| Objective: Diabetic cognitive dysfunction is such a disease that affectsthe ability of learning and memory, eventually ends in dementia.Itspathogenesis has not been elucidated, there are no effective measures that canprevent and treatment. Synaptic is the base of the structure and function ofnervous system, which is also key for information passage among neurons.The plasticity of synaptic is closely correlated with memory and learningability. MAPK/ERK signaling plays a key role in the plasticity of hippocampal,which is a highly conserved three kinase cascade signaling, that isRas/MEK/ERK, among which MEK1, an important component in the pathway,is required for activation of ERK. The present study focuses on the changes ofMEK1in hippocampal area of diabetic cognition disabled rat and the impactof Butylphthalide on cognitive function, possibly finding new target ofButylphthalide, which may be of great theoretical and practical importance forthe prevention and cure of diabetic cognition dysfunction.Methods: Healthy male Sprague-Dawley （SD） rats were treated withintraperitoneal injection of streptozotocin （STZ）（60mg/kg）. Three days later,those with blood glucose of the caudal vein above16.7mmol/L wereconsidered as the successful models. Then the diabetic rats were randomlydivided into3groups: diabetic control group, low-dose Butylphthalide grouptreated with Butylphthalide(60mg.kg-1.d-1), high-dose Butylphthalide grouptreated with Butylphthalide(120mg.kg-1.d-1). The treatment of intervention waslasted for12weeks. Body weight and blood glucose were measured weekly,during which those blood glucose of the caudal vein below16.7mmol/L wereeliminated from the study. At the end of the study, Morris water maze test wasexecuted respectively, after which body weight and blood glucose weremeasured.Then the rats were anesthesiaed subsequent to sacrifice and hippocampus of the cerebrum were kept in liquid nitrogen. The morphology ofthe hippocampus were examined with HE staining. RT-PCR and Western-blotwere employed to examine the expression of MEK1and p-MEK1levelrespectively.Results:1General stateThe body weight of rats in normal control group grew steadily, in goodspirit, and the color of fur was normal.The diabetic rats appeared marasmus,cachexia, yellow and lacklustre fur. The urine output, food intake and waterintake of diabetic rats were significantly increased.2The changes of blood glucose and body weight levelsBefore the injection of STZ, there were no significant differences inblood glucose between normal control group and diabetic model group（7.19±0.92mmol/L vs7.34±0.77mmol/L, P>0.05）. There were no significantdifferences in body weight between normal control group and diabetic modelgroup（148.06±7.53g vs149.28±4.29g, P>0.05）. At week6W and12W afterSTZ injection,compared with normal control group, the blood glucose wassignificantly higher in diabetic control group, low-dose Butylphthalide groupand high-dose Butylphthalide group（P<0.01）. The body weight wassignificantly lower compared with coeval normal control group（P<0.01）.However, there were no significant differences in blood glucose,body weight between diabetic control group, low-dose Butylphthalide groupand high-dose Butylphthalide group （P>0.05）.3Results of Morris water tests3.1Results of rats’ basic swimming speed testsBefore the Morris water tests, five rats of each group were randomlyselected from the normal control group, diabetic control group, low-doseButylphthalide group, and high-dose Butylphthalide group to swim60s freelyto test the basic swimming speed. The results showed that the swimmingspeed of rats between four groups was not significantly different（28.23±1.64cm/s,29.79±2.34cm/s,30.10±3.19cm/s vs30.77±2.60cm/s, P> 0.05）, so the experiment does not consider the contrast of rats’ individualswimming speed.3.2Results of Place navigation testsCompared with normal control group, the escape latency wassignificantly longer in diabetic control group, low-dose Butylphthalide groupand high-dose Butylphthalide group（P<0.05）, this reslut suggested there wascognitive impairment in diabetic rats. Rats in diabetic control group spentmuch longer time to find the platform than those in low-dose Butylphthalidegroup and high-dose