乳铁蛋白调控PI3K/Akt信号通路抑制骨关节炎软骨细胞凋亡的机制研究

Osteoarthritis (OA) is the most common articular disease that greatly compromises the health of the elderly. OA is pathologically characterized as chondrocytes apoptosis in lesion area of human articular cartilage. The main biological function of PI3K/Akt signal pathway is to promote cell growth and inhibit cell apoptosis, which is considered as the primary pathway for survival of chondrocytes. Our previous studies have shown that PI3K/Akt pathway was in a inhibitory state in OA chondrocytes. Lactoferrin (LF) has significant antimicrobial, antioxidant, anti-cancer and immune regulatory functions. The latest research in recent years and the preliminary studies of our project team have both shown that LF has an efficient anabolic function that exerts double protective action on chondrocytes through significantly promoting proliferation and inhibiting apoptosis. Therefore, we propose that LF can significantly inhibit OA chondrocytes apoptosis via regulating PI3K/Akt signal pathway. This current topic will be investigated in animal model plus cell experiment levels to verify our hypothesis by using specific inhibitor and siRNA to block the key genes function and expression around the PI3K/Akt anti-apoptotic pathway. Technologies like molecular biology detection methods are employed to explore the signal transduction pathway of LF on regulation of OA chondrocytes apoptosis and elucidate the molecular mechanism of its anti-OA treatment. In short, our study will lay the experimental and theoretical basis for subsequent exploration of the anti-OA drug targets and development of new anti-OA drugs.

骨关节炎（OA）是一种严重危害老年人健康的骨关节疾病，软骨细胞凋亡是OA发生的重要病理学特征。PI3K/Akt主要生物学功能为促进细胞生长，抑制细胞凋亡，是软骨细胞存活的首要通路。前期研究发现OA软骨细胞中该通路处于抑制状态。乳铁蛋白（LF）具有显著的抗菌、抗氧化、抗癌及免疫调节功能，近几年最新的研究及本项目组前期研究均表明LF具有高效的合成代谢（anabolic）作用，即促进软骨细胞增殖和抑制凋亡的双重作用。故提出假说：LF调控PI3K/Akt通路抑制OA软骨细胞凋亡。为验证假说，本课题拟在动物模型＋细胞实验基础上，采用LF对OA软骨细胞凋亡进行干预，围绕PI3K/Akt抗凋亡途径，通过特异性抑制剂和siRNA反向阻断技术，应用分子生物学检测，探讨LF调控OA软骨细胞凋亡的信号转导通路，阐明其抗OA治疗的分子机制，并为后续寻找抗OA药物靶标，研制和开发抗OA治疗新药，奠定实验和理论基础。

骨关节炎（OA）是一种严重危害老年人健康的骨关节疾病，机制不明，需要寻求新的治疗靶点。PI3K/Akt信号通路在软骨细胞存活中发挥重要作用，前期研究发现OA 软骨细胞中该通路处于抑制状态。最新研究报道乳铁蛋白（LF）具有高效的合成代谢（anabolic）作用，即促进软骨细胞增殖和抑制凋亡的双重作用，但LF与PI3K/Akt信号通路在OA软骨细胞凋亡中的相互关系，未见报道。.本研究拟通过细胞实验及动物实验，检测细胞增殖力及活力、细胞凋亡和信号通路关键基因表达水平等等，本课题获得了以下重要实验结果：(1) LF 能够显著抑制IL-1β诱导的OA 软骨细胞凋亡，表现为Annexin V-FITC/PI法检测凋亡细胞显著减少，western blot法检测凋亡关键基因cleaved-caspase3和 cleaved-PARP的表达显著下降；(2) LF可激活关键基因CREB的表达进而抑制IL-1β诱导的软骨细胞凋亡。IL-1β可使HACs的CREB磷酸化水平明显减低，而LF可以促进其表达，软骨细胞凋亡减少，而 siRNA敲除CREB基因可以显著干扰上述抑制作用，揭示了CREB基因在LF的保护机制中起着重要的作用；(3) LF通过AKT信号通路调节CREB基因的活化。研究采用AKT选择性抑制剂triciribine和siRNA阻断信号通路，软骨细胞活力下降，凋亡细胞数量增加，p-CREB表达下降，cleaved Caspase-3 和 PARP表达增加；(4)LF可以显著促进关节软骨的修复。研究通过建立大鼠OA模型，采用LF关节腔注射可显著抑制软骨细胞过度肥大，增厚大鼠关节软骨层，同时p-AKT, p-CREB 和 Col II基因表达显著增加。上述结果证实了我们提出的：“LF通过调控Akt信号通路抑制OA 软骨细胞凋亡。”这一假说，为LF在抗OA治疗中的作用提供实验和理论基础。