ERK对缺血缺氧性脑损伤后神经元凋亡的影响

目的研究在缺血缺氧性脑损伤后细胞外信号调节激酶(ERKs)对神经元凋亡的影响。方法建立光化学法诱导大鼠局灶性脑缺血模型,随机分为脑缺血组(缺血组和干预组)和假手术组,干预组于缺血前30min尾静脉注入U0126溶液,缺血组尾静脉注入相同体积不含U0126的DMSO稀释溶液;2,3,5-氯化(或溴化)三苯四氮唑(TTC)染色显示梗死灶;应用免疫组织荧光化学法检测梗死灶周围神经元核心抗原(NeuN)的表达及通过TUNEL方法检测神经元凋亡,并做NeuN与TUNEL的双标;用免疫印迹(Westernblot)方法观察损伤侧皮层NeuN、细胞周期蛋白D1(CyclinD1)和细胞周期蛋白E(CyclinE)蛋白的表达。结果缺血组的梗死体积明显大于干预组,在假手术组未见梗死灶;缺血组大鼠NeuN阳性细胞数和NeuN蛋白表达明显少于假手术组,TUNEL阳性细胞数和CyclinD1和CyclinE蛋白表达明显高于假手术组(P<0.05)。干预组NeuN阳性细胞数和NeuN蛋白表达亦少于假手术组,但多于缺血组,TUNEL阳性细胞数和CyclinD1及CyclinE蛋白表达亦多于假手术组,但少于缺血组(P<0.05)。结论ERK可通过对细胞周期的调控而对神经元凋亡产生影响,抑制脑缺血引起的pERK1/2磷酸化可部分抑制神经元凋亡,减少缺血梗死灶,对缺血性脑损伤起一定的保护作用。     

马来酸桂哌齐特对脑缺血大鼠模型胞外信号调节激酶表达的影响

目的观察马来酸桂哌齐特预处理对大鼠脑缺血半暗带区胞外信号调节激酶(ERK1/2)磷酸化表达的影响,探讨其对脑缺血的保护作用及可能的作用机制。方法采用改良线栓法建立大鼠永久性大脑中动脉阻塞(pMCAO)模型。SD大鼠随机分为1马来酸桂哌齐特组(pMCAO模型大鼠,n=57。尾静脉注射马来酸桂哌齐特3.0 mg·kg-1,×5d);2对照组(pMCAO模型大鼠,n=57。尾静脉注射生理盐水0.5 mL,×5 d);3假手术组(不插线栓,n=50。尾静脉注射生理盐水0.5 mL,×5 d);4正常组(n=3,不做任何处理)。应用TTC染色法测定梗死体积,蛋白印迹法和免疫组化法检测不同时间点(术后3、6、24、48和72 h)缺血半暗带区ERK蛋白的表达。结果马来酸桂哌齐特组与对照组比较,梗死体积减少17.91%(P=0.001)。马来酸桂哌齐特组缺血半暗带pERK1/2表达于3 h开始增加,24 h达高峰[为正常的(5.75±0.70)倍]后逐渐降低,72 h仍为正常的(2.89±0.51)倍,各时间点与正常组比较,均差异有显著统计学意义(P0.01)。6、24和48 h 3个时间点,马来酸桂哌齐特组缺血半暗带内pERK1/2表达较对照组增加(P0.05)。pERK的免疫组化检测示马来酸桂哌齐特预处理能上调缺血半暗带区内ERK磷酸化表达(P0.05)。结论马来酸桂哌齐特预处理可减少脑梗死体积,并能上调缺血半暗带区的ERK磷酸化表达;参与MAPK信号通路、上调缺血半暗带区内ERK磷酸化的表达,可能是其保护缺血性脑损伤的机制之一。     

