Rho激酶抑制剂法舒地尔对Aβ诱导痴呆大鼠模型的保护作用及其机制研究
|
摘要
背景与目的
阿尔茨海默病(Alzheimer's disease, AD)即老年性痴呆,是一种老年慢性进展性中枢神经系统退行性疾病,以认知损害为主要临床表现。随着人类平均寿命的延长和社会人口老龄化的迅猛发展,AD对人类健康的危害变得日益严重。据统计,全球的老年痴呆病人超过2600万,其中中国已超过500万。在发达国家,AD是继心脏病、癌症、中风后的第4位死因。另外,AD治疗和护理费用昂贵。因此,AD的防治已成为当前亟待解决的重大医学和社会问题。然而目前AD的研究现状是：虽然关于AD的发病机制已提出了多种学说,但尚无定论,对AD的治疗也就更加缺乏有力的手段。因此,进一步探讨AD形成和发生、发展的病理过程,并根据发病机制寻找更加有效的治疗方法,是医学工作者面临的有力挑战。
在众多的发病机制中,β淀粉样蛋白(β-amyloid protein, Aβ)异常沉积所导致的级联反应损伤学说得到了广泛的支持和论证；而在这一病理过程中,炎症扮演了重要角色。有研究证实,沉积的Aβ激活小胶质细胞和星形胶质细胞,释放炎症因子并产生活性氧簇(reactive oxygen specie, ROS),从而拉开了炎症和氧化应激损伤恶性循环的序幕。上述炎症反应和氧化应激过程互相加重,并通过肿瘤坏死因子(TNF)受体信号级联途径激活核因子-κb(NF-κb),启动凋亡因子和炎症因子转录,使得炎症损伤进一步加重,从而诱导神经元凋亡或死亡。因此基于上述的理论,如果能够阻断AD病理机制中Aβ诱导的炎症反应及其继发的病理过程,就有可能打破这种恶性循环,对AD起到保护和治疗的作用。
Rho激酶抑制剂(Rho-kinase inhibitor)盐酸法舒地尔(Fasudil)最初是作为解除脑血管痉挛的药物应用于临床。而近年来研究发现,多种细胞均表达Rho激酶。因此,Rho激酶抑制剂除具有经典的抑制平滑肌收缩、扩张血管、改善脑循环的作用外,还具有抑制炎症、神经保护、维持血-脑屏障完整性、减少氧自由基形成和增加清除,以及调节和控制细胞的运动、移行、增生和分化、生成和死亡等多种作用。目前已有Rho激酶抑制剂治疗血管性痴呆的临床和动物实验研究,发现其对血管性认知功能障碍有改善作用；有Rho激酶抑制剂显著提高老年大鼠的学习、工作能力的动物实验：有Rho激酶抑制剂改善遗忘型轻度认知障碍(aMCI)患者记忆功能的研究。
本研究制备Aβ1-42诱导的海马损伤大鼠模型,模拟AD的病理过程,两个剂量的Rho激酶抑制剂Fasudil治疗干预,观察Fasudil是否能够对抗Aβ1-42诱导的海马神经损伤、改善认知、对AD起到保护作用,并探讨其可能的抗炎、抗氧化及继发的抗凋亡机制；同时进一步验证了Aβ诱导AD的炎症机制。本实验拓宽了Rho激酶抑制剂的适用范围,为发现AD新的药物作用靶点奠定了基础,有可能部分逆转AD的病理过程。
材料与方法
80只成年雄性Wistar大鼠(体重260-300g)随机分为对照组(假手术组)(Control)、模型组(Model)、Fasudil低剂量组(Fasudil-L) (5mg/kg/d)、Fasudil高剂量组(Fasudil-H) (lOmg/kg/d),每组各20只。模型组和两个治疗组采用侧脑室注射Aβ1-42的方法制备海马损伤模拟AD大鼠模型。无菌生理盐水将Aβ1-42溶解稀释成5ug/ul,37℃孵育72h变为聚集状态。通过脑立体定向仪向大鼠侧脑室注入聚集态的Aβ1-42 2μl(10μg)；对照组侧脑室内注射无菌生理盐水2ul。术前即分组开始给药,连续14天。Fasudil低、高剂量组分别每天给予Fasudil5、10 mg/kg,分两次腹腔注射给药；模型组、对照组给予等容积生理盐水。
术后第9天进行Morris水迷宫训练,连续5天,术后第14天进行测试,观察各组大鼠学习、记忆功能的变化。水迷宫测试后,部分大鼠麻醉后4%多聚甲醛心脏灌注、取脑,制作病理标本。行HE染色、Nissl染色和电镜观察海马区神经细胞形态结构的变化,TUNEL染色观察神经元凋亡。免疫组织化学方法检测海马区NF-κb的核内表达。部分大鼠断头取脑制备海马匀浆,酶联免疫吸附法(ELISA)测定海马区炎性因子IL-1β、TNF-α的水平。比色法检测海马组织匀浆中超氧化物歧化酶(SOD)、谷胱苷肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)含量。
数据以均数±标准差(mean±SD)表示,采用SPSS17.0统计软件进行单因素方差分析(one-way ANOVA),两两比较采用LSD-t检验。以P<0.05认定差异有显著性。
结果
1.水迷宫实验
模型组和对照组相比较,学习和记忆成绩均低于对照组,差异有统计学意义(P<0.01)。Fasudil高剂量治疗组与模型组大鼠相比较,学习和记忆成绩均明显改善(P<0.01)。Fasudil低剂量组与模型组比较,大鼠的学习、记忆能力差异无统计学意义(P>0.05)。
2.海马区病理形态学
2.1 HE染色：术后14d,对照组海马齿状回(DG)区神经细胞排列紧密,细胞层次多,染色分布均匀,细胞核大而圆,核仁清晰可辨。模型组和低剂量Fasudil组海马神经细胞层次明显减少,排列稀疏,部分细胞空泡变,可见细胞核深染、固缩,核仁偏移,胶质细胞明显增生。高剂量Fasudil组海马神经细胞较模型组排列整齐,细胞呈椭圆形或圆形,细胞脱失较少见,胶质细胞增生减轻,核仁清晰。
2.2 Nissl染色：与假手术对照组相比,模型组大鼠脑海马CAl区Nissl染色浅,Nissl小体数量少,健康神经元密度低、神经细胞的活性较弱,神经细胞损伤较严重(P<0.01)。高剂量Fasudil (10 mg/kg)治疗14天后,能够明显逆转Aβ对神经元的损伤(P<0.05)。
2.3超微电镜：对照组大部分神经元结构正常,胞核以常染色质为主,核圆,胞浆内可见丰富的细胞器。AD模型组大部分神经元衰退,细胞固缩,核膜凹凸或断裂,细胞器肿胀、减少、结构破坏。Fasudil高剂量治疗组与模型组比较,神经元损伤较轻,衰退的神经元较少；而低剂量Fasudil治疗组改善不明显。
3.海马区TUNEL染色显示细胞凋亡的结果
模型组大鼠海马DG区存在较多的TUNEL染色阳性细胞,即凋亡细胞(P<0.01)。Fasudil高、低剂量治疗后,大鼠海马DG区阳性细胞均较模型组有所减少,其中Fasudil高剂量组偶见阳性细胞(P<0.01),低剂量组可见少量阳性细胞(P<0.05)。
4.海马区细胞炎性因子水平的检测
模型组侧脑室注射Aβ1-42造成海马区细胞IL-1β、TNF-α显著升高(P<0.01)；Fasudil低剂量组治疗后,IL-1β、TNF-α水平有所下降(P<0.05),差异有统计学意义；Fasudil高剂量组治疗后,IL-1β、TNF-α水平明显下降(P<0.01),差异均有统计学意义。
5.海马区NF-κb的表达
模型组海马区细胞NF-κb的核内表达明显升高(P<0.01),Fasudil低剂量组治疗后,海马细胞核内NF-κb的表达有所下降,但尚无统计学意义(P>0.05)；高剂量组治疗后,海马细胞核内NF-κb的表达明显下降(P<0.01),差异有显著性。
6.海马组织SOD、GSH-Px活力和MDA含量测定
与对照组相比较,AD模型组大鼠海马组织中GSH-Px(P<0.01)和SOD(P<0.05)的活性均有不同程度的下降,而MDA含量明显增加(P<0.05)。Fasudil高剂量(10mg/kg)治疗后,大鼠海马的GSH-Px(P<0.05)和SOD(P<0.05)这两种抗氧化酶的活性明显增加,而低剂量Fasudil (5mg/kg)对两种酶的活性均无明显影响。同时,高、低剂量Fasudil治疗后,有降低MDA含量的趋势,但差异无统计学意义。
结论
1.Rho激酶抑制剂Fasudil能够改善Aβ1-42诱导的痴呆模型大鼠空间学习和记忆能力。
2.侧脑室注射Aβ1-42后,大鼠海马区健康神经元密度明显减少,凋亡细胞数量明显增多;连续应用高剂量Rho激酶抑制剂Fasudil干预14d后,能够显著逆转Aβ诱导的神经细胞损伤。
3.Aβ1-42侧脑室注射后,大鼠海马区IL-1β、TNF-α显著升高；Rho激酶抑制剂Fasudil治疗后,大鼠海马区的上述炎性指标明显下降,且高剂量组优于低剂量组,证明Rho激酶抑制剂Fasudil剂量依赖性的对抗Aβ诱导的炎性反应,具有较强的抗炎功能。
4.Aβ1-42造成大鼠海马区的氧化应激损伤；Rho激酶抑制剂Fasudil能够增加抗氧化酶GSH-Px和SOD的活性,具有抗氧化、保护大鼠海马区氧化还原功能的作用。
5.侧脑室注射Aβ1-42导致大鼠海马区与炎症和氧化应激相关的转录因子NF-κb核内表达明显增加；而高剂量的Rho激酶抑制剂Fasudil能够有效的抑制NF-κb的核内表达,从而抑制NF-κb的核转位和激活,减轻由其激活导致的神经损伤。
6.综上,Rho激酶抑制剂Fasudil具有多重作用机制,有可能部分逆转AD的病理过程,成为抗AD药物研究的新方向。
Background and purpose:
Alzheimer's disease (AD) is a chronic, progressive, degenerative disease of central nervous system, characterized by cognitive impairment. With the average life expectancy of human and social development of population aging rapidly, the harm of AD to human health has become increasingly serious. According to statistics, there are more than 26 millions of Alzheimer's patients in the world, of which China has more than 5 millions. In developed countries, AD is the fourth cause of death following heart disease, cancer and stroke. In addition, AD causes expensive treatment and care. Thus, AD prevention and treatment has become a major medical and solved social problems. However, the current research on AD as followed: There is no confirmed conclusion on the pathogenesis of AD, although a variety of theories have been proposed. In the present, it is also lack of effective means on treatment of AD. Therefore, it is need us to further explore the pathological process of AD and to find more effective treatments on AD. It is also a strong challenge for the medical workers.
In the pathogenesis, the theory of damage cascade caused byβ-amyloid (β-amyloid protein, Aβ) abnormal deposition has been widely supported and demonstrated, with inflammation playing an important role in the above process. Studies have confirmed that the deposition of Aβcan activate microglia and astrocytes, release inflammatory cytokines and generate reactive oxygen species (ROS), which opened the inflammation and oxidative stress off a vicious cycle. The process of inflammation and oxidative stress increased with each other, and nuclear factor-Kb (NF-κb) is activated by tumor necrosis factor (TNF) receptor signaling cascades. Then the transcription of apoptotic factors and inflammatory cytokines is started, which further aggravating the the inflammation injury and inducing neuronal apoptosis or death. Therefore, based on the above theory, if we can block the pathogenesis of Aβ-induced inflammatory reaction and the secondary pathological process in AD, this vicious cycle may be broken. Then it will play a protection and treatment role on AD.
