Unveiling the Mechanisms of Mechanical Loading-Induced Knee Osteoarthritis Through Transcriptomics

Aims：To establish a novel model of mechanical loading-induced knee osteoarthritis (KOA) and explore its regulatory mechanisms through transcriptomics. Methods：Knee joints of Sprague-Dawley (SD) rats were mechanically loaded with 13 N, 20 N and 27 N for 2 or 4 weeks to construct mechanically-induced KOA model. Immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, Safranin O/fast green staining, enzyme-linked immunosorbent assay (ELISA), micro-computed tomography (Micro-CT) and behavioral analysis were used to evaluate damage on the right knee joint. Transcriptomic analysis combined with validation experiments were performed to explore the regulatory mechanism of excessive mechanical loading on KOA development. Results：A vertical load of 27 N resulted in calf fractures, whereas a 13 N load did not cause remarkable pathological alteration in the knee joint. Notably, applying compression at a load of 20 N for 4 weeks (w) significantly promoted the levels of pro-inflammatory factors IL-6, IL-β and TNF-α in serum and joint fluids and markedly minimized the levels of anti-inflammatory factors IL-10 and TGF-β. Immunohistochemistry, qRT-PCR and Western blot analyses suggested that the 20 N load reduced the expression of anabolism markers (ACAN and COL2A1) and escalated the expression of catabolism markers (MMP13 and ADAMTS4). KEGG analysis and validation results showed that the PIEZ1 channel, Ca2+ signaling pathway, PI3K-AKT signaling pathway and NF-κB signaling pathway were significantly activated in the 20N-4 w group. Conclusion：Continuous loading with 20 N for 4 weeks can induce significant OA-like damage to the cartilage of the right knee in rats, which might be induced through the piezo1-Ca2+/PI3K-AKT/ NF-κB signaling pathway.研究目的：建立一种新型机械加载诱导的膝骨关节炎（mechanical loading-induced knee osteoarthritis, KOA）模型，并通过转录组学（transcriptomics）探讨其调控机制。研究方法：选取斯普拉格-道利（Sprague-Dawley, SD）大鼠膝关节，分别以13 N、20 N、27 N的机械载荷加载2周或4周，构建机械诱导的KOA模型。采用免疫组织化学（immunohistochemistry, IHC）、实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）、蛋白质印迹（Western blot）、番红O-固绿染色、酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）、显微计算机断层扫描（micro-computed tomography, Micro-CT）及行为学分析，评估大鼠右侧膝关节的损伤情况。通过转录组学分析结合验证实验，探讨过度机械载荷调控KOA发生发展的机制。研究结果：27 N的垂直载荷可导致大鼠小腿骨折，而13 N载荷未引起膝关节出现明显病理改变。值得注意的是，以20 N载荷持续加压4周可显著升高血清及关节液中促炎因子IL-6、IL-1β及TNF-α的水平，并显著降低抗炎因子IL-10及TGF-β的水平。免疫组织化学、qRT-PCR及蛋白质印迹分析结果显示，20 N载荷可下调合成代谢标志物ACAN与COL2A1的表达，而上调分解代谢标志物MMP13与ADAMTS4的表达。KEGG富集分析及验证结果表明，20 N-4周组中PIEZO1通道、Ca²⁺信号通路、PI3K-AKT信号通路及NF-κB信号通路均被显著激活。研究结论：连续以20 N载荷加载4周可诱导大鼠右侧膝关节软骨出现显著的OA样损伤，该过程可能通过PIEZO1-Ca²⁺/PI3K-AKT/NF-κB信号通路介导。