实验性癫痫大鼠凋亡机制的探讨及重组人促红细胞生成素的影响
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摘要
癫痫是由于脑部兴奋性过高的某些神经元突然异常放电引起的脑功能的异常。癫痫是中枢神经系统最常见的症状和疾病之一。癫痫持续状态是神经科的急症,常常伴发10%–12%的死亡率,严重影响了病人的生活质量。在颞叶癫痫的病人及各种各样的试验性癫痫模型、癫痫持续状态模型上用形态学和生物化学等办法均证实损伤的神经元具有典型的凋亡特征。虽然对凋亡启动的具体的信号传导通路还不是很清楚,但凋亡在癫痫持续状态中的影响却引来了许多的关注。
凋亡的发生激活了包括线粒体、线粒体相关调控因子及Caspsae家族等许多的信号途径,是细胞死亡的主要过程,最终导致细胞核染色质的凝聚、DNA的断裂、凋亡小体的形成。凋亡的发生通过三条主要的途径:线粒体依赖途径、死亡受体依赖途径、内质网依赖途径。线粒体凋亡途径是主要的凋亡途径,它诱导细胞色素C从线粒体释放并激活了死亡信号。细胞色素C从线粒体释放出来,与凋亡活化因子-1 (apoptosis activated factor-1,Apaf-1)及Caspase-9结合形成复合体。Caspase-9的激活将引起其下游胱天蛋白酶的级联反应,导致该细胞凋亡。Bcl-2家族蛋白、Caspase家族蛋白、凋亡抑制蛋白(IAPs)家族都是重要的线粒体依赖凋亡途径的相关调控因子。Bcl-2家族蛋白通过调控线粒体膜的通透性和细胞色素C的释放来介导凋亡,Bcl-2蛋白家族包括促凋亡成员Bax、Bad等,和抗凋亡成员Bcl-2、Bcl-xl等。尽管Bax和Bad大多数时候存在于细胞质,但它们可以易位到线粒体和Bcl-2形成促凋亡的复合物,然后释放细胞色素C。与促凋亡蛋白相对应的是抗凋亡蛋白,抗凋亡蛋白Bcl-2存在于线粒体膜的外部,通过多种机制阻断促凋亡分子释放到线粒体,这些机制包括维持线粒体膜的完整性和联合Bcl-2家族中的促凋亡蛋白。Bcl-2/Bax的比率也是决定细胞生存与死亡的关键。在仅含BH3区蛋白的成员中,Bad是最早被鉴别出来,最具有特征性的蛋白。Bad通过在线粒体膜的外部与Bcl-2和Bcl-xl结合引起细胞色素C释放到细胞质来诱导凋亡的发生。Bad的促凋亡功能主要是通过与Bcl-2和Bcl-xl结合来阻断他们的抗凋亡作用。例如:Bad可以阻断Bcl-2和Bcl-xl在线粒体外膜上形成通道的能力,或者是阻断他们结合和稳定电压依从性阴离子通道的能力。Bad是一个可以被磷酸化调节的典型的促凋亡蛋白,激活的Akt使Bad中的Ser136磷酸化,导致14-3-3蛋白与Bad的结合,稳定与细胞之中,使它不能在线粒体外膜上与Bcl-2和Bcl-xl相互作用,抑制了Bad所诱导的细胞凋亡。Caspase-9,就像其他的Caspase成员一样,以一种没有活性的酶原形式存在并通过蛋白酶解加工激活。细胞色素C一旦释放到细胞质就会与Apaf-1相互作用,导致Caspase-9酶原的激活,激活的Caspase-9进而激活Caspase-3,随后引起其他的Caspase的级联反应,导致该细胞凋亡。IAPs家族是细胞内抗凋亡蛋白家族,在杆状病毒中首先被鉴定出来,在一个后线粒体水平通过调节死亡信号途径发挥着重要的促细胞存活作用。IAPs家族包括XIAP、Hiap-1、Survivin、Livin等等。XIAP是首先被克隆出来的哺乳动物IAPs家族成员之一,是该家族中最具有抗凋亡能力的成员之一。XIAP可以直接阻断Caspase-3 and Caspase-9,通过对Caspase-9的阻断来调节Bax/细胞色素C介导的线粒体凋亡途径。
PI3K是促进细胞存活的信号传导途径中的重要激酶,起到十分关键的作用。在各种细胞中发挥着调整细胞分裂周期、生长、新陈代谢、抑制凋亡的作用。众所周知,激活的Akt通过调整一些重要的调节因子介导细胞的存活,包括Bad、Bax、Bcl-2、Caspase-9和XIAP等等,这些调节因子同时也是线粒体依赖凋亡途径的相关调控因子。第一个被鉴定出来的Akt的下游分子是促凋亡蛋白Bad,Akt可以磷酸化Bad的Ser136位点,导致Bad的钝化。磷酸化的Bad具有抗凋亡的作用,因为非磷酸化的Bad可以和Bcl-2和Bcl-xl形成异二聚体,阻断他们的抗凋亡作用。另外,研究显示在Akt依赖的情况下BaxSer184可以被Akt磷酸化,阻断了Bax和Bcl-2家族中抗凋亡蛋白在线粒体膜形成异二聚体的能力,Akt也磷酸化CREB的Ser133位点,增加Bcl-2等抗凋亡因子的转录水平。Akt也可以磷酸化IKK的Thr-23位点,释放NF-кB,然后NF-кB可以上调Bcl-2的表达。最近,有研究显在体内、体外的实验中Akt在生理条件下也可以磷酸化XIAP的Ser87位点。这些研究显示了Akt和它的下游靶分子构成了主要的细胞存活途径。
EPO最初被了解是因为它通过保护红系祖细胞对抗凋亡来增多红细胞的作用。现在EPO和它的受体在人类和啮齿类动物的中枢神经系统中的各种类型的细胞中被发现,例如星形胶质细胞、少突胶质细胞、小胶质细胞和内皮细胞。在中枢和外周神经系统损伤如:创伤、缺血和炎症等体内、体外的各种模型中均证实外源性的EPO系统性给药具有神经保护的作用。另外,还有研究显示在啮齿类动物颞叶癫痫的模型中,rHuEPO已经显示出明显的对抗癫痫持续状态发生发展的作用,但在癫痫持续状态中不能确定rHuEPO是否具有神经保护作用。系统性给予rHuEPO使其发挥神经保护作用要求rHuEPO能够穿过血脑屏障,现在已有研究显示rHuEPO系统性给药可以通过受体介导的方式穿过血脑屏障。而且,预处理或者后处理rHuEPO在一些研究中也被证实在癫痫持续状态损伤中均具有神经保护作用。EPO和EPO受体结合后激活JAK2,引起下游包括转录激活因子-5 (STAT-5)、PI3K/Akt和Ras/丝裂素激活的蛋白激酶(MA PK)等信号通路的激活,来发挥抗凋亡的作用。PI3K通过激活Akt来实现其促存活的作用,Akt又称为蛋白激酶B,是PI3K最具有特征性的下游靶分子,PI3K的抑制剂可以阻断Akt的激活。许多实验通过观察磷酸化Akt的表达来确定PI3K/Akt信号通路的激活情况,在许多研究中rHuEPO已被证实具有神经保护作用。然而,研究也显示EPO还可以通过JAK/ STAT、ERK 1/2、NF-κB等信号途径发挥抗凋亡的神经保护作用。因此,我们在rHuEPO治疗前给予PI3K抑制剂LY294002进行阻断,去观察在体内试验模型中,rHuEPO是否经PI3K/Akt信号途径发挥神经保护作用。有鉴于此,我们的研究目的是观察后处理的外源性rHuEPO对PTZ诱导的癫痫持续状态大鼠的抗凋亡的神经保护作用,探讨rHuEPO是否通过PI3K/Akt信号途径对线粒体依赖凋亡途径的相关调控因子进行调节借以发挥抗凋亡的神经保护作用。
第一部分癫痫持续状态大鼠海马神经元凋亡和p-Akt表达的变化及重组人红细胞生成素的影响
目的:建立戊四氮点燃癫痫持续状态大鼠模型,观察大鼠海马区神经元的凋亡及p-Akt表达的变化及重组人红细胞生成素(rHuEPO)的影响。
方法:选用健康雄性SD大鼠,建立动物模型:戊四氮(PTZ)20 mg·kg-1大鼠腹腔注射,然后每10min注射10 mg·kg-1,直至达到足够强度和SE持续发作,Ⅳ、Ⅴ级发作即为全身性惊厥大发作,持续30 min及以上者为SE。筛选合格后随机分为五组:PTZ组:腹腔注射PTZ点燃SE发作后30分钟腹腔注射生理盐水; rHuEPO组:腹腔注射PTZ点燃SE发作后30分钟腹腔注射rHuEPO 5000U. kg-1;LY294002组:腹腔注射PTZ点燃SE发作后10分钟脑室注射给予5μl LY294002,SE发作后30分钟腹腔注射rHuEPO 5000 U.kg-1;DMSO对照组:腹腔注射PTZ点燃SE发作后10分钟脑室注射给予5μlDMSO, SE发作后30分钟腹腔注射rHuEPO 5000U.kg-1;Control组:腹腔注射相同体积、次数的生理盐水。造模成功后描记脑电图(electroencephalogram, EEG)变化。每组大鼠各取10只,10%水合氯醛麻醉,4%多聚甲醛灌注固定,取含海马脑段,石蜡包埋,冠状切片,采用TUNEL方法观察神经元的凋亡和免疫组化染色SP法测定p-Akt免疫反应阳性细胞的数目。每组大鼠各取10只,迅速分离海马,Western blot方法检测海马Akt、p-Akt蛋白表达的变化。
结果:EEG表现:Control组大鼠脑电图以α、β波,背景正常,无异常放电现象;PTZ组大鼠在基础波的背景上阵发出现大量高幅尖波、棘波、棘慢复合波、尖慢复合波,持续1-2S;rHuEPO组、LY294002组和DMSO组大鼠痫性放电明显受到抑制,在基础波的背景下,可现散在少量尖波、棘波,波幅降低。Tunel染色结果:与Control组相比,PTZ组、rHuEPO组、LY294002组和DMSO组海马区凋亡细胞数目明显增多,差异均有统计学意义(P<0.05);与rHuEPO组、LY294002组和DMSO组相比,PTZ组凋亡细胞数目明显增多,差异均有统计学意义(P <0.05);然而,与LY294002组比较,rHuEPO组、DMSO组凋亡细胞数目明显减少,差异均有统计学意义(P<0.05);DMSO组凋亡细胞数目虽多于rHuEPO组,但与rHuEPO组相比无显著性差异(P>0.05)。免疫组化染色结果:与Control相比,PTZ组、rHuEPO组、LY294002组、DMSO组p-Akt的阳性细胞数均显著增多,差异具有统计学意义(P<0.05);rHuEPO组、LY294002组、DMSO组p-Akt的阳性细胞数均较PTZ组明显增多,差异具有统计学意义(P<0.05);与rHuEPO组、DMSO组比较,LY294002组p-Akt阳性细胞数明显减少,差异具有统计学意义(P<0.05);rHuEPO组p-Akt的阳性细胞数虽多于DMSO组,但差异不具有统计学意义(P﹥0.05)。Western blot结果:Akt、p-Akt蛋白在各组大鼠海马表达水平分别以Akt/β-actin和p-Akt/β-actin表示,与Control组相比,PTZ组、rHuEPO组、LY294002组、DMSO组p-Akt蛋白的表达水平明显增多(P<0.05);与PTZ组比较,rHuEPO组、LY294002组、DMSO组p-Akt蛋白的表达水平明显增多(P<0.05);与rHuEPO组、DMSO组相比,LY294002组p-Akt蛋白的表达水平明显减少,差异具有统计学意义(P<0.05);rHuEPO组p-Akt蛋白的表达水平虽多于DMSO组,但差异不具有统计学意义(P>0.05)。PTZ、rHuEPO、LY294002对Akt没有明显的影响,各实验组Akt的表达水平无显著性差异(P>0.05)。
结论:本实验建立的戊四氮点燃癫痫持续状态大鼠模型诱导了神经元的凋亡,是研究癫痫后神经元损伤比较理想的动物模型。rHuEPO对癫痫持续状态大鼠具有抗凋亡的神经保护作用,PI3K/Akt途径可能是rHuEPO发挥神经保护的作用通路之一,与提高Akt的活性有关,借以发挥抗凋亡的神经保护作用。
第二部分癫痫持续状态大鼠海马Bcl-2家族蛋白的变化及重组人促红细胞生成素的影响
目的:观察戊四氮点燃癫痫持续状态大鼠海马Bcl-2、Bax、Bad表达的变化及rHuEPO的影响,探讨rHuEPO是否通过PI3K/Akt途径对线粒体相关调控因子Bcl-2、Bax、Bad进行调节,借以发挥抗凋亡的神经保护作用。
方法:每组大鼠各取10只,10%水合氯醛麻醉,4%多聚甲醛灌注固定,取含海马脑段,石蜡包埋,冠状切片,采用SP法行Bcl-2、Bax、Bad免疫组化染色,测定Bcl-2、Bax、Bad免疫反应阳性细胞的数目。每组大鼠各取10只,迅速分离海马,Trizol提取总RNA,应用反转录-聚合酶链反应(RT-PCR)方法检测定大鼠海马Bcl-2mRNA、BaxmRNA、BadmRNA表达的变化。每组大鼠各取10只,迅速分离海马,Western blot方法检测海马Bcl-2、Bax、Bad蛋白表达的变化。
