Sirt1/Sirt3信号轴介导的线粒体自噬对运动性骨关节炎的调控作用及纳米药物治疗机制研究

In recent years, the sports osteoarthritis pathogenesis and intervention research has become a hotspot in the area of sports medicine and rehabilitation medicine. In this study, the Sirt1 / Sirt3 signal axis in the regulation of mitochondrial autophagy and the connection the inflammatory response was selected as the breakthrough point.Firstly, we design and prepare a biodegradable polylactic acid (PLA) nanofiber which was used as drug carrier, and the anti-inflammation drug resveratrol (Res) was used as a model drug. Then the efficacy of the drug release behavior and transdermal absorption was evaluated. Secondly, the safety of nanodrugs and skin irritation were evaluated, and then its promoting drug transdermal absorption mechanism was studied, under different methods of administration, comparing the metabolic changes in the animal bodies. In the end, by constructing cartilage inflammation cell model and sports traumatic osteoarthritis animal model, using the cellular and molecular biology technology, to investigate the PLA/Res nano-drug how to intervention Sirt1 / Sirt3 signal axis regulating AMPK mitochondrial autophagy pathways and NF-κB inflammatory signaling pathways, and the molecular mechanism between the mitochondrial autophagy and inflammatory responds using the methods of co-immunoprecipitation，siRNA.，RNA-SEQ and so on. So as to reveal the biological mechanisms which PLA/Res nanodrugs play its anti-inflammatory effects by mitochondrial autophagy pathways. The Expected results can provide the theory basis for osteoarthritis treatment using nano drug and provide the train of thought and theory bas for the application of nanotechnology used in the field of sports medicine and rehabilitation medicine.

运动性骨关节炎发病机制及干预治疗研究在运动医学、运动康复领域已成为热点。本研究以Sirt1/Sirt3信号轴在调控线粒体自噬和炎症反应之间的联系为切入点。首先设计合成出一种生物可降解的聚乳酸(PLA)纳米纤维作为药物载体，抗炎药物白藜芦醇（Res）作为药物模型，评价药物释放行为和透皮吸收效果；其次评价纳米药物的安全性和皮肤刺激性，考察其促进药物透皮吸收的机理，比较不同给药途径下药物在体内的代谢变化情况；进一步通过构建软骨细胞炎症模型和运动损伤性骨关节炎动物模型，结合免疫共沉淀、siRNA干扰、RNA-SEQ等技术深入研究PLA/Res纳米药物是如何干预Sirt1/Sirt3信号轴调控AMPK线粒体自噬途径与NF-κB炎症信号通路，从而揭示PLA/Res纳米药物通过线粒体自噬途径发挥其抗炎作用的生物学机制。预期成果为运动性骨关节炎的纳米药物治疗和纳米技术在运动医学领域的应用提供理论依据。

本研究以Sirt1/Sirt3信号轴在调控线粒体自噬和运动性骨关节炎症之间的联系为切入点，以生物可降解纳米气凝胶材料作为药物载体，抗炎药物白藜芦醇（Res）作为药物模型，成功构建了白藜芦醇/气凝胶纳米载药体系（RSA），在发挥白藜芦醇抗炎作用的同时达到缓释药物，降低毒副作用，提高药物利用率的目的。通过红外分析、电镜分析、热重分析等手段对载药体系的组成、结构、形貌、载药量和体外缓释效果进行表征测试。. 通过构建炎症软骨细胞模型，RSA可以有效抑制细胞的炎症反应，并且可以缓解炎症条件下对II型胶原合成的抑制，且效果明显优于单纯药物组，采用免疫荧光染色法检测II型胶原蛋白的表达，RSA能有效解除II型胶原合成的限制，且随着其浓度的增加，效果增强。通过关节注射木瓜蛋白酶破坏软骨基质，并联合跑台运动对大鼠进行中等强度的运动训练，成功获得运动性骨关节炎动物模型。通过组织切片，Micro-CT扫描软骨结构的三维重建模型及关节软骨微结构的观察，纳米药物组介入后软骨细胞的细胞器丰富,核仁清晰,粗面内质网发达,说明在纳米药物可以使得损伤性关节软骨保持较旺盛的胶原与合成代谢能力。Western blot结果显示参与软骨降解和分解代谢因子包括IL-1、IL-6、TNF-α，在药物介入后均低于造模组（OA），RSA-h组炎症因子水平最低接近空白对照组（Con）水平。受SIRT-1介导的软骨细胞凋亡的影响，造模组（OA）SIRT-1水平略低于空白对照组，MMP-13, ADMATS-5, NF-kB，Ⅱ型胶原的表达均显著升高。给予药物介入后，（RSA-h，RSA-l，Res）各组关节软骨中MMP-13, ADMATS-5, NF-kB，Ⅱ型胶原的表达不同程度降低。研究表明药物干预实验揭示了白藜芦醇及其纳米药物体系在运动性骨关节炎所致软骨损伤治疗中的优异作用，RSA通过药物缓释的作用能有效控制病情发展，延缓关节软骨退变过程，效果好于单纯用药，这一过程通过激活SIRT-1去乙酰化NF-κB，使其相应的下游靶点（如ADAMTS-5和MMP-13）受抑制来实现，进而发挥其抗炎作用。研究成果可为运动性骨关节炎的康复治疗和纳米技术在运动医学领域的应用提供理论依据。