001),提示 LOV 显著减少神经元细胞的凋亡。

  • A:各组MAP⁃2免疫荧光染色结果;B、C:各组树突长度和数量的定量比较;两组比较,***P <0.001(n=3)。

  • 图1 MAP⁃2染色检测神经元形态

  • Figure1 Neuronal morphology assay with MAP⁃2 staining

  • A:各组TUNEL染色结果;B:各组TUNEL染色阳性细胞的定量比较;两组比较,***P <0.001(n=3)。

  • 图2 TUNEL染色检测细胞凋亡

  • Figure2 Cell apoptosis analyzed by TUNEL assay

  • 2.2 LOV减少NMDA诱导的NR2B下调及细胞表面 NR2B的丢失

  • 使用蛋白免疫印迹法同步检测 NMDA 和 Glu 对 NR2B 蛋白表达的影响。结果显示,NMDA 和 Glu减少NR2B的表达,在NMDA或Glu处理前加用 LOV或 APV 预处理,NR2B 的表达增加(图3)。接着使用蛋白免疫印迹检测 LOV 对 NMDA 受体 (NR1 和 NR2B)和 GluR2 的影响。结果显示,与未处理组相比,NMDA 处理显著抑制NR2B蛋白表达 (P <0.001),LOV 预处理后能够增加 NR2B 表达 (P <0.05,图4A~B)。生物素化法检测细胞表面受体的表达,结果显示,NMDA 处理导致细胞表面大部分 NR2B 丢失(P <0.001),而不改变细胞表面NR1、Glu2R 的蛋白水平,LOV 预处理能显著减少 NMDA 诱导的细胞表面 NR2B 丢失(P <0.05)。进一步研究表明,随着NMDA 处理时间的延长,NR2B 的表达逐渐下降直至丢失,提示 NMDA 对 NR2B 的影响呈时间依赖性(图4C)。

  • 图3 蛋白免疫印迹检测各组NR2B蛋白表达

  • Figure3 NR2B expression analyzed byimmunoblot analysis

  • A:不同受体亚基NR1、NR2B和GluR2的总分布和细胞表面分布的代表性免疫印迹图像;B:各组总NR2B及细胞表面NR2B水平的定量比较;两组比较,* P <0.05,***P <0.001(n=3);C:蛋白免疫印迹结果显示NMDA处理不同时间后NR2B的表达。

  • 图4 蛋白免疫印迹法和生物素化法检测NMDA受体亚单位总的蛋白水平和细胞表面分布

  • Figure4 Measurement of total level and surface distribution of NMDA receptor subunits by immunoblot analysis and bioti⁃ nylation assay

  • 2.3 LOV 通过调节 Tyr1472 位点的磷酸化影响 NR2B的表面分布

  • 随后,我们应用蛋白合成抑制剂100 μmol/L环己酰胺抑制NR2B的合成,以观察LOV是否能影响 NR2B 的降解。研究结果显示,在 NMDA 处理后, NR2B 迅速减少,LOV 预处理能阻止 NR2B 的降解 (图5A)。由于 NR2B 的磷酸化与 NR 的功能定位和内吞密切相关,并且 NR2B 在 Tyr1472 位点去磷酸化与其表面分布有关。为了验证 LOV 神经保护机制,我们进行相应研究。结果显示,NMDA 处理后,NR2B 在Tyr1472位点的磷酸化下降(P <0.05), LOV 预处理显著恢复 Tyr1472 位点的磷酸化(P <0.05);使用NMDA处理后,NR2B 也降低Tyr1336位点的磷酸化(P <0.05),但 LOV 预处理不能恢复 Tyr1336位点的磷酸化(图5B~C),提示LOV很可能通过Tyr1472位点的磷酸化影响NR2B的表面分布。

  • A:加入蛋白合成抑制剂100 μmol/L环己酰胺不同时间下,观察加入NMDA和洛伐他汀后NR2B的蛋白表达;B:洛伐他汀对NR2B不同磷酸化位点的影响;C:洛伐他汀对NR2B不同磷酸化位点的影响比较;两组比较,* P <0.05(n=3)。

  • 图5 蛋白免疫印迹法检测各组NR2B蛋白水平及磷酸化

  • Figure5 NR2B and its phosphorylation were by immunoblot analysis

  • 3 讨论

  • 由于兴奋性毒性损害在急性脑卒中和慢性神经退行性疾病中的重要作用[16-17],减轻兴奋性毒性损害成为神经保护的基本策略。本研究应用NMDA 诱导大鼠皮质神经元的兴奋性毒性损害,发现LOV 对NMDA诱导的兴奋性毒性损害有神经保护作用。