Butylphthalide group（P<0.05）.There were no significantdifferences in escape latency between low-dose Butylphthalide group andhigh-dose Butylphthalide group in the first and second day;but from the thirdto fifth day, rats in high-dose Butylphthalide group spent much shorter time tofind the platform than those in low-dose Butylphthalide group（P<0.05）. Fivedays consecutive tests suggested the escape latency of four groups weregradually reduced, the frist day> the second day> the third day> the forthday> the fifth day, there were statistically significant differences（P<0.05）.3.3Results of Spatical probe testsCompared with normal control group, the times of rats crossing theplatform were markedly reduced in diabetic control group, low-doseButylphthalide group and high-dose Butylphthalide group（3.17±0.61,9.17±1.72,12.67±1.78vs16.33±2.71, P<0.05）; Compared with diabeticcontrol group,the times of rats crossing the platform were markedly increasedin low-dose Butylphthalide group and high-dose Butylphthalidegroup（P<0.05）; Compared with low-dose Butylphthalide group,the times ofrats crossing the platform were markedly increased in high-doseButylphthalide group（P<0.05）.4HE stainingIn normal control group, cells of hippocampal CA1region lined up inorder,neuronal nucleus were large and round. Neurons of the hippocampalCA1in diabetes control group rats arranged disorder, the number reduced. Wefound the neurons were coloring deepened. Some neurons a qualitative nuclear ambiguity, even vacuolar degeneration. The change of Butylphthalide groupwas significantly reduced compared with the diabetic group, and120mg.kg-1Butylphthalide was better than60mg.kg-1Butylphthalide.5Immunohistochemical staining resultsCompared with normal control group, the expression of p-MEK1proteinwas significantly decreased in the hippocampus in diabetic control group,low-dose Butylphthalide group and high-dose Butylphthalide group （theaverage optical density value:0.14±0.00,0.25±0.02,0.34±0.02vs0.41±0.02, P<0.05）. Compared with diabetic control group, the expression ofp-MEK1protein was significantly increased in the hippocampus in low-doseButylphthalide group and high-dose Butylphthalide group（P＜0.05）. Theexpression of p-MEK1protein in high-dose Butylphthalide group was highlyincreased than low-dose Butylphthalide group（P＜0.05）.6Results of gene expressionCompared with normal control group, the expression of MEK1mRNAwas significantly decreased in the hippocampus in diabetic control group,low-dose Butylphthalide group and high-dose Butylphthalide group （0.16±0.00,0.25±0.00,0.33±0.01vs0.40±0.01, P＜0.05）. Compared withdiabetic control group, the expression of MEK1mRNA was significantlyincreased in the hippocampus in low-dose Butylphthalide group and high-doseButylphthalide group（P＜0.05）. The expression of MEK1mRNA in high-doseButylphthalide group was highly increased than low-dose Butylphthalidegroup（P＜0.05）.7Results of protein expressionCompared with normal control group, the expression of p-MEK1proteinwas significantly decreased in the hippocampus in diabetic control group,low-dose Butylphthalide group and high-dose Butylphthalide group （0.21±0.00,0.41±0.00,0.52±0.01vs64±0.01, P＜0.05）. Compared with diabeticcontrol group, the expression of p-MEK1protein was significantly increasedin the hippocampus in low-dose Butylphthalide group and high-doseButylphthalide group（P＜0.05）. The expression of p-MEK1protein in high-dose Butylphthalide group was highly increased than low-doseButylphthalide group（P＜0.05）.Conclusion:1At week12, diabetic rats occurred significant decline in learning andmemory function, and the expression of p-MEK1in the hippocampusdecreased. This suggested that the reduction of the expression ofp-MEK1could be a potential mechanism of diabetic cognitivedysfunction.2Butylphthalide could increase the expression of p-MEK1in thehippocampus, improved learning and memory, and prevented the ratsfrom getting cognitive impairment. It suggested that Butylphthalidemight increase the expression of p-MEK1hippocampal, then activatedMAPK/ERK signaling pathway.3In addition, the results showed that, the prevention of120mg.kg-1Butylphthalide is better than that of60mg.kg-1. |
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