艾地苯醌对帕金森病模型小鼠行为学及脑组织线粒体自噬水平的影响

目的探讨艾地苯醌是否通过抑制素2(prohibitin 2,PHB2)介导的线粒体自噬改善帕金森病(Parkinson disease,PD)模型小鼠行为学障碍。方法第一个小实验选取30只小鼠按照随机数字表法随机分为正常对照组、模型组和治疗组,每组10只,观测艾地苯醌对帕金森模型小鼠行为学的影响。第二个小实验选取20只小鼠按照随机数字法分为空白对照组、MPTP组、shRNA-PHB2组及shRNA-PHB2+MPTP组4组,每组5只,免疫荧光实验检测脑组织酪氨酸脱氢酶(TH)的改变。第三个小实验选取30只小鼠按照随机数字表法分为空白对照组、shRNA-PHB2组、MPTP+idebenone组、shRNA-PHB2+MPTP组、shRNA-PHB2+idebenone组和shRNA-PHB2+MPTP+idebenone组,每组5只,观测艾地苯醌对小鼠脑组织线粒体自噬的作用机制。将C57BL-6小鼠腹腔注射MPTP构建帕金森病动物模型,然后给予200 mg/kg艾地苯醌灌胃21 d,侧脑室显微注射腺相关病毒9(AAV9)shRNA-抑制素2(PHB2),抑制脑内PHB2的表达。Morris水迷宫实验检测小鼠行为学变化,免疫组化检测抑制PHB2对酪氨酸脱氢酶(TH)的改变,Western blot检测LC3、PHB2在小鼠中脑黑质中的蛋白表达。采用GraphPad 7.0和SPSS 22.0对数据进行统计分析。结果(1)第一个小实验中的水迷宫测试数据使用重复测量方差分析,测试1~7天小鼠逃避潜伏期组别-时间交互效应显著(F时间×组别=20.51,P<0.01);通过简单效应分析,与模型组相比,治疗组小鼠第5、6、7天逃避潜伏期明显缩短,差异有统计学意义(均P<0.05)。测试第7天,通过单因素方差分析比较,对照组与模型组,模型组与治疗组,对照组与治疗组之间均差异有统计学意义(t=-49.95,-21.81,28.14;均P<0.001)。第三个小实验中水迷宫测试数据通过重复测量方差分析显示,时间及组别交互作用显著(F时间×组别=42.11,P<0.01)。通过简单效应分析,与MPTP+idebenone组相比,shRNA-PHB2+MPTP+idebenone组潜伏期均明显延长(均P<0.05);与shRNA-PHB2+MPTP组相比,除第4天(P<0.05)外,均差异无统计学意义(均P>0.05);第7天,通过单因素方差分析,与MPTP+idebenone组小鼠相比,shRNA-PHB2+MPTP+idebenone组停留时间明显增加(t=-34.36,P<0.01),而shRNA-PHB2+MPTP+idebenone组与shRNA-PHB2+MPTP差异无统计学意义(t=2.94,P>0.05)。(2)免疫组化结果显示,对照组、MPTP组、shRNA-PHB2组、shRNA-PHB2+MPTP组四组小鼠中脑组织中TH的相对表达值分别为(41.03±3.01)、(24.20±4.18)、(38.39±3.31)和(13.12±2.65);与对照组小鼠相比,MPTP组小鼠TH的表达明显下调,差异具有统计学意义(t=7.98,P<0.01);与MPTP组小鼠相比,shRNA-PHB2抑制组中的TH的表达下调(t=-6.73,P<0.05)。(3)Western blot实验结果显示,对照组、shRNA-PHB2组、MPTP+idebenone组、shRNA-PHB2+MPTP组、shRNA-PHB2+idebenone组和shRNA-PHB2+MPTP+idebenone组小鼠中脑组织中LC3的相对表达值分别为(0.86±0.07)、(0.77±0.08)、(0.42±0.05)、(0.21±0.05)、(0.66±0.09)和(0.27±0.07);PHB2的相对表达值分别为(1.13±0.14)、(0.56±0.11)、(1.08±0.14)、(0.27±0.07)、(0.68±0.14)和(0.24±0.10)。与MPTP+idebenone组相比,shRNA-PHB2+MPTP+idebenone组LC3和PHB2的相对表达量明显减少(t=3.54,11.06,均P<0.01)。结论艾地苯醌能够通过PHB2增加PD小鼠线粒体自噬水平,从而改善行为学障碍。

