Effects Of L-3-n-butylphthalide On Cognitive Dysfunction And NR2B-containing NMDA Receptors Expression In Hippocampus Of STZ-induced Diabetic Rats

Objective: Diabetes mellitus is a known cause of cognitive dysfunctionthat involves a decline of learning and memory, eventually ends in dementia.Studies have shown that NR2B subunit of the N-methyl-D-aspartate receptor(NMDAR), which is ionic glutamate receptor, has been proved to be animportant factor for cognitive function.L-3-n-Butylphthalide (NBP) is a nerve protective drug, which canimprove microcirculation, protect mitochondria and has been widely used inclinical treatment of ischemic cerebrovascular diseases, meanwhile it canimprove cognitive dysfunction induced by diabetes mellitus, but itspathogenesis is not well-established. The aim of this study was to investigatethe preventive effects of NBP on cognitive dysfunction and NR2B expressionin hippocampus of STZ-induced diabetic rats, which may provide aninteresting direction for diabetic cognitive dysfunction in the future.Method:1STZ-induced diabetic rat modelSingle intraperitoneal injection (IP) of streptozotocin (60mg/kg) wasused for induction of diabetes in male Sprague Dawley (SD) rats. The controlgroup received the equal volume of citrate buffer solution by intrapertonealinjection. The induction of diabetes was confirmed after72h by monitoringthe blood glucose of tail-vein sampling. The rats with blood glucose above16.7mmol/L were enrolled in the T1DM group.2GroupsAfter adaptive feed about one week, fifty-five SD rats were randomlydivided into the normal control group (NC, n=10) and the diabetes mellitus group (DM, n=45); DM group again were randomly divided into3groups: i)diabetic control group (DC, n=15), ii) diabetic+low-dose NBP group (DL,administered NBP60mg/(kg· d), gavage for12weeks, n=15), iii) diabetic+high-dose NBP group (DH, administered NBP120mg/(kg· d), gavage for12weeks, n=15). NBP was dissolved with corn oil. The rats of NC and DC groupwere given the same volume corn oil for12weeks.3General observationsThe body weight and the blood glucose of the rats were monitored onceevery week. General situations of rats were observed during the research, suchas mental state, food intake, water intake, urine volume, etc.4Behavioral testsAll rats accepted the Morris water maze test at the12week (w) point,including place navigation tests and space probe tests. The escape latency andthe number of crossing platform were recorded in order to analyzing learningand memory ability of different group rats.5Morphology and observation indicatorsThe rats were sacrificed after Morris water maze test and thehippocampus tissues were removed for biochemical assays. Morphology ofthe hippocampus tissue was observed by hematoxylin-eosin staining (HE), theexpressions of NR2B mRNA and protein were assessed by Real-Time PCR、Western-blot and immunohistochemical staining(IHC).6Statistical analysisAll analyses were performed using SPSS for Windows, version13.0,which includes One-way analysis of variance (ANOVA). In the results ofMorris water maze, we use repeated-measure analysis of variance. Themethod of Bonferroni should be used to do pairwise comparisons of therepeatedly measured data in different measurement time of each treated group.With multivariate ANOVA, data in different treated group of eachmeasurement time could be compared pairwise. As the data passed thenormality test (P>0.10), the results were expressed as the means±standarddeviation. P<0.05was considered to indicate a statistically significant difference.Results:1General situationThe rats of NC group: good mental state, agility, white and glossy fur,drinking, eating and urine volume were normal; the rats of DM group: poormental state, depression, irritability, yellow, sparse and lusterless fur,polyphagia, polydipsia, polyuria.Compare blood glucose and body weight of different groups before beingmade into model and at the end of12w:Before being made into model: there were no significant differences inthe blood glucose (mmol/L) between the NC group and the DM group(7.19±0.54VS7.18±0.46, P>0.05); the body Weight (g) change also had nostatistical significance(130.46±4.98VS132.08±4.36，P>0.05).At the end of12w:Compared to NC group, the blood glucose of DM group increasedsignificantly (30.13±2.08,29.35±2.57,28.18±2.19VS7.44±0.47, P<0.01) andthe body weight decreased significantly (237.77±14.87,242.73±14.75,245.00±13.07VS340.14±6.65, P<0.01); while