Olanzapine激活AMPK上调自噬减轻Rotenone对多巴胺能神经元的损伤

发布时间：2018-03-02 16:26

本文选题：帕金森病　切入点：Olanzapine　出处：《苏州大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：研究奥氮平（Olanzapine, OLA）对rotenone诱导多巴胺（dopamine, DA）能神经元损伤的保护作用及其可能的机制。方法：（1）细胞实验：首先检测olanzapine对PC12细胞的毒性作用，，采用Western blot检测olanzapine对p-AMPK以及自噬相关蛋白LC3，p62，p70-s6k的影响，免疫荧光染色检测LC3的变化。采用0.5μM rotenone处理PC12细胞24h建立PD细胞模型，治疗组在rotenone给药前24h加入olanzapine（20μM）预处理，实验组以终浓度为2.5μM的AMPK选择性抑制剂compound C预处理PC12细胞3h后再加入olanzapine及rotenone。采用CCK-8法检测细胞活性；Hoechst33258染色法观察细胞核形态变化；Western blot法观察凋亡蛋白PARP及α-synuclein的表达情况。（2）动物实验：雄性Lewis大鼠52只随机分为4组：rotenone注射组的大鼠于颈背部皮下注射rotenone（1.5mg/kg/day）8周，治疗组在每天灌胃0.9mg/kg olanzapine，八周后观察其行为学变化，处死动物取脑、切片，免疫组化染色观察酪氨酸羟化酶（TH）、LC3及α-synuclein的表达。结果：Western blot结果显示olanzapine能够显著激活AMPK并上调LC3II的表达，下调p62以及p70-s6k的水平，表明olanzapine能够激活自噬。CCK-8及Hoechst33258结果提示olanzapine能够减轻rotenone诱导的PC12细胞的凋亡，降低由rotenone引起的α-synuclein和PARP的增高。当抑制AMPK以后，自噬的诱导被阻碍并且保护作用消失。体内实验结果显示，olanzapine能够上调大鼠脑内LC3表达。治疗组的大鼠的行为计分显著低于模型组，治疗组的黑质致密部TH阳性细胞数较模型组明显增加，且残存的TH阳性细胞内的α-synuclein显著减少。结论：olanzapine可以减轻rotenone对多巴胺能神经元的损伤，这种保护作用可能是通过激活AMPK通路，上调自噬实现的。
[Abstract]:Objective: To study the protective effect of Olanzapine (OLA) on rotenone induced dopamine (dopamine, DA) neuron injury and its possible mechanism.
Methods: (1) cell toxicity experiment: first detection of olanzapine in PC12 cells, using Western blot and p-AMPK olanzapine detection of autophagy related protein LC3, p62, p70-s6k, LC3 changes were detected by immunofluorescence staining. Using 0.5 M rotenone PC12 cell 24h establishment of PD cell model, the treatment group in rotenone administration of 24h before joining olanzapine (20 M) pretreatment, the experimental group with a final concentration of 2.5 M AMPK selective inhibitor of compound C pretreatment of PC12 cells after 3H olanzapine and rotenone. CCK-8 was used to detect cell activity; to observe morphological changes of nucleus Hoechst33258 staining; Western blot method was used to observe the expression of apoptosis protein PARP alpha and -synuclein. (2) animal experiment: 52 male Lewis rats were randomly divided into 4 groups: Group rotenone injection the rats in the subcutaneous injection of rotenone (1.5mg/kg/day) for 8 weeks, treatment The rats in each group were fed 0.9mg/kg olanzapine for eight days, then their behavioral changes were observed. The animals were killed and the brains were sectioning. Immunohistochemical staining was used to observe the expression of tyrosine hydroxylase (TH), LC3 and -synuclein.
Results: Western blot showed that olanzapine could activate the expression of AMPK and upregulation of LC3II, downregulation of p62 and p70-s6k, indicated that olanzapine could activate autophagy in.CCK-8 and Hoechst33258 indicated olanzapine can reduce the rotenone induced apoptosis of PC12 cells, decrease caused by rotenone alpha -synuclein and PARP. When the inhibition of autophagy after AMPK blocked and induced protective effect disappeared. In vivo results showed that olanzapine can upregulate the expression of LC3 in brain of rats. Rats in the treatment group behavior score was significantly lower than the model group, the number of TH positive cells in substantia nigra in the treatment group were significantly increased compared with the model group, the alpha -synuclein TH positive cells and the remnants of the significant reduced.
Conclusion: Olanzapine can reduce the damage of rotenone to dopaminergic neurons. This protective effect may be achieved by activating the AMPK pathway to increase the realization of autophagy.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

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