  • 他汀类药物,尤其是辛伐他汀和 LOV,表现出兴奋毒性保护和记忆挽救作用[12-1318-20]。然而,其中的机制尚不清楚。NMDA受体是一种配体门控离子型Glu受体,在中枢神经系统信号传递以及神经系统的可塑性方面具有重要的作用。NR2 亚单位决定了NR的突触定位和功能,一般认为NR2A与突触活性相关,NR2B 与突触外活性相关。最近的研究表明突触NR2A通常与细胞存活有密切的联系,而突触外NR2B与细胞死亡信号有关[5],并且NR2B被认为是导致脑缺血后神经元兴奋性损伤的最主要成分[621]。本研究显示,NMDA 导致 NR2B 表达下降,而且,随着NMDA 处理时间的延长,NR2B的表达逐渐下降直至丢失,LOV可显著缓解NMDA引起的NR2B表达下降,提示LOV很可能通过调节NR2B 发挥神经保护作用。

  • 既往研究[18] 表明,辛伐他汀通过上调NMDA受体亚单位NR2B的表面分布来增强NMDA介导的突触传递,改善海马依赖的空间记忆,挽救阿尔茨海默病小鼠模型的记忆缺陷。跟大多数膜蛋白一样, NR 的细胞表面定位与富含胆固醇的脂筏有关[22]。他汀能显著减少脂筏介导的大量膜蛋白的内吞[23-24]。本研究表明 NMDA 处理不改变细胞表面 NR1、 Glu2R的蛋白水平,而是选择性地导致细胞表面大部分NR2B 丢失,LOV预处理能显著减少NMDA诱导的细胞表面NR2B 丢失,提示LOV的神经保护与 NR2B 的细胞表面分布有关。结合本课题组先前的研究,他汀预处理3~5 d,而不是1 d,对大鼠皮质神经元的兴奋性损害有神经保护作用,这与他汀降低胆固醇的时程一致[23],本文推测LOV介导的兴奋性保护具有降低胆固醇作用,进而限制脂筏介导的膜受体NR2B内吞。

  • NR2B 的磷酸化与 NR 的功能定位和内吞密切相关[25-26]。NR2B 在羧基端包含网格蛋白适配器(clathrin adaptor)AP ⁃2 结合位点及内吞基序 YEKL[27]。非受体酪氨酸激酶Src家族在YEKL基序的 Tyr1472 位点磷酸化 NR2B。NR2B Tyr1472 位点磷酸化抑制AP⁃2结合,进而促进NR的表面受体表达,并且NR2B在Tyr1472位点去磷酸化影响受体的内吞[728]。本研究显示,NMDA降低NR2B在Tyr1472 和 Tyr1336位点的磷酸化,但LOV预处理只选择性地恢复 Tyr1472 位点的磷酸化,提示 LOV 很可能通过Tyr1472位点的磷酸化影响NR2B的内吞。

  • 由于细胞表面受体内吞增加和胞内降解增加均可导致 NMDA 处理后亚单位 NR2B 表面表达减少。本研究结果显示,在存在环己酰亚胺的情况下,NR2B表达在30 min内被NMDA迅速消除,LOV 预处理能稳定NR2B,提示LOV阻止NR2B的降解。

  • 综上,本研究结果表明 NR2B 是导致脑缺血后神经元兴奋性损伤的最主要成分,LOV 能减轻 NMDA诱导的兴奋性毒性损害,其神经保护作用很可能与 LOV 通过影响 NMDA 受体亚单位 NR2B 的细胞内吞或/和胞内降解进而调节 NR2B 表面分布有关。

  • 参考文献

    • [1] ABOLHASANPOUR N,ALIHOSSEINI S,GOLIPOURKH⁃ ALILI S,et al.Effect of melatonin on endoplasmic reticu⁃ lum⁃mitochondrial crosstalk in stroke[J].Arch Med Res,2021,52(7):673-682

    • [2] LI L L,KE X Y,JIANG C,et al.Na+ ,K+ ⁃ATPase partici⁃ pates in the protective mechanism of rat cerebral ischemia⁃ reperfusion through the interaction with glutamate trans⁃ porter⁃1[J].Fundam Clin Pharmacol,2021,35(5):870-881

    • [3] ANDERSEN J V,MARKUSSEN K H,JAKOBSEN E,et al.Glutamate metabolism and recycling at the excitatory synapse in health and neurodegeneration[J].Neurophar⁃ macology,2021,196:108719