细胞周期调控对局灶性缺血性脑损伤后的保护作用

目的通过抑制细胞周期素依赖激酶(Cyclindependentkinases,CDKS)来对神经元凋亡进行干预,以探讨细胞周期调控与细胞凋亡的关系。方法建立光化学法诱导大鼠局灶性脑缺血模型,并随机分为脑缺血组(对照组和干预组)和假手术组,采用HE染色显示梗死灶,并测定其面积占脑片面积百分率的平均值;通过TUNEL方法检测神经元凋亡;免疫印迹(Westernblot)观察损伤侧皮层周期素蛋白A(CyclinA)和周期素蛋白B1(CyclinB1)的表达。结果缺血后24h对照组梗死灶面积占脑片面积百分率的平均值明显大于干预组(P<0.05),缺血后梗死灶周围可见大量TUNEL阳性染色细胞,且对照组数量明显多于干预组(P<0.05),二者均多于假手术组(P<0.05);缺血后24h干预组大鼠NeuN TUNAL双标阳性表达率明显弱于对照组大鼠(P<0.05);Westernblot显示对照组CyclinA和CyclinB1的表达明显高于干预组(P<0.05)。结论细胞周期抑制剂可部分抑制缺血边缘区神经元的凋亡及减小脑梗死面积,这提示细胞周期调控可能参与了神经细胞的凋亡过程。     

短暂性脑缺血预处理诱导脑缺血耐受大鼠VEGF和Ang-1的表达变化

目的观察血管内皮生长因子(VEGF)和血管生成素-1(Ang-1)在短暂性脑缺血预处理(IP)诱导脑缺血耐受中的作用。方法 Wistar大鼠随机分成假手术对照组(9只)、非缺血预处理组(NIP,45只)、缺血预处理组(IP,45只),后两组再随机分成5个亚组。线栓法阻塞大脑中动脉建立局灶性缺血预处理模型,分别在缺血预处理后1、3、7、14、21d进行再次缺血2h再灌注22h,取脑检查。TTC染色测定脑梗死体积,免疫组织化学法检测VEGF、Ang-1蛋白表达。结果 (1)组间比较:IP组1、3、7d亚组梗死体积较NIP组明显减小(P<0.01),其神经行为缺损评分也明显降低(P<0.05);IP组1、3、7d亚组VEGF蛋白表达明显增高;而IP组7d亚组Ang-1蛋白表达明显增高(P<0.05)。(2)组内比较:IP组3d亚组梗死体积减小最为明显;3、7d亚组VEGF蛋白表达明显增高(P<0.05)。结论短暂性脑缺血预处理诱导脑缺血耐受的产生,VEGF和Ang-1在缺血预处理产生脑缺血耐受的相应时间窗内表达有所增加。     

抑制ERK1/2对大鼠弥漫性脑损伤后细胞凋亡的影响

目的 探讨抑制细胞外信号调节激酶1/2（ERK1/2）对大鼠弥漫性脑损伤（DBI）后脑组织细胞凋亡的影响。方法 按随机数字表法将228只成年SD大鼠随机分为假手术组（n=12）、DBI组（n=72）、阻滞剂组（n=72）、对照组（n=72）,后三组按动物处死时间分为30 min、3 h、24 h、48 h、72 h和7 d六个亚组,每亚组12只。参照Mamarou自由落体方法制作重型DBI模型。阻滞剂组损伤后尾静脉注射ERK1/2特异性阻滞剂U0126（0.05 mg/kg）,对照组注射等量溶剂二甲基亚砜。免疫印迹法法检测脑组织磷酸化ERK1/2（pERK1/2）的表达水平,免疫组化法检测Caspase-3表达,流式细胞术检测细胞凋亡率。结果 伤后30 min,脑组织pERK1/2表达水平显著增高（P<0.05）,并持续高水平表达至72 h,伤后7 d与假手术组无统计学差异（P>0.05）。伤后3 h,脑组织Caspase-3表达水平和细胞凋亡率均明显增高,72 h达到高峰,伤后7 d仍明显高于假手术组（P<0.05）。伤后30 min、3 h、24 h、48 h、72 h和7 d,阻滞剂组脑组织Caspase-3表达水平和细胞凋亡率均明显低于DBI组和对照组（P<0.05）,而DBI组和对照组均无统计学差异（P>0.05）。结论 阻滞ERK1/2通路,可显著抑制DBI大鼠脑组织Caspase-3的表达,降低细胞凋亡率。     

