耐力训练对帕金森病模型小鼠黑质神经细胞自噬和血浆外泌体分泌的影响

Effects of endurance training on autophagy of substantia nigra neurons and secretion of plasma exosomes in Parkinson disease mice

目的探讨耐力训练对帕金森病(Parkinson disease,PD)模型小鼠的保护作用以及通过AMPK/mTOR通路对自噬和外泌体途径的影响。方法选用10周龄雄性C57BL/6小鼠32只,随机分为安静组、运动组、PD安静组及PD运动组,每组8只。运动组及PD运动组进行为期4周跑台耐力训练。训练结束后,PD安静组和PD运动组小鼠给予鱼藤酮(30 mg·kg^(-1)·d^(-1))灌胃制备PD小鼠模型,1次/d,连续56 d;安静组和运动组小鼠给予等体积0.5%羧甲基纤维素盐溶液灌胃。灌胃结束后,运动组及PD运动组小鼠继续进行为期4周跑台耐力训练。训练结束后测定各组小鼠的行为学指标,免疫组化测定各组小鼠黑质致密部酪氨酸羟化酶(tyrosine hydroxylase,TH)含量,Western blot测定各组血浆外泌体中α-突触核蛋白(α-syn)和外泌体表面标记蛋白CD9、CD63的表达及黑质微管相关蛋白轻链3-Ⅱ(light chain 3-Ⅱ,LC3-Ⅱ)、α-syn、腺嘌呤核糖核苷酸依赖的蛋白激酶(adenine ribonucleotide dependent protein kinase,AMPK)、磷酸化AMPK(phosphorylated AMPK,p-AMPK)、雷帕霉素的哺乳动物靶点(mammalian target of rapamycin,mTOR)、磷酸化mTOR(phosphorylated mTOR,p-mTOR)的表达。应用SPSS 20.0软件进行统计分析,多组间比较采用单因素方差分析,进一步两两比较采用LSD法。结果(1)四组小鼠转棒仪上停留时间差异有统计学意义(F=2618.20,P<0.01)。PD安静组小鼠转棒仪上停留时间[(110.34±8.20)s,(186.20±6.83)s,P<0.01]低于安静组,PD运动组小鼠转棒仪上停留时间[(160.56±8.30)s,P<0.01]高于PD安静组。(2)四组小鼠血浆外泌体标记蛋白CD9、CD63的表达差异无统计学意义(F=1.57,1.26,均P>0.05)。(3)四组小鼠血浆外泌体中α-syn的表达差异有统计学意义(F=1303.99,P<0.01)。PD安静组血浆外泌体中α-syn的表达[(180.57±8.20),(100.00±0.00),P<0.01]高于安静组,PD运动组血浆外泌体中α-syn的表达[(150.23±7.30),P<0.01]低于PD安静组。(4)四组小鼠黑质TH阳性神经元数量差异有统计学意义(F=447.09,P<0.01)。PD安静组小鼠黑质TH阳性神经元数量[(48.23±6.30),(100.00±0.00),P<0.01]少于安静组,PD运动组黑质TH阳性神经元数量[(68.62±8.20),P<0.01]高于PD安静组。(5)Western blot结果显示,四组小鼠黑质α-syn、p-mTOR、p-AMPK、LC3-Ⅱ的表达差异有统计学意义(F=753.62,361.48,261.95,248.07,均P<0.01)。与安静组相比,PD安静组黑质α-syn的表达增加[(184.16±15.31),(100.00±0.00),P<0.01],黑质p-mTOR的表达升高[(156.77±3.99),(100.00±0.00),P<0.01],p-AMPK的表达降低[(70.65±8.43),(100.00±0.00),P<0.01],黑质LC3-Ⅱ的表达降低[(72.25±7.86),(100.00±0.00),P<0.01]。与PD安静组相比,PD运动组黑质α-syn的表达降低[(158.23±9.30),P<0.01],黑质p-mTOR的表达降低[(123.61±16.86),P<0.01],p-AMPK的表达升高[(96.35±9.45),P<0.01],黑质LC3-Ⅱ的表达升高[(108.89±10.67),P<0.01]。结论耐力训练通过AMPK/mTOR信号通路调节PD小鼠黑质神经细胞自噬和血浆外泌体的表达,保护小鼠中脑多巴胺能神经元,改善运动功能。