结果:免疫组化染色结果:与Control相比,PTZ组、rHuEPO组、LY294002组、DMSO组Bcl-2阳性细胞数均显著增多,差异具有统计学意义(P < 0.05);rHuEPO组、LY294002组、DMSO组Bcl-2阳性细胞数均较PTZ组明显增多,差异具有统计学意义(P < 0.05);与rHuEPO组、DMSO组比较,LY294002组Bcl-2阳性细胞数明显减少,差异具有统计学意义(P<0.05);rHuEPO组Bcl-2的阳性细胞数虽多于DMSO组,但差异不具有统计学意义(P﹥0.05)。PTZ组、rHuEPO组、LY294002组、DMSO组Bax、Bad的阳性细胞数均较Control组显著增多,差异具有统计学意义(P <0.05);与rHuEPO组、LY294002组、DMSO组相比,PTZ组Bax、Bad的阳性细胞数均明显增多,差异具有统计学意义(P <0.05);而rHuEPO组、DMSO组Bax、Bad的阳性细胞数也均少于LY294002组,差异具有统计学意义(P <0.05);rHuEPO组Bax、Bad的阳性细胞数虽少于DMSO组,但差异不具有统计学意义(P﹥0.05)。RT-PCR结果:各组大鼠海马Bcl-2mRNA、BaxmRNA、BadmRNA的表达水平以Bcl-2mRNA/β-actinmRNA、BaxmRNA/β-actinmRNA、BadmRNA/β-actin mRNA表示。与Control组比较,PTZ组、rHuEPO组、LY294002组、DMSO组Bcl-2mRNA表达水平明显增多,差异具有统计学意义(P<0.05);与PTZ组比较,HuEPO组、LY294002组、DMSO组Bcl-2mRNA表达水平明显增多,差异具有统计学意义(P<0.05);与HuEPO组、DMSO组比较,LY294002组Bcl-2mRNA表达水平明显下降,差异具有统计学意义(P<0.05);与DMSO组相比,HuEPO组Bcl-2mRNA表达水平虽然增多,但无明显统计学差异(P>0.05)。与Control组比较,PTZ组、rHuEPO组、LY294002组、DMSO组BaxmRNA、BadmRNA表达水平明显增多,差异具有统计学意义(P<0.05);与HuEPO组、LY294002组、DMSO组比较,PTZ组BaxmRNA、BadmRNA表达水平明显增多,差异具有统计学意义(P<0.05);与LY294002组比较,HuEPO组、DMSO组BaxmRNA、BadmRNA表达水平明显下降,差异具有统计学意义(P<0.05);与DMSO组相比,HuEPO组BaxmRNA、BadmRNA表达水平虽然减少,但无明显统计学差异(P>0.05)。Western blot结果:Bcl-2、Bax、Bad蛋白在各组大鼠海马表达水平以Bcl-2/β-actin、Bax/β-actin、Bad/β-actin表示,与Control组相比,PTZ组、rHuEPO组、LY294002组、DMSO组Bcl-2蛋白的表达水平明显增多(P<0.05);与PTZ组比较,rHuEPO组、LY294002组、DMSO组Bcl-2蛋白的表达水平明显增多(P<0.05);与rHuEPO组、DMSO组相比,LY294002组p-Akt蛋白的表达水平明显减少,差异具有统计学意义(P<0.05);rHuEPO组p-Akt蛋白的表达水平虽多于DMSO组,但差异不具有统计学意义(P>0.05)。与Control组比较,PTZ组、rHuEPO组、LY294002组、DMSO组Bax、Bad蛋白表达水平明显增多,差异具有统计学意义(P<0.05);与HuEPO组、LY294002组、DMSO组比较,PTZ组Bax、Bad蛋白表达水平明显增多,差异具有统计学意义(P<0.05);与LY294002组比较,HuEPO组、DMSO组Bax、Bad蛋白表达水平明显下降,差异具有统计学意义(P<0.05);与DMSO组相比,HuEPO组Bax、Bad蛋白表达水平虽然减少,但无明显统计学差异(P>0.05)。
结论:PTZ点燃癫痫持续状态后大鼠海马组织Bcl-2、Bad、Bax蛋白及其mRNA水平均升高,提示线粒体凋亡途经相关调控因子Bad、Bcl-2、Bax可能参与了癫痫持续状态后海马神经元凋亡的损伤机制。rHuEPO后处理可以提高癫痫持续状态大鼠海马组织Bcl-2蛋白及其mRNA水平,降低Bad、Bax蛋白及其mRNA表达水平,进而改善癫痫持续状态后大鼠海马神经元的凋亡损伤。rHuEPO后处理改善癫痫持续状态后大鼠海马神经元的凋亡损伤可能通过PI3K/Akt信号传导途径对线粒体凋亡途经相关调控因子Bad、Bcl-2、Bax进行调控,进而介导线粒体凋亡途经,发挥抗凋亡神经保护作用。
第三部分癫痫持续状态大鼠海马Caspase-9的变化及重组人促红细胞生成素的影响
目的:观察戊四氮点燃癫痫持续状态大鼠海马Caspase-9表达的变化及rHuEPO的影响,探讨rHuEPO是否通过PI3K/Akt途径对线粒体相关调控因子Caspase-9进行调节,借以发挥抗凋亡的神经保护作用。
方法:每组大鼠各取10只,10%水合氯醛麻醉,4%多聚甲醛灌注固定,取含海马脑段,石蜡包埋,冠状切片,采用SP法行Caspase-9免疫组化染色,测定Caspase-9免疫反应阳性细胞的数目。每组大鼠各取10只,迅速分离海马,Trizol提取总RNA,应用反转录-聚合酶链反应(RT-PCR)方法检测定大鼠海马Caspase-9mRNA表达的变化。每组大鼠各取10只,迅速分离海马,Western blot方法检测海马Caspase-9蛋白表达的变化。
结果:Caspase-9免疫组化染色结果:PTZ组、rHuEPO组、LY294002组、DMSO组Caspase-9的阳性细胞数均较Control组显著增多,差异具有统计学意义(P <0.05);与rHuEPO组、LY294002组、DMSO组相比,PTZ组Caspase-9的阳性细胞数均明显增多,差异具有统计学意义(P <0.05);而rHuEPO组、DMSO组Caspase-9的阳性细胞数也均少于LY294002组,差异具有统计学意义(P <0.05);rHuEPO组Caspase-9的阳性细胞数虽少于DMSO组,但差异不具有统计学意义(P﹥0.05)。RT-PCR结果:各组大鼠海马Caspase-9mRNA的表达水平以Caspase-9mRNA/β-actinmRNA表示。结果显示:与Control组比较,PTZ组、rHuEPO组、LY294002组、DMSO组Caspase-9mRNA表达水平明显增多,差异具有统计学意义(P<0.05);与HuEPO组、LY294002组、DMSO组比较,PTZ组Caspase-9mRNA表达水平明显增多,差异具有统计学意义(P<0.05);与LY294002组比较,HuEPO组、DMSO组Caspase-9mRNA表达水平明显下降,差异具有统计学意义(P<0.05);与DMSO组相比,HuEPO组Caspase-9mRNA表达水平虽然减少,但无明显统计学差异(P>0.05)。Western blot结果:Caspase-9蛋白在各组大鼠海马表达水平以Caspase-9/β-actin表示,结果显示:与Control组比较,PTZ组、rHuEPO组、LY294002组、DMSO组Caspase-9蛋白表达水平明显增多,差异具有统计学意义(P<0.05);与HuEPO组、LY294002组、DMSO组比较,PTZ组Caspase-9蛋白表达水平明显增多,差异具有统计学意义(P<0.05);与LY294002组比较,HuEPO组、DMSO组Caspase-9表达水平明显下降,差异具有统计学意义(P<0.05);与DMSO组相比,HuEPO组Caspase-9蛋白表达水平虽然减少,但无明显统计学差异(P>0.05)。
结论:PTZ点燃癫痫持续状态后大鼠海马组织Caspase-9蛋白及其mRNA水平均升高,提示线粒体凋亡途经相关调控因子Caspase-9可能参与了癫痫持续状态后海马神经元凋亡的损伤机制。rHuEPO后处理可以降低癫痫持续状态大鼠海马组织Caspase-9蛋白及其mRNA表达水平,进而改善癫痫持续状态后大鼠海马神经元的凋亡损伤。rHuEPO后处理改善癫痫持续状态后大鼠海马神经元的凋亡损伤可能通过PI3K/Akt信号传导途径对线粒体凋亡途经相关调控因子Caspase-9进行调控,进而介导线粒体凋亡途经,发挥抗凋亡神经保护作用。
第四部分癫痫持续状态大鼠海马XIAP的变化及重组人促红细胞生成素的影响
目的:观察戊四氮点燃癫痫持续状态大鼠海马XIAP表达的变化及rHuEPO的影响,探讨rHuEPO是否通过PI3K/Akt途径对线粒体相关调控因子XIAP进行调节,发挥抗凋亡的神经保护作用。
方法:每组大鼠各取10只,10%水合氯醛麻醉,4%多聚甲醛灌注固定,取含海马脑段,石蜡包埋,冠状切片,采用SP法行XIAP免疫组化染色,测定XIAP免疫反应阳性细胞的数目。每组大鼠各取10只,迅速分离海马,Trizol提取总RNA,应用反转录-聚合酶链反应(RT-PCR)方法检测定大鼠海马XIAPmRNA表达的变化。每组大鼠各取10只,迅速分离海马,Western blot方法检测海马XIAP蛋白表达的变化。
结果:XIAP免疫组化染色结果:与Control相比,PTZ组、rHuEPO组、LY294002组、DMSO组XIAP阳性细胞数均显著增多,差异具有统计学意义(P < 0.05);rHuEPO组、LY294002组、DMSO组XIAP阳性细胞数均较PTZ组明显增多,差异具有统计学意义(P < 0.05);与rHuEPO组、DMSO组比较,LY294002组XIAP阳性细胞数明显减少,差异具有统计学意义(P<0.05);rHuEPO组XIAP的阳性细胞数虽多于DMSO组,但差异不具有统计学意义(P﹥0.05)。RT-PCR结果:各组大鼠海马XIAPmRNA的表达水平以XIAPmRNA/β-actinmRNA表示。结果显示:与Control组比较,PTZ组、rHuEPO组、LY294002组、DMSO组XIAPmRNA表达水平明显增多,差异具有统计学意义(P<0.05);与PTZ组比较,HuEPO组、LY294002组、DMSO组XIAPmRNA表达水平明显增多,差异具有统计学意义(P<0.05);与HuEPO组、DMSO组比较,LY294002组XIAPmRNA表达水平明显下降,差异具有统计学意义(P<0.05);与DMSO组相比,HuEPO组XIAPmRNA表达水平虽然增多,但无明显统计学差异(P>0.05)。Western blot结果:XIAP蛋白在各组大鼠海马表达水平以XIAP/β-actin表示,结果显示:与Control组相比,PTZ组、rHuEPO组、LY294002组、DMSO组XIAP蛋白的表达水平明显增多(P<0.05);与PTZ组比较,rHuEPO组、LY294002组、DMSO组XIAP蛋白的表达水平明显增多(P<0.05);与rHuEPO组、DMSO组相比,LY294002组XIAP蛋白的表达水平明显减少,差异具有统计学意义(P<0.05);rHuEPO组XIAP蛋白的表达水平虽多于DMSO组,但差异不具有统计学意义(P>0.05)。
结论:PTZ点燃的癫痫持续状态后大鼠海马组织XIAP蛋白及其mRNA水平均升高,提示线粒体凋亡途经相关调控因子XIAP可能参与了癫痫持续状态后海马神经元凋亡的损伤机制。rHuEPO后处理可以降低癫痫持续状态大鼠海马组织XIAP蛋白及其mRNA表达水平,进而改善癫痫持续状态后大鼠海马神经元的凋亡损伤,其抗凋亡的机制可能通过PI3K/Akt信号传导途径对线粒体凋亡途经相关调控因子XIAP进行调控,进而介导线粒体凋亡途经,发挥抗凋亡神经保护作用。
Epilepsy, which is essentially an abnormality of cerebral function resulting from the discharges of some cerebral neuron in over high excitement state. Epilepsy is one of the most common diseases or syndromes of central nervous system. Status Epilepticus (SE) is a neurological emergency with an associated mortality rate of 10%–12%, which many affect the patients’life greatly. In patients with temporal lobe epilepsy (TLE), various experimental epilepsy and SE models, the lesions of neurons representing typical apoptotic features were verified by morphological and biochemical methods. The effect of apoptosis on SE has drawn much attention recently though it is still unclear about the detailed signaling pathways that trigger apoptosis is incomplete.