大鼠脑缺血预处理后脑组织Caspase-3蛋白的表达

目的探讨凋亡相关基因caspase-3蛋白在脑缺血预处理过程中的表达和意义.方法用免疫组织化学染色技术分别检测假手术对照组(S组)、缺血预处理对照组(A组)、缺血组(B组)和缺血预处理组(C组)大鼠脑组织石蜡切片中caspase-3蛋白的表达情况.结果缺血组(B组)和缺血预处理组(C组)各组CA1区caspase-3蛋白免疫反应强度明显高于假手术对照组(S组)(P＜0.01);末次再灌注48h的B2组和C2组以及末次再灌注72 h的B3和C3组比较caspase-3蛋白表达均有显著性增高(P＜0.05,P＜0.01).结论脑缺血预处理的保护机理可能与抑制caspase-3蛋白表达、减少脑细胞凋亡有关.     

细胞外信号调节激酶的磷酸化水平对星形胶质细胞增殖及其细胞周期的影响

目的 探讨细胞外信号调节激酶(ERK1/2)的磷酸化水平对体外星形胶质细胞(Ast)增殖及其细胞周期的影响.方法 将培养成熟的大鼠原代Ast分为两组,其中一组用ERK1/2磷酸化的特异性抑制剂U0126进行处理(U0126组),另一组不做处理,作为对照组.通过Western Blot技术、Click-iT Edu技术和流式细胞术分别检测两组Ast中ERK1/2磷酸化水平、Ast增殖细胞百分比及各期细胞百分比.结果 U0126组ERK1/2的磷酸化水平(39.13％±6.71％)明显低于对照组(100％).U0126处理24 h后,U0126组Ast增殖细胞百分比[(2.63±1.14)％]低于对照组[(21.43±3.81)％](t=21.13,P＜0.01).G1期U0126组Ast[(93.67±0.68)％]高于对照组[(84.63±1.00)％](t=12.91,P＜0.01);S期U0126组Ast[(2.90±0.23)％]低于对照组[(14.21±1.14)％] (t=16.87,P＜0.01);G2期U0126组Ast[(3.43±0.88)％]高于对照组[(2.08±0.21％)％](t=4.35,P＜0.05).结论 降低ERK1/2的磷酸化水平可抑制Ast的增殖,并将其阻断在G1期,抑制Ast从G1期向S期的转化,同时抑制其分裂将其阻断在G2期.     

ERK1/2信号途径对大鼠弥漫性脑创伤后神经细胞凋亡的调控机制

目的 探讨脑创伤后ERK1/2信号调控神经细胞凋亡的分子机制.方法 SD大鼠分为正常对照组、模型组、抑制剂U0126高、低剂量组.Marmarou's法制作弥漫性脑创伤模型.光镜下观察伤后神经细胞形态变化;免疫组化和Western Blot法检测伤后磷酸化ERK1/2水平和Bax表达;TUNEL法检测凋亡细胞.结果 与正常对照组比较,模型组海马区部分神经细胞出现变性坏死和凋亡改变,磷酸化ERK1/2、Bax表达增高,神经细胞凋亡数目增多(P<0.05);U0126治疗后,脑组织形态损伤程度、磷酸化ERK1/2和Bax表达、神经细胞凋亡数目回降,上述变化在U0126高剂量组中更为显著.结论 脑创伤后活化的ERK1/2信号通过调控Bax表达在神经细胞凋亡过程中发挥重要作用.     