Effects of idebenone on behavior and mitochondrial autophagy of brain tissue in Parkinson disease model mice

Objective To investigate whether idebenone can improve behavioral disorders in mice with Parkinson disease(PD)by increasing PHB2 mediated mitophagy.Methods In the first small experiment,thirty mice were randomly divided into normal control group,model group and treatment group according to the random number table method,with 10 animals in each group.The aim of this study was to observe the effect of idebenone on the behavior of Parkinson disease model mice.In the second experiment,20 mice were randomly divided into blank control group,MPTP group,shRNA-PHB2 group and shRNA-PHB2+MPTP group,with 5 mice in each group.The changes of tyrosine dehydrogenase(TH)in brain tissue were detected by immunofluorescence assay.In the third experiment,30 mice were randomly divided into blank control group,shRNA-PHB2 group,MPTP+idebenone group,shRNA-PHB2+MPTP group,shRNA-PHB2+idebenone group and shRNA-PHB2+MPTP+idebenone group,with 5 animals in each group.The aim of this study was to investigate the effect of idebenone on mitochondrial autophagy in mouse brain.C57BL-6 mice were intraperitoneally injected with MPTP to establish the animal model of chronic PD.Then 200 mg/kg idebenone was given by gavage for 21 days.And the expression of PHB2 in brain was inhibited by microinjection of adeno-associated virus 9(AAV9)shRNA inhibin 2(PHB2)into lateral ventricle.The behavioral changes of the PD mice were detected by Morris water maze,and the changes of tyrosine dehydrogenase(TH)induced by inhibiting PHB2 were detected by immunohistochemistry.The protein expression of LC3 and PHB2 in substantia nigra of midbrain was detected by Western blot.The data were analyzed by GraphPad 7.0 and SPSS 22.0.Results(1)In the water maze test data of the first small experiment,the repeated measurement ANOVA showed that the group-time interaction effects of latency of mice from 1 to 7 days were significant(Ftime×group=20.51,P<0.05).Simple effect analysis showed that on the 5th,6th and 7th day,the incubation period of the treatment group was significantly shortened(all P<0.05).Univariate analysis of variance showed that on the 7th day of the test,the differences between the control group and the model group,the model group and the treatment group,the control group and the treatment group were all statistically significant(t=-49.95,-21.81,28.14;all P<0.01).In the third small experiment,repeated measurement analysis of variance showed that the interaction between time and group was significant(Ftime×group=42.11,P<0.01).Simple effect analysis showed that compared with MPTP+idebenone group,the latency of shRNA-PHB2+MPTP+idebenone group was significantly prolonged(all P<0.05).There were no significant difference between shRNA-PHB2+MPTP+idebenone group and shRNA-PHB2+MPTP group except the 4th day(P<0.05).On the 7th day,compared with MPTP+idebenone group,the residence time of shRNA-PHB2+MPTP+idebenone group was significantly increased(t=-34.36,P<0.001),but there was no significant difference between shRNA-PHB2+MPTP group and shRNA-PHB2+MPTP+idebenone group(t=2.94,P>0.05).(2)The results of immunofluorescence experiment showed that the relative expression of TH in the control group,model group,shRNA-PHB2 group and shRNA-PHB2+MPTP group were(41.03±3.01),(24.20±4.18),(38.39±3.31)and(13.12±2.65),respectively.Compared with the control group,the expression of TH in the midbrain of the MPTP group was significantly down-regulated,the difference was statistically significant(t=7.98,P<0.01).Compared with the MPTP group,the expression of TH in shRNA-PHB2 group was down regulated(t=-6.73,P<0.05).(3)Western blot results showed that the relative expression of LC3 in midbrain tissue of control group,shRNA-PHB2 group,MPTP+idebenone group,shRNA-PHB2+MPTP group,shRNA-PHB2+idebenone group and shRNA-PHB2+MPTP+idebenone group were(0.86±0.07),(0.77±0.08),(0.42±0.05),(0.21±0.05),(0.66±0.09)and(0.27±0.07).The relative expression of PHB2 were(1.13±0.14),(0.56±0.11),(1.08±0.14),(0.27±0.07),(0.68±0.14)and(0.24±0.10).Compared with MPTP+idebenone group,the relative expression of LC3 and PHB2 in shRNA-PHB2+MPTP+idebenone group was significantly decreased(F=1.96,P<0.01).Conclusion Idebenone can increase the level of mitophagy in PD mice through PHB2,thus improving the behavioral disorder.