【作者】 高明；

【导师】 张松筠；

【作者基本信息】 河北医科大学 ， 内科学， 2017， 硕士

【摘要】 目的:糖尿病可以导致认知功能障碍,成为目前研究的热点。研究表明氧化应激是糖尿病认知功能障碍的重要发病机制之一,NADPH氧化酶(NADPH oxidase,NOX)在氧化应激中起中枢作用。超氧化物歧化酶(Superoxide dismutase,SOD)、丙二醛(Malondialdehyde,MDA),是检验体内氧化应激水平的重要指标。丁苯酞可以改善糖尿病认知功能障碍,但其具体机制尚不十分清楚。本实验旨在对糖尿病db/db小鼠预防性应用丁苯酞,观察丁苯酞对糖尿病db/db小鼠认知功能及其海马组织中SOD、MDA及NADPH氧化酶p67phox亚基、gp91phox亚基的影响。方法:1分组6周龄雄性db/m小鼠8只作为正常对照组(NC,n=8只)。雄性db/db小鼠16只,随机分为糖尿病对照组(DM-C,n=8只),糖尿病丁苯酞组(DM-NBP,n=8只)。2药物干预实验动物适应性喂养1周后,给予DM-NBP组小鼠丁苯酞灌胃(丁苯酞剂量为120mg/Kg/d),NC组及DM-C组小鼠给予等量的植物油灌胃,每日1次,连续干预6周。3一般观察每周监测各组小鼠的血糖、体重变化。观察各组小鼠的精神状态、进食情况、摄水量及尿量等一般情况。4行为学检测丁苯酞干预6周后,进行水迷宫测试,分析各组小鼠学习、记忆能力。包括连续5天的定位航行实验,记录各组小鼠的逃避潜伏期,于第6天进行空间探索实验,记录各组小鼠穿越平台次数。5 ELISA、Western-Blot、Real Time PCR检测水迷宫实验结束后,处死各组小鼠,取其海马组织,应用酶联免疫吸附法(Enzyme Linked Immunosorbent Assay,ELISA)检测SOD与MDA的含量。蛋白免疫印迹法(Western-Blot,WB)检测NADPH氧化酶p67phox亚基、gp91phox亚基蛋白的表达。实时荧光定量-聚合酶链反应(Real Time Polymerase Chain Reaction,Real Time PCR)检测NADPH氧化酶p67phox亚基、gp91phox亚基m RNA的表达水平。6统计学方法实验数据应用SPSS 21.0统计软件进行统计分析,数据描述用均数±标准差(x±s)表示,应用完全随机设计的单因素方差分析、重复测量方差分析进行数据统计,三组间两两比较应用SNK-q检验,P<0.05表示差异有统计学意义。结果:1一般情况NC组小鼠精神状态良好,皮毛光顺,进食水量、尿量正常。DM-C组及DM-NBP组小鼠出现多尿、多饮、多食症状,毛发稀疏无光泽,精神萎靡。干预过程中,DM-NBP组小鼠一般情况优于DM-C组。丁苯酞干预6周后,测定各组小鼠体重(g)。结果显示,与NC组(26.39±1.66)相比,DM-C组(58.06±2.38)与DM-NBP组(57.34±2.17)小鼠体重显著增加(P<0.05)。与DM-C组相比,DM-NBP组小鼠体重无明显差异(P>0.05)。丁苯酞干预6周后,取尾静脉血测定各组小鼠的血糖(mmol/L)。结果显示,与NC组(7.21±0.91)相比,DM-C组(20.94±1.71)与DM-NBP组(20.03±1.66)小鼠血糖明显升高(P<0.05)。与DM-C组相比,DM-NBP组小鼠血糖无显著差异(P>0.05)。2行为学观察:Morris水迷宫测试2.1基础游泳速度测定(cm/s)实验开始前,为排除个体差异,让各组小鼠自由游泳60s,测定基础游泳速度。结果显示NC组(138.33±7.40)、DM-C组(134.70±8.28)与DM-NBP组(136.64±7.14)三组小鼠间基础游泳速度无显著差异(P>0.05)。2.2逃避潜伏期测定(s)定位航行第1天,各组小鼠间逃避潜伏期无明显差异(P>0.05)。