细胞自噬在孕期咖啡因暴露所致关节软骨发育不良中的作用及发生机制

The quality of articular cartilage in adult life is closely related with the development of cartilage in embryonic period. We previously demonstrated in rats that prenatal caffeine exposure (PCE) induced fetal articular cartilage dysplasia and increased the susceptibility to osteoarthritis in offspring. And we found it would be related to over-expose fetuses to maternal glucocorticoid (GC). Recently, we found that PCE enhaced autophagy of articular cartilage, however, the effects and mechanism are still unclear. Autophagy is a fundamental stress response under severe condition to ensure the survive of cells. It is suggested that the transcriptional activity of insulin-like growth factor1 (IGF1) signaling pathway is retroegulated by sirtuin 6 (SIRT6), and the activity of autophagy is inhibited by IGF1/PI3K/AKT1 signaling pathway via activating the phosphorylation of mTOR. According to the results of pre-experiments, it could be speculated that high fetal blood GC level under PCE condition would upregulate SIRT6 to decrease the deacetylation and expression of IGF1 signaling pathway, and then enhance cell autophagy, which would alleviate caffeine-induced fetal articular cartilage dysplasia. This project is aimed to illuminate the epigenetic modification mechanism of chondrocytes autophagy induced by high GC level, and clarify the biological significance of autophagy in caffeine-induced fetal articular cartilage dysplasia. This study would provide theoretical and experimental basis for exploring the early intervening strategy of developmental originated osteoarthritis.

胚胎关节软骨发育对于成年关节质量至关重要。我们前期证实孕期咖啡因暴露可引起子代大鼠胎软骨发育不良及成年骨关节炎易感，并提出可能与宫内母源性糖皮质激素（GC）过暴露有关。近期我们发现，胎软骨局部存在细胞自噬增强，然而其发生机制及作用不明。自噬是细胞在恶劣环境下确保其生存的基本应激反应。文献提示，IGF1/PI3K/AKT1通路可激活mTOR磷酸化而抑制细胞自噬；而去乙酰化酶SIRT6可抑制IGF1信号通路转录活性。结合预实验结果，我们推测，孕期咖啡因暴露下的胎血高GC很可能通过上调SIRT6表达，使IGF1通路组蛋白去乙酰化及表达降低，继而诱发细胞自噬，从而代偿咖啡因所致的胎关节软骨发育不良。本项目旨在阐明高GC诱导软骨细胞自噬发生的表遗传调控机制，并确定细胞自噬在咖啡因所致胎软骨发育不良中的重要作用，为探寻胎源性骨关节炎的早期干预措施提供实验与理论依据。

胚胎关节软骨发育对于成年关节质量至关重要。我们以前的研究表明，孕期咖啡因暴露（PCE）可引起子代大鼠胎软骨发育不良，并在胎软骨局部发现细胞自噬增强，然而其发生机制及作用尚不明确。本项目发现PCE可引起子代胎鼠关节软骨细胞自噬增强，其发生机制与咖啡因所致母源性糖皮质激素（GC）过暴露的表遗传编程有关。通过整体和细胞水平实验证实，孕期咖啡因暴露下的胎血高GC，通过激活糖皮质激素受体（GR）调控miR-421-3p，使其表达升高，而miR-421-3p可靶向结合mTOR启动子3’-UTR区，抑制mTOR表达，继而诱发细胞自噬。通过细胞水平自噬诱导/抑制实验证实，皮质酮所致胎软骨细胞自噬增强可促进软骨基质蛋白聚糖合成，部分代偿PCE所致软骨基质合成功能降低。同时，咖啡因还可直接促进胎软骨细胞自噬增强，咖啡因与皮质酮联合作用可使自噬效应更为显著。本项目首次阐明细胞自噬在胎源性骨关节炎中的作用和发生机制，证实高CG通过GR-miR-421-3p-mTOR调控胎软骨细胞自噬增强的表观遗传机制。为解析国际前沿问题——“健康与疾病的发育起源”（DOHaD学说）提供了理论与实验依据。基于此项目我们获得了一些原创性研究成果，包括发表SCI论文3篇，中文核心论文1篇（均标注）。