Apoptosis is a fundamental process of cell death that occurs via activation of distinct signaling pathways involving mitochondria, mitochondrial regulatory proteins, and activation of Caspases. Ultimately, cells undergo nuclear chromatin condensation, DNA fragmentation, and formation of apoptotic bodies. Apoptosis occurs through three main pathways: the mitochondrial- dependent pathway; the death receptor dependent pathway and the endoplasmic reticulum (ER) pathway. The more important pathway is the mitochondrial-dependent pathway, which induces the leads to the release of Cytochrome c from mitochondria and activation of the death signal. The Cytochrome c quickly combines with the Apaf-1 and Caspase-9, which forms a complex called apoptosome. The activation of Caspase-9 leads to a series of activating of its downstream Caspases, which finally results in programmed cell death.Bcl-2 family proteins, Caspase family proteins and inhibitor of apoptosis proteins (IAPs) are the principal regulators of mitochondrial- dependent apoptotic pathway. The Bcl-2 family of proteins regulates apoptosis by modulating mitochondrial permeability and the release of Cytochrome c. The Bcl-2 family of proteins includes proapoptotic members such as Bax, Bad, and antiapoptotic members such as Bcl-2, Bcl-xL. Although Bad and Bax are located in the cytosol , but translocate to the mitochondria and form a proapoptotic complex with Bcl-2,and then release Cytochrome c. Proteins opposing these pro-apoptotic proteins are antiapoptosis proteins, The antiapoptotic protein Bcl-2 resides in the outer mitochondrial wall, which inhibits the release of pro-apoptotic molecules to mitochondria by multiple mechanisms, including maintenance of mitochondrial membrane integrity and binding to pro-apoptosis members of the Bcl-2 family. The ratio of Bcl-2/Bax protein has also been suggested to determine cell survival or death. Among the BH3-only class of proteins, Bad was the first identified and the best characterized. Bad is a pro-death member of Bcl-2 family that initiates apoptosis by binding to Bcl-2 and Bcl-xL on the outer mitochondrial membrane, causing the release of Cytochrome c into cytosol. The function of Bad is to bind Bcl-2 and Bcl-xl, and block the antiapoptotic action of these proteins. For example: Bad may also inhibit the ability of Bcl-2 and Bcl-xL from forming channels in the mitochondrial membrane, or bind and stabilize the voltage dependent anion channel (VDAC). Bad is an example of a proapoptotic protein that is regulated by phosphorylation, It is phosphorylated on ser-136 by Akt in the PI3K/Akt pathway, Phosphorylation at the site promotes the binding of Bad to 14-3-3 proteins. When bound to 14-3-3 proteins, Bad is sequestered in the cytosol and unable to interact with Bcl-2 or Bcl-xL at the outer mitochondrial membrane. Caspase-9, as well as the other Caspases, exists in an inactive zymogen pro-Caspase state that is activated through proteolytic processing. Cytochrome c, once released in cytosol, interacts with Apaf-1, leading to the activation of Caspase-9 proenzymes. Active Caspase-9 then activates Caspase-3, which subsequently activates the rest of the Caspase cascade and leads to apoptosis. The IAPs are a family of intracellular anti-apoptotic proteins, first identified in Baculovirus, which play a key role in cell survival by modulating death-signaling pathways at a post-mitochondrial level. They currently include X-linked IAP (XIAP), human IAP-1 (Hiap-1), Survivin, Livin and so on. Xchromosome-linked inhibitor of apoptosis protein (XIAP), one of the first mammalian IAP family members cloned, is the most potent inhibitor of apoptosis in IAP family members. It has been shown that XIAP is a direct inhibitor of Caspase-3 and Caspase-9 and can modulate the Bax/Cytochrome c pathway by inhibiting Caspase-9.
PI3K (phosphatidylinositol 3-kinase) is a kinase that plays a critical role in signaling pathways important to cell survival. PI3K/Akt signaling pathway is utilized by many cell types for regulating cell cycle, growth, metabolism and inhibition of apoptosis. Akt is known to mediate cell survival by regulating several effectors including Bad, Bcl-2, Bax, Caspase-9, XIAP and so on, which are the important regulatory factors of the mitochondrial-dependent apoptotic pathway meanwhile. The first anti-apoptotic Akt target identified was the pro-apoptotic protein Bad. Activated Akt can phosphorylate Bad specifically at Ser136 and thus can inactivate Bad. Phosphorylated Bad prevents apoptosis, because unphosphorylated Bad can form heterodimers with the anti-apoptotic proteins Bcl-xl or Bcl-2, and then antagonize their anti-apoptotic functions. In addition, researches have also shown that Bax is regulated by phosphorylation of Ser184 in an Akt-dependent manner and that phosphorylation inhibits Bax effects on the mitochondria by maintaining the protein level in the cytoplasm, heterodimerized with antiapoptotic Bcl-2 family members. Akt also phosphorylates and activates the cyclic AMP-response element-binding protein (CREB) on Ser-133, which increases the transcription of anti-apoptotic genes, such as Bcl-2. Akt also phosphorylates the IKK on Thr-23 and releases NF-кB, and then NF-кB up-regulates the expression of Bcl-2. Akt phosphorylates XIAP at residue serine 87 in vitro and in vivo, and interacts with XIAP at physiological protein concentration. These evidences show that Akt and its downstream targets constitute a major cell survival pathway.
Erythropoietin (EPO) was originally described for its role in hematopoiesis, which can increase red blood cells by protecting erythroid progenitors against apoptosis. We now know that there are EPO and its receptor (EPO-R) on many cell types of central nervous system in rodent and human brain, such as astrocytes, oligodendrocytes, microglia, and endothelial cells. Exogenous administration of EPO revealed neuroprotective activity in vitro and in vivo in models of central and peripheral neuronal injury occurred in the contexts of trauma, stroke and inflammation. In addition, studies in rodent models of TLE revealed significant effects of rHuEPO(recombinant Human erythropoietin, rHuEPO) in antagonizing the development of SE, but did not determine whether rHuEPO was neuroprotective. Neuroprotective effect induced by rHuEPO applied systemically requires that it crosses the blood–brain barrier (BBB), and many researches have showed rHuEPO administered peripherally crosses the BBB via a receptor-mediated mechanism. In addition, many researches show that pretreatment or even post-treatment of rHuEPO provides protection to neurons after SE. EPO binds to EPO receptor first and then activates janus tyrosine kinase 2 (JAK2), which triggers at least three intracellular signaling cascades through the anti-apoptotic role played by EPO: signal transducer and activator of transcription 5 (STAT5); phosphatidylinositol-3 kinase (PI3K)/Akt and RAS/mitogen-activated protein kinase (MAPK). Pro-survival actions of PI3K occur through activation of the anti-apoptotic effector Akt. Akt activation leads to phosphorylation of certain proteins that promote cell survival. Akt, also known as protein kinase B, is the most well characterized target of PI3K, whose catastaltica would inhibit the activation of Akt. Many experiments presumed the activation of PI3K/Akt signaling pathway through detecting the expression of p-Akt, which showed neuroprotective effects and confirmed by many researches. however, documents demonstrated that EPO could also play neuroprotective effect through the signal pathway of JAK/ STAT; ERK 1/2; NF-κB. To clarify whether the PI3K/Akt signaling pathway involved in the neuroprotective progression originated by rHuEPO in vivo model, we administered LY294002 before rHuEPO treatment, an inhibitor of PI3K. Based on the above mentioned facts, the purpose of our research is to evaluate the anti-apoptosis neuroprotective effect of exogenous rHuEPO post-treatment in PTZ kindled SE rats, and discuss whether rHuEPO modulates the regulatory factors of mitochondrial-dependent apoptotic pathway to mediate the anti-apoptosis neuron protective effects through PI3K/Akt signaling pathway.
Part I Changes of p-Akt expression levels and neurons apoptosis in hippocampus of statural epilepticus rats kindled by PTZ and the effect of rHuEPO
Objective: To establish a status epilepticus rat model kindled by PTZ and observe the changes of p-Akt expression levels and neurons apoptosis in hippocampus of status epilepticus rat and the effect of rHuEPO.
Methods: Adult male Sprague–Dawley (SD) rats were selected to establish a status epilepticus rat model kindled by PTZ. First, a dose of 20 mg/kg was intraperitoneal injection (i.p.), then followed additional doses of 10mg/kg were administered to the rat every 10 min until the onset of SE. Rats were regarded as fully kindled when they exhibited seizure stages 4-5, this procedure lasted for at least 30 minutes. Rats were divided into 5 groups randomly. In the PTZ group, rats were subjected to PTZ i.p.; in the rHuEPO group, rats were subjected to rHuEPO 5000U/kg i.p., the procedure were performed 30 minutes after SE kindled by the PTZ; in LY294002 group, rats were subjected to intracerebral ventricle (ICV) infusion of LY294002 5μl and rHuEPO 5000U/kg i.p., and the procedure were performed 10 min and 30min respectively after SE kindled by PTZ; in DMSO Control group, rats were subjected to ICV infusion of DMSO5μl and rHuEPO 5 000U/kgi.p., and the procedure were performed 10 min and 30min respectively after the SE kindled by the PTZ; in Control group, animals received the same volume of physiological saline i.p. as their paired PTZ- exposed congeners. Then those rats were recorded with electroencephalogram (EEG). 10 rats in each group were anesthetized by 10% chloral hydrate and perfused with 4% paraformaldehyde solution. The brain tissue containing hippocampus was cut and embedded with paraffin. The apoptotic neurons of the hippocampal region were detect through TUNEL method and the positive cell numbers of p-Akt were detected through immunohistochemical SP methods. 10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total protein for p-Akt was extracted. The expression levels of p-Akt protein were detected by Western blot.
Results: EEG changes: the EEG records of the rats in Control group were dominant withαwaves andβwaves and the background was normal without any discharges. The rats in PTZ group were recorded in EEG as sharps, spike-slow waves and most polyspike discharge, with high potential on a relatively normal background. The EEG records of the rats in rHuEPO group, LY294002 group and DMSO group were demonstrated the repressed discharges. The results of TUNEL stains: compared with the Control group, a significant increases for the number of TUNEL-positive cells were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P <0.05). Meanwhile, the same increase were also found for the number of TUNEL-positive cells in the PTZ group compared with those in the rHuEPO group, the LY294002 group and the DMSO group (P <0.05). However, compared with the LY294002 group, a significant decrease for the number of TUNEL-positive cells were observed in the rHuEPO group and the DMSO group (P<0.05). The number of TUNEL-positive cells in the DMSO group were higher than in the rHuEPO group, but with no significant difference (P>0.05). The results of immunohistochemical stains: compared with the Control group, the significant increases for the number of p-Akt-IR (immunohistochemical reaction) cells were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group( P<0.05). Meanwhile, the same increases were also found for the number of p-Akt-IR cells in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05) compared with those in the PTZ group. However, there were the significant decreases in the number of p-Akt-IR cells in the LY294002 group compared with those in the rHuEPO group and the DMSO group (P<0.05). The numbers of p-Akt-IR cells in the rHuEPO group were higher than in the DMSO group, but the difference was insignificant (P>0.05). The results of Western blot: the protein levels of p-Akt in hippocampus of rats in each group were determined by p-Akt/β-actin. compared with the Control group, the significant increases for the protein levels of p-Akt were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). At the same time, the same increase were also found for the protein levels of P-Akt in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05) compared with those in the PTZ group. However, there were significant decrease for the protein levels of P-Akt in the LY294002 group compared with those in the rHuEPO group and the DMSO group (P<0.05). The protein levels of p-Akt in the rHuEPO group were higher than those in the DMSO group, but the difference were insignificant (P>0.05).