第2-5天,与NC组相比,DM-C组与DM-NBP组小鼠逃避潜伏期均明显延长(P<0.05);与DM-C组相比,DM-NBP组小鼠逃避潜伏期明显缩短(P<0.05)。各组小鼠随着天数增加逃避潜伏期逐渐缩短:Day1>Day2>Day3>Day4>Day5。2.3空间探索实验:穿越平台次数(次/60 s)与NC组(7.00±0.76)相比,DM-NBP组(5.13±0.64)、DM-C组(2.38±0.52)小鼠穿越平台次数均明显减少(P<0.05)。与DM-C组相比,DM-NBP组小鼠穿越平台次数明显增加(P<0.05)。3 Real Time PCR结果NADPH氧化酶p67phox亚基m RNA的表达:与NC组(0.686±0.015)相比,DM-C组(1.003±0.026)、DM-NBP组(0.802±0.023)小鼠海马组织中NADPH氧化酶p67phox亚基m RNA的表达水平均明显升高(P<0.05)。与DM-C组相比,DM-NBP组小鼠海马组织中NADPH氧化酶p67phox亚基m RNA的表达水平明显降低(P<0.05)。NADPH氧化酶gp91phox亚基m RNA的表达:与NC组(0.629±0.054)相比,DM-C组(1.020±0.019)、DM-NBP组(0.784±0.020)小鼠海马组织中NADPH氧化酶gp91phox亚基m RNA的表达水平均明显升高(P<0.05)。与DM-C组相比,DM-NBP组小鼠海马组织中NADPH氧化酶gp91phox亚基m RNA的表达水平明显降低(P<0.05)。4 WB结果NADPH氧化酶p67phox亚基蛋白的表达:与NC组(0.574±0.055)相比,DM-C组(1.310±0.019)、DM-NBP组(1.071±0.023)小鼠海马组织内NADPH氧化酶p67phox亚基蛋白的表达均明显增强(P<0.05)。与DM-C组相比,DM-NBP组小鼠海马组织内NADPH氧化酶p67phox亚基蛋白的表达明显减弱(P<0.05)。NADPH氧化酶gp91phox亚基蛋白的表达:与NC组(0.690±0.028)相比,DM-C组(1.516±0.024)、DM-NBP组(1.258±0.026)小鼠海马组织中NADPH氧化酶gp91phox亚基蛋白的表达明显增强(P<0.05)。与DM-C组相比,DM-NBP组小鼠海马组织中NADPH氧化酶gp91phox亚基蛋白的表达明显减弱(P<0.05)。5 ELISA结果SOD含量(ng/ml):与NC组(112.311±3.078)相比,DM-C组(43.602±1.808)、DM-NBP组(51.544±1.482)小鼠海马组织中SOD含量均明显降低(P<0.05)。与DM-C组相比,DM-NBP组小鼠海马组织中SOD含量明显升高(P<0.05)。MDA含量(pg/ml):与NC组(0.858±0.047)相比,DM-C组(1.353±0.111)、DM-NBP组(1.024±0.043)小鼠海马组织中MDA的含量均明显升高(P<0.05)。与DM-C组相比,DM-NBP组小鼠海马组织中MDA的含量明显降低(P<0.05)。结论:1 13周龄的糖尿病db/db小鼠学习、记忆能力明显下降。2丁苯酞可以改善糖尿病db/db小鼠学习、记忆能力。3 13周龄的糖尿病db/db小鼠海马组织中MDA的含量增加,SOD的含量降低,提示氧化应激水平增强,抗氧化应激能力减弱。其中在氧化应激反应中起中枢作用的NADPH氧化酶的亚基p67phox与gp91phox的蛋白及m RNA表达增强。4丁苯酞可以增加SOD含量,降低MDA含量,降低NADPH氧化酶p67phox亚基及gp91phox亚基的m RNA及蛋白的表达。考虑丁苯酞通过降低氧化应激反应,提高抗氧化应激能力,从而改善糖尿病认知功能障碍。更多还原

【Abstract】 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.更多还原