Rho kinase inhibitor—fasudil hydrochloride (Fasudil) started as a lifting of cerebral vasospasm in clinical medicine. In recent years studies have found that many cells expressed Rho-kinase. Thus, Rho kinase inhibitors inhibit the addition to the classic smooth muscle contraction, dilation of blood vessels, improving the role of the cerebral circulation, but also inhibit inflammation, neuroprotection, maintenance of the blood - brain barrier integrity, reducing oxygen free radical formation and increasing clearance, and regulating cell movement, migration, proliferation and differentiation, and death, et al. At present, Rho kinase inhibitor treatment of vascular dementia in clinical and animal studies found that there was improvement in vascular cognitive impairment.A Rho kinase inhibitor significantly improving learning and working ability in aged rats has been reported. Rho kinase inhibitor Fasudil has been demostrated to improve amnestic mild cognitive impairment (aMCI) patients with memory function studies.
Therefore, this study prepared Aβ1-42 induced rat model of AD, two doses of Rho kinase inhibitor Fasudil therapeutic intervention. It observed whether the Fasudil against Aβ1-42 induced nerve injury in the hippocampus and improving the cognitive impairment which may have protective effect on AD. Furthermore, it was discussed the involed possible anti-inflammatory, anti-oxidant and anti-apoptotic mechanisms. The experiment broadened the scope of Rho kinase inhibitor for the prevention and treatment of AD which may find new drug targets and partly reverse the pathological process of AD.
Materials and methods:
80 adult male Wistar rats (body weight 260-300g) were randomly divided into control group (sham group), model group, Fasudil low-dose group (5mg/kg/d), Fasudil high-dose group (10mg/kg/d),20 in each group. Aβ1-42 sterile saline solution was diluted into 5ug/ul and then incubated into aggregation state for 72 h at 37℃. Rats model were established via intracerebroventricular injection of Aβ1-42 while the control group intracerebroventricular injected of sterile saline 2ul. Before the operation, Fasudil low and high-dose treatment groups were daily given Fasudil 5,10 mg/kg, twice intraperitoneal injection for 14 consecutive days, with equal volume of saline for the control group.
On the 9th day after operation, Morris water maze training were excuted for 5 days.On the 14th day we observed the learning and memory changes in each group. After that, some rats were anesthetized with normal saline and 4% paraformaldehyde perfusion to find the brain producing pathological specimens. HE staining, Nissl staining and electron microscopy of nerve cells in hippocampus morphology changes were observed. TUNEL staining of neuronal apoptosis and the immunohistochemistry of nuclear NF-Kb expression in hippocampus were detected. The other rats were prepared hippocampus homogenate to determine IL-1β, TNF-a levels through enzyme-linked immunosorbent assay (ELISA). The superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) were evaluated with colorimetry.
The SPSS 17.0 computer programme was used for all calculations and statistical evaluations. Differences among different groups were tested by one-way ANOVA with LSD test,. Results were presented as means±SD and a level of p< 0.05 was considered significant.
Results:
1. Morris water maze experiment
In the present study, learning and memory ability in AD group was significant lower than those in control group (p< 0.01). When compared with AD group, high dose of Fasudil group (10 mg/kg/d) exhibited higher levels of learning and memory ability (p< 0.01), meanwhile, low dose of Fasudil group (5 mg/kg/d) was not differed with AD group.
2. Histopathological analysis
2.1 HE staining
After 14d, hippocampal neurons in the control group were oval-shaped, tightly packed neatly, evenly stained, round nucleus, large and clear nucleolus. Hippocampal neurons in model group and low dose of Fasudil group exhibit sparse arrangement, reduced cellular level, deeply stained nuclei and condensation. Meanwhile the glial cell proliferation was significant increased. When compared with model group, hippocampal neurons in high dose of Fasudil group arranged in neat rows, oval or round, rare cell depigmentation, reduced glial cell proliferation and clearly visible nucleolus.
2.2 Nissl staining
Compared with the control group rats, Nissl staining of hippocampal CA1 region in model group was lighter. Meanwhile there were smaller number of Nissl body, low density of healthy neurons, weak activity of nerve cells and more serious nerve cell damage (p< 0.01). High dose of Fasudil could significantly reverse the neuronal damage of AZβafter treatment for 14 days (p<0.05).
2.3 Electron microscopy
The structure of most neurons in the control group was normal, which mainly contain euchromatin nucleus and round nucleus. These cells were also rich in the organelle. Neurons recession, cell shrinkage, membrane bump, reduced organelles and structural damage could be seen in AD group. Compared with the control group, more glial cells could be seen. In high dose Fasudil group, there were lighter neuronal injury, less depression of neurons and proliferation of glial cells when compared with AD group.
3. TUNEL staining to evaluate apoptosis in hippocampal
TUNEL staining showed that there were more TUNEL-positive cells in the hippocampal DG region of model rats, which are also called apoptotic cells (p< 0.01). The TUNEL-positive cells in hippocampal DG region of high dose Fasudil group p (< 0.01) and low dose Fasudil group (p< 0.05) are significantly reduced when compared with model group.
4. The secretion of IL-1βand TNF-αin hippocampal region
The rats in the group treated with 10 mg/kg/d Fasudil and in the group treated with 5 mg/kg Fasudil showed lower levels of IL-1βand TNF-a in hippocampus region than those in the model group (p< 0.01 and p< 0.05).
5.The nuclear expression of NF-κb in hippocampus region.
The nuclear expression of NF-κb in hippocampus of the model group was obviously increased than the control(p＜0.01). The rats of the group treated with 10 mg/kg/d Fasudil showed lower levels of NF-κb in nucleus than those of the model group (p< 0.01). Although there was no statistical difference between 5 mg/kg/d Fasudil group and model group, the nuclear expression of NF-κb tended to reduce in 5 mg/kg/d Fasudil group.
6. SOD, GSH-Px activity and MDA in hippocampus tissue
The rats in the model group showed lower activity of GSH-Px, SOD and higher levels of MDA in hippocampus region than those in the control group (P< 0.01, P< 0.05 and P< 0.05) The rats in the group treated with 10 mg/kg/d Fasudil showed higher activity of GSH-Px and SOD(P< 0.05, respectively). The activity of GSH-Px and SOD was not increased in 5 mg/kg/d Fasudil group. While the change of MDA was not obviously in the two treating groups compared with the AD group.
Conclusions:
1. Rho kinase inhibitor Fasudil can improve the ability of spatial learning and memory in Aβ1-42-induced AD rats.
2. After intracerebroventricular injection of Aβ1-42, reduced density of healthy neurons and increased apoptotic cells have been shown in the hippocampus region of rats. While the neuronal injury induced by Aβcan be reversed after application of high dose Fasudil for 14 days.
3. The expression of IL-1βand TNF-a in hippocampus region are significantly increased after intracerebroventricular injection of Aβ1-42.After Rho kinase inhibitor Fasudil treatment, the previous inflammatory parameters of the rat hippocampus region are significantly decreased, and the high dose group is better than the low dose group. It is indicated that Rho kinase inhibitor with a strong anti-inflammatory function can dose-dependently inhibit Aβ-induced inflammatory response.
4. The oxidative stress injury in hippocampus region of rats can be caused by Aβ1-42·Fasudil can dose-dependently increase the activity of antioxidant enzyme GSH-Px and SOD, which have the function of antioxidant for the rat hippocampus region.
5. The nuclear expression of NF-Kb in hippocampus region of rats is significantly increased after intracerebroventricular injection of Aβ1-42.Large dose of Rho kinase inhibitor Fasudil can effectively inhibit the nuclear expression of NF-Kb. Then the nuclear translocation and activation of NF-κb can be inhibited and the nerve injury can be reduced.
6. In summary, Rho kinase inhibitor Fasudil has multiple mechanisms. It may be considered as a new drug which can partly reverse the pathological process of AD.
阿尔茨海默病(Alzheimer's disease, AD)即老年性痴呆,是一种老年慢性进展性中枢神经系统退行性疾病,以认知损害为主要临床表现。随着人类平均寿命的延长和社会人口老龄化的迅猛发展,AD对人类健康的危害变得日益严重。据统计,全球的老年痴呆病人超过2600万,其中中国已超过500万。在发达国家,AD是继心脏病、癌症、中风后的第4位死因。另外,AD治疗和护理费用昂贵。因此,AD的防治已成为当前亟待解决的重大医学和社会问题。然而目前AD的研究现状是：虽然关于AD的发病机制已提出了多种学说,但尚无定论,对AD的治疗也就更加缺乏有力的手段。因此,进一步探讨AD形成和发生、发展的病理过程,并根据发病机制寻找更加有效的治疗方法,是医学工作者面临的有力挑战。
在众多的发病机制中,β淀粉样蛋白(β-amyloid protein, Aβ)异常沉积所导致的级联反应损伤学说得到了广泛的支持和论证；而在这一病理过程中,炎症扮演了重要角色。有研究证实,沉积的Aβ激活小胶质细胞和星形胶质细胞,释放炎症因子并产生活性氧簇(reactive oxygen specie, ROS),从而拉开了炎症和氧化应激损伤恶性循环的序幕。上述炎症反应和氧化应激过程互相加重,并通过肿瘤坏死因子(TNF)受体信号级联途径激活核因子-κb(NF-κb),启动凋亡因子和炎症因子转录,使得炎症损伤进一步加重,从而诱导神经元凋亡或死亡。因此基于上述的理论,如果能够阻断AD病理机制中Aβ诱导的炎症反应及其继发的病理过程,就有可能打破这种恶性循环,对AD起到保护和治疗的作用。