Conclusion: The status epilepticus rat model kindled by PTZ in the experiment did have impairments of neuronal apoptosis. This model is the perfect animal model which is feasible to study the damaged neuronal apoptosis after status epilepticus. The rHuEPO showed the effect of antiapoptosis neuroprotection in SE rats, the pathway of PI3K/Akt may be one of the neuroprotective ways of rHuEPO. The possible mechanism is rHuEPO activated the PI3K/Akt pathway and then up-regulated the expression of p-Akt to displayed neuroprotective effect.
Part II Changes of the Bcl-2 proteins family expression levels in hippocampus of statural epilepticus rats kindled by PTZ and the effect of rHuEPO
Objective: To observe the changes of Bcl-2 proteins family expression levels in hippocampus of status epilepticus rat kindled by PTZ and the effect of rHuEPO, and to explore whether the rHuEPO regulated the expression of Bcl-2、Bax、Bad that are the regulatory factors of mitochondrial-dependent apoptotic pathway through PI3K/Akt signaling pathway, and played anti-apoptotic neuroprotective effect.
Methods: 10 rats in each group were anesthetized by 10% chloral hydrate and perfused with 4% paraformaldehyde solution. The brain tissue containing hippocampus was cut and embedded with paraffin. the immunohistochemical reaction positive cell numbers of Bcl-2、Bax、Bad were detected through immunohistochemical SP methods. 10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total RNA was extracted strictly by Trizol. The expression of Bcl-2mRNA、BaxmRNA、BadmRNA was measured by RT-PCR.10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total protein for Bcl-2、Bax、Bad was extracted. The expression levels of Bcl-2、Bax、Bad protein were detected by Western blot.
Results: The results of immunohistochemical stains: compared with the Control group, the significant increases for the number of Bcl-2-IR cells were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group( P<0.05). Meanwhile, the same increases were also found for the number of Bcl-2-IR cells in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05) compared with those in the PTZ group. However, there were the significant decreases in the number of Bcl-2-IR cells in the LY294002 group compared with those in the rHuEPO group and the DMSO group (P<0.05). The numbers of Bcl-2-IR cells in the rHuEPO group were higher than in the DMSO group, but the difference was insignificant (P>0.05). Compared with the Control group, the significant increases for the number of Bax-IR、Bad-IR cells were observed in the PTZ group(P<0.05), the rHuEPO group, the LY294002 group and the DMSO group. Meanwhile, the same increase were also found for the number of Bax-IR、Bad-IR cells in the PTZ group compared with those in the rHuEPO group, the LY294002 group and the DMSO group(P<0.05). However, compared with the LY294002 group, a significant decrease in the number of Bax-IR、Bad-IR cells were found in the rHuEPO group and the DMSO group(P<0.05). The number of Bax-IR、Bad-IR cells in the rHuEPO group were lower than in the DMSO group, but the difference was insignificant (P>0.05).The results of RT-PCR: the expression levels of Bcl-2mRNA、BaxmRNA、BadmRNA were determined by calculating the density ratio of Bcl-2mRNA/β-actin mRNA、BaxmRNA/β-actinmRNA、BadmRNA/β-actinmRNA. Compared with the Control group, the significant increases for Bcl-2mRNA were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). Meanwhile, the same increase were also found for Bcl-2mRNA in the rHuEPO group, the LY294002 group and the DMSO group compared with those in the PTZ group (P<0.05). However, there were significant decrease for Bcl-2mRNA in the LY294002 group compared with those in the rHuEPO group and the DMSO group (P<0.05). The expression level of Bcl-2mRNA in the rHuEPO group were higher than in the DMSO group, but the difference was insignificant (P>0.05). Compared with the Control group, significant increases for BaxmRNA、BadmRNA were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group(P<0.05). Meanwhile, the same increase were also found for BaxmRNA、BadmRNA in the PTZ group compared with those in the rHuEPO group, the LY294002 group and the DMSO group(P<0.05). However, compared with the LY294002 group, a significant decrease for BaxmRNA、BadmRNA were found in the rHuEPO group and the DMSO group(P<0.05). The expression level of BaxmRNA、BadmRNA in the rHuEPO group were lower than in the DMSO group, but the difference was insignificant(P>0.05).The results of Western blot: The protein levels of Bcl-2、Bax、Bad in hippocampus of rats in each group were determined by Bcl-2/β-actin、Bax/β-actin、Bad/β-actin respectively. compared with the Control group, the significant increases for the protein levels of Bcl-2 were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). At the same time, the same increase were also found for the protein levels of Bcl-2 in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05)compared with those in the PTZ group. However, there were significant decrease for the protein levels of Bcl-2 in the LY294002 group compared with those in the rHuEPO group and the DMSO group( P<0.05). The protein levels of Bcl-2 in the rHuEPO group were higher than those in the DMSO group, but the difference was insignificant (P>0.05).Compared with the Control group, the significant increase for the protein levels of Bax and Bad were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group(P<0.05). Meanwhile, the same increase were also found for the protein levels of Bax and Bad in the PTZ group compared with those in the the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). However, compared with the LY294002 group, the significant decrease for the protein levels of Bax and Bad were found in the rHuEPO group and the DMSO group (P<0.05). The protein levels of Bax and Bad in the rHuEPO group were lower than those in the DMSO group, but the difference was insignificant (P>0.05).
Conclusion: There were elevation in expression levels of Bcl-2、Bax and Bad protein and Bcl-2mRNA、BaxmRNA and BadmRNA in the hippocampus of status epilepticus rat kindled by PTZ, which suggests that Bcl-2、Bax and Bad might participate in the mechanism of hippocampus neuronal apoptosis lesion after status epilepticus. rHuEPO posttreament could increase the expression levels of Bcl-2、Bcl-2mRNA and decrease the expression levels of Bax、BaxmRNA、Bad、BadmRNA, the possible mechanism is the rHuEPO regulated the expression of Bcl-2、Bax、Bad that are the regulatory factors of mitochondrial-dependent apoptotic pathway through PI3K/Akt signaling pathway, and played anti-apoptotic neuroprotective effect.
Part III Changes of the Caspase-9 expression levels in hippocampus of statural epilepticus rats kindled by PTZ and the effect of rHuEPO
Objective: To observe the changes of Caspase-9 expression levels in hippocampus of status epilepticus rat kindled by PTZ and the effect of rHuEPO, and to explore whether the rHuEPO regulated the expression of Caspase-9 that are the regulatory factors of mitochondrial-dependent apoptotic pathway through PI3K/Akt signaling pathway, and played anti-apoptotic neuroprotective effect.
Methods: 10 rats in each group were anesthetized by 10% chloral hydrate and perfused with 4% paraformaldehyde solution. The brain tissue containing hippocampus was cut and embedded with paraffin, the immunohistochemical reaction positive cell numbers of Caspase-9 were detected through immunohistochemical SP methods. 10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total RNA was extracted strictly by Trizol. The expression of Caspase-9mRNA were measured by RT-PCR.10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total protein for Caspase-9 was extracted. The expression levels of Caspase-9 protein were detected by Western blot.
Results: The results of immunohistochemical stains: compared with the Control group, the significant increases for the number of Caspase-9-IR cells were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group ( P<0.05), Meanwhile, the same increase were also found for the number of Caspase-9-IR cells in the PTZ group compared with those in the rHuEPO group, the LY294002 group and the DMSO group(P<0.05). However, compared with the LY294002 group, a significant decrease in the number of Caspase-9-IR were found in the rHuEPO group and the DMSO group(P<0.05). The number of Caspase-9-IR cells in the rHuEPO group were lower than in the DMSO group, but the difference was insignificant (P>0.05).The results of RT-PCR: the expression levels of Caspase-9mRNA were determined by calculating the density ratio of Caspase-9mRNA/β-actin mRNA. Compared with the Control group, the significant increases for Caspase-9mRNA were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). Meanwhile, the same increase were also found for Caspase-9mRNA in the PTZ group compared with those in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). However, compared with the LY294002 group, a significant decrease for Caspase-9mRNA were found in the rHuEPO group and the DMSO group(P<0.05). The expression level of Caspase-9mRNA in the rHuEPO group were lower than in the DMSO group, but the difference was insignificant(P>0.05).The results of Western blot: The protein levels of Caspase-9 in hippocampus of rats in each group were determined by Caspase-9/β-actin. Compared with the Control group, the significant increase for the protein levels of Caspase-9 were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group(P<0.05). Meanwhile, the same increase were also found for the protein levels of Caspase-9 in the PTZ group compared with those in the the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). However, compared with the LY294002 group, the significant decrease for the protein levels of Caspase-9 were found in the rHuEPO group and the DMSO group (P<0.05). The protein levels of Caspase-9 in the rHuEPO group were lower than those in the DMSO group, but the difference was insignificant (P>0.05).
Conclusion: There were elevation in expression levels of Caspase-9 protein and Caspase-9mRNA in the hippocampus of status epilepticus rat kindled by PTZ, which suggests that Caspase-9 might participate in the mechanism of hippocampus neuronal apoptosis lesion after status epilepticus. rHuEPO posttreament could increase the expression levels of Caspase-9、Caspase-9mRNA, the possible mechanism is the rHuEPO regulated the expression of Caspase-9 that are the regulatory factors of mitochondrial-dependent apoptotic pathway through PI3K/Akt signaling pathway, and played anti-apoptotic neuroprotective effect.
Part IV Changes of the XIAP expression levels in hippocampus of statural epilepticus rats kindled by PTZ and the effect of rHuEPO
Objective: To observe the changes of XIAP expression levels in hippocampus of status epilepticus rat kindled by PTZ and the effect of rHuEPO, and to explore whether the rHuEPO regulated the expression of XIAP that are the regulatory factors of mitochondrial-dependent apoptotic pathway through PI3K/Akt signaling pathway, and played anti-apoptotic neuroprotective effect.
Methods: 10 rats in each group were anesthetized by 10% chloral hydrate and perfused with 4% paraformaldehyde solution. The brain tissue containing hippocampus was cut and embedded with paraffin. the immunohistochemical reaction positive cell numbers of XIAP were detected through immunohistochemical SP methods. 10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total RNA was extracted strictly by Trizol. The expression of XIAPmRNA was measured by RT-PCR.10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total protein for XIAP were extracted. The expression levels of XIAP protein were detected by Western blot.
Results: The results of immunohistochemical stains: compared with the Control group, the significant increases for the number of XIAP-IR cells were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group( P<0.05). Meanwhile, the same increases were also found for the number of XIAP-IR cells in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05) compared with those in the PTZ group. However, there were the significant decreases in the number of XIAP-IR cells in the LY294002 group compared with those in the rHuEPO group and the DMSO group (P<0.05). The numbers of XIAP-IR cells in the rHuEPO group were higher than in the DMSO group, but the difference was insignificant (P>0.05). The results of RT-PCR: the expression levels of XIAPmRNA were determined by calculating the density ratio of XIAPmRNA/β-actin mRNA. Compared with the Control group, the significant increases for XIAPmRNA were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). Meanwhile, the same increase were also found for XIAPmRNA in the rHuEPO group, the LY294002 group and the DMSO group compared with those in the PTZ group (P<0.05). However, there were significant decrease for XIAPmRNA in the LY294002 group compared with those in the rHuEPO group and the DMSO group (P<0.05). The expression level of XIAPmRNA in the rHuEPO group were higher than in the DMSO group, but the difference was insignificant (P>0.05). The results of Western blot: The protein levels of XIAP in hippocampus of rats in each group were determined by XIAP/β-actin. compared with the Control group, the significant increases for the protein levels of XIAP were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). At the same time, the same increase were also found for the protein levels of XIAP in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05)compared with those in the PTZ group. However, there were significant decrease for the protein levels of XIAP in the LY294002 group compared with those in the rHuEPO group and the DMSO group( P<0.05). The protein levels of XIAP in the rHuEPO group were higher than those in the DMSO group, but the difference was insignificant (P>0.05).
Conclusion: There were elevation in expression levels of XIAP protein and XIAPmRNA in the hippocampus of status epilepticus rat kindled by PTZ, which suggests that XIAP might participate in the mechanism of hippocampus neuronal apoptosis lesion after status epilepticus. rHuEPO posttreament could increase the expression levels of XIAP、XIAPmRNA, the possible mechanism is the rHuEPO regulated the expression of XIAP that are the regulatory factors of mitochondrial-dependent apoptotic pathway through PI3K/Akt signaling pathway, and played anti-apoptotic neuroprotective effect.
