| Objective: It has been shown that Diabetes mellitus(DM)can cause cognitive impairment which is associated with learning and memory deficits,eventually develops dementia.The hippocampus is believed to function as learning and memory.Previous studies demonstrated cholinergic system and neuronal apoptosis in hippocampus are involved in cognitive dysfunction.The levels of ACh in cholinergic system are dependent on the balance of choline acetyltransferase(ChAT)and acetylcholinesterase(AChE)activity and its activities as a marker was to evaluate the function of the cerebral cortex and hippocampus.Caspase-3 is the most critical executioner molecules in the apoptosis.Bcl-2 is recognized as an inhibitor of apoptosis.L-3-n-butylphthalide(NBP)can improve cognitive function.However,so far,very few studies has been done to show the ameliorative effects of NBP on diabetes-associated-cognitive-declie(DACD).In this study,we sought to investigate the influence of NBP on the expression and activity of ChAT?AChE?Caspase-3 and Bcl-2 in hippocampal neurons of diabetic db/db mice treated with NBP and reveal the potential pathogenesis of DACD.Method:1 groups of animalsThe experimental group will consist of 3 groups of 8 mice(six weeks old)in each group(total 24 animals).Normal control group: eight male db/m mice(NC).Sixteen male diabetic db/db mice(six weeks old)were randomly divided into: diabetic control group(DM-C,n=8)and diabetic group administrated with NBP(DM-NBP,n=8).2 Drug interventionAfter maintaining one week,the mice in DM-NBP group were given with NBP dissolved in vegetable oil by a gavage of at a dose of 120mg/kg per dayfor six weeks.The NC group and DM-C group were fed with vegetable oil120mg/kg once a day for six consecutive weeks.3 General observationsGeneral conditions such as: mental status,fur color,food intake,water intake,urine volume of the mice were observed and recorded.The body weight and the blood glucose of the mice were detected per week.4 Ethological testAfter six weeks of NBP intervention,Morris water maze tests were performed to evaluate the spatial learning and memory ability of mice in each group.Morris water maze tests include place navigation test and space probe test.The place navigation test was used to test the escape latency of mice in the first five days.The number of crossing the platform of mice from different groups were recorded at the sixth day during the space probe test.5 ELISA,Real Time PCR and Western Blot assayAfter the space probe test,the mice were sacrificed and the hippocampal tissues from different groups were carefully removed.The levels of ChAT?AChE were determined by ELISA.The Real Time PCR were used to examine the expression of Caspase-3 and Bcl-2 mRNA.The levels of Caspase-3 and Bcl-2 protein were detected by Western Blot.6 Statistical analysesAll the experimental data were analyzed by SPSS 21.0 software and expressed as mean±SEM.The statistical significance of the differences were conducted using repeated measure ANOVA and One-way ANOVA.Comparisons between two groups were assessed using SNK.Statistical significance was considered as a level of P<0.05.Results:1 General observation(the body weight :g,the blood glucose :mmol/L)The mice from DM-C group and DM-NBP group showed sparse and lusterless fur,unresponsive,polyuria,polydipsia and polyphagia.However,the mice from NC group possessed good mental state and no symptoms of polyuria,polydipsia and polyphagia.After six-week NBP intervention,compared with NC group,the body weight and the levels of blood glucose from the DM-C group and DM-NBP group of mice were increased significiantly(the body weight : 58.06±2.38,57.34±2.17 vs 26.39±1.66;blood glucose: 20.94±1.71,20.03±1.66 vs 7.21±0.91,P<0.05).However,there was no significant difference in the body weight and blood glucose between DM-C group and DM-NBP group were observed(body weight: 58.06±2.38 vs57.34±2.17;blood glucose : 20.94±1.71 vs 20.03±1.66,P>0.05)(Table 1).2 Results of ethological test2.1 The basic swimming speed(cm/s)Before the beginning of Morris water maze test,all mice swam freely for60 s and the swimming speed were recorded.The results among the three groups were not significantly different(138.33±7.40 vs 134.70±8.28 vs 136.64±7.14,P>0.05)(Fig.1,Table 2).2.2 Results of the place navigation test(s)On the first day of the test,these were no statistically significant difference among three group in the escape latency(58.06±1.82 vs 56.57±2.85 vs 58.11±1.57,P>0.05).From the second day to the fifth day,the DM group showed longer escape latency than the NC group(P<0.05)(Fig.1,Table3).However,compared to DM-C group,DM-NBP group remarkably reduced the escape latency,but still longer than NC group(P<0.05)(Fig.2,Table 3).2.3 Results of the space probe test(times/60s)Even though the number of crossing the former platform were reduced in DM-C group compared with NC group(P<0.05).While,NBP intervention dramatically enhanced the number of crossing the platform in DM-NBP group compared with DM-C group,but still less than NC group(P<0.05)(Fig.3,Table 4).The numbers in each group crossing the platform: NC group>DM-NBP group>DM-C group,with statistical difference(7.00 ± 0.76 vs 5.13 ± 0.64 vs2.38 ± 0.52,P<0.05).3 Effects of NBP on hippocampal ChAT and ACh E(pg/ml)The levels of hippocampal ChAT were markedly declined in DM-Cgroup,compared with NC group(32.24±4.28 vs 104.21±4.50,P<0.05).NBP administration upregulated the expression of hippocampal Ch AT in DM-NBP group,compared with DM-C group(49.88±6.05 vs 32.24±4.28,P<0.05)(Fig.4,Table 5).The levels of hippocampal AChE was dramatically increased in DM-C group compared with NC group(108.92±5.64 vs 58.64±6.71,P<0.05).NBP treatment showed lower levels of AChE in DM-NBP group than those in DM-C group with statistical difference(83.70±4.95 vs 108.92±5.64,P<0.05)(Fig.5,Table 6).4 Effects of NBP on Caspase-3 and Bcl-2Compared with NC group,the increased expression of hippocampal Caspase-3 mRNA and protein,and downregulation of Bcl-2 mRNA as well as protein were observed in DM-C group(P<0.05).Interestingly,NBP intervention for 6 weeks significantly reduced expression of Caspase-3 mRNA and protein,concomitantly upregulated Bcl-2 mRNA and protein expression in DM-NBP group,and there was significantly statistical difference between DM-NBP group and DM-C group(P<0.05)(Caspase-3: Fig.6,Fig.8,Fig.10,Table 7,Table 9;Bcl-2: Fig.7,Fig.9,Fig.10,Table 8,Table 10).Conclusion1 The diabetic db/db mice developed significant decline in learning and memory ability at 13 weeks old.2 The expression of hippocampal AChE and Caspase-3 increased significantly;the level of hippocampal ChAT and Bcl-2 decreased markedly.These results indicated that impaired cholinergic system and increased apoptosis might contribute to the pathogenesis of DACD.3 NBP ameliorated cognitive impairment of the diabetic db/db mice.NBP could decreased the expression of hippocampal AChE and Caspase-3 and upregulated the expression of hippocampal ChAT and Bcl-2.We speculate that NBP can inhibit the occurrence of cognitive impairment by decreasing cholinergic system damage and apoptosis. |
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