Rho激酶抑制剂(Rho-kinase inhibitor)盐酸法舒地尔(Fasudil)最初是作为解除脑血管痉挛的药物应用于临床。而近年来研究发现,多种细胞均表达Rho激酶。因此,Rho激酶抑制剂除具有经典的抑制平滑肌收缩、扩张血管、改善脑循环的作用外,还具有抑制炎症、神经保护、维持血-脑屏障完整性、减少氧自由基形成和增加清除,以及调节和控制细胞的运动、移行、增生和分化、生成和死亡等多种作用。目前已有Rho激酶抑制剂治疗血管性痴呆的临床和动物实验研究,发现其对血管性认知功能障碍有改善作用；有Rho激酶抑制剂显著提高老年大鼠的学习、工作能力的动物实验：有Rho激酶抑制剂改善遗忘型轻度认知障碍(aMCI)患者记忆功能的研究。
本研究制备Aβ1-42诱导的海马损伤大鼠模型,模拟AD的病理过程,两个剂量的Rho激酶抑制剂Fasudil治疗干预,观察Fasudil是否能够对抗Aβ1-42诱导的海马神经损伤、改善认知、对AD起到保护作用,并探讨其可能的抗炎、抗氧化及继发的抗凋亡机制；同时进一步验证了Aβ诱导AD的炎症机制。本实验拓宽了Rho激酶抑制剂的适用范围,为发现AD新的药物作用靶点奠定了基础,有可能部分逆转AD的病理过程。
材料与方法
80只成年雄性Wistar大鼠(体重260-300g)随机分为对照组(假手术组)(Control)、模型组(Model)、Fasudil低剂量组(Fasudil-L) (5mg/kg/d)、Fasudil高剂量组(Fasudil-H) (lOmg/kg/d),每组各20只。模型组和两个治疗组采用侧脑室注射Aβ1-42的方法制备海马损伤模拟AD大鼠模型。无菌生理盐水将Aβ1-42溶解稀释成5ug/ul,37℃孵育72h变为聚集状态。通过脑立体定向仪向大鼠侧脑室注入聚集态的Aβ1-42 2μl(10μg)；对照组侧脑室内注射无菌生理盐水2ul。术前即分组开始给药,连续14天。Fasudil低、高剂量组分别每天给予Fasudil5、10 mg/kg,分两次腹腔注射给药；模型组、对照组给予等容积生理盐水。
术后第9天进行Morris水迷宫训练,连续5天,术后第14天进行测试,观察各组大鼠学习、记忆功能的变化。水迷宫测试后,部分大鼠麻醉后4%多聚甲醛心脏灌注、取脑,制作病理标本。行HE染色、Nissl染色和电镜观察海马区神经细胞形态结构的变化,TUNEL染色观察神经元凋亡。免疫组织化学方法检测海马区NF-κb的核内表达。部分大鼠断头取脑制备海马匀浆,酶联免疫吸附法(ELISA)测定海马区炎性因子IL-1β、TNF-α的水平。比色法检测海马组织匀浆中超氧化物歧化酶(SOD)、谷胱苷肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)含量。
数据以均数±标准差(mean±SD)表示,采用SPSS17.0统计软件进行单因素方差分析(one-way ANOVA),两两比较采用LSD-t检验。以P<0.05认定差异有显著性。
结果
1.水迷宫实验
模型组和对照组相比较,学习和记忆成绩均低于对照组,差异有统计学意义(P<0.01)。Fasudil高剂量治疗组与模型组大鼠相比较,学习和记忆成绩均明显改善(P<0.01)。Fasudil低剂量组与模型组比较,大鼠的学习、记忆能力差异无统计学意义(P>0.05)。
2.海马区病理形态学
2.1 HE染色：术后14d,对照组海马齿状回(DG)区神经细胞排列紧密,细胞层次多,染色分布均匀,细胞核大而圆,核仁清晰可辨。模型组和低剂量Fasudil组海马神经细胞层次明显减少,排列稀疏,部分细胞空泡变,可见细胞核深染、固缩,核仁偏移,胶质细胞明显增生。高剂量Fasudil组海马神经细胞较模型组排列整齐,细胞呈椭圆形或圆形,细胞脱失较少见,胶质细胞增生减轻,核仁清晰。
2.2 Nissl染色：与假手术对照组相比,模型组大鼠脑海马CAl区Nissl染色浅,Nissl小体数量少,健康神经元密度低、神经细胞的活性较弱,神经细胞损伤较严重(P<0.01)。高剂量Fasudil (10 mg/kg)治疗14天后,能够明显逆转Aβ对神经元的损伤(P<0.05)。
2.3超微电镜：对照组大部分神经元结构正常,胞核以常染色质为主,核圆,胞浆内可见丰富的细胞器。AD模型组大部分神经元衰退,细胞固缩,核膜凹凸或断裂,细胞器肿胀、减少、结构破坏。Fasudil高剂量治疗组与模型组比较,神经元损伤较轻,衰退的神经元较少；而低剂量Fasudil治疗组改善不明显。
3.海马区TUNEL染色显示细胞凋亡的结果
模型组大鼠海马DG区存在较多的TUNEL染色阳性细胞,即凋亡细胞(P<0.01)。Fasudil高、低剂量治疗后,大鼠海马DG区阳性细胞均较模型组有所减少,其中Fasudil高剂量组偶见阳性细胞(P<0.01),低剂量组可见少量阳性细胞(P<0.05)。
4.海马区细胞炎性因子水平的检测
模型组侧脑室注射Aβ1-42造成海马区细胞IL-1β、TNF-α显著升高(P<0.01)；Fasudil低剂量组治疗后,IL-1β、TNF-α水平有所下降(P<0.05),差异有统计学意义；Fasudil高剂量组治疗后,IL-1β、TNF-α水平明显下降(P<0.01),差异均有统计学意义。
5.海马区NF-κb的表达
模型组海马区细胞NF-κb的核内表达明显升高(P<0.01),Fasudil低剂量组治疗后,海马细胞核内NF-κb的表达有所下降,但尚无统计学意义(P>0.05)；高剂量组治疗后,海马细胞核内NF-κb的表达明显下降(P<0.01),差异有显著性。
6.海马组织SOD、GSH-Px活力和MDA含量测定
与对照组相比较,AD模型组大鼠海马组织中GSH-Px(P<0.01)和SOD(P<0.05)的活性均有不同程度的下降,而MDA含量明显增加(P<0.05)。Fasudil高剂量(10mg/kg)治疗后,大鼠海马的GSH-Px(P<0.05)和SOD(P<0.05)这两种抗氧化酶的活性明显增加,而低剂量Fasudil (5mg/kg)对两种酶的活性均无明显影响。同时,高、低剂量Fasudil治疗后,有降低MDA含量的趋势,但差异无统计学意义。
结论
1.Rho激酶抑制剂Fasudil能够改善Aβ1-42诱导的痴呆模型大鼠空间学习和记忆能力。
2.侧脑室注射Aβ1-42后,大鼠海马区健康神经元密度明显减少,凋亡细胞数量明显增多;连续应用高剂量Rho激酶抑制剂Fasudil干预14d后,能够显著逆转Aβ诱导的神经细胞损伤。
3.Aβ1-42侧脑室注射后,大鼠海马区IL-1β、TNF-α显著升高；Rho激酶抑制剂Fasudil治疗后,大鼠海马区的上述炎性指标明显下降,且高剂量组优于低剂量组,证明Rho激酶抑制剂Fasudil剂量依赖性的对抗Aβ诱导的炎性反应,具有较强的抗炎功能。
4.Aβ1-42造成大鼠海马区的氧化应激损伤；Rho激酶抑制剂Fasudil能够增加抗氧化酶GSH-Px和SOD的活性,具有抗氧化、保护大鼠海马区氧化还原功能的作用。
5.侧脑室注射Aβ1-42导致大鼠海马区与炎症和氧化应激相关的转录因子NF-κb核内表达明显增加；而高剂量的Rho激酶抑制剂Fasudil能够有效的抑制NF-κb的核内表达,从而抑制NF-κb的核转位和激活,减轻由其激活导致的神经损伤。
6.综上,Rho激酶抑制剂Fasudil具有多重作用机制,有可能部分逆转AD的病理过程,成为抗AD药物研究的新方向。
Background and purpose:
Alzheimer's disease (AD) is a chronic, progressive, degenerative disease of central nervous system, characterized by cognitive impairment. With the average life expectancy of human and social development of population aging rapidly, the harm of AD to human health has become increasingly serious. According to statistics, there are more than 26 millions of Alzheimer's patients in the world, of which China has more than 5 millions. In developed countries, AD is the fourth cause of death following heart disease, cancer and stroke. In addition, AD causes expensive treatment and care. Thus, AD prevention and treatment has become a major medical and solved social problems. However, the current research on AD as followed: There is no confirmed conclusion on the pathogenesis of AD, although a variety of theories have been proposed. In the present, it is also lack of effective means on treatment of AD. Therefore, it is need us to further explore the pathological process of AD and to find more effective treatments on AD. It is also a strong challenge for the medical workers.
In the pathogenesis, the theory of damage cascade caused byβ-amyloid (β-amyloid protein, Aβ) abnormal deposition has been widely supported and demonstrated, with inflammation playing an important role in the above process. Studies have confirmed that the deposition of Aβcan activate microglia and astrocytes, release inflammatory cytokines and generate reactive oxygen species (ROS), which opened the inflammation and oxidative stress off a vicious cycle. The process of inflammation and oxidative stress increased with each other, and nuclear factor-Kb (NF-κb) is activated by tumor necrosis factor (TNF) receptor signaling cascades. Then the transcription of apoptotic factors and inflammatory cytokines is started, which further aggravating the the inflammation injury and inducing neuronal apoptosis or death. Therefore, based on the above theory, if we can block the pathogenesis of Aβ-induced inflammatory reaction and the secondary pathological process in AD, this vicious cycle may be broken. Then it will play a protection and treatment role on AD.
Rho kinase inhibitor—fasudil hydrochloride (Fasudil) started as a lifting of cerebral vasospasm in clinical medicine. In recent years studies have found that many cells expressed Rho-kinase. Thus, Rho kinase inhibitors inhibit the addition to the classic smooth muscle contraction, dilation of blood vessels, improving the role of the cerebral circulation, but also inhibit inflammation, neuroprotection, maintenance of the blood - brain barrier integrity, reducing oxygen free radical formation and increasing clearance, and regulating cell movement, migration, proliferation and differentiation, and death, et al. At present, Rho kinase inhibitor treatment of vascular dementia in clinical and animal studies found that there was improvement in vascular cognitive impairment.A Rho kinase inhibitor significantly improving learning and working ability in aged rats has been reported. Rho kinase inhibitor Fasudil has been demostrated to improve amnestic mild cognitive impairment (aMCI) patients with memory function studies.
Therefore, this study prepared Aβ1-42 induced rat model of AD, two doses of Rho kinase inhibitor Fasudil therapeutic intervention. It observed whether the Fasudil against Aβ1-42 induced nerve injury in the hippocampus and improving the cognitive impairment which may have protective effect on AD. Furthermore, it was discussed the involed possible anti-inflammatory, anti-oxidant and anti-apoptotic mechanisms. The experiment broadened the scope of Rho kinase inhibitor for the prevention and treatment of AD which may find new drug targets and partly reverse the pathological process of AD.
Materials and methods:
80 adult male Wistar rats (body weight 260-300g) were randomly divided into control group (sham group), model group, Fasudil low-dose group (5mg/kg/d), Fasudil high-dose group (10mg/kg/d),20 in each group. Aβ1-42 sterile saline solution was diluted into 5ug/ul and then incubated into aggregation state for 72 h at 37℃. Rats model were established via intracerebroventricular injection of Aβ1-42 while the control group intracerebroventricular injected of sterile saline 2ul. Before the operation, Fasudil low and high-dose treatment groups were daily given Fasudil 5,10 mg/kg, twice intraperitoneal injection for 14 consecutive days, with equal volume of saline for the control group.
On the 9th day after operation, Morris water maze training were excuted for 5 days.On the 14th day we observed the learning and memory changes in each group. After that, some rats were anesthetized with normal saline and 4% paraformaldehyde perfusion to find the brain producing pathological specimens. HE staining, Nissl staining and electron microscopy of nerve cells in hippocampus morphology changes were observed. TUNEL staining of neuronal apoptosis and the immunohistochemistry of nuclear NF-Kb expression in hippocampus were detected. The other rats were prepared hippocampus homogenate to determine IL-1β, TNF-a levels through enzyme-linked immunosorbent assay (ELISA). The superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) were evaluated with colorimetry.
The SPSS 17.0 computer programme was used for all calculations and statistical evaluations. Differences among different groups were tested by one-way ANOVA with LSD test,. Results were presented as means±SD and a level of p< 0.05 was considered significant.
Results:
1. Morris water maze experiment
In the present study, learning and memory ability in AD group was significant lower than those in control group (p< 0.01). When compared with AD group, high dose of Fasudil group (10 mg/kg/d) exhibited higher levels of learning and memory ability (p< 0.01), meanwhile, low dose of Fasudil group (5 mg/kg/d) was not differed with AD group.