凋亡的发生激活了包括线粒体、线粒体相关调控因子及Caspsae家族等许多的信号途径,是细胞死亡的主要过程,最终导致细胞核染色质的凝聚、DNA的断裂、凋亡小体的形成。凋亡的发生通过三条主要的途径:线粒体依赖途径、死亡受体依赖途径、内质网依赖途径。线粒体凋亡途径是主要的凋亡途径,它诱导细胞色素C从线粒体释放并激活了死亡信号。细胞色素C从线粒体释放出来,与凋亡活化因子-1 (apoptosis activated factor-1,Apaf-1)及Caspase-9结合形成复合体。Caspase-9的激活将引起其下游胱天蛋白酶的级联反应,导致该细胞凋亡。Bcl-2家族蛋白、Caspase家族蛋白、凋亡抑制蛋白(IAPs)家族都是重要的线粒体依赖凋亡途径的相关调控因子。Bcl-2家族蛋白通过调控线粒体膜的通透性和细胞色素C的释放来介导凋亡,Bcl-2蛋白家族包括促凋亡成员Bax、Bad等,和抗凋亡成员Bcl-2、Bcl-xl等。尽管Bax和Bad大多数时候存在于细胞质,但它们可以易位到线粒体和Bcl-2形成促凋亡的复合物,然后释放细胞色素C。与促凋亡蛋白相对应的是抗凋亡蛋白,抗凋亡蛋白Bcl-2存在于线粒体膜的外部,通过多种机制阻断促凋亡分子释放到线粒体,这些机制包括维持线粒体膜的完整性和联合Bcl-2家族中的促凋亡蛋白。Bcl-2/Bax的比率也是决定细胞生存与死亡的关键。在仅含BH3区蛋白的成员中,Bad是最早被鉴别出来,最具有特征性的蛋白。Bad通过在线粒体膜的外部与Bcl-2和Bcl-xl结合引起细胞色素C释放到细胞质来诱导凋亡的发生。Bad的促凋亡功能主要是通过与Bcl-2和Bcl-xl结合来阻断他们的抗凋亡作用。例如:Bad可以阻断Bcl-2和Bcl-xl在线粒体外膜上形成通道的能力,或者是阻断他们结合和稳定电压依从性阴离子通道的能力。Bad是一个可以被磷酸化调节的典型的促凋亡蛋白,激活的Akt使Bad中的Ser136磷酸化,导致14-3-3蛋白与Bad的结合,稳定与细胞之中,使它不能在线粒体外膜上与Bcl-2和Bcl-xl相互作用,抑制了Bad所诱导的细胞凋亡。Caspase-9,就像其他的Caspase成员一样,以一种没有活性的酶原形式存在并通过蛋白酶解加工激活。细胞色素C一旦释放到细胞质就会与Apaf-1相互作用,导致Caspase-9酶原的激活,激活的Caspase-9进而激活Caspase-3,随后引起其他的Caspase的级联反应,导致该细胞凋亡。IAPs家族是细胞内抗凋亡蛋白家族,在杆状病毒中首先被鉴定出来,在一个后线粒体水平通过调节死亡信号途径发挥着重要的促细胞存活作用。IAPs家族包括XIAP、Hiap-1、Survivin、Livin等等。XIAP是首先被克隆出来的哺乳动物IAPs家族成员之一,是该家族中最具有抗凋亡能力的成员之一。XIAP可以直接阻断Caspase-3 and Caspase-9,通过对Caspase-9的阻断来调节Bax/细胞色素C介导的线粒体凋亡途径。
PI3K是促进细胞存活的信号传导途径中的重要激酶,起到十分关键的作用。在各种细胞中发挥着调整细胞分裂周期、生长、新陈代谢、抑制凋亡的作用。众所周知,激活的Akt通过调整一些重要的调节因子介导细胞的存活,包括Bad、Bax、Bcl-2、Caspase-9和XIAP等等,这些调节因子同时也是线粒体依赖凋亡途径的相关调控因子。第一个被鉴定出来的Akt的下游分子是促凋亡蛋白Bad,Akt可以磷酸化Bad的Ser136位点,导致Bad的钝化。磷酸化的Bad具有抗凋亡的作用,因为非磷酸化的Bad可以和Bcl-2和Bcl-xl形成异二聚体,阻断他们的抗凋亡作用。另外,研究显示在Akt依赖的情况下BaxSer184可以被Akt磷酸化,阻断了Bax和Bcl-2家族中抗凋亡蛋白在线粒体膜形成异二聚体的能力,Akt也磷酸化CREB的Ser133位点,增加Bcl-2等抗凋亡因子的转录水平。Akt也可以磷酸化IKK的Thr-23位点,释放NF-кB,然后NF-кB可以上调Bcl-2的表达。最近,有研究显在体内、体外的实验中Akt在生理条件下也可以磷酸化XIAP的Ser87位点。这些研究显示了Akt和它的下游靶分子构成了主要的细胞存活途径。
EPO最初被了解是因为它通过保护红系祖细胞对抗凋亡来增多红细胞的作用。现在EPO和它的受体在人类和啮齿类动物的中枢神经系统中的各种类型的细胞中被发现,例如星形胶质细胞、少突胶质细胞、小胶质细胞和内皮细胞。在中枢和外周神经系统损伤如:创伤、缺血和炎症等体内、体外的各种模型中均证实外源性的EPO系统性给药具有神经保护的作用。另外,还有研究显示在啮齿类动物颞叶癫痫的模型中,rHuEPO已经显示出明显的对抗癫痫持续状态发生发展的作用,但在癫痫持续状态中不能确定rHuEPO是否具有神经保护作用。系统性给予rHuEPO使其发挥神经保护作用要求rHuEPO能够穿过血脑屏障,现在已有研究显示rHuEPO系统性给药可以通过受体介导的方式穿过血脑屏障。而且,预处理或者后处理rHuEPO在一些研究中也被证实在癫痫持续状态损伤中均具有神经保护作用。EPO和EPO受体结合后激活JAK2,引起下游包括转录激活因子-5 (STAT-5)、PI3K/Akt和Ras/丝裂素激活的蛋白激酶(MA PK)等信号通路的激活,来发挥抗凋亡的作用。PI3K通过激活Akt来实现其促存活的作用,Akt又称为蛋白激酶B,是PI3K最具有特征性的下游靶分子,PI3K的抑制剂可以阻断Akt的激活。许多实验通过观察磷酸化Akt的表达来确定PI3K/Akt信号通路的激活情况,在许多研究中rHuEPO已被证实具有神经保护作用。然而,研究也显示EPO还可以通过JAK/ STAT、ERK 1/2、NF-κB等信号途径发挥抗凋亡的神经保护作用。因此,我们在rHuEPO治疗前给予PI3K抑制剂LY294002进行阻断,去观察在体内试验模型中,rHuEPO是否经PI3K/Akt信号途径发挥神经保护作用。有鉴于此,我们的研究目的是观察后处理的外源性rHuEPO对PTZ诱导的癫痫持续状态大鼠的抗凋亡的神经保护作用,探讨rHuEPO是否通过PI3K/Akt信号途径对线粒体依赖凋亡途径的相关调控因子进行调节借以发挥抗凋亡的神经保护作用。
第一部分癫痫持续状态大鼠海马神经元凋亡和p-Akt表达的变化及重组人红细胞生成素的影响
目的:建立戊四氮点燃癫痫持续状态大鼠模型,观察大鼠海马区神经元的凋亡及p-Akt表达的变化及重组人红细胞生成素(rHuEPO)的影响。
方法:选用健康雄性SD大鼠,建立动物模型:戊四氮(PTZ)20 mg·kg-1大鼠腹腔注射,然后每10min注射10 mg·kg-1,直至达到足够强度和SE持续发作,Ⅳ、Ⅴ级发作即为全身性惊厥大发作,持续30 min及以上者为SE。筛选合格后随机分为五组:PTZ组:腹腔注射PTZ点燃SE发作后30分钟腹腔注射生理盐水; rHuEPO组:腹腔注射PTZ点燃SE发作后30分钟腹腔注射rHuEPO 5000U. kg-1;LY294002组:腹腔注射PTZ点燃SE发作后10分钟脑室注射给予5μl LY294002,SE发作后30分钟腹腔注射rHuEPO 5000 U.kg-1;DMSO对照组:腹腔注射PTZ点燃SE发作后10分钟脑室注射给予5μlDMSO, SE发作后30分钟腹腔注射rHuEPO 5000U.kg-1;Control组:腹腔注射相同体积、次数的生理盐水。造模成功后描记脑电图(electroencephalogram, EEG)变化。每组大鼠各取10只,10%水合氯醛麻醉,4%多聚甲醛灌注固定,取含海马脑段,石蜡包埋,冠状切片,采用TUNEL方法观察神经元的凋亡和免疫组化染色SP法测定p-Akt免疫反应阳性细胞的数目。每组大鼠各取10只,迅速分离海马,Western blot方法检测海马Akt、p-Akt蛋白表达的变化。
结果:EEG表现:Control组大鼠脑电图以α、β波,背景正常,无异常放电现象;PTZ组大鼠在基础波的背景上阵发出现大量高幅尖波、棘波、棘慢复合波、尖慢复合波,持续1-2S;rHuEPO组、LY294002组和DMSO组大鼠痫性放电明显受到抑制,在基础波的背景下,可现散在少量尖波、棘波,波幅降低。Tunel染色结果:与Control组相比,PTZ组、rHuEPO组、LY294002组和DMSO组海马区凋亡细胞数目明显增多,差异均有统计学意义(P<0.05);与rHuEPO组、LY294002组和DMSO组相比,PTZ组凋亡细胞数目明显增多,差异均有统计学意义(P <0.05);然而,与LY294002组比较,rHuEPO组、DMSO组凋亡细胞数目明显减少,差异均有统计学意义(P<0.05);DMSO组凋亡细胞数目虽多于rHuEPO组,但与rHuEPO组相比无显著性差异(P>0.05)。免疫组化染色结果:与Control相比,PTZ组、rHuEPO组、LY294002组、DMSO组p-Akt的阳性细胞数均显著增多,差异具有统计学意义(P<0.05);rHuEPO组、LY294002组、DMSO组p-Akt的阳性细胞数均较PTZ组明显增多,差异具有统计学意义(P<0.05);与rHuEPO组、DMSO组比较,LY294002组p-Akt阳性细胞数明显减少,差异具有统计学意义(P<0.05);rHuEPO组p-Akt的阳性细胞数虽多于DMSO组,但差异不具有统计学意义(P﹥0.05)。Western blot结果:Akt、p-Akt蛋白在各组大鼠海马表达水平分别以Akt/β-actin和p-Akt/β-actin表示,与Control组相比,PTZ组、rHuEPO组、LY294002组、DMSO组p-Akt蛋白的表达水平明显增多(P<0.05);与PTZ组比较,rHuEPO组、LY294002组、DMSO组p-Akt蛋白的表达水平明显增多(P<0.05);与rHuEPO组、DMSO组相比,LY294002组p-Akt蛋白的表达水平明显减少,差异具有统计学意义(P<0.05);rHuEPO组p-Akt蛋白的表达水平虽多于DMSO组,但差异不具有统计学意义(P>0.05)。PTZ、rHuEPO、LY294002对Akt没有明显的影响,各实验组Akt的表达水平无显著性差异(P>0.05)。
结论:本实验建立的戊四氮点燃癫痫持续状态大鼠模型诱导了神经元的凋亡,是研究癫痫后神经元损伤比较理想的动物模型。rHuEPO对癫痫持续状态大鼠具有抗凋亡的神经保护作用,PI3K/Akt途径可能是rHuEPO发挥神经保护的作用通路之一,与提高Akt的活性有关,借以发挥抗凋亡的神经保护作用。
第二部分癫痫持续状态大鼠海马Bcl-2家族蛋白的变化及重组人促红细胞生成素的影响
目的:观察戊四氮点燃癫痫持续状态大鼠海马Bcl-2、Bax、Bad表达的变化及rHuEPO的影响,探讨rHuEPO是否通过PI3K/Akt途径对线粒体相关调控因子Bcl-2、Bax、Bad进行调节,借以发挥抗凋亡的神经保护作用。
方法:每组大鼠各取10只,10%水合氯醛麻醉,4%多聚甲醛灌注固定,取含海马脑段,石蜡包埋,冠状切片,采用SP法行Bcl-2、Bax、Bad免疫组化染色,测定Bcl-2、Bax、Bad免疫反应阳性细胞的数目。每组大鼠各取10只,迅速分离海马,Trizol提取总RNA,应用反转录-聚合酶链反应(RT-PCR)方法检测定大鼠海马Bcl-2mRNA、BaxmRNA、BadmRNA表达的变化。每组大鼠各取10只,迅速分离海马,Western blot方法检测海马Bcl-2、Bax、Bad蛋白表达的变化。
