Runx2与NGF-TrkA信号通路对话介导膝骨关节炎进展的分子机制及补肾活血除痹方干预研究

There is a clinical discrepancy that the degree of cartilage degeneration from radiographic and histological evidence does not correlate with perceived levels of pain sensation. Only 60% of patients with severe radiographic changes in knee joints suffering symptomatic joint pain. The mechanisms of cartilage degenaration, inflammatory joint pain and by which OA develops from an asymptomatic condition to a painful disease represent a critical gap in our knowledge. . In this study, conditional cartilage specific Runx2 gene KO mice will be generated, and knee OA will be induced in these mice by destabilization of the medial meniscus (DMM) surgery to clarify the role of Runx2 and NGF-TrkA signaling in OA progression.. Besides, to clarify whether VEGF mediate the signaling talking between Runx2 and NGF-TrkA signaling in OA progression, anti-VEGF antibody will be intra-articular administrated in addition to cell and molecular biology experiments.. Finally, DMM induced OA mice will be treated with Bu Shen Huo Xue Chu Bi formula (BSHXCBf) to evaluate the efficacies of BSHXCBf for curing OA, such as joint pain relieving, joint function improving, and delaying of OA progression. We will also try to investigate the regulation mechanism of BSHXCBf on Runx2 and NGF-TrkA signaling pathways in OA knee by cell and molecular biology experiments.. This study is supposed to provide new treatment strategy for OA and experimental foundation for developing of anti-OA compound prescription of TCM.

膝OA患者关节疼痛严重程度与关节退变程度不成正比，严重关节退变的膝OA患者中只有约60%的患者表现出严重的关节炎性疼痛。膝OA软骨退变、关节疼痛以及由单纯软骨退变向症状性膝OA进展的分子机制尚不完全明确。本项目拟①构建可诱导软骨细胞特异性Runx2基因敲除小鼠，DMM手术后诱导敲除软骨细胞Runx2基因，评价小鼠膝OA改变，阐明Runx2和NGF-TrkA信号通路在OA进展过程中的分子机制；②通过膝关节腔注射抗VEGF抗体治疗小鼠膝OA模型，结合细胞、分子实验阐明VEGF参与介导关节软骨细胞Runx2与滑膜NGF-TrkA信号通路对话的分子机制；③采用补肾活血除痹方干预小鼠膝OA模型，明确其对Runx2和NGF-TrkA信号通路的干预作用，评价该方缓解膝OA关节疼痛、改善关节功能、延缓OA进展等综合性治疗效果。本研究为膝OA治疗提供新策略，为抗膝OA中药复方新药开发提供实验依据。

膝骨关节炎（osteoarthritis， OA）是一种非常常见的运动系统退变性疾病，OA患者关节疼痛严重程度与关节退变程度不成正比，严重关节退变的膝OA患者中只有约60%的患者表现出严重的关节炎性疼痛。膝OA软骨退变、关节疼痛以及由单纯软骨退变向症状性膝OA进展的分子机制尚不完全明确。. 本研究构建可诱导软骨细胞特异性Runx2基因敲除小鼠(Agc1-CreER; Runx2fx/fx)，DMM手术后诱导敲除软骨细胞Runx2基因，通过行为学、组织学等研究观察OA进展情况。结果发现：行DMM 诱导 OA 造模后 8 周和 12 周，Cre+小鼠热板实验平均延迟延迟时 间和旷场试验平均水平穿越次数显著优于Cre-小鼠，Cre+小鼠膝关节软骨平均 OARSI 评分和关节滑膜OARSI评分显著优于Cre-小鼠，Cre+小鼠膝关节胫骨内侧平台关节软骨细胞平均 Runx2 和MMP-13阳性率显著低于Cre-小鼠，关节内侧滑膜平均 NGF 表达水平显著低于 Cre-小鼠。. 本研究自拟补肾活血除痹方，采用HPLC 法建立补肾活血除痹方汤药的HPLC 指纹图谱。于DMM手术造模后4周开始，连续灌胃给药8周。于治疗后 4w 和治疗后 8w 分别对各组小鼠进行热板试验和旷场试验，并在第8周完成试验后处死各组小鼠，留取小鼠膝关节样本进行组织病理学及免疫组化研究。结果显示：采用补肾活血除痹方干预 OA 小鼠 4 周和 8 周后，干预组的热板试验和旷场试验结果均优于OA对照组，但劣于正常组小鼠。组织病理学研究结果也显示补肾活血除痹方同样具有延缓关节软骨丢失、抑制关节内滑膜炎症及末梢神经生长。细胞实验表明Runx2和MMP-13蛋白在软骨细胞具有明显的共表达现象，VEGF和NGF蛋白在滑膜细胞具有明显的共表达现象。. 本研究结果表明表明，抑制软骨细胞Runx2 基因能通过延缓关节软骨丢失、抑制关节内滑膜炎症及末梢神经生长，达到缓解OA小鼠的疼痛、改善OA小鼠下肢的活动功能。补肾活血除痹方中药能通过干预关节软骨细胞 Runx2—MMP-13 轴以及滑膜细胞 VEGF-NGF 表达，有减轻疼痛、改善功能、延缓 OA 进展的治疗作用。本研究为中医药治疗膝 OA 提供新的靶点和理论基础，为抗膝OA中药复方新药的开发提供实验依据。