2. Histopathological analysis
2.1 HE staining
After 14d, hippocampal neurons in the control group were oval-shaped, tightly packed neatly, evenly stained, round nucleus, large and clear nucleolus. Hippocampal neurons in model group and low dose of Fasudil group exhibit sparse arrangement, reduced cellular level, deeply stained nuclei and condensation. Meanwhile the glial cell proliferation was significant increased. When compared with model group, hippocampal neurons in high dose of Fasudil group arranged in neat rows, oval or round, rare cell depigmentation, reduced glial cell proliferation and clearly visible nucleolus.
2.2 Nissl staining
Compared with the control group rats, Nissl staining of hippocampal CA1 region in model group was lighter. Meanwhile there were smaller number of Nissl body, low density of healthy neurons, weak activity of nerve cells and more serious nerve cell damage (p< 0.01). High dose of Fasudil could significantly reverse the neuronal damage of AZβafter treatment for 14 days (p<0.05).
2.3 Electron microscopy
The structure of most neurons in the control group was normal, which mainly contain euchromatin nucleus and round nucleus. These cells were also rich in the organelle. Neurons recession, cell shrinkage, membrane bump, reduced organelles and structural damage could be seen in AD group. Compared with the control group, more glial cells could be seen. In high dose Fasudil group, there were lighter neuronal injury, less depression of neurons and proliferation of glial cells when compared with AD group.
3. TUNEL staining to evaluate apoptosis in hippocampal
TUNEL staining showed that there were more TUNEL-positive cells in the hippocampal DG region of model rats, which are also called apoptotic cells (p< 0.01). The TUNEL-positive cells in hippocampal DG region of high dose Fasudil group p (< 0.01) and low dose Fasudil group (p< 0.05) are significantly reduced when compared with model group.
4. The secretion of IL-1βand TNF-αin hippocampal region
The rats in the group treated with 10 mg/kg/d Fasudil and in the group treated with 5 mg/kg Fasudil showed lower levels of IL-1βand TNF-a in hippocampus region than those in the model group (p< 0.01 and p< 0.05).
5.The nuclear expression of NF-κb in hippocampus region.
The nuclear expression of NF-κb in hippocampus of the model group was obviously increased than the control(p＜0.01). The rats of the group treated with 10 mg/kg/d Fasudil showed lower levels of NF-κb in nucleus than those of the model group (p< 0.01). Although there was no statistical difference between 5 mg/kg/d Fasudil group and model group, the nuclear expression of NF-κb tended to reduce in 5 mg/kg/d Fasudil group.
6. SOD, GSH-Px activity and MDA in hippocampus tissue
The rats in the model group showed lower activity of GSH-Px, SOD and higher levels of MDA in hippocampus region than those in the control group (P< 0.01, P< 0.05 and P< 0.05) The rats in the group treated with 10 mg/kg/d Fasudil showed higher activity of GSH-Px and SOD(P< 0.05, respectively). The activity of GSH-Px and SOD was not increased in 5 mg/kg/d Fasudil group. While the change of MDA was not obviously in the two treating groups compared with the AD group.
Conclusions:
1. Rho kinase inhibitor Fasudil can improve the ability of spatial learning and memory in Aβ1-42-induced AD rats.
2. After intracerebroventricular injection of Aβ1-42, reduced density of healthy neurons and increased apoptotic cells have been shown in the hippocampus region of rats. While the neuronal injury induced by Aβcan be reversed after application of high dose Fasudil for 14 days.
3. The expression of IL-1βand TNF-a in hippocampus region are significantly increased after intracerebroventricular injection of Aβ1-42.After Rho kinase inhibitor Fasudil treatment, the previous inflammatory parameters of the rat hippocampus region are significantly decreased, and the high dose group is better than the low dose group. It is indicated that Rho kinase inhibitor with a strong anti-inflammatory function can dose-dependently inhibit Aβ-induced inflammatory response.
4. The oxidative stress injury in hippocampus region of rats can be caused by Aβ1-42·Fasudil can dose-dependently increase the activity of antioxidant enzyme GSH-Px and SOD, which have the function of antioxidant for the rat hippocampus region.
5. The nuclear expression of NF-Kb in hippocampus region of rats is significantly increased after intracerebroventricular injection of Aβ1-42.Large dose of Rho kinase inhibitor Fasudil can effectively inhibit the nuclear expression of NF-Kb. Then the nuclear translocation and activation of NF-κb can be inhibited and the nerve injury can be reduced.
6. In summary, Rho kinase inhibitor Fasudil has multiple mechanisms. It may be considered as a new drug which can partly reverse the pathological process of AD.
引文
[1]Aguzzi A, O Connor T.Protein aggregation diseases:pathogenicity and therapeutic perspectives[J].Nat Rev Drug Discov,2010,9:237-248.
[2]Citron M. Alzheimer's disease:Strategies for disease modification[J]. Nat Rev Drug Discov,2010,9:387-398.
[3]Wimo A, Winblad B. Economic aspects on drug therapy of dementia[J]. Curr Pharm Des,2004,10(3):295-301.
[4]Gray SL,Anderson ML,Crane PK,et al.Antioxidant vitamin supplement use and risk of dementia or Alzheimers disease in older adults[J]. Am Geriatr Soc,2008,56(2):291-295.
[5]Mattsson N, Zetterberg H, Hansson O, et al. CSF biomarkers and incipient Alzheimer disease in patients with mild cognitive impairment[J]. J Am Med Assoc, 2009,302(4):385-393.
[6]Roberson ED, Scearce-Levie K, Palop JJ, et al. Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model [J]. Science,2007,316(5825):750-754.
[7]Perry R T, Collins J S, Wiener H, et al. The role of TNF and it s receptors in Alzheimer's disease[J]. Neurobiol Aging,2001,22 (6):873-883.
[8]Cole GM, Morihara T'Lim GP,Yang F, et al.NSAID and antioxidant prevention of Alzheimer's disease:lessons from in vitro and animal models. Ann N Y Acad Sci,2004,1035:68-84.
[9]Li R, Yang L, Lindholm K, et al. Tumor necrosis factor death receptor signaling cascade is required for amyloid-beta protein-induced neuron death[J] Neurosci,2004,24 (7):1760-1771.
[10]Combs CK, Karlo JC, Kao SC, et al. Beta-Amyloid stimulation ofmicroglia and monocytes results in TNF alpha - dependant exp ression of inducible nitric oxide synthase and neuronal apop tosis [J]. Neurosci,2001,21 (6):114-121.
[11]Zhou Y,Su Y,Li B, et al.Nonsteroidal anti-inflammatory drugs can lower amyloidogenic by inhibiting Rho. Science,2003,302:1215-1217.
[12]Huentelman MJ,Stephan DA,Talboom J,et al.Peripheral Delivery of a ROCK Inhibitor Improves Learning and Working Memory.Behav Neurosci,2009,123(1): 218-223.
[13]Petersen RC, Negash S. Mild cognitive impairment: an overview[J].CNS Spectr, 2008,13(1):45-53.
[14]Riento K,Ridley AJ.Rocks:multifunctional kenases in cell behaviour[J].Nat Rev Mol Cell Biol,2003,4(6):446-456.
[15]Kikuchi Y, Yamada M, Imakiire T, et al. A Rho—kinase inhibitor, fasudil, prevents development of diabetes and nephropathy in insulin—resistant diabetic rats. J Endocrinol,2007,192:595-603.
[16]Yamashita K, Kotani Y, Nakajima Y, et al. Fasudil, a Rho kinase(ROCK)inhibitor,protects against ischemic neuronal damage in vitro and in vivo by acting directly on neurons[J]. Brain Res,2007,1154:215-224.
[17]Butterfield DA, Reed L Newman SF,Sultana R.Roles of amyloid beta-peptide-associated oxidative stress and brain protein modifications in the pathogenesis of AIzheimeCs disease and mild cognitive impairment. Free Radic Bioi Med,2007,43(5):658-77.
[18]Tanzi RE, Bertram L. Twenty years of the Alzheimer's disease amyloid hypothesis:a genetic perspective [J]. Cell,2005,120(4):545-555.
[19]Reddy PH, Beal MF. Amyloid beta, mitochondrial dysfunction and synaptic damage:implications for cognitive decline in aging and Alzheimer's disease[J]. Trends Mol Med,2008,14(2):45-53.
[20]O'Barr S1CooperNR1The C5a comp lement activation pep tide increases IL21beta and IL26 release from amyloid2beta p rimed human monocytes:imp lications for Alzheimer's disease[J].Neuroimmunol,2000,109(2):872-941.
[21]DeWitt DA, Perry G, Cohen M, et al.Astrocytes regulate microglial phagocytosis of senile p laque cores of Alzheimer's disease.Exp Neurol,1998,149 (2):329-401.
[22]Wallin AK, Blennow K, Andreasen N, et al. CSF biomarkers for Alzheimer's disease:levels of beta-amyloid, tau, phosphorylated tau relate to clinical symptoms and survival[J]. Dement Geriatr Cogn Disord,2006,21(1):131-138.
[23]Halliday G, Robinson SR, Shepherd C.Alzheimer's disease and inflam2 mation: a review of cellular and therapeutic mechanisms[J].Clin Exp Pharmacol Physiol,2000, 27(122):1281-1290.
[24]Floden AM, Li S, Combs CK. Beta-amyloid—stimulated microglia induce neuron death via synergistic stimulation of tumor necrosis factor alpha and NMDA receptors. J Neurosci,2005,25(10):2566—75.
[25]Liao H, Bu WY Wang TH, Ahmed S, et al. Tenascin-R plays a role in neuroprotection via its distinct domains that coordinate to modulate the microglia function. J Biol Chem,2005,280(9):8316-23.
[26]Von Bemhardi R. Glial cell dysregulation:a new perspective on Aizheimer disease. Neurotox Res,2007,12(4):215-32.
[27]Yang X, Wang J, Yang ES. Coenzyme Q1O attenuates beta-amyloid pathology in the aged transgenic mice with Alzheimer presenilin 1 mutation. J Mol Neurosci, 2008,34(2):165-71.
[28]Markesbery W.Oxidative stress hypothesis in Alzheimer's disease. Free Radio Biol Med,1997,23(1):134-47.
[29]Rosales Corral S, Tan DX, Reiter RJ,et al.Kinetics of the neuroinflammation-oxidative stress correlation in the brain following the injection of fibrillar amyloid-beta onto the hippocampus in vivo. J Neuroimmuno,2004,150(1-2): 20-8.
[30]Abramov AY,Canevari L,Duchen M. Changes in intracellular calcium and glutathione in astrocytes as the primary mechanism of amyloid neurotoxicity. J Neurosci,2003,23(12):5088-95.
[31]Zhou J, Fonseca MI, Pisalyaput K. Complement C3 and C4 expression in Clq sufficient and deficient mouse models of Alzheimer's disease. J Neurochem,2008,106(5):2080-92.
[32]Rosales,Corral S, Tan DX, et al. Kinetics of the neuroinflammation-oxidative stress correlation in the brain following the injection of fibrillar amyloid-beta onto the hippocampus in vivo. J Neuroimmunol,2004,150(1-2):20-8.
[33]Puig B, Gomez-lsla T, Ribe E,et al. Expression of stress-activated kinases c-Jun N-terminal kinase(SAPK / JNK-P)and p38 kinase(p38-P), and tau hyperphosphorylation in neurites surrounding betaA plaques in APP T92576 mice. Neuropathol Appl Neurobiol,2004,30(5):491-502.
[34]Yumi Y,Richard B G. Therapeutic potential of inhibition of the NF-κ B pathway in the treatment of inflammation and cancer [J]. J Clin Invest,2001,107(2):135-142.
[35]Young JS,Kyung SC,Hyun HC, et al.Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals:down-regulation of COX-2 and iNOS through suppression of NF-κ B activation [J].Mutat Res,2001,9 (480-481):243-268.