结果:免疫组化染色结果:与Control相比,PTZ组、rHuEPO组、LY294002组、DMSO组Bcl-2阳性细胞数均显著增多,差异具有统计学意义(P < 0.05);rHuEPO组、LY294002组、DMSO组Bcl-2阳性细胞数均较PTZ组明显增多,差异具有统计学意义(P < 0.05);与rHuEPO组、DMSO组比较,LY294002组Bcl-2阳性细胞数明显减少,差异具有统计学意义(P<0.05);rHuEPO组Bcl-2的阳性细胞数虽多于DMSO组,但差异不具有统计学意义(P﹥0.05)。PTZ组、rHuEPO组、LY294002组、DMSO组Bax、Bad的阳性细胞数均较Control组显著增多,差异具有统计学意义(P <0.05);与rHuEPO组、LY294002组、DMSO组相比,PTZ组Bax、Bad的阳性细胞数均明显增多,差异具有统计学意义(P <0.05);而rHuEPO组、DMSO组Bax、Bad的阳性细胞数也均少于LY294002组,差异具有统计学意义(P <0.05);rHuEPO组Bax、Bad的阳性细胞数虽少于DMSO组,但差异不具有统计学意义(P﹥0.05)。RT-PCR结果:各组大鼠海马Bcl-2mRNA、BaxmRNA、BadmRNA的表达水平以Bcl-2mRNA/β-actinmRNA、BaxmRNA/β-actinmRNA、BadmRNA/β-actin mRNA表示。与Control组比较,PTZ组、rHuEPO组、LY294002组、DMSO组Bcl-2mRNA表达水平明显增多,差异具有统计学意义(P<0.05);与PTZ组比较,HuEPO组、LY294002组、DMSO组Bcl-2mRNA表达水平明显增多,差异具有统计学意义(P<0.05);与HuEPO组、DMSO组比较,LY294002组Bcl-2mRNA表达水平明显下降,差异具有统计学意义(P<0.05);与DMSO组相比,HuEPO组Bcl-2mRNA表达水平虽然增多,但无明显统计学差异(P>0.05)。与Control组比较,PTZ组、rHuEPO组、LY294002组、DMSO组BaxmRNA、BadmRNA表达水平明显增多,差异具有统计学意义(P<0.05);与HuEPO组、LY294002组、DMSO组比较,PTZ组BaxmRNA、BadmRNA表达水平明显增多,差异具有统计学意义(P<0.05);与LY294002组比较,HuEPO组、DMSO组BaxmRNA、BadmRNA表达水平明显下降,差异具有统计学意义(P<0.05);与DMSO组相比,HuEPO组BaxmRNA、BadmRNA表达水平虽然减少,但无明显统计学差异(P>0.05)。Western blot结果:Bcl-2、Bax、Bad蛋白在各组大鼠海马表达水平以Bcl-2/β-actin、Bax/β-actin、Bad/β-actin表示,与Control组相比,PTZ组、rHuEPO组、LY294002组、DMSO组Bcl-2蛋白的表达水平明显增多(P<0.05);与PTZ组比较,rHuEPO组、LY294002组、DMSO组Bcl-2蛋白的表达水平明显增多(P<0.05);与rHuEPO组、DMSO组相比,LY294002组p-Akt蛋白的表达水平明显减少,差异具有统计学意义(P<0.05);rHuEPO组p-Akt蛋白的表达水平虽多于DMSO组,但差异不具有统计学意义(P>0.05)。与Control组比较,PTZ组、rHuEPO组、LY294002组、DMSO组Bax、Bad蛋白表达水平明显增多,差异具有统计学意义(P<0.05);与HuEPO组、LY294002组、DMSO组比较,PTZ组Bax、Bad蛋白表达水平明显增多,差异具有统计学意义(P<0.05);与LY294002组比较,HuEPO组、DMSO组Bax、Bad蛋白表达水平明显下降,差异具有统计学意义(P<0.05);与DMSO组相比,HuEPO组Bax、Bad蛋白表达水平虽然减少,但无明显统计学差异(P>0.05)。
结论:PTZ点燃癫痫持续状态后大鼠海马组织Bcl-2、Bad、Bax蛋白及其mRNA水平均升高,提示线粒体凋亡途经相关调控因子Bad、Bcl-2、Bax可能参与了癫痫持续状态后海马神经元凋亡的损伤机制。rHuEPO后处理可以提高癫痫持续状态大鼠海马组织Bcl-2蛋白及其mRNA水平,降低Bad、Bax蛋白及其mRNA表达水平,进而改善癫痫持续状态后大鼠海马神经元的凋亡损伤。rHuEPO后处理改善癫痫持续状态后大鼠海马神经元的凋亡损伤可能通过PI3K/Akt信号传导途径对线粒体凋亡途经相关调控因子Bad、Bcl-2、Bax进行调控,进而介导线粒体凋亡途经,发挥抗凋亡神经保护作用。
第三部分癫痫持续状态大鼠海马Caspase-9的变化及重组人促红细胞生成素的影响
目的:观察戊四氮点燃癫痫持续状态大鼠海马Caspase-9表达的变化及rHuEPO的影响,探讨rHuEPO是否通过PI3K/Akt途径对线粒体相关调控因子Caspase-9进行调节,借以发挥抗凋亡的神经保护作用。
方法:每组大鼠各取10只,10%水合氯醛麻醉,4%多聚甲醛灌注固定,取含海马脑段,石蜡包埋,冠状切片,采用SP法行Caspase-9免疫组化染色,测定Caspase-9免疫反应阳性细胞的数目。每组大鼠各取10只,迅速分离海马,Trizol提取总RNA,应用反转录-聚合酶链反应(RT-PCR)方法检测定大鼠海马Caspase-9mRNA表达的变化。每组大鼠各取10只,迅速分离海马,Western blot方法检测海马Caspase-9蛋白表达的变化。
结果:Caspase-9免疫组化染色结果:PTZ组、rHuEPO组、LY294002组、DMSO组Caspase-9的阳性细胞数均较Control组显著增多,差异具有统计学意义(P <0.05);与rHuEPO组、LY294002组、DMSO组相比,PTZ组Caspase-9的阳性细胞数均明显增多,差异具有统计学意义(P <0.05);而rHuEPO组、DMSO组Caspase-9的阳性细胞数也均少于LY294002组,差异具有统计学意义(P <0.05);rHuEPO组Caspase-9的阳性细胞数虽少于DMSO组,但差异不具有统计学意义(P﹥0.05)。RT-PCR结果:各组大鼠海马Caspase-9mRNA的表达水平以Caspase-9mRNA/β-actinmRNA表示。结果显示:与Control组比较,PTZ组、rHuEPO组、LY294002组、DMSO组Caspase-9mRNA表达水平明显增多,差异具有统计学意义(P<0.05);与HuEPO组、LY294002组、DMSO组比较,PTZ组Caspase-9mRNA表达水平明显增多,差异具有统计学意义(P<0.05);与LY294002组比较,HuEPO组、DMSO组Caspase-9mRNA表达水平明显下降,差异具有统计学意义(P<0.05);与DMSO组相比,HuEPO组Caspase-9mRNA表达水平虽然减少,但无明显统计学差异(P>0.05)。Western blot结果:Caspase-9蛋白在各组大鼠海马表达水平以Caspase-9/β-actin表示,结果显示:与Control组比较,PTZ组、rHuEPO组、LY294002组、DMSO组Caspase-9蛋白表达水平明显增多,差异具有统计学意义(P<0.05);与HuEPO组、LY294002组、DMSO组比较,PTZ组Caspase-9蛋白表达水平明显增多,差异具有统计学意义(P<0.05);与LY294002组比较,HuEPO组、DMSO组Caspase-9表达水平明显下降,差异具有统计学意义(P<0.05);与DMSO组相比,HuEPO组Caspase-9蛋白表达水平虽然减少,但无明显统计学差异(P>0.05)。
结论:PTZ点燃癫痫持续状态后大鼠海马组织Caspase-9蛋白及其mRNA水平均升高,提示线粒体凋亡途经相关调控因子Caspase-9可能参与了癫痫持续状态后海马神经元凋亡的损伤机制。rHuEPO后处理可以降低癫痫持续状态大鼠海马组织Caspase-9蛋白及其mRNA表达水平,进而改善癫痫持续状态后大鼠海马神经元的凋亡损伤。rHuEPO后处理改善癫痫持续状态后大鼠海马神经元的凋亡损伤可能通过PI3K/Akt信号传导途径对线粒体凋亡途经相关调控因子Caspase-9进行调控,进而介导线粒体凋亡途经,发挥抗凋亡神经保护作用。
第四部分癫痫持续状态大鼠海马XIAP的变化及重组人促红细胞生成素的影响
目的:观察戊四氮点燃癫痫持续状态大鼠海马XIAP表达的变化及rHuEPO的影响,探讨rHuEPO是否通过PI3K/Akt途径对线粒体相关调控因子XIAP进行调节,发挥抗凋亡的神经保护作用。
方法:每组大鼠各取10只,10%水合氯醛麻醉,4%多聚甲醛灌注固定,取含海马脑段,石蜡包埋,冠状切片,采用SP法行XIAP免疫组化染色,测定XIAP免疫反应阳性细胞的数目。每组大鼠各取10只,迅速分离海马,Trizol提取总RNA,应用反转录-聚合酶链反应(RT-PCR)方法检测定大鼠海马XIAPmRNA表达的变化。每组大鼠各取10只,迅速分离海马,Western blot方法检测海马XIAP蛋白表达的变化。
结果:XIAP免疫组化染色结果:与Control相比,PTZ组、rHuEPO组、LY294002组、DMSO组XIAP阳性细胞数均显著增多,差异具有统计学意义(P < 0.05);rHuEPO组、LY294002组、DMSO组XIAP阳性细胞数均较PTZ组明显增多,差异具有统计学意义(P < 0.05);与rHuEPO组、DMSO组比较,LY294002组XIAP阳性细胞数明显减少,差异具有统计学意义(P<0.05);rHuEPO组XIAP的阳性细胞数虽多于DMSO组,但差异不具有统计学意义(P﹥0.05)。RT-PCR结果:各组大鼠海马XIAPmRNA的表达水平以XIAPmRNA/β-actinmRNA表示。结果显示:与Control组比较,PTZ组、rHuEPO组、LY294002组、DMSO组XIAPmRNA表达水平明显增多,差异具有统计学意义(P<0.05);与PTZ组比较,HuEPO组、LY294002组、DMSO组XIAPmRNA表达水平明显增多,差异具有统计学意义(P<0.05);与HuEPO组、DMSO组比较,LY294002组XIAPmRNA表达水平明显下降,差异具有统计学意义(P<0.05);与DMSO组相比,HuEPO组XIAPmRNA表达水平虽然增多,但无明显统计学差异(P>0.05)。Western blot结果:XIAP蛋白在各组大鼠海马表达水平以XIAP/β-actin表示,结果显示:与Control组相比,PTZ组、rHuEPO组、LY294002组、DMSO组XIAP蛋白的表达水平明显增多(P<0.05);与PTZ组比较,rHuEPO组、LY294002组、DMSO组XIAP蛋白的表达水平明显增多(P<0.05);与rHuEPO组、DMSO组相比,LY294002组XIAP蛋白的表达水平明显减少,差异具有统计学意义(P<0.05);rHuEPO组XIAP蛋白的表达水平虽多于DMSO组,但差异不具有统计学意义(P>0.05)。
结论:PTZ点燃的癫痫持续状态后大鼠海马组织XIAP蛋白及其mRNA水平均升高,提示线粒体凋亡途经相关调控因子XIAP可能参与了癫痫持续状态后海马神经元凋亡的损伤机制。rHuEPO后处理可以降低癫痫持续状态大鼠海马组织XIAP蛋白及其mRNA表达水平,进而改善癫痫持续状态后大鼠海马神经元的凋亡损伤,其抗凋亡的机制可能通过PI3K/Akt信号传导途径对线粒体凋亡途经相关调控因子XIAP进行调控,进而介导线粒体凋亡途经,发挥抗凋亡神经保护作用。
Epilepsy, which is essentially an abnormality of cerebral function resulting from the discharges of some cerebral neuron in over high excitement state. Epilepsy is one of the most common diseases or syndromes of central nervous system. Status Epilepticus (SE) is a neurological emergency with an associated mortality rate of 10%–12%, which many affect the patients’life greatly. In patients with temporal lobe epilepsy (TLE), various experimental epilepsy and SE models, the lesions of neurons representing typical apoptotic features were verified by morphological and biochemical methods. The effect of apoptosis on SE has drawn much attention recently though it is still unclear about the detailed signaling pathways that trigger apoptosis is incomplete.
