mTOR信号通路和线粒体-内质网自噬在大骨节病软骨细胞损伤中的分子机制

As the pathogenesis of Kashin-Beck disease (KBD) is still unknown, this research will take full advantages of unique KBD resources in China, based on the mitochondria-endoplasmic reticulum autophagy observed in the KBD cartilage tissues and chondrocytes of cell intervention experiments, the mTOR signal pathway (related to mitochondria-endoplasmic reticulum autophagy) was selected from the analysis of proteomics results. First of all, this research will detect the expression levels of key proteins and their interaction in mTOR signal pathway from human cartilage tissues, in order to investigate the molecular mechanism of mTOR signal pathway in mitochondria-endoplasmic reticulum autophagy of KBD. Furthermore, single or combined interventions of T-2 toxin and low selenium (environmental suspected pathogenic factors) or supplementation of selenium -chondroitin sulfate A nanoparticles (SeCSA) will be conducted in cellular and animal experiments, as to observe the relationship between environmental suspected pathogenic factors induced mitochondria-endoplasmic reticulum autophagy and the expression levels of key genes and proteins in mTOR signal pathway; Besides, the relationship between the protective effects of SeCSA in T-2 toxin induced KBD chondrocytes damages and mTOR signal pathway or mitochondria-endoplasmic reticulum autophagy will be investigated. This research will promote pathogenesis research of KBD and provide theoretical and experimental basis for the prevention and treatment of KBD.

针对大骨节病(KBD)发病机制不明的关键问题，本项研究利用我国独有KBD资源，基于KBD患者软骨组织和软骨细胞干预实验中观察到的线粒体-内质网自噬现象，结合蛋白质组学筛选出与线粒体-内质网自噬密切相关的mTOR信号通路。本项研究将首先从人体采集的软骨组织中检测mTOR信号通路关键蛋白的表达水平及其相互作用，探索mTOR信号通路在KBD软骨细胞线粒体-内质网自噬中的分子机制。继而在细胞和动物实验中开展环境可疑致病因素T-2毒素、低硒/补充硫酸软骨素A纳米硒(SeCSA)单一或联合干预实验，观察T-2毒素、低硒单一或联合诱导软骨细胞线粒体-内质网自噬与mTOR信号通路关键基因和蛋白表达的关系；同时探究补充SeCSA对T-2毒素诱导的KBD软骨细胞损伤的保护作用与线粒体内质网自噬和mTOR信号通路的关系。本项研究可推动KBD发病机制研究，为KBD预防和治疗提供理论和实验依据。

大骨节病（Kashin-Beck disease，KBD）软骨细胞中自噬水平有争议，且其分子机制至今尚未阐明，而既往研究表明T-2毒素和硒有调控细胞自噬的作用。本项目基于在KBD患者软骨组织中发现的细胞自噬现象，开展蛋白质组学和糖基化蛋白质组学检测，结果提示mTOR信号通路在KBD软骨中差异表达。进一步实验验证发现mTOR通路和自噬标志物在 KBD软骨中存在差异表达，AMPK/mTOR/ULK1通路呈现下调趋势，提示KBD软骨细胞内自噬起始复合物形成被抑制。为探究细胞自噬和AMPK/mTOR/ULK1信号通路在KBD软骨病变进程中的变化，利用T-2毒素和低硒构建KBD动物模型，实验结果表明大鼠软骨组织mTOR信号通路的表达改变与软骨细胞损伤和自噬有关。短期作用的低硒和低浓度的T-2毒素可能通过上调AMPK/mTOR/ULK1通路起到促进自噬的作用，软骨细胞可能通过自噬来清除部分胞内受损细胞器或变性的物质，从而起到一定代偿作用。长期作用的低硒或高浓度的T-2毒素可能下调AMPK/mTOR/ULK1通路，起到抑制自噬的作用。相较于低硒干预，补充SeCSA呈现上调AMPK/mTOR/ULK1通路的趋势，SeCSA可能通过调节mTOR信号通路和自噬活性起到保护作用。进一步研究体外条件下T-2毒素在对软骨细胞作用过程中的自噬活性变化及相关机制，T-2毒素干预C28/I2软骨细胞并检测T-2毒素对软骨细胞作用自噬水平的影响，发现T-2毒素短时间作用时就能引起AMPK/mTOR/ULK1通路上调，自噬标志物表达水平升高，自噬水平升高。为明确补硒对KBD软骨细胞起到保护作用时的自噬水平变化及相关机制，补硒和T-2毒素干预KBD软骨细胞，结果显示补硒可适当降低T-2毒素所致的KBD软骨细胞自噬，SeCSA比亚硒酸钠体现出更强的保护作用，SeCSA可能通过调节AMPK/mTOR/ULK1通路缓解T-2毒素引起的KBD软骨细胞自噬而起到保护作用。目前，本项目已发表相关论文17篇，其中SC1论文14篇，中文核心期刊2篇，并申请专利2项，作出特邀报告2人次，培养博硕士研究生22名。