    • [4] TATSUROU Y,YASUHIRO Y,HIROMI K.Pathophysio⁃ logical roles of intracellular proteases in neuronal devel⁃ opment and neurological diseases[J].Mol Neurobiol,2019,56(5):3090-3112

    • [5] ATILLA E,BASAK E A.N⁃methyl⁃D⁃aspartate receptorsignaling ⁃ protein kinases crosstalk in cerebral ischemia [J].Adv Exp Med Biol,2021,1275:259-283

    • [6] ELENA A P,MAR P,VERÓNICA B,et al.Cdk5 contrib⁃ utes to Huntington’s disease learning and memory defi⁃ cits via modulation of brain region⁃specific substrates[J].Mol Neurobiol,2017,55(8):6250-6268

    • [7] KWON H H,NEUPANE C,SHIN J,et al.Calpain⁃2 as a treatment target in prenatal stress ⁃ induced epileptic spasms in infant rats[J].Exp Neurobiol,2019,28(4):529-536

    • [8] FLACH C,ELSTAD M,MURUET W,et al.The impact of pre⁃and post⁃stroke statin use on stroke severity and long⁃ term outcomes:a population⁃based cohort study[J].Cere⁃ brovasc Dis,2019,47(5-6):260-267

    • [9] ZHAO W,XIAO Z J,ZHAO S P.The benefits and risks of statin therapy in ischemic stroke:a review of the litera⁃ ture[J].Neurol India,2019,67(4):983-992

    • [10] TRAMACERE I,BONCORAGLIO G B,BANZI R T,et al.Comparison of statins for secondary prevention in patients with ischemic stroke or transient ischemic attack:a sys⁃ tematic review and network meta⁃analysis[J].BMC Med,2019,17(1):1-12

    • [11] GEORGINA T,BRANIGAN G L,FRANCESCA V,et al.Statin therapy and risk of Alzheimer’s and age ⁃ related neurodegenerative diseases[J].Alzheimer’s Dement(N Y),2020,6(1):e12108

    • [12] 马涛,许著一,姚晴宇,等.洛伐他汀通过抑制 Cal⁃ pain 和CDK5的过度激活减轻NMDA的毒性损害[J].中华神经医学杂志,2012,11(2):109-113

    • [13] MA T,ZHAO Y B,KWAK Y D,et al.Statin’s excitopro⁃ tection is mediated by sAPP and the subsequent attenua⁃ tion of calpain ⁃induced truncation events,likely via rho ⁃ ROCK signaling[J].J Neurosci,2009,29(36):11226-11236

    • [14] LI R,XU D,MA T.Lovastatin suppresses the aberrant tau phosphorylation from FTDP⁃17 mutation and okadaic acid⁃ induction in rat primary neurons[J].Neuroscience,2015,294:14-20

    • [15] HAWASLI A H,BENAVIDES D R,CHAN N,et al.Cy⁃ clin ⁃ dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation[J].Nat Neu⁃ rosci,2007,10(7):880-886

    • [16] SHEN Z H,XIANG M,CHEN C,et al.Glutamate excito⁃ toxicity:potential therapeutic target for ischemic stroke [J].Biomed Pharmacother,2022,151:113125

    • [17] OCTAVIO B,JORDI O.Excitotoxicity as a target against neurodegenerative processes[J].Curr Pharm Des,2020,26(12):1251-1262

    • [18] LILLA B,WALTER F R,ANDRÁS H,et al.Simvastatin,edaravone and dexamethasone protect against kainate⁃in⁃ duced brain endothelial cell damage[J].Fluids Barriers CNS,2020,17(1):5

    • [19] EKLADIOUS S T,EL NESRINE S.Effect of pioglitazone and simvastatin in lipopolysaccharide ⁃induced amyloido⁃ genesis and cognitive impairment in mice:possible role of glutamatergic pathway and oxidative stress[J].Behav Pharmacol,2019,30(1):5-15

    • [20] HU X Q,SONG C W,FANG M,et al.Simvastatin inhibits the apoptosis of hippocampal cells in a mouse model of Alzheimer’s disease[J].Exp Ther Med,2018,15(2):1795-1802

    • [21] DI Z,GUO Q,ZHANG Q A.Neuroprotective effect of moxibustion on cerebral ischemia/reperfusion injury in rats by downregulating NR2B expression[J].Evid Based Complementary Altern Med,2021,2021:5370214

    • [22] TAKESHI K,TAKUMI N,KAORU S,et al.Lipid rafts act as a common platform for amyloid⁃β oligomer⁃induced Al⁃ zheimer’s disease pathology[J].J Alzheimer’s Dis JAD,2022,87(3):1189-1203

