Antirheumatic Drug Response in Human Chondrocytes: Potential Molecular Targets to Stimulate Cartilage Regeneration. Homo sapiens

Rheumatoid arthritis (RA) leads to progressive destruction of articular structures. Despite recent progress in controlling inflammation and pain, little cartilage repair has yet been observed. This in vitro study aims to determine the role of chondrocytes in RA-related cartilage destruction and antirheumatic drug-related regenerative processes. Human chondrocytes were three-dimensionally cultured in alginate beads. To determine the RA-induced gene expression pattern, human chondrocytes were stimulated with supernatant of RA synovial fibroblasts (RASF) and normal donor synovial fibroblasts (NDSF), respectively. To examine antirheumatic drug response signatures, human chondrocytes were stimulated with supernatant of RASF that have been treated with disease-modifying antirheumatic drugs (DMARD; azathioprine, sodium aurothiomalate, chloroquine phosphate, methotrexate), non-steroidal anti-inflammatory drugs (NSAID; piroxicam, diclofenac) or steroidal anti-inflammatory drugs (SAID; methylprednisolone, prednisolone). Genome-wide expression profiling with oligonucleotide microarrays was used to determine differentially expressed genes. Real-time RT-PCR and ELISA were performed for validation of microarray data. Following antirheumatic treatment, microarray analysis disclosed a reverted expression of 94 RA-induced chondrocyte genes involved in inflammation/NF-κB signalling, cytokine/chemokine activity, immune response, proliferation/differentiation and matrix remodelling. Hierarchical clustering analysis showed that treatment of RASF with the DMARD azathioprine, gold sodium thiomalate and methotrexate resulted in chondrocyte gene expression signatures that were closely related to the “healthy” pattern. Treatment with the SAID methylprednisolone and prednisolone strongly reverted the RA-related chondrocyte gene expression, in particular the expression of genes involved in inflammation/NF-κB and cytokine/chemokine activity. The NSAID piroxicam and diclofenac and the DMARD chloroquine phosphate had only moderate to marginal effects. Pathway analysis determined major mechanisms of drug action, for example pathways of cytokine-cytokine receptor interaction, TGF-β/TLR/Jak-STAT signalling and ECM-receptor interaction were targeted. This in vitro study provides a comprehensive molecular insight into the antirheumatic drug response signatures in human chondrocytes, thereby revealing potential molecular targets, pathways and mechanisms of drug action involved in chondrocyte regeneration. Thus, the present study may contribute to the development of novel therapeutic chondro-protective compounds and strategies. Keywords: drug response Overall design: Drug-related suppression of gene expression in activated chondrocytes was determined by genome-wide microarray analysis. Chondrocytes were stimulated with supernatant of RASF and NDSF. Effect of treatment with DMARDs, NSAIDs and glucocorticoids was tested by treating RASF prior to collection of supernatant. Two RNA pools were analyzed for each group (RASF-stimulated NDSF stimulated and RASF-treated), each pool consisting of equal amounts of RNA from three different donors.

类风湿关节炎（RA）可导致关节结构进行性破坏。尽管当前在炎症与疼痛管控领域已取得一定进展，但尚未观察到明显的软骨修复现象。本体外研究旨在探究软骨细胞（chondrocytes）在RA相关软骨破坏及抗风湿药物调控的再生过程中的作用。
研究人员采用海藻酸微球对人软骨细胞进行三维培养。为探究RA诱导的基因表达谱特征，分别采用类风湿关节炎滑膜成纤维细胞（RA synovial fibroblasts, RASF）与正常供体滑膜成纤维细胞（normal donor synovial fibroblasts, NDSF）的培养上清刺激人软骨细胞。为检测抗风湿药物应答特征，研究人员将经改善病情抗风湿药（disease-modifying antirheumatic drugs, DMARD：硫唑嘌呤、金硫苹果酸钠、磷酸氯喹、甲氨蝶呤）、非甾体抗炎药（non-steroidal anti-inflammatory drugs, NSAID：吡罗昔康、双氯芬酸）或甾体抗炎药（steroidal anti-inflammatory drugs, SAID：甲泼尼龙、泼尼松龙）处理后的RASF培养上清，用于刺激人软骨细胞。
本研究采用寡核苷酸微阵列进行全基因组表达谱分析，以筛选差异表达基因，并通过实时荧光定量逆转录聚合酶链反应（real-time RT-PCR）与酶联免疫吸附试验（ELISA）对微阵列数据进行验证。
经抗风湿药物处理后，微阵列分析显示，94个RA诱导的软骨细胞基因的表达发生逆转，这些基因涉及炎症/核因子κB（NF-κB）信号通路、细胞因子/趋化因子活性、免疫应答、增殖分化及基质重塑。分层聚类分析显示，经DMARD类药物硫唑嘌呤、金硫苹果酸钠与甲氨蝶呤处理RASF后，软骨细胞的基因表达谱与"healthy"状态的表达模式高度相似。采用SAID类药物甲泼尼龙与泼尼松龙处理，则可显著逆转RA相关的软骨细胞基因表达，尤其对炎症/NF-κB通路及细胞因子/趋化因子活性相关基因的表达逆转效果显著。而NSAID类药物吡罗昔康、双氯芬酸及DMARD类药物磷酸氯喹仅表现出轻度至极微弱的调控效果。
通路分析揭示了药物作用的核心机制：例如细胞因子-细胞因子受体相互作用、转化生长因子β（TGF-β）/Toll样受体（TLR）/Janus激酶-信号转导与转录激活因子（Jak-STAT）信号通路及细胞外基质（ECM）-受体相互作用通路均为药物作用靶点。本体外研究全面解析了人软骨细胞的抗风湿药物应答特征，揭示了参与软骨细胞再生的潜在分子靶点、通路及药物作用机制。因此，本研究可为新型软骨保护治疗性化合物及治疗策略的开发提供理论依据。
关键词：药物应答
整体实验设计：本研究通过全基因组微阵列分析，探究活化软骨细胞中药物相关的基因表达抑制效应。研究将软骨细胞分别以RASF与NDSF的培养上清进行刺激；通过在收集培养上清前对RASF施加DMARDs、NSAIDs及糖皮质激素处理，以检测上述药物的调控效应。每组设置两份RNA混合样本，每份混合样本均由3名不同供体的等量RNA混合制备而成。