硫化氢对短暂脑缺血/再灌注老年大鼠海马神经元Camkkβ、Bcl-2和Bax表达的影响

目的探讨硫化氢对短暂脑缺血再灌注老年大鼠海马神经元Camkkβ、Bcl-2和Bax表达的影响。方法健康雄性SD老龄大鼠120只,随机分为对照组(C组)、缺血/再灌注组(IR组)、H2S组(H组)和生理盐水组(S组),采用Pusinelli四动脉阻断法建立全脑缺血模型。H组缺血前10 min腹腔内注射H2S外源性供体NaHS;S组注入等量生理盐水;C组不行任何处理。大鼠脑缺血/再灌注后24 h进行神经行为学评分;脑缺血/再灌注后1、3、5 d采用TUNEL法检测神经元的凋亡情况,采用Western-blot法测定大鼠海马内Camkkβ、Bcl-2和Bax蛋白表达。结果与对照组比较,缺血/再灌注组老年大鼠脑缺血/再灌注24 h后神经行为学评分升高,海马TUNEL阳性神经元计数升高,Camkkβ表达升高,Bcl-2表达下调,Bax表达上调(P<0.05);与缺血/再灌注组比较,H2S组老年大鼠脑缺血/再灌注24 h后神经行为学评分降低、海马区TUNEL阳性神经元计数降低、Camkkβ表达降低,Bcl-2表达上调、Bax表达下调(P<0.05)。结论硫化氢可通过降低神经元海马Camkkβ蛋白表达、上调Bc1-2蛋白表达、抑制Bax蛋白表达来减轻短暂脑缺血/再灌注损伤。     

腺苷预处理对大鼠局灶性脑缺血再灌注损伤的脑保护作用

目的 观察腺苷预处理对大鼠局灶性脑缺血再灌注损伤的改善作用及脑保护机制.方法 线栓法建立大鼠局灶性脑缺血2 h后再灌注损伤模型;SD大鼠60只,随机分为假手术组(F组),缺血再灌注组(IR组),腺苷预处理组(AP组),每组20只;通过HE 染色在光镜下观察神经细胞损伤变化,免疫组化法检测Bax蛋白的阳性表达.结果 光镜下,假手术组(F组)神经细胞结构正常,腺苷预处理组(AP组)和缺血再灌注组(IR组)均有不同程度的缺血再灌注损伤,腺苷预处理组较缺血再灌注组损伤轻;假手术组Bax蛋白表达极弱,缺血再灌注组和腺苷预处理组在脑缺血再灌注后2 h接近缺血核心区出现Bax蛋白阳性表达,6 h后表达增多,24 h达到高峰,72 h开始减少.与假手术组相比,腺苷预处理组和缺血再灌注组Bax蛋白阳性细胞数显著增多(P＜0.05);与缺血再灌注组相比,腺苷预处理组Bax蛋白阳性细胞数显著减少(P＜0.05).结论 腺苷对大鼠局灶性脑缺血再灌注损伤有保护作用,腺苷可通过下调Bax蛋白的表达发挥脑保护作用.     

阻滞弥漫性脑损伤ERK1/2信号通路对星形胶质细胞反应的作用

目的研究阻滞弥漫性脑损伤急性期ERKI／2信号通路过度激活对星形胶质细胞反应的影响。方法制作大鼠外伤性弥漫性脑损伤模型，打击损伤前30min自尾静脉注射U0126。Westemblot法检测损伤脑皮层pERKl／2表达水平，免疫组化染色法检测pERKl／2和GFAP在损伤脑组织中的表达。结果pERKl／2表达在损伤后迅速、显著升高，5min为表达高峰，其后下降，但直到损伤后72h都有高水平表达，至7d下降至基础水平。损伤后各个时间点，U0126组pERKl／2水平较DBI组明显降低（P〈0．05）。U0126组与DBI组比较，12～72h各时间点GFAP阳性细胞平均光密度值降低（P〈0.05）。结论弥漫性脑损伤诱导了强烈的ERKl／2信号通路激活和星形胶质细胞反应。U0126能够剂量依赖性抑制GFAP的表达，抑制急性期星形胶质细胞反应。     

Inhibition of MEK/ERK 1/2 pathway reduces pro-inflammatory cytokine interleukin-1 expression in focal cerebral ischemia