[36]陈云波,张大鹏,冯梅,等.人参皂苷Rg1对Aβ 25235诱导的神经细胞核因子2K B活化的影响[J].中国药理学通报,2007,23(5)：612-7.
[37]Li KYang L, Lindholm K, Konishi Y, et al.Tumor necrosis factor death receptor signaling cascade is required for amyloid-beta protein-induced neuron death. J Neurosci,2004,24(7):1760-71.
[38]Valerio A,Boroni F,Benarese M, et al. NF-kappaB pathway:a target for preventing beta-amyloid(Abeta)-induced neuronal damage and Abeta42 production. Eur J Neurosci,2006,23(7):1711-20.
[39]Rohn TT, Rissman RA, Davis MC, et al. Caspase—9 activation and caspase cleavage of tau in the Alzheimer'S disease brain[J]. Neurobiol Dis,2002,11(2): 341-354.
[40]Wyss-Coray T,Mucke L. Inflammation in neurodegenerative disease-a double-edged sword[J]. Neuron,2002,35(3):419-432.
[41]Walter Adriani, Elisa Ognibene, Emilie Heuland. Motor impulsivity in APP-SWE mice: a model of Alzheimer's Disease. Behavioural Pharmacology[J],2006,17:525-533.
[42]Eikelenboom P, Van Gool W,et al. Neuroinflammatory perspectives on the two faces of Alzheimer'S disease. Neural Transm.2004, 11:281-294.
[43]Lee sv,Lee JW,LeeH Yoo HS, et al. Inhibitory effect of green tea extract on beta-amyloid-induced PCI2 cell death by inhibition ofthe activation of NF-kappaB and ERK/p38 MAP kinase pathway through antioxidant mechanisms. Brain Res Mol Brain Res,2005,140(1-2):45-54.
[44]Yamaguchi Y,Kawashima S. Effects of amyloid—beta-(25-35)on passive avoidance, radial-armmaze learning and choline acetyltransferase activity in the rat. Eur J Pharmacol,2001,41,2(3):265—72.
[45]Yamada H, Aimi Y Nagatsu I, Arai R. Immunohistochemical detection of L— DOPA--derived dopamine within serotonergic fibers in the striatum and the substantia nigra pars reticulata in Parkinsonian model rats. Neurosci Res,2007,59(1):1-7.
[46]Townsend KP,Pratico D. Novel therapeutic opportunities for Alzheimer's disease:focus on nonsteroidal anti—inflammatory drugs. Faseb J,2005,19(12): 1592-601.
[47]Kuhad A,Bishnoi M, Tiwari,et al. Suppression of NF-kappabeta signaling pathway by tocotrienol Can prevent diabetes associated clgnitive deficits. Pharmacol Biochem Behav,2009,92(2):251-9.
[48]Etminan M, Gill S, Samii A. Effect of nonsteroidal anti-inflammatory drugs on risk of Alzheimer's disease:systematic review and meta-analysis of observational studies. BMJ,2003,327(7407):128-142.
[49]Szekely CA, Thome JE, Zandi PP, et al. Nonsteroidal anti-inflammatory drugs for the prevention of Alzheimer's disease:a systematic review. Neuroepidemiology, 2004,23(4):159-69.
[50]Zhou Y, Su Y, Li B, Liu F, et al. Nonsteroidal anti-inflammatory drugs can lower amyloidogenic A β 42 by inhibiting Rho. Science,2003,302:1215-1217.
[51]Mueller BK, Mack H, Teusch N. Rho kinase, a promising drug target for neurological disorders [J]. Nat Rev Drug Discov,2005,4:387-398.
[52]Tanaka T, Nishimura D, Wu RC, et al. Nuclear Rho kinase, ROCK2, targets p300 acetyltransferase [J]. J Biol Chem,2006,281:15320-15329.
[53]Ishizaki T. Rho-mediated signal transduction and its physiological roles [J]. Nippon Yakurigaku Zasshi,2003,121:153-162.
[54]Kioiler L, Hall A. Signaling to Rho GTPase[J].Exp Cell Res,1999,253(1): 166-179.
[55]Matozaki T. Nakanishi H. Takai Y. Small G-protein networks:their crosstalk and signal cascades[J]. Cell signal.2000,12(8):515-524.
[56]Cole GM, Morihara T, Lim GP,et al. NSAID and antioxidant prevention of Alzheimer's disease:lessons from in vitro and animal models. Ann N Y Acad Sci,2004,1035:68-84.
[57]Li KYang L, Lindholm K,et al. Tumor necrosis factor death receptor signaling cascade is required for amyloid-beta protein-induced neuron death. J Neurosci, 2004,24(7):1760-71.
[58]Valerio A,Boroni F,Benarese M, et al. NF-kappaB pathway:a target for preventing beta-amyloid(Abeta)-induced neuronal damage and Abeta42 production. Eur J Neurosci,2006,23(7):1711-20.
[59]Meffen Mk,Chang JM, wiltgen BJ, et al. NF-kappa B functions in synaptic signaling and behavior. Nat Neurosci,2003,6(10):1072-8.
[60]Zhang Z, Fauser U, Schluesener HJ. Dexamethasone suppresses infiltration of RhoA(+)cells into early lesions of rat traumatic brain injury. Acta Neuropathol,2008, 115(3):335-43.
[61]Conrad S, Schluesener HJ, Trautmann K, et al. Prolonged lesional expression of RhoA and RhoB following spinal cord injury. J Comp Neurol,2005,27,487(2): 166-75.
[62]Paintlia AS, Paintlia MK, Khan M, et al. HMCoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis[J]. Faseb J,2005,19:1407-1421.
[63]Sun X, Minohara M, Kikuehi H. The selective Rho-kinase inhibitor Fasudil is protective and therapeutic in experimental autoimmune[J]. J Neuroimmunol,2006, 180(1-2):126—34.
[64]Shema R, Sacktor TC, Dudai Y. Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKM zeta. Science,2007,317(5840):951-953.
[65]王晓明,赵晴,莽靖等.法舒地尔对慢性脑缺血大鼠认知功能及皮层自由基代谢的影响.中国老年学杂志.2008,10(28).
[66]Johansson R, Persson K. Phenotypic modulation of cultured bladder smooth muscle cells and the expression of inducible nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol,2004,286:R642-R648.
[67]Dobashi K, Araki S, Kubo K,et al. Hydroxymethylglutaryl-CoA reductase inhibitor inhibits induction of nitric oxide synthase in T3-L1 preadipocytes. Life Sci,2008,82:85-90
[68]Cory, S, Adams, J.M. The Bcl-2 family: regulators of cellular life-ordeath switch. Nat Rev Cancer,2002,2:647-656.
[69]Lud Cadet, Harrington B, Ordonez S. Bcl-2 overexpression attenuates dopamine-induced apoptosis in an immortalized neural cell line by suppressing the production of reactive oxygen species. Synapse,2000,35:228-233.
[70]Kirkland RA, Windelborn JA, Kasprzak JM,et al.Bax induced pro-oxidant state is critical for cytochrome c release during programmed neuronal death. Neurosci,2002, 22:6480-6490.
[71]Fukushima M, Nakamuta M, Kohjima M, et al. Fasudil hydrochloride hydrate, a Rho-kinase (ROCK) inhibitor, suppresses collagen production and enhances collagenase activity in hepatic stellate cells. Liver Int,2005,25(4):829-38.
[72]Del Re DP, Miyamoto S, Brown JH. RhoA/Rho kinase up-regulate Bax to activate a mitochondrial death pathway and induce cardiomyocyte apoptosis. J Biol Chem,2007,16,282(11):8069-78.
[73]Satoh S, Toshima Y, Ikegaki I,et al. Wide therapeutic time window for fasudil neuroprotection against ischemia-induced delayed neuronal death in gerbils. Brain Res,2007,1128(1):175-180.
[74]Leung T,Manser E,Tan L,et al.A novel serine/threonine kinase binding the Ras-related Rho A GTPase which translocates the kinase to peripheral membranes.J Biol Chem,1995,270:29051-29054.
[75]Ishizaki T. Rho-mediated signal transduction and its physiological roles[J].Nippon Yakurigaku Zasshi.2003,121:153-162.
[76]Dobashi K, Araki S, Kubo K,et al. Hydroxymethylglutaryl--CoA reductase inhibitor inhibits induction of nitric oxide synthase in 3T3--L1 preadipocytes. Life Sci,2008,82:85-90.
[77]Rikitake Y, Kim H, Huang Z,et al. Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection. Stroke,2005,36:2251-2257.
[78]Yamashita K, Kotani Y, Nakajima Y, et al. Fasudil, a Rho kinase (ROCK) inhibitor, p rotects against ischemic neuronal damage in vitro and in vivo by acting directly on neurons [J]. Brain Res,2007,1154:215-224.
[79]Yagita Y, Kitagawa K, Sasaki T, et al. Rho - kinase activation in endothelial cells contributes to expansion of infarction after for cerebral ischemia [J]. J Neurosci Res,2007,85 (11):2460-2469.
[80]Shin HK, Salomone S, Potts EM, et al. Rho-kinase inhibition acutely augments blood flow in focal cerebral ischemia via endo-thelialmechanisms [J]. J Cereb Blood Flow Metab,2007,27(5):998-1009.
[81]Yan Z, Yuan S, Baolin L, et al. Nonsteroidal Anti-Inflammatory Drugs Can Lower Amyloidogenic A β 42 by Inhibiting Rho[J]. Science,2003, 302(5648):1215-17.
[82]vramovich Y, Amit T, Youdim M B. Nonsteroidal Anti-inflammatory Drugs Stimulate Secretion of Non-amyloidogenic Precursor Protein[J]. J B iol Chem,2002, 277(35):31466-73.
[83]Weggen S, Eriksen J L, Das P, et al. A subset of NSA IDs lower amyloidogenic A β 42 independently of cyclooxygenase activity[J].N ature,2001,414(6860):212-6.
[84]Leo A,Berezovska O, HerlL, et al. Nonsteroidal anti-inflammatory drugs lower Abeta42 and change presenilin 1 conformation[J]. N atM ed,2004,10(10):1065-6.
[85]Persidsky Y, Heilman D, Haorah J, et al. Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis. Blood,2006,180:126-134.
[86]Greenwood J,Walters CE, Pryee G, et al. RhoA expression in EAE and MS.Blackwell Publishing Ltd. Neuropathology and Applied Neuroblology.2007, 34,231-240.
[87]Negishi M, Katoh H. Rho family GTPases as key regulators for neuronal network formation. J Bilchem Tokyo,2002,132:157-166.
[88]Schmandke A,Strittmatter SM. ROCK and Rho:biochemistry and neuronal functions of Rho-associated protein kinases. Neuroscientist,2007,13:454-469.
[89]Zhou Y,Su Y,Li B.et al.Nonsteroidal anti-inflammatory drugs can lower amyloidogenic by inhibiting Rho. Science,2003,302:1215-1217.
[90]Dubreuil CI, Winton MJ, McKerraeher L, et al. Rho activation patterns after spinal cord inj ury and the role of activated Rho in apoptosis in the central neurous system[J]. J Cell Biol,2003,162:233-36.
[91]Dergham P, Ellezam B, Essagian C, et al. Rho signaling pathway targeted to promote spinal cord repair[J]. J Neurosci,2002,22:6570-6573.
[92]Huentelman MJ,Stephan DA,et al.Peripheral Delivery of a ROCK Inhibitor Improves Learning and Working Memory.Behav Neurosci,2009,123(1):218-223.
