The Influences Of L-3-n-butylphthalide On Cognitive Damage And The Expression Of Ampar In Hippocampus Of Streptozotocin(STZ)-induced Diabetic Rats

Objective: Cognitive dysfunction is one of most challenging aspects of diabetes complication,whose main feature is the declined ability of learning and memory.Synaptic plasticity is the molecular basis of learning and memory,and AMPA receptors(AMPARs) are critically important in mediating the synaptic plasticity.Changes in AMPAR number and/or function is a core feature of learning and memory.L-3-n-butylphthalide(L-NBP)has been usd in ischemic cerebrovascular disease for its potent neuroprotective effects.The aim of this study was to observe the influences of L-NBP on cognitive damage and the expression of AMPAR in hippocampus of STZ-induced Diabetic Rats.Methods:1 Model and Drug administrationFifty-five healthy male Sprague-Dawley rats(120±10g)were divided into two groups:normal control group(NC, n=10) and diabetic group(n=45).To induce experimental diabetes, STZ was injected once intraperitoneally at a dose of 60 mg/kg to the fasting overnight mouse. Blood sample was collected from the tail vein,we chose the animals with the fasting PGL level >16.7 mmol/L 3 days after STZ treatment.Then the STZ-induced diabetes were randomly didided into three groups:STZ-diabetic control(DC, n=15);low L-NBP(DL, L-NBP 60 mg/kg perday, n=15)-treated STZ-diabetic;and high L-NBP(DH, L-NBP 120 mg/kg perday, n=15)-treated STZ-diabetes.L-NBP was dissolved in corn oil,DL and DH groups were given 60 and 120 mg/kg L-NBP respectively by oral gavage once a day for 5 consecutive days starting on Day 1. NC and DC rats received vegetable oil only.2 General observationsThe weight and blood glucose of the rats were monitored every week.At the same time,the general state of rats such as mental state,hair color,food intake and urine volume were recored also.3 Behavioral testsAll the rats accepted Morris water maze test at the end of 12 week.First,spatial training of the hidden platform in the water maze was performed for five consecutive days.For each training trial, the latency to escape was recorded.Then the spatial probe test was conducted immediately after the last trial on training day 5.In probe trial, the mice were allowed to swim for 60 s,and the number of crossing platform was recorded.4 Index detectionAfter the behavioral experiments,the hippocampus were rapidly removed and hematoxylineosin(HE)-staining for morphology observation;The expression of AMPAR m RNA was assessed by Real-Time PCR;Western-bolt and immunohistochemical staining were used to assessed the expressinon of AMPAR protein.5 Statistical analysisAll statistical analyses were used SPSS for Windows, version 13.0.The datas were analyzed by using One-way analysis of variance(ANOVA), Bonferroni and multibariate ANOVA.The results are expressed as mean +S.E.M.Statistical significance was accepted at P <0.05.Results: 1 General observationThe rats of NC group appeared good condition,sensitive reaction,white and gossy fur,gain steady growth.While the DC group rats were slow to respond,polydipsia,polyphagia,yellow and Iusterless fur.In the process of intervention,the general conditions of DH group were better than DL group,but still worse comparied with NC group.Before being injected STZ,the blood glucose level in NC group and DC group were normal(7.20±0.54 vs 7.19±0.68, P>0.05);the body weight also hadAt the 12 week after being injected STZ, the blood glucose level in NC group increased significantly compared to DM group(P<0.01);the body weight decreased significantly(P<0.01).But among the subgroups in DM group,there had no significance in blood glucose and body weight(P>0.05).2 Behavioral tests 2.1 Basic swimming speedIn order to eliminate the individual differences of rats, test the basic swimming speed,a 60-second free swim trial was run before the training.The result showed that there was no statistical significance in swimming speed among each group(P>0.05). 2.2 Spatial learning trainingIn the first day,there was no obvious difference among groups.From day2 to day5,the escape latency of DC,DL and DH were extended compared with NC(P<0.05),which demonstrated that diabete rats had cognitive dysfunction at the end of 12 w,and the escape latency of DL and DH obviously shortened than DC(P<0.05).From day3 to day5,we observed that DH significantly lower than DL in escape latency(P<0.05)In addition to DC group,NC,DL and DH had a significant changes in the escape latency from the fifth day to the first day(P<0.05).The trend is Day1>Day2>Day3>Day4>Day5. 2.3 Probe testThe l-NBP-treated rats have more times than DC rats(P<0.05; one-sample t test),but still have few times than NC rats(P<0.05; one-sample t test). Compared with DL group,the DH group showed better(P<0.05; one-sample t test). 3 HE stainingIn NC group,no pathological abnormalities were observed in the hippocampal CA1 area, the cells showed normal structure,arranged in rows and clear in layers,cell nucleus was large and round.In DC group,the cells become disorganized in rows and vaguer in layers,cell loss,nuclei shrinkage, cerebral edema and dark staining of neurons were seen.Administration of l-NBP can attenuated the neuronal damage,and DH wan better than DL. 4 RT-PCRCompared with NC group,the AMPAR m RNA were significantly decre- ased in DC,DL and DH(P<0.05).While,L-NBP treated groups were better than DC group in AMPAR m RNA,and DH group significantly increased AMPAR m RNA in the hippocampus. 5 Western blotCompared with NC group,declined expression of AMPAR protein were observed in hippocampal CA1 regions in the DC,DL and DH groups(P<0.05).Repeated administration of 60 and 120 mg/kg L-NBP significantly enhanced the leval of AMPAR protein, especially in 120 mg/kg L-NBP- treated group(P<0.05). 6 Immunohistochemical stainingThe number of AMPAR were rare in the hippocampus of rats in DC group. However, in L-NBP treated groups,AMPAR were markedly increased in the hippocampus, and administration of 120 mg/kg L-NBP was more prominent.Conclusion:1 STZ-induced diabetic rats developed cognitive dysfunction at 12-week point, had significant decilned learning and memory function.Meanwhile the expression of AMPAR was decreased.2 Butylphthalide could improve learning and memory function in diab- etic rats,upregulate AMPAR expression,and prevent diabetic cognitive dysfuncition.And the preventive effect of high dose was better than the low dose.