Abstract：
Vitamin K2 (VK2) is an effective compound for anti-ferroptosis and anti-osteoporosis, and Semen sojae praeparatum (Dandouchi in Chinese) is the main source of VK2. Chondrocyte ferroptosis and extracellular matrix (ECM) degradation playing a role in the pathogenesis of osteoarthritis (OA). Glutathione peroxidase 4 (GPX4) is the intersection of two mechanisms in regulating OA progression. But no studies have elucidated the therapeutic effects and mechanisms of VK2 on OA. This study utilized an in vivo rat OA model created via anterior cruciate ligament transection (ACLT) and an in vitro chondrocyte oxidative damage model induced by TBHP to investigate the protective effects and mechanisms of action of VK2 in OA. Knee joint pain in mice was evaluated using the Von Frey test. Micro-CT and Safranin O-Fast Green staining were employed to observe the extent of damage to the tibial cartilage and subchondral bone, while immunohistochemistry and PCR were used to examine GPX4 levels in joint cartilage. The effects of VK2 on rat chondrocyte viability were assessed using CCK-8 and flow cytometry assays, and chondrocyte morphology was observed with toluidine blue and alcian blue staining. The impact of VK2 on intracellular ferroptosis-related markers was observed using fluorescent staining and flow cytometry. Protein expression changes were detected by immunofluorescence and Western blot analysis. Furthermore, specific protein inhibitors were applied to confirm the dual-regulatory effects of VK2 on GPX4. VK2 can increase bone mass and cartilage thickness in the subchondral bone of the tibia, and reduce pain and the OARSI score induced by OA. Immunohistochemistry results indicate that VK2 exerts its anti-OA effects by regulating GPX4 to delay ECM degradation. VK2 can inhibit the activation of the MAPK/NFκB signaling pathway caused by reduced expression of intracellular GPX4, thereby decreasing ECM degradation. Additionally, VK2 can reverse the inhibitory effect of RSL3 on GPX4, increase intracellular GSH content and the GSH/GSSG ratio, reduce MDA content, and rescue chondrocyte ferroptosis. The protective mechanism of VK2 may involve its dual-target regulation of GPX4, reducing chondrocyte ferroptosis and inhibiting the MAPK/NFκB signaling pathway to decelerate the degradation of the chondrocyte extracellular matrix.
摘要：
维生素K2（VK2）是一种有效的抗铁细胞凋亡和抗骨质疏松的化合物，Semensojaepraeparatum（中文为Dandouchi）是VK2的主要来源。软骨细胞铁性凋亡和细胞外基质（ECM）降解在骨关节炎（OA）的发病机理中起作用。谷胱甘肽过氧化物酶4(GPX4)是调节OA进展的两种机制的交叉点。但尚无研究阐明VK2对OA的治疗作用和机制。本研究利用通过前交叉韧带横切（ACLT）创建的体内大鼠OA模型和TBHP诱导的体外软骨细胞氧化损伤模型来研究VK2在OA中的保护作用和作用机制。使用VonFrey测试评价小鼠的膝关节疼痛。采用Micro-CT和SafraninO-FastGreen染色观察胫骨软骨和软骨下骨的损伤程度,而免疫组织化学和PCR用于检测关节软骨中的GPX4水平。使用CCK-8和流式细胞术测定评估VK2对大鼠软骨细胞活力的影响。甲苯胺蓝和阿辛蓝染色观察软骨细胞形态。使用荧光染色和流式细胞术观察VK2对细胞内铁凋亡相关标志物的影响。通过免疫荧光和Westernblot分析检测蛋白表达变化。此外，特异性蛋白质抑制剂用于证实VK2对GPX4的双重调节作用。VK2可以增加胫骨软骨下骨的骨量和软骨厚度，减轻OA引起的疼痛和OARSI评分。免疫组织化学结果表明，VK2通过调节GPX4延迟ECM降解来发挥其抗OA作用。VK2可以抑制细胞内GPX4表达降低引起的MAPK/NFκB信号通路的激活,从而减少ECM降解。此外,VK2可以逆转RSL3对GPX4的抑制作用，增加细胞内GSH含量和GSH/GSSG比值，降低MDA含量，和拯救软骨细胞的铁性凋亡。VK2的保护机制可能涉及其对GPX4的双靶点调控，减少软骨细胞的铁凋亡，抑制MAPK/NFκB信号通路，减缓软骨细胞细胞外基质的降解。