[93]Shema R, Sacktor TC, Dudai Y. Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKM zeta. Science,2007,317(5840):951-953.
[1]Ishizaki T. Rho-mediated signal transduction and its physiological roles [J]. Nippon Yakurigaku Zasshi,2003,121:153-162.
[2]Kioiler L, Hall A. Signaling to RhoGTPase[J].Exp Cell Res,1999,253(1):166 —179.
[3]Mueller BK, Mack H, Teusch N. Rho kinase, a promising drug target for eurological disorders[J]. Nat Rev Drug Discov,2005,4:387-398.
[4]Etienne-Manneville S, Hall A. Rho GTPases in cell biology[J]. Nature,2002, 420(6916):629—635.
[5]Tanaka T, Nishimura D, Wu RC, et al. Nuclear Rho kinase, ROCK2, targets p300 acetyltransferase [J]. J Biol Chem,2006,281:15320-15329.
[6]Moon SY, Zang H, Zheng Y. Characterization of a brain-specific Rho GTPase-activating protein, p200 RhoGAP[J]. J Biol Chem,2003,278 (6): 4151—4159.
[7]Chang J, Xie M, Shah VR, et al. Activation of Rho-associated coiled-coil protein kinase-1 (ROCK-1) by caspase-3 cleavage p lays an essential role in cardiac myocyte apop tosis [J]. Proc Natl Acad Sci USA,2006,103:14495-14500.
[8]Sebbagh M, Hamelin J, Bertoglio J, et al. Direct cleavage of ROCK II by granzyme B induces target cell membrane blebbing in a caspase-independent manner[J]. J Exp Med,2005,201:465-471.
[9]Anwar KN, Fazal F, Malik AB, et al. RhoA/Rho-associated kinase pathway selectively regulates thrombin-induced intercellular adhesion molecule expression in endothelial cells via activation of I kappa B kinase beta and phosphorylation of RelA /p65 [J]. J Immunol,2004,173:6965-6972.
[10]TorsoniAS, Marin TM, Velloso LA, et al. RhoA/ROCK signaling is critical to FAK activation by cyclic stretch in cardiac myocytes [J]. Am J Physiol Heart Circ Physiol,2005,289:H1488-1496.
[11]Watanabe Y, Faraci FM, Heistad DD. Activation of Rho-associated kinase during augmented contraction of the basilar artery to serotonin after subarachnoid hemorrhage [J]. Am J Physiol Heart Circ Physiol,2005,288:H2653-2658.
[12]Shema R, Sacktor TC, Dudai Y. Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKM zeta. Science,2007,317(5840):951-953.
[13]Tamura M, Nakao H, Yoshizaki H, et al. Development of specific Rho2kinase inhibitors and their clinical application [J]. Biochim Biophys Acta,2005,1754:245-252.
[14]Chitaley K, Webb RC, Mills TM. The ups and downs of Rho-kinase and penile erection: up stream regulators and downstream substrates of Rho-kinase and their potential role in the erectile response [J]. Int J Impot Res,2003,15:105-109.
[15]Murata T, Arii S, Nakamura T, et al. Inhibitory effect of Y227632, a ROCK inhibitor, on p regression of rat liver fibrosis in association with inactivation of hepatic stellate cells [J]. J Hepatol,2001,35:474-481.
[16]NakajimaM, Hayashi K, Egi Y, et al. Effect of Wf2536, a novel ROCK inhibitor, against metastasis of B16 melanoma [J]. Cancer Chemother Pharmacol,2003,52: 319-324.
[17]Rikitake Y, Kim HH, Huang, ZH, et al. Inhibition of Rho Kinase (ROCK) Leads to Increased Cerebral Blood Flow and Stroke Protection. Stroke,2005,36(10):2251 2257.
[18]Shin HK, Salomone S, Potts EM, et al. Rho-kinase inhibition acutely augments blood flow in focal cerebral ischemia via endothelial mechanisms[J]. J Cereb Blood Flow Metab,2007,27(5):998-1009.
[19].Yamashita K, Kotani Y, Nakajima Y, et al. Fasudil, a Rho kinase (ROCK) inhibitor, protects against ischemic neuronal damage in vitro and in vivo by acting directly on neurons[J]. Brain Res,2007,1154:215-224.
[20]Dobashi K, Araki S, Kubo K,et al. Hydroxymethylglutaryl-CoA reductase inhibitor inhibits induction of nitric oxide synthase in 3T3--L1 preadipocytes. Life Sci,2008,82:85-90.
[21]Satoh S, Toshima, Y, Ilegaki I, et al. Wide therapeutic time windows for fasudil neuroprotection against ischemia-induced delayed neuronal death in gerbils[J]. Brain Res,2007,1128(1):175-180.
[22]Zhou Y,Su Y,Li B,Liu F,et al.Nonsteroidal anti-inflammatory drugs can lower amyloidogenic by inhibiting Rho. Science,2003,302:1215-1217.
[23]Schmandke A,Strittmatter SM.ROCK and Rho:biochemistry and neuronal functions of Rho-associated protein kinases. Neuroscientist,2007,13:454-469.
[24]Sbibuya M, Hirai S, Seto M, et al. Effects of fasudil in acute ischemic Stroke: results of a prospective placebo-controlled double-blind trial[J]. J Neurol Sci, 2005,238(1-2):31-39.
[25]Shin HK, Salomone S, Potts EM, et al. Rho-kinase inhibition acutely augments blood flow in focal cerebral ischemia via endo-thelialmechanisms [J]. J Cereb Blood Flow Metab,2007,27(5):998-1009.
[26]Yan Z, Yuan S, Baolin L, et al. Nonsteroidal Anti2Inflammatory Drugs Can LowerAmyloidogenic A β 42 by Inhibiting Rho[J]. Science,2003,302 (5648):1215-17.
[27]Avramovich Y, Amit T, Youdim MB. Nonsteroidal Anti-inflammatory Drugs Stimulate Secretion of Non-amyloidogenic Precursor Protein[J]. J B iol Chem,2002, 277 (35):31466-73.
[28]Weggen S, Eriksen J L. Das P, et al. A subset of NSA IDs lower amyloidogenic A β 42 independently of cyclooxygenase activity[J].N ature,2001,414 (6860):212-6.
[29]Lleo A,Berezovska O, HerlL, et al. Nonsteroidal anti2inflammatory drugs lowerAbeta42 and change p resenilin 1 conformation[J]. Nat Med,2004,10 (10): 1065-6.
[30]Huentelman MJ,Stephan DA,Talboom J,et al.Peripheral Delivery of a ROCK Inhibitor Improves Learning and Working Memory.Behav Neurosci,2009,123(1): 218-223.
[31]Shema R, Sacktor TC, Dudai Y. Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKM zeta. Science,2007,317(5840):951-953.
[32]Eva Cernuda-Morolln, Anne J. Ridley. Rho GTPases and leukocyte adhesion receptor expression and function in endothelial cells. Cire Res,2006,98:757-767.
[33]Waiczies S, Bendix I,Zipp F. Geranylgeranylation but not GTP-loading of Rho GTPases determines T cell function[J]. Sci Signal,2008, Mar,25,1(12):pt3.
[34]Z Zhang, J Schittenhelm, R Meyermann, et al. Lesional accumulation of RhoA+cells in brains of experimental autoimmune encephalomyelitis and multiple sclerosis. Neuropathology and Applied Neurobiology,2008,34:231-240.
[35]Paintlia AS, Paintlia MK, Khan M, et al. HMCoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis[J]. Faseb J,2005,19:1407.1421.
[36]Sun X, Minohara M, Kikuehi H. The selective Rho-kinase inhibitor Fasudil is protective and therapeutic in experimental autoimmune[J]. J Neuroimmunol, 2006,180(1-2):126-34.
[37]Vollmer T, Key L, Durkalski V, et al. Oral simvastatin treatment in relapsing. remitting multiple sclerosis[J]. Lancet,2004,363:1607-1608.
[38]Greenwood J,Walters CE, Pryce G, et al. RhoA expression in EAE and MS. Neuropathology and Applied Neurobiology,2007,34:231-240.
[2]Citron M. Alzheimer's disease:Strategies for disease modification[J]. Nat Rev Drug Discov,2010,9:387-398.
[3]Wimo A, Winblad B. Economic aspects on drug therapy of dementia[J]. Curr Pharm Des,2004,10(3):295-301.
[4]Gray SL,Anderson ML,Crane PK,et al.Antioxidant vitamin supplement use and risk of dementia or Alzheimers disease in older adults[J]. Am Geriatr Soc,2008,56(2):291-295.
[5]Mattsson N, Zetterberg H, Hansson O, et al. CSF biomarkers and incipient Alzheimer disease in patients with mild cognitive impairment[J]. J Am Med Assoc, 2009,302(4):385-393.
[6]Roberson ED, Scearce-Levie K, Palop JJ, et al. Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model [J]. Science,2007,316(5825):750-754.
[7]Perry R T, Collins J S, Wiener H, et al. The role of TNF and it s receptors in Alzheimer's disease[J]. Neurobiol Aging,2001,22 (6):873-883.
[8]Cole GM, Morihara T'Lim GP,Yang F, et al.NSAID and antioxidant prevention of Alzheimer's disease:lessons from in vitro and animal models. Ann N Y Acad Sci,2004,1035:68-84.
[9]Li R, Yang L, Lindholm K, et al. Tumor necrosis factor death receptor signaling cascade is required for amyloid-beta protein-induced neuron death[J] Neurosci,2004,24 (7):1760-1771.
[10]Combs CK, Karlo JC, Kao SC, et al. Beta-Amyloid stimulation ofmicroglia and monocytes results in TNF alpha - dependant exp ression of inducible nitric oxide synthase and neuronal apop tosis [J]. Neurosci,2001,21 (6):114-121.
[11]Zhou Y,Su Y,Li B, et al.Nonsteroidal anti-inflammatory drugs can lower amyloidogenic by inhibiting Rho. Science,2003,302:1215-1217.
[12]Huentelman MJ,Stephan DA,Talboom J,et al.Peripheral Delivery of a ROCK Inhibitor Improves Learning and Working Memory.Behav Neurosci,2009,123(1): 218-223.
[13]Petersen RC, Negash S. Mild cognitive impairment: an overview[J].CNS Spectr, 2008,13(1):45-53.
[14]Riento K,Ridley AJ.Rocks:multifunctional kenases in cell behaviour[J].Nat Rev Mol Cell Biol,2003,4(6):446-456.
[15]Kikuchi Y, Yamada M, Imakiire T, et al. A Rho—kinase inhibitor, fasudil, prevents development of diabetes and nephropathy in insulin—resistant diabetic rats. J Endocrinol,2007,192:595-603.
[16]Yamashita K, Kotani Y, Nakajima Y, et al. Fasudil, a Rho kinase(ROCK)inhibitor,protects against ischemic neuronal damage in vitro and in vivo by acting directly on neurons[J]. Brain Res,2007,1154:215-224.
[17]Butterfield DA, Reed L Newman SF,Sultana R.Roles of amyloid beta-peptide-associated oxidative stress and brain protein modifications in the pathogenesis of AIzheimeCs disease and mild cognitive impairment. Free Radic Bioi Med,2007,43(5):658-77.
[18]Tanzi RE, Bertram L. Twenty years of the Alzheimer's disease amyloid hypothesis:a genetic perspective [J]. Cell,2005,120(4):545-555.
[19]Reddy PH, Beal MF. Amyloid beta, mitochondrial dysfunction and synaptic damage:implications for cognitive decline in aging and Alzheimer's disease[J]. Trends Mol Med,2008,14(2):45-53.