Objective To investigate the protective effect of endurance training on Parkinson disease(PD)mice and the effect of AMPK/mTOR pathway on autophagy and exosomes secretion.Methods Thirty-two 10-week-old male C57BL/6 mice were randomly divided into quiet group,exercise group,PD quiet group and PD exercise group,with 8 mice in each group.The mice in exercise group and PD exercise group received 4-week treadmill endurance training.After training,mice in PD quiet group and PD exercise group were given rotenone(30 mg·kg-1·d-1)dissolved in 0.5%carboxymethyl cellulose salt solution and gavaged for 56 consecutive days.The mice in quiet group and exercise group were given 0.5%carboxymethyl cellulose salt solution by gavage.Then,the mice in exercise group and PD exercise group received treadmill endurance training for 4 weeks.The behaviors of mice in each group were measured after training.The content of tyrosine hydroxylase(TH)in substantia nigra of mice in each group was measured by immunohistochemistry.Western blot was used to detect the expression of plasmaα-synuclein(α-syn),exosomes surface marker proteins CD9 and CD63,and the content of microtubule associated protein light chain 3-Ⅱ(LC3-Ⅱ),α-syn,adenine ribonucleotide dependent protein kinase(AMPK)and phosphorylated AMPK(p-AMPK),mammalian target of rapamycin(mTOR)and phosphorylated mTOR(p-mTOR)in substantia nigra of mice in each group.SPSS 20.0 software was used to analyze the data.One-way ANOVA was used for comparison among multiple groups and the LSD method was used for further pairwise comparison.Results There was significant difference in the residence time of mice in the four groups on the rotarod instrument(F=2618.20,P<0.01).Compared with the quiet group,the residence time of PD quiet group decreased((110.34±8.20)s,(186.20±6.83)s,P<0.01).Compared with the PD quiet group,the residence time of PD exercise group increased((160.56±8.30)s,P<0.01).(2)There was no significant difference in the expression of plasma exosome marker proteins CD9 and CD63 among the four groups(F=1.57,1.26,both P>0.05).(3)There was significant difference in the expression ofα-syn in plasma exosomes of the four groups(F=1303.99,P<0.01).The expression ofα-syn in plasma exosomes in PD quiet group was higher than that of quiet group((180.57±8.20),(100.00±0.00),P<0.01).Compared with the PD quiet group,the expression ofα-syn in plasma exosomes in PD exercise group decreased((150.23±7.30),P<0.01).(4)There was significant difference in the number of TH positive neurons in substantia nigra among the four groups(F=447.09,P<0.01).Compared with the quiet group,the number of TH positive neurons in the substantia nigra of PD quiet group decreased((48.23±6.30),(100.00±0.00),P<0.01).Compared with the PD quiet group,the number of TH positive neurons in the substantia nigra of PD exercise group increased((68.62±8.20),P<0.01).(5)Western blot showed that there were significant differences in the expression ofα-syn,p-mTOR,p-AMPK and LC3-Ⅱin substantia nigra of the four groups(F=753.62,361.48,261.95,248.07,all P<0.01).Compared with the quiet group,the expression ofα-syn in substantia nigra of PD quiet group increased((184.16±15.31),(100.00±0.00),P<0.01),the expression of p-mTOR in substantia nigra increased((156.77±3.99),(100.00±0.00),P<0.01),the expression of p-AMPK decreased((70.65±8.43),(100.00±0.00),P<0.01),and the expression of LC3-Ⅱin substantia nigra decreased((72.25±7.86),(100.00±0.00),P<0.01).Compared with PD quiet group,the expression ofα-syn in substantia nigra decreased((158.23±9.30),P<0.01),the expression of p-mTOR in substantia nigra decreased((123.61±16.86),P<0.01),the expression of p-AMPK increased((96.35±9.45),P<0.01),and the expression of LC3-Ⅱin substantia nigra increased((108.89±10.67),P<0.01).Conclusion Endurance training regulates autophagy and the expression of exosomes in PD mice through AMPK/mTOR signal pathway,protects dopaminergic neurons in mouse midbrain and improves motor function.