SIRT3对鱼藤酮诱导PD细胞模型的保护作用及其与自噬的关系

Parkinson's disease is the second most common neurodegenerative disease affecting approximately five million people worldwide. Aging is the most important risk factor of PD. The pathology of this disease is complex and not entirely understood. Evidence suggests that PD is related withα-synuclein accumulation,mitochondrial dysfunction and oxidative stress. Recently it is demonstrated that autophagy also play a certain role in PD and inducing autophagy could improve α-synuclein accumulation and clearance of dysfuctional mitochondria to protect PD. SIRT3 belongs to the Sirtuins family, which associated with aging closely. SIRT3 could reduce the reactive oxygen species so as to ameliorate the damage of oxidative stress. It is also suggested that SIRT3 could induce autophagy. In our previous study, we found that the expression of SIRT3 increased obviously in the cellular model of PD. All of this suggested that SIRT3 may play a protective role in PD. In order to clarify the relationship between SIRT3 and PD, we plan to use rotenone to construct PD cellular model, use siRNA to interfere the expression of SIRT3 and construct vector to overexpress SIRT3, detect the expression of SIRT3,α-synuclein accumulation, mitochondrial dysfunction, oxidative stress, autophagy and LKB1-AMPK pathway by immunofluorescence, real-time PCR, western bolt, flow cytometry,confocal microscopy and electron microscope. By this，we could demostrate the correlation between SIRT3 and PD and clarify whether SIRT3 protect PD through autophagic pathway. This work would give us a new target for PD neuroprotection.

帕金森病（PD）与α-synuclein沉积、线粒体损伤和氧化应激及自噬缺陷密切相关。诱导自噬促进α-synuclein降解和损伤线粒体清除是保护PD的有效途径。既往研究表明SIRT3有神经保护作用，可能诱导自噬，但其对PD是否有保护作用，该保护作用是否与诱导自噬相关，目前国内外尚缺乏相关证据。我们前期研究已发现PD细胞模型中SIRT3表达明显升高，下调其表达可加重氧化损伤，已初步证实SIRT3对PD细胞有保护作用，但其具体机制尚不清楚。本课题拟建立鱼藤酮诱导PD细胞模型，利用电镜、细胞免疫组化、共聚焦显微镜、荧光染色、生物化学等多种手段，采用基因转染和RNAi基因沉默技术，探讨SIRT3与自噬及PD细胞损伤的关系，明确SIRT3是否通过诱导自噬促进α-synuclein降解和损伤线粒体清除对PD产生保护作用。SIRT3对PD的保护作用及其机制的明确，将为PD防治新的思路提供科学依据。

自噬与帕金森病密切相关，诱导自噬促进α-synuclein降解和损伤线粒体清除是保护PD的有效途径。而SIRT3可能诱导自噬对PD具有保护作用。本研究建立鱼藤酮诱导SH-SY5Y细胞PD细胞模型，开展SIRT3在鱼藤酮诱导SH-SY5Y细胞PD模型中的表达研究，利用RNAi基因沉默SIRT3表达，发现SIRT3沉默后SH-SY5Y细胞对鱼藤酮损伤的耐受能力下降，α-synuclein沉积增多，线粒体损伤增加，氧化应激损伤增高，同时SIRT3沉默后自噬相关指标的表达水平均降低；为了进一步验证SIRT3的保护作用，利用基因过表达SIRT3，建立过表达SIRT3的SH-SY5Y细胞模型，结果表明过表达SIRT3的SH-SY5Y细胞对鱼藤酮的毒性耐受力增加，α-synuclein沉积，线粒体损伤及氧化应激损伤均减弱，同时自噬相关指标升高；为进一步确定自噬与保护作用的关系，利用3-MA阻断自噬后发现SIRT3的保护作用减弱，以上结果表明，SIRT3对鱼藤酮诱导的SH-SY5Y细胞PD模型有保护作用，自噬是SIRT3发挥保护作用的途径。更深入研究发现过表达SIRT3后LKB1磷酸化水平增高，AMPK磷酸化水平增高，mTOR磷酸化水平减低，LC3II的水平增高而促进自噬，而我们利用AMPK抑制剂Dorsomorphin抑制AMPK表达后发现，SIRT3所介导的自噬上调作用减弱，表明SIRT3经由LKB1-AMPK-mTOR通路激活自噬。综上，本研究明确了SIRT3对PD细胞模型具有保护作用，自噬是SIRT3发挥神经保护作用的通路，SIRT3通过LKB1-AMPK-mTOR通路激活自噬。我们的研究为PD的发病机制研究提供了实验依据，为PD治疗寻找新的靶点提供了实验基础。