Objective

Low molecular weight compounds that reduce the expression of MMP13 at the mRNA level might serve as disease-modifying osteoarthritis (OA) drugs (DMOADs). The objective of this study was to identify a candidate DMOAD that targets MMP13 expression.

Design

High-throughput screening was performed to identify compounds that suppress inflammatory cytokine-induced MMP13 expression. Ingenuity pathway analysis (IPA) using isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analysis was conducted to identify signaling pathways related to cytokines. MMP13 expression in chondrocytes was evaluated through RT-qPCR and western blotting analyses. Additionally, 10-week-old mice were subjected to destabilization of the medial meniscus (DMM) surgery to induce OA and were sacrificed 12 weeks post-surgery for pathological examination. OA was evaluated using the OARSI scoring system.

Results

Colchicine was identified as a DMOAD candidate as it inhibited inflammatory cytokine-induced MMP13 expression in vitro, and the colchicine-administered mice with DMM presented significantly lower OARSI scores (adjusted P: 0.0242, mean difference: 1.6, 95% confidence interval (CI) of difference: 0.1651–3.035) and significantly lower synovial membrane inflammation scores (adjusted P: 0.0243, mean difference: 0.6, 95% CI of difference: 0.06158–1.138) than mice with DMM. IPA further revealed that components of the Rho signaling pathways are regulated by cytokines and colchicine. IL-1β and TNF-α activate RAC1 and SRC signals, respectively, leading to the phosphorylation of PLC-γ1 and synergistic induction of MMP13 expression. Most notably, colchicine abrogates inflammatory cytokine-induced phosphorylation of PLC-γ1, leading to the induction of MMP13 expression.

Conclusions

Colchicine is a potential DMOAD candidate that inhibits MMP13 expression and consequent cartilage degradation by disrupting the SRC/RAC1-phospho-PLCγ1-Ca²⁺ signaling pathway.

中文翻译：

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秋水仙碱通过 PLC-γ1 磷酸化抑制 MMP13 表达，防止软骨退化

 客观的

在 mRNA 水平上降低MMP13表达的低分子量化合物可能作为缓解骨关节炎 (OA) 疾病的药物 (DMOAD)。本研究的目的是确定一种针对MMP13表达的候选 DMOAD。

 设计

进行高通量筛选以确定抑制炎症细胞因子诱导的MMP13表达的化合物。使用同量异序标签进行基于相对和绝对定量 (iTRAQ) 的蛋白质组分析的独创性途径分析 (IPA) 来鉴定与细胞因子相关的信号传导途径。通过 RT-qPCR 和蛋白质印迹分析评估软骨细胞中 MMP13 的表达。此外，对 10 周大的小鼠进行内侧半月板不稳定 (DMM) 手术以诱导 OA，并在术后 12 周处死小鼠进行病理检查。使用 OARSI 评分系统评估 OA。

 结果

秋水仙碱被确定为 DMOAD 候选药物，因为它在体外抑制炎症细胞因子诱导的MMP13表达，并且给予 DMM 的秋水仙碱小鼠的 OARSI 评分显着降低（调整后的 P：0.0242，平均差：1.6，95% 置信区间 (CI)）与DMM小鼠相比，差异的差异：0.1651–3.035）并且滑膜炎症评分显着降低（调整后的P：0.0243，平均差异：0.6，差异的95％CI：0.06158–1.138）。 IPA 进一步揭示 Rho 信号通路的组成部分受到细胞因子和秋水仙碱的调节。 IL-1β和TNF-α分别激活RAC1和SRC信号，导致PLC-γ1磷酸化并协同诱导MMP13表达。最值得注意的是，秋水仙碱可消除炎症细胞因子诱导的 PLC-γ1 磷酸化，从而诱导 MMP13 表达。

 结论

Colchicine 是一种潜在的 DMOAD 候选药物，可通过破坏 SRC/RAC1-磷酸-PLCγ1-Ca ²⁺信号通路来抑制 MMP13 表达和随后的软骨降解。