自噬对帕金森病小鼠的影响及TFEB的相关调控作用

目的观察调节自噬对C57BL／6帕金森病模型小鼠行为学、多巴胺能神经元存活数目、α-突触核蛋白（α-Syn）的影响，并探讨转录因子EB（TFEB）的相关调控作用。方法C57BL／6雄性小鼠采用1-甲基-4-苯基-1，2，3，6-四氢吡啶（MPTP）腹腔注射建立帕金森病模型，分别采用雷帕霉素、3-甲基腺嘌吟（3-MA）进行干预，设立正常对照。各组小鼠在最后1次MPTP或生理盐水注射后1d、7d、14d进行行为学测试，14d处死取中脑黑质脑组织行酪氨酸羟化酶（TH）免疫组化染色，Western Blot检测α-Syn、TFEB和自噬蛋白LC3的蛋白表达。结果（1）雷帕霉素干预可使帕金森病小鼠的爬杆时间延长，悬挂分值增加，旷场试验总距离及平均速度增加，而3-MA干预则相反，造模后14d时最明显。（2）雷帕霉素干预可增加帕金森病小鼠黑质TH免疫阳性神经元数目，减少帕金森痛小鼠黑质α-Syn的蛋白表达，增加自噬蛋白LC3-Ⅱ水平以及TFEB的表达，3-MA干预则使得黑质TH免疫阳性神经元数目进一步减少，α-Syn蛋白表达进一步增加，并减少LC3-Ⅱ水平以及TFEB的表达。结论诱导自噬可促进α-Syn的清除，保护多巴胺能神经元，从而改善帕金森病小鼠的运动障碍，相反抑制自噬则会导致α-Syn异常聚集增加进而加重帕金森病小鼠的运动障碍，TFEB在此过程中可能发挥重要调控作用。

Objective To explore the effect of autophagy on behavioral manifestation of Parkinson disease animal model in C57BL/6 mice, survival of dopaminergic neuron and aggregation of α-Syn, and to discuss the regulative role of TFEB in this process. Methods Parkinson＇s disease model was established by intraperitoneal injection of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine （ MPTP ） in C57BL/6 male mice, and intervened with rapamycin,3-methyladenine （ 3-MA ） respectively, normal mice were used as controls. The mice in each group were subjected to behavioral test on first day, 7th day, 14th day after the last MPTP or saline injection.On the 14th day, we took out the mice＇ s substantia nigra of midbrain for tyrosine hydroxylase （ TH ） immunohistochemical staining, Western Blot detection of alpha-Syn, expression of TFEB and autophagy protein LC3 protein. Results （ 1 ） Rapamycin prolonged the climbing time,increase the suspension score,total distance and average velocity of open-field test in Parkinson＇s disease mice the most obvious fourteenth days after modeling,while intervention with 3-MA is the opposite. （ 2 ） Rapamycin increased the number ofTH immunopositive neurons in substantia nigra, the expression of TFEB and the levels of autophagy protein LC3- II ,reduced the expression of substantia nigra alpha-Syn protein in Parkinson＇s disease mice,while intervention with 3-MA is the opposite. Conclusion The induction of autophagy can promote the removal of alpha-Syn and protect dopaminergic neurons, thereby improving dyskinesia in Parkinson＇s disease mice.On the contrary, inhibition of autophagy will lead to abnormal aggregation of alpha-Syn increased and aggravate dyskinesia in Parkinson＇s disease mice, during which TFEB may play an important regulatory role in this process.