The Influences Of L-3-n-Butylphthalide On Cognitive Damage And The Synaptic Plasticity Related Signaling Pathway Of Db/db Mice

Objective: Diabetes mellitus(DM) can cause dementia and cognition dysfunction, but the specific mechanism is not clear. Hippocampal synaptic plasticity is considered to be the basis in neural biology of learning and memory. LTP is an important index to evaluate the plasticity of synaptic transmission in the hippocampus. Reports specify that diabetes prevent enhancements of synaptic transmission and reduce the magnitude of LTP in the the hippocampus. The molecular mechanisms of LTP induction and formation can be summarized as: release of glutamate, activation of the NMDA glutamate receptors, Ca2+ entry, and activations CaMKⅡ.Consequently, AMPA glutamate receptors were activated by phosphorylation by CaMKⅡ, the conductance enhances and LTP produces. L-NBP showed potent neuroprotective effects by improving microcirculation, decreasing inflammatory response, protecting mitochondria function and has been widely used in the treatment of ischemic stroke. In addition, studies have showed that L-NBP also can improve diabetic cognitive deficits. In the present study, we investigated the effect of L-NBP treatment on cognitive impairment and the expression of hippocampus NMDAR-CaMKⅡ-AMPAR in db/db mice.Methods:1 GroupsSixteen healthy male db/db mice(38g~42g) were randomly divided into two groups: diabetic control group(DM, n=8) and L-NBP group(DM-NBP,n=8). Same ageing male lean and nondiabetic db/m mice(17g~19g) were used as control group(NC, n=8).2 Drug interventionsAfter adaptive feeding about one week, DM-NBP group was given L-NBP dissolved in vegetable oil by oral gavage once a day at a dose of 120 mg/kg body weight. NC and DM group received vegetable oil only.Treatment was started when the mice were 7 weeks old and continued for 6 weeks.3 General observationsThe food intake, fur color, urine volume and mental state of mice were observed and recorded. Meanwhile, the weight and blood glucose of mice were detected weekly.4 Behavioral testsEach group was tested in Morris water maze(MWM) at the end of 6 weeks of drug treatment. The water maze test consists of place navigation test and space probe test. The place navigation test was performed for five consecutive days and the escape latency was recorded. Then the number of crossing the platform during the free swimming was recorded in the space probe test.5 Electrophysiological testsThe day after finishing MWM test, mice were given LTP test. First, the anesthetic mice were placed in a stereotaxic frame. Then the recording and stimulating electrodes were lowered into PP and DG regions, respectively.The amplitude of PS were measured for the pre- and post-high frequency stimulation.6 Index detectionAfter the electrophysiological experiment, the hippocampi of mice were rapidly removed. The expression of NMDAR2 B, CaMKⅡ and AMPAR1 mRNA were assessed by Real-Time PCR; Western-bolt was used to assessd the expressinon of NMDAR2 B, CaMKⅡ and AMPAR1 protein.7 Statistical analysesAll data were presented as mean±SEM. The analyses were performed using SPSS 13.0 software. Escape latencies in place navigation were compared using repeated measure ANOVA. Data for spatial probe and LTP recording were compared using one-way ANOVA. Comparisons beween means were assessed using S-N-K. Significance levels were established at alevel of P<0.05.Results:1 General observationThe mice of NC group appeared good mental state, sensitive reaction and bright fur. The drinking, eating and urine were normal. Nevertheless, the mice of DM group and DM-NBP group gradually showed unresponsive, sparse fur,polyuria, polydipsia and polyphagia.After 6 week of drug treatment, the blood glucose level and body weight of mice were measured. The blood glucose level and body weight in DM and DM-NBP group were increased significantly compared to NC group(P<0.05);But DM and DM-NBP group had no significance in blood glucose and body weight(P>0.05).2 Behavioral tests2.1 Spatial learning trainingOn day1, each group was no obvious difference in the escape latencies(P>0.05).From day2 to day5: the escape latency of NC group were less than the DM group and DM-NBP group, and the difference was statistically significant(P<0.05). Compared with the DM group, the DM-NBP group was less in escape latency(P<0.05).2.2 Probe testCompared with the NC group, the numbers of DM group were decreased in 60 s of crossing platform(P<0.05).The number in 60 s of crossing platform of DM-NBP group were more than the DM group, but still less than the NC group(P<0.05).The order of the numbers: NC group >DM-NBP group >DM group.2.3 Basic swimming speedBefore the water maze, a 60-second free swimming test was carried out.The result showed that DM group and DM-NBP group were no statistical difference in swimming speed(P>0.05).3 Electrophysiological testsThe amplitude of PS of DM group was lower than NC group(P<0.05).Compared with the DM group, DM-NBP was high in the amplitude of PS and the difference was statistically significant(P<0.05).The order of PS amplitude: NC group >DM-NBP group >DM group.4 RT-PCR4.1 The expression of hippocampal NMDAR2 B mRNA in DM group was decreased than NC group(P<0.05). The expression of mRNA was higher in DM-NBP group than DM group, and the difference was statistically significant(P<0.05).4.2 The expression of hippocampal CaMKⅡ hjgjkmRNA in DM group was decreased than NC group(P<0.05). The expression of mRNA was higher in DM-NBP group than DM group, and the difference was statistically significant(P<0.05).4.3 The expression of hippocampal AMPAR1 mRNA in DM group was decreased than NC group(P<0.05). The expression of mRNA was higher in DM-NBP group than DM group, and the difference was statistically significant(P<0.05).5 Western blot5.1 The expression of hippocampal NMDAR2 B protein in DM group was lower than that in NC group(P<0.05). Compared with DM group, the increased protein expression were observed in DM-NBP group, and the difference was statistically significant(P<0.05).5.2 The expression of hippocampal CaMKⅡ protein in DM group was lower than that in NC group(P<0.05). Compared with DM group, the increased protein expression were observed in DM-NBP group, and the difference was statistically significant(P<0.05).5.3 The expression of hippocampal AMPAR1 protein in DM group was lower than that in NC group(P<0.05). Compared with DM group, the increased protein expression were observed in DM-NBP group, and the difference was statistically significant(P<0.05).Conclusion:1 The expression of hippocampal NMDAR-CaMKⅡ-AMPAR and LTP were decreased in 13 weeks of age diabetic db/db mice, which were consistent with the decline of learning and memory. Those suggested that the lower expression of NMDAR-CaMKⅡ-AMPAR and impaired LTP might participate in the pathogenesis of diabetic cognitive dysfunction.2 Application of butylphthalide can increase the expression of hippocampal NMDAR-CaMKⅡ-AMPAR and enhance the LTP level. These further improve cognitive dysfunction in diabetic db/db mice.