[20]O'Barr S1CooperNR1The C5a comp lement activation pep tide increases IL21beta and IL26 release from amyloid2beta p rimed human monocytes:imp lications for Alzheimer's disease[J].Neuroimmunol,2000,109(2):872-941.
[21]DeWitt DA, Perry G, Cohen M, et al.Astrocytes regulate microglial phagocytosis of senile p laque cores of Alzheimer's disease.Exp Neurol,1998,149 (2):329-401.
[22]Wallin AK, Blennow K, Andreasen N, et al. CSF biomarkers for Alzheimer's disease:levels of beta-amyloid, tau, phosphorylated tau relate to clinical symptoms and survival[J]. Dement Geriatr Cogn Disord,2006,21(1):131-138.
[23]Halliday G, Robinson SR, Shepherd C.Alzheimer's disease and inflam2 mation: a review of cellular and therapeutic mechanisms[J].Clin Exp Pharmacol Physiol,2000, 27(122):1281-1290.
[24]Floden AM, Li S, Combs CK. Beta-amyloid—stimulated microglia induce neuron death via synergistic stimulation of tumor necrosis factor alpha and NMDA receptors. J Neurosci,2005,25(10):2566—75.
[25]Liao H, Bu WY Wang TH, Ahmed S, et al. Tenascin-R plays a role in neuroprotection via its distinct domains that coordinate to modulate the microglia function. J Biol Chem,2005,280(9):8316-23.
[26]Von Bemhardi R. Glial cell dysregulation:a new perspective on Aizheimer disease. Neurotox Res,2007,12(4):215-32.
[27]Yang X, Wang J, Yang ES. Coenzyme Q1O attenuates beta-amyloid pathology in the aged transgenic mice with Alzheimer presenilin 1 mutation. J Mol Neurosci, 2008,34(2):165-71.
[28]Markesbery W.Oxidative stress hypothesis in Alzheimer's disease. Free Radio Biol Med,1997,23(1):134-47.
[29]Rosales Corral S, Tan DX, Reiter RJ,et al.Kinetics of the neuroinflammation-oxidative stress correlation in the brain following the injection of fibrillar amyloid-beta onto the hippocampus in vivo. J Neuroimmuno,2004,150(1-2): 20-8.
[30]Abramov AY,Canevari L,Duchen M. Changes in intracellular calcium and glutathione in astrocytes as the primary mechanism of amyloid neurotoxicity. J Neurosci,2003,23(12):5088-95.
[31]Zhou J, Fonseca MI, Pisalyaput K. Complement C3 and C4 expression in Clq sufficient and deficient mouse models of Alzheimer's disease. J Neurochem,2008,106(5):2080-92.
[32]Rosales,Corral S, Tan DX, et al. Kinetics of the neuroinflammation-oxidative stress correlation in the brain following the injection of fibrillar amyloid-beta onto the hippocampus in vivo. J Neuroimmunol,2004,150(1-2):20-8.
[33]Puig B, Gomez-lsla T, Ribe E,et al. Expression of stress-activated kinases c-Jun N-terminal kinase(SAPK / JNK-P)and p38 kinase(p38-P), and tau hyperphosphorylation in neurites surrounding betaA plaques in APP T92576 mice. Neuropathol Appl Neurobiol,2004,30(5):491-502.
[34]Yumi Y,Richard B G. Therapeutic potential of inhibition of the NF-κ B pathway in the treatment of inflammation and cancer [J]. J Clin Invest,2001,107(2):135-142.
[35]Young JS,Kyung SC,Hyun HC, et al.Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals:down-regulation of COX-2 and iNOS through suppression of NF-κ B activation [J].Mutat Res,2001,9 (480-481):243-268.
[36]陈云波,张大鹏,冯梅,等.人参皂苷Rg1对Aβ 25235诱导的神经细胞核因子2K B活化的影响[J].中国药理学通报,2007,23(5)：612-7.
[37]Li KYang L, Lindholm K, Konishi Y, et al.Tumor necrosis factor death receptor signaling cascade is required for amyloid-beta protein-induced neuron death. J Neurosci,2004,24(7):1760-71.
[38]Valerio A,Boroni F,Benarese M, et al. NF-kappaB pathway:a target for preventing beta-amyloid(Abeta)-induced neuronal damage and Abeta42 production. Eur J Neurosci,2006,23(7):1711-20.
[39]Rohn TT, Rissman RA, Davis MC, et al. Caspase—9 activation and caspase cleavage of tau in the Alzheimer'S disease brain[J]. Neurobiol Dis,2002,11(2): 341-354.
[40]Wyss-Coray T,Mucke L. Inflammation in neurodegenerative disease-a double-edged sword[J]. Neuron,2002,35(3):419-432.
[41]Walter Adriani, Elisa Ognibene, Emilie Heuland. Motor impulsivity in APP-SWE mice: a model of Alzheimer's Disease. Behavioural Pharmacology[J],2006,17:525-533.
[42]Eikelenboom P, Van Gool W,et al. Neuroinflammatory perspectives on the two faces of Alzheimer'S disease. Neural Transm.2004, 11:281-294.
[43]Lee sv,Lee JW,LeeH Yoo HS, et al. Inhibitory effect of green tea extract on beta-amyloid-induced PCI2 cell death by inhibition ofthe activation of NF-kappaB and ERK/p38 MAP kinase pathway through antioxidant mechanisms. Brain Res Mol Brain Res,2005,140(1-2):45-54.
[44]Yamaguchi Y,Kawashima S. Effects of amyloid—beta-(25-35)on passive avoidance, radial-armmaze learning and choline acetyltransferase activity in the rat. Eur J Pharmacol,2001,41,2(3):265—72.
[45]Yamada H, Aimi Y Nagatsu I, Arai R. Immunohistochemical detection of L— DOPA--derived dopamine within serotonergic fibers in the striatum and the substantia nigra pars reticulata in Parkinsonian model rats. Neurosci Res,2007,59(1):1-7.
[46]Townsend KP,Pratico D. Novel therapeutic opportunities for Alzheimer's disease:focus on nonsteroidal anti—inflammatory drugs. Faseb J,2005,19(12): 1592-601.
[47]Kuhad A,Bishnoi M, Tiwari,et al. Suppression of NF-kappabeta signaling pathway by tocotrienol Can prevent diabetes associated clgnitive deficits. Pharmacol Biochem Behav,2009,92(2):251-9.
[48]Etminan M, Gill S, Samii A. Effect of nonsteroidal anti-inflammatory drugs on risk of Alzheimer's disease:systematic review and meta-analysis of observational studies. BMJ,2003,327(7407):128-142.
[49]Szekely CA, Thome JE, Zandi PP, et al. Nonsteroidal anti-inflammatory drugs for the prevention of Alzheimer's disease:a systematic review. Neuroepidemiology, 2004,23(4):159-69.
[50]Zhou Y, Su Y, Li B, Liu F, et al. Nonsteroidal anti-inflammatory drugs can lower amyloidogenic A β 42 by inhibiting Rho. Science,2003,302:1215-1217.
[51]Mueller BK, Mack H, Teusch N. Rho kinase, a promising drug target for neurological disorders [J]. Nat Rev Drug Discov,2005,4:387-398.
[52]Tanaka T, Nishimura D, Wu RC, et al. Nuclear Rho kinase, ROCK2, targets p300 acetyltransferase [J]. J Biol Chem,2006,281:15320-15329.
[53]Ishizaki T. Rho-mediated signal transduction and its physiological roles [J]. Nippon Yakurigaku Zasshi,2003,121:153-162.
[54]Kioiler L, Hall A. Signaling to Rho GTPase[J].Exp Cell Res,1999,253(1): 166-179.
[55]Matozaki T. Nakanishi H. Takai Y. Small G-protein networks:their crosstalk and signal cascades[J]. Cell signal.2000,12(8):515-524.
[56]Cole GM, Morihara T, Lim GP,et al. NSAID and antioxidant prevention of Alzheimer's disease:lessons from in vitro and animal models. Ann N Y Acad Sci,2004,1035:68-84.
[57]Li KYang L, Lindholm K,et al. Tumor necrosis factor death receptor signaling cascade is required for amyloid-beta protein-induced neuron death. J Neurosci, 2004,24(7):1760-71.
[58]Valerio A,Boroni F,Benarese M, et al. NF-kappaB pathway:a target for preventing beta-amyloid(Abeta)-induced neuronal damage and Abeta42 production. Eur J Neurosci,2006,23(7):1711-20.
[59]Meffen Mk,Chang JM, wiltgen BJ, et al. NF-kappa B functions in synaptic signaling and behavior. Nat Neurosci,2003,6(10):1072-8.
[60]Zhang Z, Fauser U, Schluesener HJ. Dexamethasone suppresses infiltration of RhoA(+)cells into early lesions of rat traumatic brain injury. Acta Neuropathol,2008, 115(3):335-43.
[61]Conrad S, Schluesener HJ, Trautmann K, et al. Prolonged lesional expression of RhoA and RhoB following spinal cord injury. J Comp Neurol,2005,27,487(2): 166-75.
[62]Paintlia AS, Paintlia MK, Khan M, et al. HMCoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis[J]. Faseb J,2005,19:1407-1421.
[63]Sun X, Minohara M, Kikuehi H. The selective Rho-kinase inhibitor Fasudil is protective and therapeutic in experimental autoimmune[J]. J Neuroimmunol,2006, 180(1-2):126—34.
[64]Shema R, Sacktor TC, Dudai Y. Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKM zeta. Science,2007,317(5840):951-953.
[65]王晓明,赵晴,莽靖等.法舒地尔对慢性脑缺血大鼠认知功能及皮层自由基代谢的影响.中国老年学杂志.2008,10(28).
[66]Johansson R, Persson K. Phenotypic modulation of cultured bladder smooth muscle cells and the expression of inducible nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol,2004,286:R642-R648.
[67]Dobashi K, Araki S, Kubo K,et al. Hydroxymethylglutaryl-CoA reductase inhibitor inhibits induction of nitric oxide synthase in T3-L1 preadipocytes. Life Sci,2008,82:85-90
[68]Cory, S, Adams, J.M. The Bcl-2 family: regulators of cellular life-ordeath switch. Nat Rev Cancer,2002,2:647-656.
[69]Lud Cadet, Harrington B, Ordonez S. Bcl-2 overexpression attenuates dopamine-induced apoptosis in an immortalized neural cell line by suppressing the production of reactive oxygen species. Synapse,2000,35:228-233.
[70]Kirkland RA, Windelborn JA, Kasprzak JM,et al.Bax induced pro-oxidant state is critical for cytochrome c release during programmed neuronal death. Neurosci,2002, 22:6480-6490.
[71]Fukushima M, Nakamuta M, Kohjima M, et al. Fasudil hydrochloride hydrate, a Rho-kinase (ROCK) inhibitor, suppresses collagen production and enhances collagenase activity in hepatic stellate cells. Liver Int,2005,25(4):829-38.
[72]Del Re DP, Miyamoto S, Brown JH. RhoA/Rho kinase up-regulate Bax to activate a mitochondrial death pathway and induce cardiomyocyte apoptosis. J Biol Chem,2007,16,282(11):8069-78.
[73]Satoh S, Toshima Y, Ikegaki I,et al. Wide therapeutic time window for fasudil neuroprotection against ischemia-induced delayed neuronal death in gerbils. Brain Res,2007,1128(1):175-180.
[74]Leung T,Manser E,Tan L,et al.A novel serine/threonine kinase binding the Ras-related Rho A GTPase which translocates the kinase to peripheral membranes.J Biol Chem,1995,270:29051-29054.