It has been proposed that mitogen-activated protein kinase (MAPK) pathways may play a role in the regulation of pro-inflammatory cytokines, such as interlukine-1, during cerebral ischemia. Our previous study showed that extracellular-signal-regulated kinases 1 and 2 (ERK 1/2) were activated during focal cerebral ischemia in mice [J. Cereb. Blood Flow Metab. 20 (2000) 1320]. However, the effect of ERK 1/2 activation in focal cerebral ischemia is still unclear. In this study we reported that in vivo phospho-ERK 1/2 expression increased following 30 min of middle cerebral artery occlusion (MCAO) in the mouse brain in both the ischemic core and perifocal regions. Western blot analysis and immunohistochemistry demonstrated that pro-treatment with 1,4-diamino-2,3-dicyano-1,4-bis butadiene (U0126) [J. Biol. Chem. 273 (1998) 18623] could significantly inhibit mouse brain phospho-MEK 1/2 and phospho-ERK 1/2 expression after 1-2 h of MCAO (p<0.05). Compared to the control group of mice, brain infarct volume was significantly decreased after 24 h of MCAO in the U0126-treated mice (27+/-6 vs. 46+/-9 mm(2), p<0.05). Inhibition of the MEK/ERK 1/2 pathway also prevented downstream kinase Elk-1 phosphorylation, and further reduced cytokine IL-1beta mRNA, but not TNFalpha, IL-1alpha, or chemokine MIP-1alpha mRNA expression. Our data demonstrates that in vivo the close linking of MEK 1/2, ERK 1/2, Elk-1, and IL-1 mRNA expression in the cerebral ischemia animals suggests that ERK 1/2 pathway activation is important in pro-inflammatory cytokine IL-1beta signaling, which induces an inflammatory response and exacerbates ischemic brain injury. Inhibiting the ERK 1/2 pathway may therefore provide a novel approach for the reduction of ischemia-induced IL-1beta overexpression.     

Activation of NF-kB and ERK1/2 after permanent focal ischemia is abolished by simvastatin treatment

We investigated the effects of simvastatin treatment on the expression of IL-1beta and MCP-1, the activity of NF-kB, and the signaling pathways related to NF-kB activation in a rat model of permanent middle cerebral artery occlusion (pMCAO). IL-1beta and MCP-1 expression, determined using RT-PCR, was enhanced by pMCAO; this effect was inhibited by the administration of simvastatin before ischemia. Pre-treatment with simvastatin abolished the ischemia-induced activation of NF-kB observed in vehicle-treated animals. The evaluation of signal transduction pathways, including extracellular signal-regulated kinase (ERK1/2), SAPK/JNK 46/54 and p38, indicated that only ERK1/2 phosphorylation was enhanced by ischemia, and this activation was prevented by simvastatin. ERK1/2-inhibitor, U0126, reduced brain ischemia but not cytokine induction. These results provide evidence that the HMG-CoA reductase inhibitor induces its effect in the protection of ischemic brain damage with a more complex mechanism which also involve anti-inflammatory properties rather than simple inhibition of ERK1/2 signaling pathway.     

U0126介导T细胞亚型增殖在缺血性脑卒中的神经保护作用及机制研究

目的探究U0126在缺血性脑卒中的神经保护效应及其介导的T细胞在其中的作用。方法分别采用成年雄性C57BL/6J野生型(WT)小鼠和严重联合免疫缺陷(SCID)小鼠,随机分为sham组、sham+U0126 (2 g/L)组、MCAO组及MCAO+U0126组,每组各10只,经线栓法构建大脑中动脉短暂性闭塞模型,60 min后取出线栓,恢复血流。观察在有或无免疫缺陷的情况下U0126对脑梗死范围和脾脏增殖情况的影响。结果 U0126可显著减小小野生型小鼠脑缺血再灌注后的脑梗死体积(P 0. 05);减轻脑缺血再灌注损伤对脾脏增殖的破坏(P 0. 05)。但在T细胞缺陷的SCID小鼠中U0126的应用并未减小梗死面积,反而扩大了梗死范围(P 0. 05),而当ConA刺激T细胞增殖后U0126的保护作用得到恢复(P 0. 05),这表明T细胞可能是U0126的作用靶点。结论 U0126可能通过抑制ERK1/2信号通路并选择性促进T细胞增殖来介导其在缺血性脑卒中中的神经保护效应。     