Apoptosis is a fundamental process of cell death that occurs via activation of distinct signaling pathways involving mitochondria, mitochondrial regulatory proteins, and activation of Caspases. Ultimately, cells undergo nuclear chromatin condensation, DNA fragmentation, and formation of apoptotic bodies. Apoptosis occurs through three main pathways: the mitochondrial- dependent pathway; the death receptor dependent pathway and the endoplasmic reticulum (ER) pathway. The more important pathway is the mitochondrial-dependent pathway, which induces the leads to the release of Cytochrome c from mitochondria and activation of the death signal. The Cytochrome c quickly combines with the Apaf-1 and Caspase-9, which forms a complex called apoptosome. The activation of Caspase-9 leads to a series of activating of its downstream Caspases, which finally results in programmed cell death.Bcl-2 family proteins, Caspase family proteins and inhibitor of apoptosis proteins (IAPs) are the principal regulators of mitochondrial- dependent apoptotic pathway. The Bcl-2 family of proteins regulates apoptosis by modulating mitochondrial permeability and the release of Cytochrome c. The Bcl-2 family of proteins includes proapoptotic members such as Bax, Bad, and antiapoptotic members such as Bcl-2, Bcl-xL. Although Bad and Bax are located in the cytosol , but translocate to the mitochondria and form a proapoptotic complex with Bcl-2,and then release Cytochrome c. Proteins opposing these pro-apoptotic proteins are antiapoptosis proteins, The antiapoptotic protein Bcl-2 resides in the outer mitochondrial wall, which inhibits the release of pro-apoptotic molecules to mitochondria by multiple mechanisms, including maintenance of mitochondrial membrane integrity and binding to pro-apoptosis members of the Bcl-2 family. The ratio of Bcl-2/Bax protein has also been suggested to determine cell survival or death. Among the BH3-only class of proteins, Bad was the first identified and the best characterized. Bad is a pro-death member of Bcl-2 family that initiates apoptosis by binding to Bcl-2 and Bcl-xL on the outer mitochondrial membrane, causing the release of Cytochrome c into cytosol. The function of Bad is to bind Bcl-2 and Bcl-xl, and block the antiapoptotic action of these proteins. For example: Bad may also inhibit the ability of Bcl-2 and Bcl-xL from forming channels in the mitochondrial membrane, or bind and stabilize the voltage dependent anion channel (VDAC). Bad is an example of a proapoptotic protein that is regulated by phosphorylation, It is phosphorylated on ser-136 by Akt in the PI3K/Akt pathway, Phosphorylation at the site promotes the binding of Bad to 14-3-3 proteins. When bound to 14-3-3 proteins, Bad is sequestered in the cytosol and unable to interact with Bcl-2 or Bcl-xL at the outer mitochondrial membrane. Caspase-9, as well as the other Caspases, exists in an inactive zymogen pro-Caspase state that is activated through proteolytic processing. Cytochrome c, once released in cytosol, interacts with Apaf-1, leading to the activation of Caspase-9 proenzymes. Active Caspase-9 then activates Caspase-3, which subsequently activates the rest of the Caspase cascade and leads to apoptosis. The IAPs are a family of intracellular anti-apoptotic proteins, first identified in Baculovirus, which play a key role in cell survival by modulating death-signaling pathways at a post-mitochondrial level. They currently include X-linked IAP (XIAP), human IAP-1 (Hiap-1), Survivin, Livin and so on. Xchromosome-linked inhibitor of apoptosis protein (XIAP), one of the first mammalian IAP family members cloned, is the most potent inhibitor of apoptosis in IAP family members. It has been shown that XIAP is a direct inhibitor of Caspase-3 and Caspase-9 and can modulate the Bax/Cytochrome c pathway by inhibiting Caspase-9.
PI3K (phosphatidylinositol 3-kinase) is a kinase that plays a critical role in signaling pathways important to cell survival. PI3K/Akt signaling pathway is utilized by many cell types for regulating cell cycle, growth, metabolism and inhibition of apoptosis. Akt is known to mediate cell survival by regulating several effectors including Bad, Bcl-2, Bax, Caspase-9, XIAP and so on, which are the important regulatory factors of the mitochondrial-dependent apoptotic pathway meanwhile. The first anti-apoptotic Akt target identified was the pro-apoptotic protein Bad. Activated Akt can phosphorylate Bad specifically at Ser136 and thus can inactivate Bad. Phosphorylated Bad prevents apoptosis, because unphosphorylated Bad can form heterodimers with the anti-apoptotic proteins Bcl-xl or Bcl-2, and then antagonize their anti-apoptotic functions. In addition, researches have also shown that Bax is regulated by phosphorylation of Ser184 in an Akt-dependent manner and that phosphorylation inhibits Bax effects on the mitochondria by maintaining the protein level in the cytoplasm, heterodimerized with antiapoptotic Bcl-2 family members. Akt also phosphorylates and activates the cyclic AMP-response element-binding protein (CREB) on Ser-133, which increases the transcription of anti-apoptotic genes, such as Bcl-2. Akt also phosphorylates the IKK on Thr-23 and releases NF-кB, and then NF-кB up-regulates the expression of Bcl-2. Akt phosphorylates XIAP at residue serine 87 in vitro and in vivo, and interacts with XIAP at physiological protein concentration. These evidences show that Akt and its downstream targets constitute a major cell survival pathway.
Erythropoietin (EPO) was originally described for its role in hematopoiesis, which can increase red blood cells by protecting erythroid progenitors against apoptosis. We now know that there are EPO and its receptor (EPO-R) on many cell types of central nervous system in rodent and human brain, such as astrocytes, oligodendrocytes, microglia, and endothelial cells. Exogenous administration of EPO revealed neuroprotective activity in vitro and in vivo in models of central and peripheral neuronal injury occurred in the contexts of trauma, stroke and inflammation. In addition, studies in rodent models of TLE revealed significant effects of rHuEPO(recombinant Human erythropoietin, rHuEPO) in antagonizing the development of SE, but did not determine whether rHuEPO was neuroprotective. Neuroprotective effect induced by rHuEPO applied systemically requires that it crosses the blood–brain barrier (BBB), and many researches have showed rHuEPO administered peripherally crosses the BBB via a receptor-mediated mechanism. In addition, many researches show that pretreatment or even post-treatment of rHuEPO provides protection to neurons after SE. EPO binds to EPO receptor first and then activates janus tyrosine kinase 2 (JAK2), which triggers at least three intracellular signaling cascades through the anti-apoptotic role played by EPO: signal transducer and activator of transcription 5 (STAT5); phosphatidylinositol-3 kinase (PI3K)/Akt and RAS/mitogen-activated protein kinase (MAPK). Pro-survival actions of PI3K occur through activation of the anti-apoptotic effector Akt. Akt activation leads to phosphorylation of certain proteins that promote cell survival. Akt, also known as protein kinase B, is the most well characterized target of PI3K, whose catastaltica would inhibit the activation of Akt. Many experiments presumed the activation of PI3K/Akt signaling pathway through detecting the expression of p-Akt, which showed neuroprotective effects and confirmed by many researches. however, documents demonstrated that EPO could also play neuroprotective effect through the signal pathway of JAK/ STAT; ERK 1/2; NF-κB. To clarify whether the PI3K/Akt signaling pathway involved in the neuroprotective progression originated by rHuEPO in vivo model, we administered LY294002 before rHuEPO treatment, an inhibitor of PI3K. Based on the above mentioned facts, the purpose of our research is to evaluate the anti-apoptosis neuroprotective effect of exogenous rHuEPO post-treatment in PTZ kindled SE rats, and discuss whether rHuEPO modulates the regulatory factors of mitochondrial-dependent apoptotic pathway to mediate the anti-apoptosis neuron protective effects through PI3K/Akt signaling pathway.
Part I Changes of p-Akt expression levels and neurons apoptosis in hippocampus of statural epilepticus rats kindled by PTZ and the effect of rHuEPO
Objective: To establish a status epilepticus rat model kindled by PTZ and observe the changes of p-Akt expression levels and neurons apoptosis in hippocampus of status epilepticus rat and the effect of rHuEPO.
Methods: Adult male Sprague–Dawley (SD) rats were selected to establish a status epilepticus rat model kindled by PTZ. First, a dose of 20 mg/kg was intraperitoneal injection (i.p.), then followed additional doses of 10mg/kg were administered to the rat every 10 min until the onset of SE. Rats were regarded as fully kindled when they exhibited seizure stages 4-5, this procedure lasted for at least 30 minutes. Rats were divided into 5 groups randomly. In the PTZ group, rats were subjected to PTZ i.p.; in the rHuEPO group, rats were subjected to rHuEPO 5000U/kg i.p., the procedure were performed 30 minutes after SE kindled by the PTZ; in LY294002 group, rats were subjected to intracerebral ventricle (ICV) infusion of LY294002 5μl and rHuEPO 5000U/kg i.p., and the procedure were performed 10 min and 30min respectively after SE kindled by PTZ; in DMSO Control group, rats were subjected to ICV infusion of DMSO5μl and rHuEPO 5 000U/kgi.p., and the procedure were performed 10 min and 30min respectively after the SE kindled by the PTZ; in Control group, animals received the same volume of physiological saline i.p. as their paired PTZ- exposed congeners. Then those rats were recorded with electroencephalogram (EEG). 10 rats in each group were anesthetized by 10% chloral hydrate and perfused with 4% paraformaldehyde solution. The brain tissue containing hippocampus was cut and embedded with paraffin. The apoptotic neurons of the hippocampal region were detect through TUNEL method and the positive cell numbers of p-Akt were detected through immunohistochemical SP methods. 10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total protein for p-Akt was extracted. The expression levels of p-Akt protein were detected by Western blot.
Results: EEG changes: the EEG records of the rats in Control group were dominant withαwaves andβwaves and the background was normal without any discharges. The rats in PTZ group were recorded in EEG as sharps, spike-slow waves and most polyspike discharge, with high potential on a relatively normal background. The EEG records of the rats in rHuEPO group, LY294002 group and DMSO group were demonstrated the repressed discharges. The results of TUNEL stains: compared with the Control group, a significant increases for the number of TUNEL-positive cells were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P <0.05). Meanwhile, the same increase were also found for the number of TUNEL-positive cells in the PTZ group compared with those in the rHuEPO group, the LY294002 group and the DMSO group (P <0.05). However, compared with the LY294002 group, a significant decrease for the number of TUNEL-positive cells were observed in the rHuEPO group and the DMSO group (P<0.05). The number of TUNEL-positive cells in the DMSO group were higher than in the rHuEPO group, but with no significant difference (P>0.05). The results of immunohistochemical stains: compared with the Control group, the significant increases for the number of p-Akt-IR (immunohistochemical reaction) cells were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group( P<0.05). Meanwhile, the same increases were also found for the number of p-Akt-IR cells in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05) compared with those in the PTZ group. However, there were the significant decreases in the number of p-Akt-IR cells in the LY294002 group compared with those in the rHuEPO group and the DMSO group (P<0.05). The numbers of p-Akt-IR cells in the rHuEPO group were higher than in the DMSO group, but the difference was insignificant (P>0.05). The results of Western blot: the protein levels of p-Akt in hippocampus of rats in each group were determined by p-Akt/β-actin. compared with the Control group, the significant increases for the protein levels of p-Akt were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). At the same time, the same increase were also found for the protein levels of P-Akt in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05) compared with those in the PTZ group. However, there were significant decrease for the protein levels of P-Akt in the LY294002 group compared with those in the rHuEPO group and the DMSO group (P<0.05). The protein levels of p-Akt in the rHuEPO group were higher than those in the DMSO group, but the difference were insignificant (P>0.05).