among subgroups of DMgroup, the blood glucose and body weight had no statistical significance(P>0.05).2Behavioral observations (Morris water maze)2.1The basic swimming speedIn order to exclude individual swimming speed difference, we randomlyselected from five rats of each group to swim60s freely to test the basicswimming speed. The result showed that the swimming speed of rats amongfour groups was not significantly different (30.97±1.96,30.73±2.04,29.72±2.95and30.00±2.36, P>0.05).2.2place navigation testsOn the first day, there were no significant differences in the escapelatencies between DM group and NC group (P>0.05);From the second day to the fifty day, the escape latencies of DL group and DH group were significantly shorter than DC group, but were still longerthan NC group (P<0.05). Meanwhile, the escape latency of DH group wassignificantly shorter than DL from the third to fifth day (P<0.05). There wasno interaction between the days and the groups (P>0.05).The escape latency variations from the fifth day to the first day: DCgroup, no significant changes in the escape latency (P>0.05); NC group, DLgroup and DH group were significantly shorter in the escape latency (P<0.05).The order of the escape latency each group: NC group<NH group<DLgroup<DC group.2.3space probe testsCompare with NC group, the numbers of rats crossing the platform weremarkedly reduced in DC group (3.00±0.64VS15.68±1.57, P<0.05).Compare with DC group, the numbers of rats crossing the platform weresignificantly increased in NBP-treated groups (9.01±1.1and11.68±1.57);however compare with NC, the numbers were still markedly reduced(P<0.05).Compare with DL group, the numbers of rats crossing the platform weresignificantly increased in DH group, with statistical difference (P<0.05).The order of the numbers of rats crossing the platform: NC group>NHgroup>DL group>DC group.3HE stainingIn NC group, the hippocampal pyramidal cells were arranged in rows andclear in layers, and its structure was integrated, cell nucleus was large andround. On the contrary, the hippocampal pyramidal cells of DC group becamedisorganized in rows and vaguer in layers, with membrane rupture, decreasingof endochylema, and karyopycnosis, even vacuolar degeneration. The changeof HE staining in NBP-treated groups were significantly reduced compare toDC group and DH group was superior to the DL group.4The expression of NR2B mRNA and protein in hippocampus4.1The expression of NR2B mRNA in hippocampus Compare with NC group, the expressions of NR2B mRNA in DC, DLand DH group were significantly declined (0.14±0.01,0.23±0.01,0.29±0.01VS0.35±0.02, P<0.05).Compare with DC group, the expressions of NR2B mRNA inNBP-treated groups were significantly increased (P<0.05).Compare with DL group, the expression of NR2B mRNA in DH groupwas significantly increased (P<0.05).4.2The expression of NR2B protein in hippocampusCompared with NC group, the expression of NR2B protein wassignificantly decreased in the hippocampus in DC, DL and DH groups(0.37±0.03,0.49±0.04,0.60±0.04VS0.68±0.05, P<0.05).Compared with NC group, the expressions of NR2B protein inNBP-treated groups were significantly increased (P<0.05).Compared with DL group, the expression of NR2B protein in DH groupwas also significantly increased (P<0.05).5Immunohistochemical stainingCompared with NC group, the expression of NR2B positive cells weresignificantly decreased in the hippocampus in DC group (the average opticaldensity value:0.16±0.01VS0.47±0.10, P<0.05).Compared with NC group, the expression of NR2B positive cells inNBP-treated groups were significantly increased (the average optical densityvalue:0.27±0.11,0.37±0.14VS0.47±0.10, P<0.05), but was stillsignificantly decreased compare to NC group (P<0.05).Compared with DL group, the expression of NR2B positive cells in DHgroup was also significantly increased, with Statistical significance (P<0.05).Conclusions:1STZ-induced diabetic rat caused learning and memory deficits at theend of12week.2The expressions of NR2B mRNA and protein in the hippocampus werealso decreased in STZ-induced diabetic rats at the end of12week, inaccordance with the cognitive dysfunction. 3NBP could improve cognitive impairment and increase the expressionof NR2B, which speculated that NBP might prevent the rats from gettingcognitive impairment by up-regulating NR2B expression, and the preventiveeffect of120mg/kg is better than that of60mg/kg.