    • [23] JOVITA P,LA OSSA NATALIA PÉREZ D,OLIVIA H,et al.Simvastatin reduces the association of NMDA recep⁃ tors to lipid rafts:a cholesterol ⁃mediated effect in neuro⁃protection[J].Stroke,2008,39(4):1269-1275

    • [24] HILL J M,ISRAEL S,MATTHEWS K E,et al.Statins lower the risk of developing Alzheimer’s disease by limit⁃ ing lipid raft endocytosis and decreasing the neuronal spread of Herpes simplex virus type 1[J].Med Hypothe⁃ ses,2005,64(1):53-58

    • [25] CHEN T T,WANG C H,SHA S,et al.Simvastatin en⁃ hances spatial memory and long⁃term potentiation in hip⁃ pocampal CA1 via upregulation of alpha 7 nicotinic ace⁃ tylcholine receptor[J].Mol Neurobiol,2016,53(6):4060-4072

    • [26] WATERS E M,MAZID S,DODOS M,et al.Effects of es⁃ trogen and aging on synaptic morphology and distribution of phosphorylated Tyr1472 NR2B in the female rat hippo⁃ campus[J].Neurobiol Aging,2019,73:200-210

    • [27] ROCHE K W,STANDLEY S,MCCALLUM J,et al.Mo⁃ lecular determinants of NMDA receptor internalization [J].Nat Neurosci,2001,4(8):794-802

    • [28] LAVEZZARI G,MCCALLUM J,LEE R,et al.Differen⁃ tial binding of the AP⁃2 adaptor complex and PSD⁃95 to the C⁃terminus of the NMDA receptor subunit NR2B reg⁃ ulates surface expression[J].Neuropharmacology,2003,45(6):729-737

  • 参考文献

    • [1] ABOLHASANPOUR N,ALIHOSSEINI S,GOLIPOURKH⁃ ALILI S,et al.Effect of melatonin on endoplasmic reticu⁃ lum⁃mitochondrial crosstalk in stroke[J].Arch Med Res,2021,52(7):673-682

    • [2] LI L L,KE X Y,JIANG C,et al.Na+ ,K+ ⁃ATPase partici⁃ pates in the protective mechanism of rat cerebral ischemia⁃ reperfusion through the interaction with glutamate trans⁃ porter⁃1[J].Fundam Clin Pharmacol,2021,35(5):870-881

    • [3] ANDERSEN J V,MARKUSSEN K H,JAKOBSEN E,et al.Glutamate metabolism and recycling at the excitatory synapse in health and neurodegeneration[J].Neurophar⁃ macology,2021,196:108719

    • [4] TATSUROU Y,YASUHIRO Y,HIROMI K.Pathophysio⁃ logical roles of intracellular proteases in neuronal devel⁃ opment and neurological diseases[J].Mol Neurobiol,2019,56(5):3090-3112

    • [5] ATILLA E,BASAK E A.N⁃methyl⁃D⁃aspartate receptorsignaling ⁃ protein kinases crosstalk in cerebral ischemia [J].Adv Exp Med Biol,2021,1275:259-283

    • [6] ELENA A P,MAR P,VERÓNICA B,et al.Cdk5 contrib⁃ utes to Huntington’s disease learning and memory defi⁃ cits via modulation of brain region⁃specific substrates[J].Mol Neurobiol,2017,55(8):6250-6268

    • [7] KWON H H,NEUPANE C,SHIN J,et al.Calpain⁃2 as a treatment target in prenatal stress ⁃ induced epileptic spasms in infant rats[J].Exp Neurobiol,2019,28(4):529-536

    • [8] FLACH C,ELSTAD M,MURUET W,et al.The impact of pre⁃and post⁃stroke statin use on stroke severity and long⁃ term outcomes:a population⁃based cohort study[J].Cere⁃ brovasc Dis,2019,47(5-6):260-267

    • [9] ZHAO W,XIAO Z J,ZHAO S P.The benefits and risks of statin therapy in ischemic stroke:a review of the litera⁃ ture[J].Neurol India,2019,67(4):983-992

    • [10] TRAMACERE I,BONCORAGLIO G B,BANZI R T,et al.Comparison of statins for secondary prevention in patients with ischemic stroke or transient ischemic attack:a sys⁃ tematic review and network meta⁃analysis[J].BMC Med,2019,17(1):1-12