Conclusion: The status epilepticus rat model kindled by PTZ in the experiment did have impairments of neuronal apoptosis. This model is the perfect animal model which is feasible to study the damaged neuronal apoptosis after status epilepticus. The rHuEPO showed the effect of antiapoptosis neuroprotection in SE rats, the pathway of PI3K/Akt may be one of the neuroprotective ways of rHuEPO. The possible mechanism is rHuEPO activated the PI3K/Akt pathway and then up-regulated the expression of p-Akt to displayed neuroprotective effect.
Part II Changes of the Bcl-2 proteins family expression levels in hippocampus of statural epilepticus rats kindled by PTZ and the effect of rHuEPO
Objective: To observe the changes of Bcl-2 proteins family expression levels in hippocampus of status epilepticus rat kindled by PTZ and the effect of rHuEPO, and to explore whether the rHuEPO regulated the expression of Bcl-2、Bax、Bad that are the regulatory factors of mitochondrial-dependent apoptotic pathway through PI3K/Akt signaling pathway, and played anti-apoptotic neuroprotective effect.
Methods: 10 rats in each group were anesthetized by 10% chloral hydrate and perfused with 4% paraformaldehyde solution. The brain tissue containing hippocampus was cut and embedded with paraffin. the immunohistochemical reaction positive cell numbers of Bcl-2、Bax、Bad were detected through immunohistochemical SP methods. 10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total RNA was extracted strictly by Trizol. The expression of Bcl-2mRNA、BaxmRNA、BadmRNA was measured by RT-PCR.10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total protein for Bcl-2、Bax、Bad was extracted. The expression levels of Bcl-2、Bax、Bad protein were detected by Western blot.
Results: The results of immunohistochemical stains: compared with the Control group, the significant increases for the number of Bcl-2-IR cells were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group( P<0.05). Meanwhile, the same increases were also found for the number of Bcl-2-IR cells in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05) compared with those in the PTZ group. However, there were the significant decreases in the number of Bcl-2-IR cells in the LY294002 group compared with those in the rHuEPO group and the DMSO group (P<0.05). The numbers of Bcl-2-IR cells in the rHuEPO group were higher than in the DMSO group, but the difference was insignificant (P>0.05). Compared with the Control group, the significant increases for the number of Bax-IR、Bad-IR cells were observed in the PTZ group(P<0.05), the rHuEPO group, the LY294002 group and the DMSO group. Meanwhile, the same increase were also found for the number of Bax-IR、Bad-IR cells in the PTZ group compared with those in the rHuEPO group, the LY294002 group and the DMSO group(P<0.05). However, compared with the LY294002 group, a significant decrease in the number of Bax-IR、Bad-IR cells were found in the rHuEPO group and the DMSO group(P<0.05). The number of Bax-IR、Bad-IR cells in the rHuEPO group were lower than in the DMSO group, but the difference was insignificant (P>0.05).The results of RT-PCR: the expression levels of Bcl-2mRNA、BaxmRNA、BadmRNA were determined by calculating the density ratio of Bcl-2mRNA/β-actin mRNA、BaxmRNA/β-actinmRNA、BadmRNA/β-actinmRNA. Compared with the Control group, the significant increases for Bcl-2mRNA were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). Meanwhile, the same increase were also found for Bcl-2mRNA in the rHuEPO group, the LY294002 group and the DMSO group compared with those in the PTZ group (P<0.05). However, there were significant decrease for Bcl-2mRNA in the LY294002 group compared with those in the rHuEPO group and the DMSO group (P<0.05). The expression level of Bcl-2mRNA in the rHuEPO group were higher than in the DMSO group, but the difference was insignificant (P>0.05). Compared with the Control group, significant increases for BaxmRNA、BadmRNA were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group(P<0.05). Meanwhile, the same increase were also found for BaxmRNA、BadmRNA in the PTZ group compared with those in the rHuEPO group, the LY294002 group and the DMSO group(P<0.05). However, compared with the LY294002 group, a significant decrease for BaxmRNA、BadmRNA were found in the rHuEPO group and the DMSO group(P<0.05). The expression level of BaxmRNA、BadmRNA in the rHuEPO group were lower than in the DMSO group, but the difference was insignificant(P>0.05).The results of Western blot: The protein levels of Bcl-2、Bax、Bad in hippocampus of rats in each group were determined by Bcl-2/β-actin、Bax/β-actin、Bad/β-actin respectively. compared with the Control group, the significant increases for the protein levels of Bcl-2 were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). At the same time, the same increase were also found for the protein levels of Bcl-2 in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05)compared with those in the PTZ group. However, there were significant decrease for the protein levels of Bcl-2 in the LY294002 group compared with those in the rHuEPO group and the DMSO group( P<0.05). The protein levels of Bcl-2 in the rHuEPO group were higher than those in the DMSO group, but the difference was insignificant (P>0.05).Compared with the Control group, the significant increase for the protein levels of Bax and Bad were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group(P<0.05). Meanwhile, the same increase were also found for the protein levels of Bax and Bad in the PTZ group compared with those in the the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). However, compared with the LY294002 group, the significant decrease for the protein levels of Bax and Bad were found in the rHuEPO group and the DMSO group (P<0.05). The protein levels of Bax and Bad in the rHuEPO group were lower than those in the DMSO group, but the difference was insignificant (P>0.05).
Conclusion: There were elevation in expression levels of Bcl-2、Bax and Bad protein and Bcl-2mRNA、BaxmRNA and BadmRNA in the hippocampus of status epilepticus rat kindled by PTZ, which suggests that Bcl-2、Bax and Bad might participate in the mechanism of hippocampus neuronal apoptosis lesion after status epilepticus. rHuEPO posttreament could increase the expression levels of Bcl-2、Bcl-2mRNA and decrease the expression levels of Bax、BaxmRNA、Bad、BadmRNA, the possible mechanism is the rHuEPO regulated the expression of Bcl-2、Bax、Bad that are the regulatory factors of mitochondrial-dependent apoptotic pathway through PI3K/Akt signaling pathway, and played anti-apoptotic neuroprotective effect.
Part III Changes of the Caspase-9 expression levels in hippocampus of statural epilepticus rats kindled by PTZ and the effect of rHuEPO
Objective: To observe the changes of Caspase-9 expression levels in hippocampus of status epilepticus rat kindled by PTZ and the effect of rHuEPO, and to explore whether the rHuEPO regulated the expression of Caspase-9 that are the regulatory factors of mitochondrial-dependent apoptotic pathway through PI3K/Akt signaling pathway, and played anti-apoptotic neuroprotective effect.
Methods: 10 rats in each group were anesthetized by 10% chloral hydrate and perfused with 4% paraformaldehyde solution. The brain tissue containing hippocampus was cut and embedded with paraffin, the immunohistochemical reaction positive cell numbers of Caspase-9 were detected through immunohistochemical SP methods. 10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total RNA was extracted strictly by Trizol. The expression of Caspase-9mRNA were measured by RT-PCR.10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total protein for Caspase-9 was extracted. The expression levels of Caspase-9 protein were detected by Western blot.
Results: The results of immunohistochemical stains: compared with the Control group, the significant increases for the number of Caspase-9-IR cells were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group ( P<0.05), Meanwhile, the same increase were also found for the number of Caspase-9-IR cells in the PTZ group compared with those in the rHuEPO group, the LY294002 group and the DMSO group(P<0.05). However, compared with the LY294002 group, a significant decrease in the number of Caspase-9-IR were found in the rHuEPO group and the DMSO group(P<0.05). The number of Caspase-9-IR cells in the rHuEPO group were lower than in the DMSO group, but the difference was insignificant (P>0.05).The results of RT-PCR: the expression levels of Caspase-9mRNA were determined by calculating the density ratio of Caspase-9mRNA/β-actin mRNA. Compared with the Control group, the significant increases for Caspase-9mRNA were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). Meanwhile, the same increase were also found for Caspase-9mRNA in the PTZ group compared with those in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). However, compared with the LY294002 group, a significant decrease for Caspase-9mRNA were found in the rHuEPO group and the DMSO group(P<0.05). The expression level of Caspase-9mRNA in the rHuEPO group were lower than in the DMSO group, but the difference was insignificant(P>0.05).The results of Western blot: The protein levels of Caspase-9 in hippocampus of rats in each group were determined by Caspase-9/β-actin. Compared with the Control group, the significant increase for the protein levels of Caspase-9 were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group(P<0.05). Meanwhile, the same increase were also found for the protein levels of Caspase-9 in the PTZ group compared with those in the the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). However, compared with the LY294002 group, the significant decrease for the protein levels of Caspase-9 were found in the rHuEPO group and the DMSO group (P<0.05). The protein levels of Caspase-9 in the rHuEPO group were lower than those in the DMSO group, but the difference was insignificant (P>0.05).
Conclusion: There were elevation in expression levels of Caspase-9 protein and Caspase-9mRNA in the hippocampus of status epilepticus rat kindled by PTZ, which suggests that Caspase-9 might participate in the mechanism of hippocampus neuronal apoptosis lesion after status epilepticus. rHuEPO posttreament could increase the expression levels of Caspase-9、Caspase-9mRNA, the possible mechanism is the rHuEPO regulated the expression of Caspase-9 that are the regulatory factors of mitochondrial-dependent apoptotic pathway through PI3K/Akt signaling pathway, and played anti-apoptotic neuroprotective effect.
Part IV Changes of the XIAP expression levels in hippocampus of statural epilepticus rats kindled by PTZ and the effect of rHuEPO
Objective: To observe the changes of XIAP expression levels in hippocampus of status epilepticus rat kindled by PTZ and the effect of rHuEPO, and to explore whether the rHuEPO regulated the expression of XIAP that are the regulatory factors of mitochondrial-dependent apoptotic pathway through PI3K/Akt signaling pathway, and played anti-apoptotic neuroprotective effect.
Methods: 10 rats in each group were anesthetized by 10% chloral hydrate and perfused with 4% paraformaldehyde solution. The brain tissue containing hippocampus was cut and embedded with paraffin. the immunohistochemical reaction positive cell numbers of XIAP were detected through immunohistochemical SP methods. 10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total RNA was extracted strictly by Trizol. The expression of XIAPmRNA was measured by RT-PCR.10 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total protein for XIAP were extracted. The expression levels of XIAP protein were detected by Western blot.
Results: The results of immunohistochemical stains: compared with the Control group, the significant increases for the number of XIAP-IR cells were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group( P<0.05). Meanwhile, the same increases were also found for the number of XIAP-IR cells in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05) compared with those in the PTZ group. However, there were the significant decreases in the number of XIAP-IR cells in the LY294002 group compared with those in the rHuEPO group and the DMSO group (P<0.05). The numbers of XIAP-IR cells in the rHuEPO group were higher than in the DMSO group, but the difference was insignificant (P>0.05). The results of RT-PCR: the expression levels of XIAPmRNA were determined by calculating the density ratio of XIAPmRNA/β-actin mRNA. Compared with the Control group, the significant increases for XIAPmRNA were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). Meanwhile, the same increase were also found for XIAPmRNA in the rHuEPO group, the LY294002 group and the DMSO group compared with those in the PTZ group (P<0.05). However, there were significant decrease for XIAPmRNA in the LY294002 group compared with those in the rHuEPO group and the DMSO group (P<0.05). The expression level of XIAPmRNA in the rHuEPO group were higher than in the DMSO group, but the difference was insignificant (P>0.05). The results of Western blot: The protein levels of XIAP in hippocampus of rats in each group were determined by XIAP/β-actin. compared with the Control group, the significant increases for the protein levels of XIAP were observed in the PTZ group, the rHuEPO group, the LY294002 group and the DMSO group (P<0.05). At the same time, the same increase were also found for the protein levels of XIAP in the rHuEPO group, the LY294002 group and the DMSO group (P<0.05)compared with those in the PTZ group. However, there were significant decrease for the protein levels of XIAP in the LY294002 group compared with those in the rHuEPO group and the DMSO group( P<0.05). The protein levels of XIAP in the rHuEPO group were higher than those in the DMSO group, but the difference was insignificant (P>0.05).
Conclusion: There were elevation in expression levels of XIAP protein and XIAPmRNA in the hippocampus of status epilepticus rat kindled by PTZ, which suggests that XIAP might participate in the mechanism of hippocampus neuronal apoptosis lesion after status epilepticus. rHuEPO posttreament could increase the expression levels of XIAP、XIAPmRNA, the possible mechanism is the rHuEPO regulated the expression of XIAP that are the regulatory factors of mitochondrial-dependent apoptotic pathway through PI3K/Akt signaling pathway, and played anti-apoptotic neuroprotective effect.
引文
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