晚期氧化蛋白产物通过ROS敏感的MAPKs信号调控PI3K/Akt/mTOR/p70S6K通路抑制成骨细胞自噬

The impaired differentiation and mineralization ability of osteoblast is a key factor of osteoporosis. Autophagy plays a crucial role in differentiation, mineralization and anti-apoptosis process of osteoblast. PI3K/Akt/mTOR/p70S6K pathway is a classical pathway which could regulate autophagy. Advanced oxidation protein products (AOPPs) was a marker of oxidative stress, which was also a kind of endogenous pathological mediator. AOPPs may induce diseases via oxidative stress sensitive signals (MAPKs et al.). Accumulation of AOPPs was positively related to the age-related bone loss. AOPPs challenge could accelerate bone loss and bone degradation in aged rats, and inhibit the cell proliferation and osteogenic differentiation in osteoblast-like cells. Our pre-experiment also revealed that AOPPs could inhibit autophagy and induce apoptosis of osteoblast. Oxidative stress has a close relationship with autophagy, but whether AOPPs could affect the autophagy progress via oxidative stress related pathway and affect the function of osteoblast still remains unknown. By in vivo and in vitro studies, the project is planned to investigate: (1) The pathologic role of AOPPs inhibit the autophagy progress and the differentiation and mineralization progress of osteoblast. (2) The molecular mechanism of the effect on autophagy progress of osteoblast induced by AOPPs. (3) Blocking the pathologic pathways of AOPPs , its effect on the autophagy progress of osteoblast and its relationship with osteoporosis. This project is aimed to find out a new sight about how AOPPs could lead to osteoporosis by inhibiting the autophagy progress of osteoblast. It would further reveal the new pathogenetic mechanism underlying osteoporosis, and provide a theoretical basis for the development of new prevention and treatment strategies in osteoporosis.

成骨细胞分化矿化受损是骨质疏松症发病的重要因素之一。自噬在成骨细胞分化矿化中有重要作用，PI3K/Akt/mTOR/p70S6K是经典的自噬调节通路。晚期氧化蛋白产物（AOPPs）是氧化应激标志物，也是内源性致病介质，可通过氧化应激敏感的信号（MAPKs等）致病。我们研究已证实：AOPPs蓄积与增龄性骨量丢失呈正相关；AOPPs加速老年大鼠骨量丢失， 抑制成骨样细胞增值分化；预实验结果表明AOPPs可以抑制成骨细胞自噬。氧化应激与自噬密切相关，但AOPPs引起的氧化应激是否抑制成骨细胞自噬，进而导致成骨细胞分化矿化能力降低，从而导致骨代谢失衡，尚不清楚。本项目通过体内外实验探讨（1）AOPPs抑制成骨细胞自噬并抑制其分化矿化的作用（2）AOPPs抑制成骨细胞自噬的分子基础（4）阻断AOPPs病理作用对成骨细胞自噬功能的影响及对骨量丢失的干预作用。从而为骨质疏松症的防治提供新靶点。

晚期氧化蛋白产物（AOPPs）是一种氧化应激标志物，也是多种疾病的致病物质之一。现有研究证实AOPPs可诱导成骨细胞中的NADPH氧化酶（NOXs）生成大量活性氧（ROS），后者可进一步激活内源性凋亡途径，进而诱导成骨细胞凋亡。. 项目研究目的：1、观察AOPPs刺激成骨细胞时是否会生成mtROS、是否会激活（或抑制）线粒体自噬；2、探究线粒体自噬能否抑制AOPPs诱导的成骨细胞凋亡；3、观察促进线粒体自噬能否遏制AOPPs蓄积所致小鼠骨量丢失和骨微结构破坏。. 结果：体外实验使用小鼠MC3T3-E1前成骨细胞系。首先使用MitoSOX检测AOPPs是否能诱导成骨细胞生成mtROS，并通过免疫印迹、溶酶体/线粒体免疫荧光、透射电镜等手段观察AOPPs是否能激活PINK1/Parkin介导的线粒体自噬。通过使用外源性试剂及shRNA进一步调控线粒体自噬水平以分析线粒体自噬对AOPPs诱导的成骨细胞凋亡的影响及相关机制。体内实验使用C57小鼠，分为对照组、MSA组、AOPPs组、AOPPs+Rapa组和Rapa组。连续腹腔注射8周后，收集各组小鼠的血浆及胫骨，检测血浆AOPPs及B-ALP浓度、骨组织中线粒体自噬及成骨细胞凋亡相关指标，并结合显微CT扫描，综合观察促进线粒体自噬对AOPPs蓄积所致小鼠骨量丢失和骨微结构破坏的影响。MitoSOX检测结果证实AOPPs能诱导成骨细胞生成mtROS。AOPPs可上调成骨细胞中线粒体自噬相关蛋白的表达并使溶酶体数量增多，电镜下亦观察到AOPPs诱导成骨细胞发生线粒体自噬的现象。流式凋亡检测及免疫印迹结果显示，促进线粒体自噬可以抑制AOPPs诱导的成骨细胞凋亡，而抑制线粒体自噬则可致成骨细胞凋亡率进一步升高。敲低PINK1可加重AOPPs诱导的成骨细胞凋亡并使rapamycin的抑制凋亡效应失效，这些结果共同提示PINK1/Parkin介导的线粒体自噬可抑制AOPPs诱导的成骨细胞凋亡。体内实验结果发现促进线粒体自噬可使AOPPs蓄积小鼠的骨量、骨密度及骨小梁相关参数得到部分改善。. 综上，PINK1/Parkin介导的线粒体自噬可通过及时清除受损线粒体及mtROS抑制AOPPs诱导的成骨细胞凋亡。促进线粒体自噬可遏制AOPPs蓄积所致小鼠骨量丢失和骨微结构破坏。