骨髓间充质干细胞旁分泌作用与脑缺血后的细胞凋亡*

背景：骨髓间充质干细胞移植治疗脑缺血的机制之一是骨髓间充质干细胞的旁分泌作用,而目前对于这一机制的研究报道较少。 目的：观察骨髓间充质干细胞旁分泌作用对脑缺血后细胞凋亡的抑制作用并探索相关机制。 方法：体外培养大鼠骨髓间充质干细胞,建立大鼠大脑中动脉缺血模型。24只SD大鼠随机数字表法分为4组,每组6只。细胞移植给药组：大鼠纹状体内移植骨髓间充质干细胞后给予ERK1/2抑制剂U0126;非移植给药组：注射等量的PBS后给予U0126;细胞移植对照组：移植骨髓间充质干细胞后给予溶剂对照;非移植对照组：注射等量的PBS后给予溶剂对照。7 d后通过Western blot检测血管内皮细胞生长因子、磷酸化ERK1/2蛋白的表达;TUNEL染色检测梗死区周围及皮质区细胞凋亡情况。 结果与结论：细胞移植组较非移植组大鼠纹状体内血管内皮细胞生长因子蛋白的表达明显增高,磷酸化ERK1/2表达增强,细胞凋亡数明显减少;经U0126处理后,血管内皮细胞生长因子的表达没有变化,而随着磷酸化ERK1/2的表达受到抑制,细胞凋亡数明显增高。提示骨髓间充质干细胞在大脑纹状体内可以旁分泌血管内皮细胞生长因子,并通过激活ERK1/2抑制了脑梗死区细胞的凋亡。     

Proteomic Expression Changes in Large Cerebral Arteries After Experimental Subarachnoid Hemorrhage in Rat Are Regulated by the MEK-ERK1/2 Pathway

Subarachnoid hemorrhage (SAH) is a serious clinical condition where leakage of blood into the subarachnoid space causes an acute rise in intracranial pressure and reduces cerebral blood flow, which may lead to delayed cerebral ischemia and poor outcome. In experimental SAH, we have previously shown that the outcome can be significantly improved by early inhibition of the MAPK/ERK kinase/extracellular signal-regulated kinase (MEK/ERK1/2) pathway. The aim of this study was to apply mass spectrometry to investigate the overall late effects of experimental SAH on cerebrovascular protein expression. SAH was induced in rats that were treated with the MEK1/2 inhibitor U0126 or vehicle. Neurological outcome was assessed using a battery of behavioral tests. Specific protein expression of large cerebral arteries was analyzed quantitatively with high-throughput tandem mass spectrometry. SAH resulted in a marked reduction of neurological scores, which was counteracted by U0126 treatment. Mass spectrometry analysis demonstrated regulation of 184 proteins after SAH, regulations that were in part prevented by U0126 treatment. Network analysis identified several protein networks including a strong structural network centered around 14-3-3. Additionally, protein networks with functions in mRNA metabolism and protein folding were identified. Treatment with U0126 inhibited cerebral vessel wall pERK1/2 expression and significantly improved outcome of the rats. In conclusion, we show that SAH induces a broad array of specific changes in the overall protein networks in cerebral artery smooth muscle cells and suggest that this is essential for understanding the vascular pathophysiology after SAH.     

Activation of ERK1/2 in spinal cord contributes to the development of acute cystic pain in rabbits

Objective To investigate the role of activated extracellular signal-regulated kinase 1/2 （ERK1/2） in spinal cord in the development of cystic pain in rabbit. Methods We observed the relationship between the activation of ERK1/2 in spinal cord and nociceptive behaviors, as well as the effect of U0126, a mitogen-activated protein kinase （MEK, upstream protein of ERK1/2） inhibitor, on cystic pain in rabbits by behavioral test, immunohistochemistry and western blot analysis. Results After injecting 0.5 ml formalin into gallbladder, the behaviors such as grasping of the cheek and licking of the abdomen increased in 30 min, with a significant increase in pERK1/2 expression in the spinal cord, as well as the pERK1/ 2 immunoreactive cells located in laminae Ⅴ-Ⅶ and X of the dorsal horn and ventral horn of T6 spinal cord. Administration of U0126 （100 -400 μg/kg body weight, i.v., 10 min before instillation of formalin） could attenuated nociceptive behaviors dose-dependently, but could not restrain the nociceptive behaviors completely even at the maximal efficient dose of 400 μg/kg body weight. Conclusion Activated ERK1/2 in the spinal cord at least partly participates in the development of acute inflammatory cystic pain induced by formalin in rabbits.