| Objective: The Diabetic encephalopathy have been widely studied.It was characterized by cognitive deficits.Studies have shown that oxidative stress is a critical factor in the progress of the cognitive function.NADPH oxidase(NOX)played an central role in oxidative stress.SOD and MDA are important indicators of the level of oxidative stress.L-3-n-Butylphthalide(NBP)can improve cognitive dysfunction induced by diabetes mellitus,but its mechanism is not well established.The aim of this study is to observe the effect of NBP on diabetic db/db mice behavior,SOD and MDA content,the subunit p67 phox of NADPH oxidase and the subunit gp91 phox.Method:1 Group6-week old male db/m mice were used as normal control group(NC,n =8).16 male db/db mice,were randomly divided into diabetic disease control group(DM-C,n = 8)and diabetic NBP group(DM-NBP,n = 8).2 Drug Intervention After raising a week,the DM-NBP group was fed with NBP,which dose was 120mg/Kg/d.The NC group and DM-C group were given the same amount of vegetable oil,the intervention last for 6 weeks.3 General observations The body weight and the blood glucose of the mice were monitored once every week.General situations of mice were observed during the research,such as mental state,food intake,water intake,urine volume,etc.4 Behavioral tests After six weeks’ drug intervention,these mice were taken in water maze test for 5 days persistently for spatial training.At the sixth day for spatial probe test.The escape latency and the number of times of crossing the platform were recorded.5 ELISA,Western-Blot,Real Time PCR detection After the water maze experiment,these mice were killed.The hippocampal tissue samples was prepared.ELISA was applied to detect the SOD and MDA content.The expression of the subutnit p67 phox and gp91 phox were detected by Real Time PCR and Western-Blot.6 Statistical analysis The experimental data using SPSS 21 statistical software for statistical analysis.The results were expressed as the mean±standard deviation.One-way analysis of variance(ANOVA)and repeated-measure analysis of variance of completely random design were used for data analysis.The SNK-q test was used in the comparison between different two groups.Statistical significance was set at P<0.05.Result:1 Generally observation The NC group: the mice were with good mental state,glossy fur.The drinking,eating and uresis were normal.The DM-C group and DM-NBP group: the mice were accompanied with polyuria,polydipsia,polyphagia,in low spirits,sparse and lusterless fur.In a process of intervention,the DM-NBP group was superior to DM-C group in generally situation.After 6 weeks of intervention,the results of body weight(g)shown that,compared to NC group(26.39±1.66),the DM-C group and DM-NBP group were markedly higher(respective 58.06±2.38,57.34±2.17,P<0.05).While there were no statistical significance between the DM-C group and DM-NBP group(P>0.05).After 6 weeks of intervention,the blood glucose(mmol/L)of mice shown that,compared to NC group(7.21±0.91),the DM-C group and DM-NBP group were markedly higher(respective 20.94±1.71,20.03±1.66,P<0.05).While there were no significant differences between DM-C and DM-NBP group(P>0.05). 2 Behavioral investigation: Morris water maze2.1 Measured of basic swimming speed(cm/s)To exclud individual differences,ahead of the experiment started,mice in each group swam freely for 60 s.The basic swimming speed shown that there was no significant difference between NC group(138.33±7.40),DM-C group(134.70±8.28)and DM-NBP group(136.64±7.14)(P>0.05).2.2 Escape latency(s)At the first day,there was no significant difference between each group(P>0.05).At the following days,the escape latency of DM-C group and DM-NBP group were significantly longer than the NC group(P<0.05).While the DM-NBP group was significantly shorter than the DM-C group(P<0.05).The escape latency in each group were gradually shortened as time went on: Day 1> Day 2> Day 3> Day 4> Day 5.2.3 Probe test:The number of crossing the platform(times/60s)Compared to NC group(7.00±0.76),the DM-C group(2.38±0.52)and DM-NBP group(5.13±0.64)were significantly decreased(P<0.05).While the DM-NBP group was significantly increased compared to DM-C group(P<0.05).3 Real Time PCR results The subunit p67 phox of NADPH oxidase expression: Compared to NC group(0.686±0.015),the DM-C group and DM-NBP group were obviously higher(respective 1.003±0.026,0.802±0.023,P<0.05).While the DM-NBP group was obviously lower than the DM-C group(P<0.05).The subunit gp91 phox expression: Compared to NC group(0.629±0.054),the DM-C group and DM-NBP group were obviously higher(respective1.020±0.019,0.784±0.020,P<0.05).While the DM-NBP group was obviously lower than the DM-C group(P<0.05).4 Results of Western-Blot The subunit p67 phox of NADPH oxidase expression: Compared to NC group(0.574±0.055),the DM-C group and DM-NBP group were obviously higher(respective 1.310±0.019,1.071±0.023,P<0.05).While the DM-NBP group was obviously lower than the DM-C group(P<0.05).The subunit gp91 phox expressionin: Compared to NC group(0.690±0.028),the DM-C group and DM-NBP group were obviously higher(respective 1.516±0.024,1.258±0.026,P<0.05).While the DM-NBP group was obviously lower than the DM-C group(P<0.05).5 Results of ELISA The content of SOD(ng /ml): Compared to NC group(112.311±3.078),the DM-C group and DM-NBP group were obviously lower(respective43.602±1.808,51.544±1.482,P<0.05).While the DM-NBP group was obviously higher than the DM-C group(P<0.05).The content of MDA(pg /ml): Compared to NC group(0.858±0.047),the DM-C group and DM-NBP group were significantly higher(respective1.353±0.111,1.024±0.043,P<0.05).While the DM-NBP group was obviously lower than the DM-C group(P<0.05).Conclusions:1 The learning and memory behavior of 13 week-old db/db mice decreased significantly.2 NBP can improve the diabetic db/db mice cognitive dysfunction.3 The content of MDA increased,and the content of SOD decreased in hippocampal of 13 week-old db/db mice.Prompting that the level of oxidative stress was increased,and the ability of antioxidative stress was decreased.Another the NADPH oxidase,which is critical in oxidative stress.The protein and m RNA expression of the subunit p67 phox and gp91 phox of it were increased.4 NBP can increase the SOD content,reduce the MDA content,reduce the protein and m RNA expression of the subunit p67 phox and gp91 phox.Therefore we thought NBP can improve the diabetic cognitive dysfunction by reducing oxidative stress,improving the antioxidative ability. |
PDF Full Text Request