[75]Ishizaki T. Rho-mediated signal transduction and its physiological roles[J].Nippon Yakurigaku Zasshi.2003,121:153-162.
[76]Dobashi K, Araki S, Kubo K,et al. Hydroxymethylglutaryl--CoA reductase inhibitor inhibits induction of nitric oxide synthase in 3T3--L1 preadipocytes. Life Sci,2008,82:85-90.
[77]Rikitake Y, Kim H, Huang Z,et al. Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection. Stroke,2005,36:2251-2257.
[78]Yamashita K, Kotani Y, Nakajima Y, et al. Fasudil, a Rho kinase (ROCK) inhibitor, p rotects against ischemic neuronal damage in vitro and in vivo by acting directly on neurons [J]. Brain Res,2007,1154:215-224.
[79]Yagita Y, Kitagawa K, Sasaki T, et al. Rho - kinase activation in endothelial cells contributes to expansion of infarction after for cerebral ischemia [J]. J Neurosci Res,2007,85 (11):2460-2469.
[80]Shin HK, Salomone S, Potts EM, et al. Rho-kinase inhibition acutely augments blood flow in focal cerebral ischemia via endo-thelialmechanisms [J]. J Cereb Blood Flow Metab,2007,27(5):998-1009.
[81]Yan Z, Yuan S, Baolin L, et al. Nonsteroidal Anti-Inflammatory Drugs Can Lower Amyloidogenic A β 42 by Inhibiting Rho[J]. Science,2003, 302(5648):1215-17.
[82]vramovich Y, Amit T, Youdim M B. Nonsteroidal Anti-inflammatory Drugs Stimulate Secretion of Non-amyloidogenic Precursor Protein[J]. J B iol Chem,2002, 277(35):31466-73.
[83]Weggen S, Eriksen J L, Das P, et al. A subset of NSA IDs lower amyloidogenic A β 42 independently of cyclooxygenase activity[J].N ature,2001,414(6860):212-6.
[84]Leo A,Berezovska O, HerlL, et al. Nonsteroidal anti-inflammatory drugs lower Abeta42 and change presenilin 1 conformation[J]. N atM ed,2004,10(10):1065-6.
[85]Persidsky Y, Heilman D, Haorah J, et al. Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis. Blood,2006,180:126-134.
[86]Greenwood J,Walters CE, Pryee G, et al. RhoA expression in EAE and MS.Blackwell Publishing Ltd. Neuropathology and Applied Neuroblology.2007, 34,231-240.
[87]Negishi M, Katoh H. Rho family GTPases as key regulators for neuronal network formation. J Bilchem Tokyo,2002,132:157-166.
[88]Schmandke A,Strittmatter SM. ROCK and Rho:biochemistry and neuronal functions of Rho-associated protein kinases. Neuroscientist,2007,13:454-469.
[89]Zhou Y,Su Y,Li B.et al.Nonsteroidal anti-inflammatory drugs can lower amyloidogenic by inhibiting Rho. Science,2003,302:1215-1217.
[90]Dubreuil CI, Winton MJ, McKerraeher L, et al. Rho activation patterns after spinal cord inj ury and the role of activated Rho in apoptosis in the central neurous system[J]. J Cell Biol,2003,162:233-36.
[91]Dergham P, Ellezam B, Essagian C, et al. Rho signaling pathway targeted to promote spinal cord repair[J]. J Neurosci,2002,22:6570-6573.
[92]Huentelman MJ,Stephan DA,et al.Peripheral Delivery of a ROCK Inhibitor Improves Learning and Working Memory.Behav Neurosci,2009,123(1):218-223.
[93]Shema R, Sacktor TC, Dudai Y. Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKM zeta. Science,2007,317(5840):951-953.
[1]Ishizaki T. Rho-mediated signal transduction and its physiological roles [J]. Nippon Yakurigaku Zasshi,2003,121:153-162.
[2]Kioiler L, Hall A. Signaling to RhoGTPase[J].Exp Cell Res,1999,253(1):166 —179.
[3]Mueller BK, Mack H, Teusch N. Rho kinase, a promising drug target for eurological disorders[J]. Nat Rev Drug Discov,2005,4:387-398.
[4]Etienne-Manneville S, Hall A. Rho GTPases in cell biology[J]. Nature,2002, 420(6916):629—635.
[5]Tanaka T, Nishimura D, Wu RC, et al. Nuclear Rho kinase, ROCK2, targets p300 acetyltransferase [J]. J Biol Chem,2006,281:15320-15329.
[6]Moon SY, Zang H, Zheng Y. Characterization of a brain-specific Rho GTPase-activating protein, p200 RhoGAP[J]. J Biol Chem,2003,278 (6): 4151—4159.
[7]Chang J, Xie M, Shah VR, et al. Activation of Rho-associated coiled-coil protein kinase-1 (ROCK-1) by caspase-3 cleavage p lays an essential role in cardiac myocyte apop tosis [J]. Proc Natl Acad Sci USA,2006,103:14495-14500.
[8]Sebbagh M, Hamelin J, Bertoglio J, et al. Direct cleavage of ROCK II by granzyme B induces target cell membrane blebbing in a caspase-independent manner[J]. J Exp Med,2005,201:465-471.
[9]Anwar KN, Fazal F, Malik AB, et al. RhoA/Rho-associated kinase pathway selectively regulates thrombin-induced intercellular adhesion molecule expression in endothelial cells via activation of I kappa B kinase beta and phosphorylation of RelA /p65 [J]. J Immunol,2004,173:6965-6972.
[10]TorsoniAS, Marin TM, Velloso LA, et al. RhoA/ROCK signaling is critical to FAK activation by cyclic stretch in cardiac myocytes [J]. Am J Physiol Heart Circ Physiol,2005,289:H1488-1496.
[11]Watanabe Y, Faraci FM, Heistad DD. Activation of Rho-associated kinase during augmented contraction of the basilar artery to serotonin after subarachnoid hemorrhage [J]. Am J Physiol Heart Circ Physiol,2005,288:H2653-2658.
[12]Shema R, Sacktor TC, Dudai Y. Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKM zeta. Science,2007,317(5840):951-953.
[13]Tamura M, Nakao H, Yoshizaki H, et al. Development of specific Rho2kinase inhibitors and their clinical application [J]. Biochim Biophys Acta,2005,1754:245-252.
[14]Chitaley K, Webb RC, Mills TM. The ups and downs of Rho-kinase and penile erection: up stream regulators and downstream substrates of Rho-kinase and their potential role in the erectile response [J]. Int J Impot Res,2003,15:105-109.
[15]Murata T, Arii S, Nakamura T, et al. Inhibitory effect of Y227632, a ROCK inhibitor, on p regression of rat liver fibrosis in association with inactivation of hepatic stellate cells [J]. J Hepatol,2001,35:474-481.
[16]NakajimaM, Hayashi K, Egi Y, et al. Effect of Wf2536, a novel ROCK inhibitor, against metastasis of B16 melanoma [J]. Cancer Chemother Pharmacol,2003,52: 319-324.
[17]Rikitake Y, Kim HH, Huang, ZH, et al. Inhibition of Rho Kinase (ROCK) Leads to Increased Cerebral Blood Flow and Stroke Protection. Stroke,2005,36(10):2251 2257.
[18]Shin HK, Salomone S, Potts EM, et al. Rho-kinase inhibition acutely augments blood flow in focal cerebral ischemia via endothelial mechanisms[J]. J Cereb Blood Flow Metab,2007,27(5):998-1009.
[19].Yamashita K, Kotani Y, Nakajima Y, et al. Fasudil, a Rho kinase (ROCK) inhibitor, protects against ischemic neuronal damage in vitro and in vivo by acting directly on neurons[J]. Brain Res,2007,1154:215-224.
[20]Dobashi K, Araki S, Kubo K,et al. Hydroxymethylglutaryl-CoA reductase inhibitor inhibits induction of nitric oxide synthase in 3T3--L1 preadipocytes. Life Sci,2008,82:85-90.
[21]Satoh S, Toshima, Y, Ilegaki I, et al. Wide therapeutic time windows for fasudil neuroprotection against ischemia-induced delayed neuronal death in gerbils[J]. Brain Res,2007,1128(1):175-180.
[22]Zhou Y,Su Y,Li B,Liu F,et al.Nonsteroidal anti-inflammatory drugs can lower amyloidogenic by inhibiting Rho. Science,2003,302:1215-1217.
[23]Schmandke A,Strittmatter SM.ROCK and Rho:biochemistry and neuronal functions of Rho-associated protein kinases. Neuroscientist,2007,13:454-469.
[24]Sbibuya M, Hirai S, Seto M, et al. Effects of fasudil in acute ischemic Stroke: results of a prospective placebo-controlled double-blind trial[J]. J Neurol Sci, 2005,238(1-2):31-39.
[25]Shin HK, Salomone S, Potts EM, et al. Rho-kinase inhibition acutely augments blood flow in focal cerebral ischemia via endo-thelialmechanisms [J]. J Cereb Blood Flow Metab,2007,27(5):998-1009.
[26]Yan Z, Yuan S, Baolin L, et al. Nonsteroidal Anti2Inflammatory Drugs Can LowerAmyloidogenic A β 42 by Inhibiting Rho[J]. Science,2003,302 (5648):1215-17.
[27]Avramovich Y, Amit T, Youdim MB. Nonsteroidal Anti-inflammatory Drugs Stimulate Secretion of Non-amyloidogenic Precursor Protein[J]. J B iol Chem,2002, 277 (35):31466-73.
[28]Weggen S, Eriksen J L. Das P, et al. A subset of NSA IDs lower amyloidogenic A β 42 independently of cyclooxygenase activity[J].N ature,2001,414 (6860):212-6.
[29]Lleo A,Berezovska O, HerlL, et al. Nonsteroidal anti2inflammatory drugs lowerAbeta42 and change p resenilin 1 conformation[J]. Nat Med,2004,10 (10): 1065-6.
[30]Huentelman MJ,Stephan DA,Talboom J,et al.Peripheral Delivery of a ROCK Inhibitor Improves Learning and Working Memory.Behav Neurosci,2009,123(1): 218-223.
[31]Shema R, Sacktor TC, Dudai Y. Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKM zeta. Science,2007,317(5840):951-953.
[32]Eva Cernuda-Morolln, Anne J. Ridley. Rho GTPases and leukocyte adhesion receptor expression and function in endothelial cells. Cire Res,2006,98:757-767.
[33]Waiczies S, Bendix I,Zipp F. Geranylgeranylation but not GTP-loading of Rho GTPases determines T cell function[J]. Sci Signal,2008, Mar,25,1(12):pt3.
[34]Z Zhang, J Schittenhelm, R Meyermann, et al. Lesional accumulation of RhoA+cells in brains of experimental autoimmune encephalomyelitis and multiple sclerosis. Neuropathology and Applied Neurobiology,2008,34:231-240.
[35]Paintlia AS, Paintlia MK, Khan M, et al. HMCoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis[J]. Faseb J,2005,19:1407.1421.
[36]Sun X, Minohara M, Kikuehi H. The selective Rho-kinase inhibitor Fasudil is protective and therapeutic in experimental autoimmune[J]. J Neuroimmunol, 2006,180(1-2):126-34.
[37]Vollmer T, Key L, Durkalski V, et al. Oral simvastatin treatment in relapsing. remitting multiple sclerosis[J]. Lancet,2004,363:1607-1608.
[38]Greenwood J,Walters CE, Pryce G, et al. RhoA expression in EAE and MS. Neuropathology and Applied Neurobiology,2007,34:231-240.