| Objective: Vascular dementia (VD) refers to dementia caused by cerebralvascular lesions that result in brain damage. It is the second most commontype of dementia except for Alzheimer’s disease (AD).Pathogenesis is thoughtto be the targets of the cerebrovascular disease involving the frontal lobe,temporal lobe and the limbic system, or the enough capacity of brain tissue aredamaged, leading to seriously destruction of the higher cognitive function. Atpresent, study on molecular mechanism of VD has become a focus issue. Themolecular mechanism of VD includes excitatory amino acids toxicity,cholinergic defects, free radical damage, inflammation, nerve cells apoptosis,etc. In recent years, the relationship between nerve cells apoptosis and VD hasbeen payed more and more attention by scientists.c-Jun amino terminal kinase (JNK) is also called stress activated proteinkinase (SAPK), which is a member of the super family named the silkcracking original activated protein kinases (MAPKs). Large numbers ofexperiments suggest that JNK signaling pathway plays an important role incell proliferation, apoptosis, oxidative stress, and the development of a varietyof human diseases. Researches have shown that the application of specificJNK inhibitors can effectively block the JNK signaling pathway and regulateapoptosis effectively.Probucol is a kind of un-statin drug that regulates blood lipid level.Researches at home and abroad show that probucol has significant brainprotection effects in AD rats, while it is rarely reported in the treatmentagainst VD.This experiment aims to detect nerve cell apoptosis and JNK andp-JNK level changes in hippocampus of VD rats, to explore the role of JNKsignaling pathways in VD and the brain protection mechanism of probucol inVD rats, to provide experimental basis for the clinical treatment of VD and the clinical application of probucol.Methods: Healthy male Sprague-Dawley (SD) rats were randomlydivided into control group, model group, and probucol group, and there were10rats in each group. Vascular dementia rat model was established with themethod of permanent ligation of bilateral common carotid artery. The rats inprobucol group were intragastrically given probucol500mg/kg/d. While therats in control group and model group were given the same volume sodiumcarboxymethyl cellulose.8weeks after administration, Morris water mazeexperiment was used to evaluate learning and memory ability of each rat, andthen flow cytometry was used to assess the apoptotic rate of hippocampusnerve cells. Meanwhile, the expression levels of JNK and p-JNK proteins inthe hippocampus were examined by immunohistochemical staining methodand western blot method.Results:1Influence of probucol on ethology of VD rats1.1The results of escape latency periodWater maze navigation experiments showed that from the second day tothe end of the training, there was a downward trend of the escape latencyperiod in all the three groups of rats, indicating that training can enhance theability to learn. However, there were differences among the groups in theability of learning and memory (F=15.34, P＜0.05). Compared with thecontrol group, the escape latency periods of rats in model group and probucolgroup were significantly prolonged within the first4days (P＜0.05);Compared with model group, the escape latency period of rats in probucolgroup was significantly shortened (P＜0.05) from the second day to the fourthday, and there was no significant difference at the first day.1.2The results of crossing platform frequencyCrossing platform frequency which represent spatial memory ability ofrats was detected on the fifth day, the more the crossing platform frequencywas, the better the memory ability was. Compared with the control group,crossing platform frequency of model group rats decreased significantly (P＜ 0.05);Compared with model group, crossing platform frequency of probucolgroup rats increased significantly (P＜0.05); There was no significantdifference between control group and probucol group on crossing platformfrequency (P＞0.05). With training time and the numbers of training timesincreasing, the ways rats looking for the platform were changing and the timerats looking for platform was shortening, indicating that study and exercisecan strengthen the ability of spatial memory.2Influnce of probucol against VD rats on cells apoptosis in hippocampalCA1region：Cell cycle quantitative analysis by flow cytometry showed that there wasno obvious apoptosis peak in the control group, although a spontaneousapoptosis was found with a very low apoptosis percentage of only (5.06±2.52)%. Apoptosis rate of model group (11.27±2.67%) was significantlyhigher than that of the control group (P＜0.05); Apoptosis rate of probucolgroup (6.57±1.47)%decreased significantly than that of the model group (P＜0.05); Compared with control group, apoptosis rate of probucol groupincreased to some extent, but there was no statistical significance (P＞0.05).3Influence of probucol against vascular dementia rats on proteinexpression of JNK and P-JNK in rat’s hippocampus3.1The results of immunohistochemical stainingThe number of JNK and p-JNK positive cells in hippocampal CA1region of rats：p-JNK positive cells in the hippocampus were rarely found inthe control group and probucol group, meanwhile, much more p-JNK positivecells were present in model group, the cells were smaller and incoherent inhippocampus. Compared with control group, the number of p-JNK positivecells in hippocampal CA1region of rats in model group increasedsignificantly (P＜0.05).Compared with model group, the number of p-JNKpositive cells in hippocampal CA1region of rats in probucol group wassignificantly reduced (P＜0.05). There was no obvious difference of thenumber of p-JNK positive cells in hippocampal CA1region between probucol group and control group,(P＞0.05).Comparison among the three groups, noobvious difference was found on the number of JNK positive cells (F=0.54,P＞0.05).3.2The results of western blotThe protein expression level of p–JNK and JNK in the hippocampus ofrats：The protein expression level of p–JNK in the hippocampus of rats inmodel group increased significantly compared with control group(P＜0.05);the protein expression level of p–JNK in the hippocampus of rats in probucolgroup significantly decreased compared with model group (P＜0.05);compared with control group, the protein expression level of p–JNK in thehippocampus of rats in probucol group significantly increased too (P＜0.05).There were no significant differences among the three groups on the proteinexpression level of JNK in the hippocampus of rats.The comparison of p-JNK/JNK value among the three groups: The p-JNK/JNK value in the hippocampus of rats increased significantly in modelgroup compared to control group (P＜0.05)； compared with modelgroup,the p-JNK/JNK value in the hippocampus of rats decreasedsignificantly in probucol group (P＜0.05)；compared with control group, thep-JNK/JNK value in the hippocampus of rats in probucol group increasedslightly, but there was no statistical significance (P＞0.05).Conclusion:1. Probucol can reduce neuron cells apoptosis of the VD rat hippocampus,and improve their ability of learning and memory.2. p-JNK/JNK value of the VD rat hippocampus increased significantly,showing that JNK signaling pathway is involved in the pathogenesis of VDprocess.3. Probucol can reduce p-JNK/JNK value of the VD rat hippocampus,which suggests that inhibiting apoptosis caused by oxidative stress may be one of the mechanisms of improving spatial learning ability and memorydisorder of VD rats by Probucol. |
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