线粒体自噬在铝抑制骨形成中的作用及ROS/PINK1/Parkin通路的调控机制

Aluminum (Al) pollution has become an international hot-spot issue. Excessive intake of Al suppresses bone formation and causes Al-induced bone diseases (AIBD) in humans and animals. Our previous research has found that mitochondrial oxidative damage of osteoblast (OB) is the pathological basis for AIBD, and activated autophagy can relieve the damage. However, whether mitophagy is involved in AIBD remains unclear. In this study, we propose a theoretical hypothesis that mitophagy mediates the bone formation disorders caused by Al and regulated by the ROS/PINK1/Parkin pathway. Primary rat OBs were used as research object, and the OBs will be exposed to a serial of aluminum chloride (AlCl3) doses over different durations for observing the changes in mitophagy, oxidative stress and bone formation in OB, to determine the dose/time-effects relationship between the mitophagy, oxidative stress and OB function disorders caused by Al, and screen out the optimal dose and exposure time of AlCl3 for subsequent OB intervention trial. Subsequently, the OBs treated with AlCl3 are intervened by ROS scavenger, mitophagy inducer/inhibitor and Parkin gene silencing/overexpression, respectively. The corresponding changes of the mitophagy level, oxidative stress and bone formation strength will be determined to determine the role of mitophagy in the bone formation disorder induced by AlCl3 and the regulation mechanism of ROS/PINK1/Parkin pathway. Finally, we used wild-type and Parkin gene knockout rats to verify the effects of the intervention of mitophagy, ROS levels and PINK1/Parkin pathway on bone formation inhibited by AlCl3, and screen out the treatment targets of AIBD. These results will provide a theoretical basis for detoxifying AIBD.

铝污染是现今国际关注的热点，过量铝摄入抑制骨形成，引发人和动物的铝骨病，申请者前期研究表明，成骨细胞（OB）线粒体氧化损伤是其病理基础，激活自噬可缓解该损伤，但线粒体自噬是否参与其中尚不清楚。本研究以线粒体自噬参与铝抑制骨形成的病理过程且受ROS/PINK1/Parkin通路调控为理论假说，以OB为研究对象，设定梯度染铝剂量及时间，检测OB氧化应激、线粒体自噬水平、成骨功能，确定氧化应激、线粒体自噬与铝抑制OB骨形成的剂量/时间-效应关系，筛选出OB干预试验的铝剂量和时间；再通过ROS清除剂、线粒体自噬及Parkin基因干预染铝OB试验，确定线粒体自噬在铝抑制骨形成中的作用及ROS/PINK1/Parkin通路的调控机制；最后采用野生型和Parkin基因敲除大鼠，验证干预线粒体自噬、ROS及PINK1/Parkin途径对铝抑制骨形成的作用，筛选铝骨病的治疗靶点，为铝骨病的解除提供理论依据。

本研究以氧化应激、自噬和骨形成三者间的关系为理论基础，以“线粒体自噬参与铝抑制骨形成的病理过程且受ROS/PINK1/Parkin信号通路调控”为理论假说，通过铝骨病小鼠在体实验和体外成骨细胞培养实验，分别对ROS、PINK1/Parkin途径和线粒体自噬进行干预，观测小鼠和成骨细胞的成骨功能、氧化应激和线粒体自噬关键因子的变化，获得了如下开创性结果：（1）铝暴露可导致股骨组织和MC3T3-E1细胞损伤，成骨功能障碍，诱发氧化应激，激活PINK1/Parkin信号通路介导的线粒体自噬；且随染铝剂量增加，成骨功能障碍逐渐加剧，氧化应激及线粒体自噬水平逐渐升高。（2）ROS是铝激活PINK1/Parkin信号通路的上游信号。（3）PINK1/Parkin途径介导的线粒体自噬通过缓解氧化应激和线粒体损伤在铝抑制骨形成中发挥保护作用。（4）初步筛选出ROS和PINK1/Parkin途径为铝骨病的防治靶点。研究结果为揭示铝骨病的发病机理和铝骨毒性的解除奠定了理论依据。