    • [11] GEORGINA T,BRANIGAN G L,FRANCESCA V,et al.Statin therapy and risk of Alzheimer’s and age ⁃ related neurodegenerative diseases[J].Alzheimer’s Dement(N Y),2020,6(1):e12108

    • [12] 马涛,许著一,姚晴宇,等.洛伐他汀通过抑制 Cal⁃ pain 和CDK5的过度激活减轻NMDA的毒性损害[J].中华神经医学杂志,2012,11(2):109-113

    • [13] MA T,ZHAO Y B,KWAK Y D,et al.Statin’s excitopro⁃ tection is mediated by sAPP and the subsequent attenua⁃ tion of calpain ⁃induced truncation events,likely via rho ⁃ ROCK signaling[J].J Neurosci,2009,29(36):11226-11236

    • [14] LI R,XU D,MA T.Lovastatin suppresses the aberrant tau phosphorylation from FTDP⁃17 mutation and okadaic acid⁃ induction in rat primary neurons[J].Neuroscience,2015,294:14-20

    • [15] HAWASLI A H,BENAVIDES D R,CHAN N,et al.Cy⁃ clin ⁃ dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation[J].Nat Neu⁃ rosci,2007,10(7):880-886

    • [16] SHEN Z H,XIANG M,CHEN C,et al.Glutamate excito⁃ toxicity:potential therapeutic target for ischemic stroke [J].Biomed Pharmacother,2022,151:113125

    • [17] OCTAVIO B,JORDI O.Excitotoxicity as a target against neurodegenerative processes[J].Curr Pharm Des,2020,26(12):1251-1262

    • [18] LILLA B,WALTER F R,ANDRÁS H,et al.Simvastatin,edaravone and dexamethasone protect against kainate⁃in⁃ duced brain endothelial cell damage[J].Fluids Barriers CNS,2020,17(1):5

    • [19] EKLADIOUS S T,EL NESRINE S.Effect of pioglitazone and simvastatin in lipopolysaccharide ⁃induced amyloido⁃ genesis and cognitive impairment in mice:possible role of glutamatergic pathway and oxidative stress[J].Behav Pharmacol,2019,30(1):5-15

    • [20] HU X Q,SONG C W,FANG M,et al.Simvastatin inhibits the apoptosis of hippocampal cells in a mouse model of Alzheimer’s disease[J].Exp Ther Med,2018,15(2):1795-1802

    • [21] DI Z,GUO Q,ZHANG Q A.Neuroprotective effect of moxibustion on cerebral ischemia/reperfusion injury in rats by downregulating NR2B expression[J].Evid Based Complementary Altern Med,2021,2021:5370214

    • [22] TAKESHI K,TAKUMI N,KAORU S,et al.Lipid rafts act as a common platform for amyloid⁃β oligomer⁃induced Al⁃ zheimer’s disease pathology[J].J Alzheimer’s Dis JAD,2022,87(3):1189-1203

    • [23] JOVITA P,LA OSSA NATALIA PÉREZ D,OLIVIA H,et al.Simvastatin reduces the association of NMDA recep⁃ tors to lipid rafts:a cholesterol ⁃mediated effect in neuro⁃protection[J].Stroke,2008,39(4):1269-1275

    • [24] HILL J M,ISRAEL S,MATTHEWS K E,et al.Statins lower the risk of developing Alzheimer’s disease by limit⁃ ing lipid raft endocytosis and decreasing the neuronal spread of Herpes simplex virus type 1[J].Med Hypothe⁃ ses,2005,64(1):53-58

    • [25] CHEN T T,WANG C H,SHA S,et al.Simvastatin en⁃ hances spatial memory and long⁃term potentiation in hip⁃ pocampal CA1 via upregulation of alpha 7 nicotinic ace⁃ tylcholine receptor[J].Mol Neurobiol,2016,53(6):4060-4072

    • [26] WATERS E M,MAZID S,DODOS M,et al.Effects of es⁃ trogen and aging on synaptic morphology and distribution of phosphorylated Tyr1472 NR2B in the female rat hippo⁃ campus[J].Neurobiol Aging,2019,73:200-210

    • [27] ROCHE K W,STANDLEY S,MCCALLUM J,et al.Mo⁃ lecular determinants of NMDA receptor internalization [J].Nat Neurosci,2001,4(8):794-802

    • [28] LAVEZZARI G,MCCALLUM J,LEE R,et al.Differen⁃ tial binding of the AP⁃2 adaptor complex and PSD⁃95 to the C⁃terminus of the NMDA receptor subunit NR2B reg⁃ ulates surface expression[J].Neuropharmacology,2003,45(6):729-737

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