Neuroprotective Effects And Mechanisms Of Rho Kinase Inhibitor Fasudil On Aβ-induced AD Rat

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 experimentIn 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 analysis2.1 HE stainingAfter 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 stainingCompared 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 microscopyThe 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 hippocampalTUNEL 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 regionThe 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 tissueThe 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.