Piezos-CaN-NFAT1轴在退变性骨关节炎中的级联调控及靶向治疗研究

Excessive mechanical load is thought to be responsible for the degenerative osteoarthritis (OA), but its mechanism is not yet fully elucidated and it is difficult for prevention and cure. It was found that the concentration of calcium ions in the cartilage from OA patients was increased, which might be related to the ion channel-mediated abnormal signal transduction. Based on the literature and our preliminary results, we speculated that the Piezo1 and Piezo2 channels might be induced to be highly expressed and activated by excessive mechanical load, which resulted in an increase of intracellular calcium concentration and activated the cascade pathway of calcineurin (CaN) and the nuclear factor of activated T-cells (NFAT1). The activated NFAT1 further promoted the transcriptional expression of TNF-α, which consequently lead to the occurrence of degenerative OA. This project will prove the abnormal activation and potential role of the Piezos-CaN-NFAT1 axis in degenerative OA basing on the animal and cell experiments. We will also confirm that the Piezo channels in cartilage can be targeted for the treatment of degenerative OA by the nanosphere of hyaluronic acid-chitosan-GsMTx4, which can block the abnormal activation of the Piezos-CaN-NFAT1 axis. This study will provide important theoretical and experimental basis for elucidating the mechanism and investigating the potential treatment strategy of degenerative OA.

过度机械负荷可导致退变性骨关节炎（OA）的发生，但机制尚不明确，临床早期防治困难。研究发现，OA患者软骨组织内钙离子浓度增加，可能与软骨细胞离子通道介导的异常信号转导有关。综合文献和预实验结果，我们推测：过度机械负荷可诱导软骨细胞Piezo1/Piezo2离子通道高表达并使之激活，引起胞内钙离子浓度升高，进而级联活化CaN-NFAT1，促进炎症因子TNF-α的转录表达，最终导致退变性OA的发生。本项目拟在整体与细胞水平，证实退变性OA软骨中Piezos-CaN-NFAT1轴异常活化现象，确证该轴在退变性OA发生中的作用机制；并在整体水平证实构建的透明质酸-壳聚糖-GsMTx4纳米微球可靶向作用于软骨Piezos离子通道，阻断Piezos-CaN-NFAT1轴异常活化，从而干预退变性OA的发生。本研究为解析OA的发生机制，探寻OA的干预及早期防治策略，提供重要理论和实验依据。

研究背景：病理性机械应力是骨关节炎的突出风险因素，会导致严重的软骨组织破坏和退行性改变。然而，骨关节炎中机械信号传导的基本分子机制仍不清楚。Piezo1是一种机械敏感性钙离子通道，在骨关节炎疾病进展中的作用尚未确定。.主要研究内容：采取前交叉韧带及半月板切除建立Wistar大鼠骨关节炎模型。于8周后处死大鼠并取材。使用苏木精-伊红染色、番红-固绿染色、TUNEL、免疫组织化学等方法从整体水平证实Piezo1-CaN-NFAT1轴异常活化能够介导退变性骨关节炎的发生。从正常大鼠和骨关节炎大鼠中获得膝关节软骨组织，建立正常和骨关节炎软骨细胞培养体系，从细胞水平确证Piezo1-CaN-NFAT1轴活化介导软骨细胞凋亡及细胞外基质降解的调控机制。向骨关节炎造模大鼠的膝关节腔内注射不同剂量的Gsmtx4多肽（一种Piezo1抑制剂），从整体水平证实Gsmtx4多肽能够靶向调控Piezo1-CaN-NFAT1轴，进而延缓骨关节炎的疾病进展。.重要结果和关键数据：在骨关节炎患者和骨关节炎造模大鼠的软骨组织中，Piezo1的表达均显著上调。使用病理性机械应力或Yoda1（一种Piezo1激活剂）激活Piezo1会导致软骨细胞凋亡。敲低Piezo1或使用Gsmtx4多肽可以保护软骨细胞免于凋亡，并在病理性机械应力下维持软骨细胞分解代谢和合成代谢平衡。体内实验表明，向骨关节炎大鼠的关节腔内注射Gsmtx4多肽有效抑制了软骨细胞凋亡，减轻了软骨损伤和基质分解，促进了软骨基质合成，显著延缓了骨关节炎疾病进展。在机制上，我们观察到软骨细胞中Piezo1在病理性机械应力干预下激活，导致钙离子内流、CaN活性升高并促进NFAT1的核转位，最终引发软骨细胞合成分解代谢失衡和细胞凋亡。使用抑制CaN或NFAT1的药物可以逆转机械应力引起的软骨细胞病理性改变。.科学意义：我们的研究结果显示，Piezo1是软骨细胞响应机械信号的重要分子，并通过CaN/NFAT1轴调节软骨细胞凋亡和细胞外基质合成分解平衡，敲低或抑制Piezo1可以在一定程度上减轻软骨细胞在病理性机械应力干预中的损伤。关节腔内注射Gsmtx4多肽能显著延缓骨关节炎大鼠的疾病进展，可能是有潜力的骨关节炎治疗方法。